CN1326997C - OmpA gene encoding R. anatipestifer outer membrane protein and application method - Google Patents

Abstract

The invention provides an OmpA gene of Riemerella anatipestifer and a protein coded by the OmpA gene, and the gene and the protein can be used for producing vaccines and diagnosing the exudative septicemia (septicemia anserinum exsudaitiva) which is an economically important disease in avian species.

Description

OmpA gene encoding R. anatipestifer outer membrane protein and application method

Background of the invention

1. Technical field

The invention relates to a R. anatipestifer gene, a purified protein coded by the gene, and vaccines, diagnostic methods and products using the gene and protein. In particular, this gene (OmpA) encodes a major outer membrane protein that is highly antigenic and therefore useful in the preparation of vaccines and in diagnostic assays for serum detection.

2. Background Domain Narrative

R. anatipestifer is a Gram-negative, non-motile, non-spore-forming rod-shaped bacterium. According to the 16s rRNA gene sequence analysis, it belongs to the rRNA superfamily V of the genus Flavobacterium. It is the causative agent of septicemia anserum exsudativa, an endemic, infectious sepsis of domestic ducklings and other birds. The disease has serious consequences for the duck farming industry and is widely distributed throughout the world. Enzootic infection is usually limited to commercial ducks and turkeys, but other species of poultry such as chicks and geese are also susceptible, including wild birds such as swans. Since 1982, R. anatipestifer infection has been a persistent problem in modern meat duck intensive production processes in Singapore and other Southeast Asian countries. Mortality and morbidity ratios usually range from 10% to 30%, with a maximum mortality of 75% in infected duck farms.

Slide and tube agglutination tests isolated 21 different R. anatipestifer serotypes. Serotypes 1, 2, 3, 5, and 15 were most common in outbreaks of exudative sepsis in Anseriformes. Ducks are infected with more than one serotype of R. anatipestifer at a time, so year after year, within the same farm, immunity from one serotype antigen is greatly reduced due to annual serotype changes. The use of homologous strains or serotypes of bacteria has been found to confer a degree of resistance to infection, but the use of heterologous strains provides little protection. In the assessment of mortality and morbidity in outbreaks, a large variability in virulence between different R. anatipestifer serotypes was found. In addition, differences in toxicity for specific serotypes have been observed. However, the molecular basis for these differences is unknown, as no virulence factors for R. anatipestifer have been identified to date. So far, fibrinolytic enzymes, erythrolysins, and a lipopolysaccharide have been postulated as virulence factors of R. anatipestifer, but the presence of these factors has not been determined.

Furthermore, limited knowledge of the immunogenic factors of R. anatipestifer limits the production and diagnosis of effective vaccines. Understanding the main components of an infectious substance is important for analyzing the molecular mechanism of toxicity, studying the mode of infection, serological diagnosis of the disease, and developing effective immune protection mechanisms and eradicating the disease.

The outer membrane proteins of pathogenic bacteria are usually immunogenic, and they play an important role in the virulence and immunity of bacterial diseases. The outer membrane proteins of Gram-negative bacteria comprise a small number of major outer membrane proteins that are usually present in high copy numbers. Among them, outer membrane protein A (OmpA) is necessary to maintain the structural integrity of the cell envelope. It is also involved in bacterial conjugation and attachment, colicin uptake, porin activity and as a receptor for specific phages. Since the OmpA deletion mutant E. coli K-1 is less toxic in juvenile murine bacteremia in rats, OmpA is also thought to elicit a strong antibody response and may play an important role in toxicity. Antibodies raised against OmpA and several OmpA family proteins are usually bactericidal, opsonic, or protective.

OmpA proteins of different bacterial genera have high homology, indicating that it plays an important role in cells and may be related to toxicity. OmpA-like proteins are ubiquitous in Gram-negative bacteria (Beher et al., J. Bacteriology, 143:906-913, 1980) and have been well characterized in E. coli. In E. coli, the N-terminus consists of a long, relatively hydrophobic sequence that spans the membrane eight times. The C-terminus, located in the periplasmic space, is highly hydrophilic and may contain major immunodominant epitopes. Antibodies against OmpA are produced during enteric bacterial infection and cross-reactivity among some species has been reported. Although the OmpA protein has been described in members of the Enterobacteriaceae family and in some Gram-positive species (e.g., Bacillus subtilis), no scientific literature has described it in the Rimmeremeria anatipestipes species or in other related Flavobacteriaceae. OmpA protein in (Flavobacteriaceae) species.

Summary of the invention

The present invention relates to the cloning and isolation of the gene encoding R. anatipestifer OmpA. The present invention also relates to an OmpA protein that is almost free of contamination by other R. anatipestifer proteins. The present invention also relates to a nucleic acid comprising nucleotide bases 82-1242 of SEQ ID NO: 17 and a polypeptide having the sequence of SEQ ID NO: 18.

In other embodiments, the present invention relates to a vector comprising the R. anatipestifer OmpA gene, a host cell transformed with the vector, a method for producing the R. anatipestifer OmpA protein and immunogenic fragments generated therefrom, anti- The antibody of the peptide, the vaccine prepared by using the peptide or DNA, and the immunodiagnosis and vaccination method by using the peptide and its antibody.

Brief description of the drawings

Figure 1 provides the structure of the 2.2 kb insert in the pJWRaOmpA15 plasmid generated in Example 1.

Figure 2 depicts total cell lysate (lane 1) and purified recombinant 6xHis-OmpA-10xHis protein (lane 2) of R. anatipestifer strain CVL110/89 serotype 15 with monospecific polyclonal anti-OmpA antiserum Western blot of the reaction.

Figure 3 depicts an immunoblot as in Figure 2, but reacted with convalescent duck sera after experimental infection with R. anatipestifer serotype 15.

Figure 4 depicts the autograph of the same blot as in Figures 2 and 3 after reaction ^{with45Ca ++} .

Figure 5 is an immunoblot of total cell lysates of several different R. anatipestifer serotypes reacted with polyclonal antiserum against 6xHis-OmpA.

Detailed Description of the Preferred Embodiment

The gene OmpA encoding a major antigenic outer membrane protein of R. anatipestifer has been found, cloned and analyzed (refer to Table 3 for sequence information). The Genbank DNA sequence accession number is AF104936. The protein encoded by R. anatipestifer OmpA is the main and specific antigen of this species. The R. anatipestifer OmpA gene and protein can be used for preparing vaccines for preventing and alleviating poultry septicemia caused by R. anatipestifer and for serological diagnosis of R. anatipestifer.

The R. anatipestiferi type strains, serotype reference strains and field isolates used to isolate and clone the OmpA gene are listed in Table 1 below. Any suitable R. anatipestifer can be used to isolate the OmpA gene. All strains were grown on Columbia agar plates for 24 hours at 37°C in an atmosphere of 5% CO ₂ . Different bacterial, mammalian, plant and insect host cells can be used for gene cloning and expression. The Escherichia coli strain used for cloning and expression in this paper is as follows: XL1-Blue (Escherichia coli K-12, recA1 endA1 gyrA96 thi-1 hsdR17 supE44relA1 lac[F′pro AB lacl ^q ZΔM15 Tn10(Tet ^r )]) (Stratagene , La Jolla, California, USA), XL1-Blue MRF' (Escherichia coli K-12, Δ(mcrA)183, Δ(mcrCB-hsdSMR-mrr)173, endA1, supE44 thi-1, recA1, gyrA96, relA1, lac [F′proABlacl ^q ZΔM15, Tn10(Tet ^r )]) (Stratagene) and XLOLR (Escherichia coli K-12, Δ(mcrA)183, Δ(mcrCB-hsdSMR-mrr)173, endA1, thi-1, recA1, gyrA96, relA1, lac[F′proAB, lacl ^q ZΔM15, Tn10(Tet ^r )]λ ^R , Su ^- ) (Stratagene), BL21(DE3) (Escherichia coli B F ^- dcm omp T hadS(r _B ^- m _B ^- )galλ(DE3 T7pol)) (Stratagene). E. coli strains were grown in Luria Broth. Those skilled in the art are well familiar with other appropriate and useful methods of culturing cells, cloning and expression.

Table 1 R. anatipestifer strains

Strain a serotype ATCC1845 ^T HPRS1795 ^R HPRS2527 ^R HPRS2554 ^R HPRS2550 ^R CVL389/89 ^R DRL27179 ^R HPRS1785 ^R CCUG25055-890822 ^R DRL28020 ^R CCUG25012-890804 ^R CVL664/83 ^R CVL743/85 ^R DRLS-4801 ^R CVL977/83 ^R CVL540/86 ^R CVL30/ 90 ^R CVL110/39 ^C ND ^b 123567911111314151617181915

^a ATCC, American Type Culture Collection; HPRS, Houghton Poultry Research Station, UK; CVL, Veterinary Central Laboratory; Singapore; DRL, Duch Research Laboratories, New York, USA; CCUG, Culture Collection, University of Gothenburg, Sweden .

^T type strain

^C field isolate

^R serotype reference strain

^bUndecided

A large number of expression and cloning vectors are well known to those skilled in the art and can be used to clone and express the R. anatipestifer OmpA gene of the present invention. The cloning and expression plasmid vectors described herein are ^{pBluescriptIISK-} (Stratagene) and pBK-CMV (Stratagene). For the expression of polyhistidine-tailed proteins, selection was performed with plasmid pETHIS-1 (a high copy number expression vector derived from ColE1 containing the bla ampicillin resistance gene). T-7 polymerase-dependent expression of cloned genes was performed using specific promoters (see Table 2). This promoter can express fusion proteins with N-terminal histidine hexamers, C-terminal histidine decamers, or both (R. Segers and J. Frey, GenBank/EMBL accession number is AF012911) .

Cloning and expression vectors containing the R. anatipestifer OmpA gene preferably contain selectable markers, as is well known in the art. In the work described here, selection of transformants and maintenance of plasmids pBluescriptIISK- and pETHIS were performed by adding 100 μg/ml ampicillin to the culture medium and cloning vector pBK-CMV by adding 50 μg/ml kanamycin. Induction of cloned genes on vector pETHIS-1 in strain BL21 by addition of 0.3 mM (final concentration) isopropyl-β-D-thiogalactopyranoside (IPTG) at mid-exponential growth and continued incubation for 3 hours Express.

The oligonucleotide primers and annealing temperatures used in this study are listed in Table 2 and the attached sequence listing. Primers pETHIS1-3' and T7 paired with fragments flanking the multiple cloning site of vector pETHIS-1 and were used to identify correctly fused genes cloned in pETHIS-1. A 50 ul reaction mixture (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl ₂ , 170 uM each deoxynucleoside triphosphate, 20 pmol each primer) was used in a DNA thermal cycler (GeneAmp 9600; Perkin Elmer Cetus) , 5ng plasmid DNA or 200ng genomic DNA and 1.5U Taq polymerase (Bowlingman)) for PCR. The thermal cycling parameters used were 94°C for 30 seconds for 35 amplification cycles, corresponding to the annealing temperature for 30 seconds (Table 2) and 72°C for 1 minute.

When DNA fragments were synthesized by PCR for further cloning and expression, or DNA sequence analysis, the extension step was increased to 72°C for 2 minutes, and Taq polymerase was replaced with 2.5 UTaq/Pfu polymerase mixture (Bowringman). Additionally, an extension step of 7 min at 72°C was added at the end of the last cycle to ensure full length of the different fragments. Digoxigenin (Dig)-labeled DNA probes were generated by supplementing the PCR reaction mixture with Digoxigenin-11-dUTP (Bowringmans) at a final concentration of 50 μM.

Table 2 Oligonucleotides used for polymerase chain reaction

Primer sequence SEQ ID NO T _m RA6OMPA-LRA6OMPA-RRAOMPAH1-RAOMPAH1RAOMPAH1-RAOMPAF1RAOMPAF2RAOMPAF3RAOMPAF4RAOMPAR1RAOMPAR2RAOMPAR3RAOMPAR4PETHIS1-T7T3B-S GCAACAGGAGCATTACAAGGTACTGTCTTTCATTCTCTCTTTC CGCTATGGATCC TTATTTTCTTTTCTT TACGGATCC TTTTCTTTTCTTTTTTACTA CGCAGCCATAT GGGTTAAAGAATTTGACTGGCAAACTTCAGTAGGTGGTCTTGGTATCCAAGGGGAATAACGGTTGCCCTTGGCCGCTGCTTTAGAAGCTAGAGGCAATGAAGCTGACGCTTGCCGCCCAGGAACTGTAGGACACGTAGCTTCAGCAGAACCAACCAACGAGCCATGCTTAGAGGCCGTCTTCAAGCTCATGTTTGTAATACGACTCACTATAGGGGCGCGCAATTAACCCTCACTAAAG   12345678910111213141516 50℃50℃48℃48℃48℃55℃55℃55℃55℃55℃55℃55℃55℃55℃55℃73℃

Underlined letters designate nucleotides added to the ompA sequence to form restriction enzyme recognition sites required for cloning. T _m = annealing temperature

DNA sequence analysis was performed using the AmpliTaq FS Dye Stop Kit (Perkin Elmer Cetus) in reactions containing approximately 500 ng of plasmid DNA and 5 pmol of oligonucleotide primers. Use T3B-S, T7, PETHIS1-3' (Table 2) primers complementary to both sides of the multiple cloning site (MCS) of the vector to sequence the ends of the DNA fragments cloned into the vectors pBKCMV pETHIS-1 and pBluescriptIISK-, and the entire length of the cloned fragments Long nucleotide sequences are determined by the movement of primers. The sequences were edited using the Sequencher 3.0 program (Genecodes, Ann Arbor, Michigan, USA) to obtain contiguous sequences. Nucleotide sequences were compared by NCBI BLASTN and BLASTX programs to find related sequences. The DNA and amino acid sequences were analyzed with the PCGENE programs PROSITE and PSORT and the GCG program. The OmpA gene was cloned and expressed in E. coli. Referring to Example 1.

A phage library of genomic DNA HindIII fragment extracted from R. anatipestifer serotype 15 strain CVL110/89 was established in Escherichia coli. The library was screened with a digoxin-labeled probe derived from the OmpA sequence and an anti-digoxigenin antibody, resulting in a highly expressed clone containing the OmpA gene. The gene was amplified by PCR and cloned into an expression vector, resulting in an OmpA coding frame fused to the 5' end of the six histidine codons, and cloned into another 5' end containing the six histidine codons and the 3' on a vector containing 10 histidine codons at the end. The plasmid was transformed into E. coli cells, and the expression of the OmpA gene construct was induced with IPTG. See Table 3 for the sequence.

Table 3. The sequence of the 2.2kb fragment and its gene product in the plasmid pJFFRaOmpA15 containing R. anatipestifer OmpA gene

acagttgcta gaaacttgaa caaggcgtta gttcttgact ggcaaacttc agtaggtaat

attgataata agagaattgg aatgggtaaa gaatttatgt tgatgactgg acttggtctt

cagcttaaat ttgcaggtct tctttttggc aacgaagatg catggtttga cccttatgta

agagttggag ccaactattt gagacacgac tatacaggtc ttacgttccc tgtgactgat

agctacaatg atgtaactta cgcggggtat agcgaaaata aaccatacac tcaaggaaga

gcggatcatt ttgctttatc aacaggttta ggtacaaaca tttggttaac taagaacttt

ggtcttggta tccaagggga ttatgtttct actccagtag ataaatctag attggctaac

ttttggcaag cgtcagcttc attgaacttt agatttggta acagagataa ggataaggat

ggagtgttag ataaagacga tttatgttca gaaacaccag gtttacctga attccaaggt

tgtccagata cagatggtga cggtgttcca gataaagatg ataactgtcc agaagtagca

ggaccagtag aaaacaatgg ttgcccttgg ccagatacag acaaagatgg tgtattggat

aaagacgatg cttgtgttga tgtagcagga ccagctgaaa ataacggttg cccttggcca

gatacggata atgatggtgt gttagataaa gatgataagt gtcctacagt tcctgggctt

ccacagtacg atggatgtcc taagccacag tctgcatttg cagctgaagc aacaggagca

ttacaaggta tattcttcaa ctttaataag gcgtctatca gatctgaatc taatactaag

ttagatcaag ctgctgaggt aattaagtct tctaacggag gtactttctt agtggtaggt

catacggatg ttaagggtaa tgctaactac aacttgaaac tttctagaga aagagctgca

tctgtagtag ctgctttaga agctagagga gttaatccat ctcagttaaa atctaaaggg

gttggttctg ctgaagctac agtaccagcg tctgcttcta acgaagagag aatgaaagac

agaaaagtgg ttgtagaagc aatcagcgga tctgcttggg aagctcttca aaagtctgac

cttccagtag tgaagaaaaa agtagtaaaa aagaaaagaa aataattagt attttctaat

cttaaaaata aacgccctct tttgaaaagg gcgttttttt attgtattaa aattagtatt

tttgcacatc taaatcatat tataattatg ggacgtgcgt ttgaatatag aaaagcctct

aagatggctc gttgggataa aatggcaaaa actttttcta aaataggaaa agatattgcg

ttagcagtaa aagctggcgg tccagatcca gactctaatc cagcgttgag aagatgtata

caaaatgcta aaggggctaa tatgcctaaa gataatgtag aaagagccat taaaaaggca

agtggtgcag atgctgagaa ctatgaggag attacttacg aaggatatgg acaaggaggt

gttgcatttt ttgtagaatg tactactaat aactcaacta gaactgtggc taatgtaaga

gctatcttta ataaatttga cggtaacctt gggaagaatg gagagctttc tttcttattc

gatagaaaag ggatatttac tttagaaaaa tctttgataa acatggattg ggaagagttt

gagatggaaa tgatagacgg aggtgcggaa gatatagact ctgatgaaac agaagttatg

gtaactacgg cgtttgagga ttttgggtct ttatcacata agttagacga gctggggata

gaggttaaga atgcagaact gcaaaggata cctaatatta gtaaatctgt atcagaagag

caatttattg cgaatatgaa aatgttacaa aggtttgagg aagatgatga tgtacagaat

gtatatcata acatggaaat tacagacgag ctaatgaaga aactataaaa tagaaaaaag

gctacttaga ataggtagcc ttttttattt tttgtttacg aaaggagtaa gccattgaga

taaacttgat aatcaatgcc gacattgggt tctaaagttt tggataccga acaatatttt

tcaaaagaaa gttgagcagc cttcaaagctt (SEQ ID NO: 17)

MGKEFMLMTGLGLQLKFAGLLFGNEDAWFDPYVRVGANYLRHDYTGLTFPVTDSYNDVTYAGYSEN

KPYTQGRADHFALSTGLGTNIWLTKNFGLGIQGDYVSTPVDKSRLANFWQASASLNFRFGNRDKDK

DGVLDKDDLCSETPGLPEFQGCPDTDGDGVPDKDDNCPEVAGPVENNGCPWPDTDKDGVLDKDDAC

VDVAGPAENNGCPWPDTDNDGVLDKDDKCPTVPGLPQYDGCPKPQSAFAAEATGALQGIFFNFNKA

SIRSESNTKLDQAAEVIKSSNGGTFLVVGHTDVKGNANYNLKLSRERAASVVAALEARGVNPSQLK

SKGVGSAEATVPASASNEERMKDRKVVVEAISGSAWEALQKSDLPVVKKKVVKKKRK (SEQ ID

NO:18)

Recombinant OmpA exhibited a molecular weight similar to the size predicted from the OmpA gene nucleotide sequence, but smaller than that observed in R. anatipestifer total cell lysates. Sequence data obtained from the insert of plasmid pJFFRaOmpA15 showed that there was an OmpA protein with an estimated molecular weight of 41,696 Daltons and a pI of 4.91, and an open reading frame (ORF) of 1163 bp encoding 387 amino acids. It is preceded by the 6 nucleotide conserved sequence of the ribosome binding site (RBS) upstream of the methionine start codon ATG. There is a typical promoter sequence upstream of the RBS, comprising a -10 box (TAATAT, SFQ ID NO: 19) and a -35 box (TTGACT, SEQ ID NO: 20) preferably separated by 16 nucleotides. This interval is characteristic of the promoter recognized by the E. coli C532 RNA polymerase. Shorter fragments further upstream do not have any homology to known nucleotide or amino acid sequences.

An inverted repeat structure is located downstream of the OmpA open reading frame. This structure represents a potential transcription termination signal. see picture 1. The promoter sequence upstream of OmpA is indicated by a black triangle, while the inverted repeat structure is indicated by a hairpin structure. A portion of the open reading frame, ORFX, immediately downstream of OmpA showed similarity to the 17 kDa spore coat protein of Bacillus subtilis. This area has the following sequence:

MAKTFSKIGKDIALAVKAGGPDPDSNPALRRCIQNAKGANMPKDN

VERAIKKASGADAENYEEITYEGYGQGGVAFFVECTTNNSTRTVA

NVRAIFNKFDGNLGKNGELSFLFDRKGIFTLEKSLINMDWEEFEM

EMIDGGAEDIDSDETEVMVTTAFEDFGSLSHKLDELGIEVKNAEL

QRIPNISKSVSEEQFIANMKMLQRFEEDDDVQNVYHNMEITDELM

KKL (SEQ ID NO: 21).

Analysis of the deduced OmpA amino acid sequence (SEQ ID NO: 18) of the OmpA gene sequences of strain CVL110/89 and strain ATCC1845 revealed the identity of the OmpA protein found in other Gram-negative bacteria. The C-terminus contains a characteristic 45 amino acid OmpA-like domain. The C-terminus of the protein, especially amino acid residues 125-229, is remarkably hydrophilic. The N-terminal region (amino acids 4-22) is highly hydrophobic and bears no similarity to other outer membrane proteins. These are features shared by OmpA proteins, but the N-termini usually differ.

Amino acid residues 5-22 form an inside-to-out transmembrane helix with the N-terminus of OmpA located intracellularly. The deletion of the alanine-proline or proline-rich region of R. anatipestifer OmpA is notable because of the junction between the periplasmic and transmembrane domains of outer membrane proteins of other bacterial species Such domains are commonly found.

The sequence also revealed the presence of an EF hand calcium binding domain between amino acids 129 and 141 and two PEST regions (amino acids 139-164 and amino acids 166-187). The presence of a calcium-binding domain and a PEST region and the absence of a proline-rich region suggest that OmpA may have a role in R. anatipestifer that is distinct from that normally associated with outer membrane proteins of other species. Calbindin is known to play an important role in intracellular signaling pathways and has a broad impact on the development of diseases. The discovery of six EF-hand calcium-binding domains in R. anatipestifer is striking because this structure is not contained in other OmpA proteins.

Adjacent to the calcium-binding domain are two PEST regions. These peptide sequences are rich in proline, glutamic acid, serine, and threonine residues and form a motif that can be targeted to the disrupted protein by an unknown mechanism. PEST sequences are found in important metabolic enzymes, transcription factors, protein kinases, protein phosphatases and cyclins and are abundant in proteins that generate immunogenic peptides presented by MHC I molecules. Since the PEST sequence is hydrophilic, it may be solution exposed. Although PEST sequences are often present in extensions at the ends of proteins, they localize in the middle of R. anatipestifer OmpA. The proximity of the two PEST regions to the calcium-binding domain of the EF hand suggests that OmpA may be a preferred calpain substrate.

The lower part of Figure 1 is a diagram of different structural domains of OmpA. The domains of the inside-out transmembrane helices are represented by small square bars. The six EF-hand calcium-binding domains are represented by vertical hatched blocks. The two gray bars represent the PEST region (amino acids 139-164 and 166-187) and the black bar represents the OmpA-like domain of the C-terminal portion of the protein.

The difference between the OmpA protein of serotype 15 strain CVL110/89 and the type strain ATCC11845 is only 7 amino acids located at the outer end of the characteristic structure and clustered between amino acids 228 and 255. Using the NCBI BLAST computer program to compare the deduced amino acid sequence of the 42kDa R. anatipestifer OmpA gene with the sequence in the Swiss Prot Databank, it was found that many amino acids were identical to the outer membrane protein sequences of known specific Gram-negative bacteria, including aeruginosa The OprF porin of Pseudomonas sp. See Table 4. The OmpA-like domains share 33-37% amino acid identity and 45-59% identity, including conservative substitutions. R. anatipestifer OmpA is most similar to Bordetella avium OmpA, the causative agent of bordetella turkey disease, a highly contagious upper respiratory disease of turkeys with characteristics similar to duck plague The appearance and symptoms of Limerella in ducks and other poultry are similar. The N-terminal portion of R. anatipestifer OmpA showed no similarity to other proteins in the Swiss ProtDatabank and GenBank/EMBLI databases.

The amino acid homology (identity) percentage of table 4 Rimmerella anatipestifer OmpA and other known bacterial proteins

bacterial protein % Homology Bordetella aeruginosa OmpA (accession number Q05146) Serafia marcescens OmpA (accession number P04845) Enterobacter aerogenes OmpA (accession number P09146) Pseudomonas aeruginosa OprF (accession number P13794) 38% 28% 28% 29%

PCR analysis with RAOMPAH1-L and RAOMPAH1A-R (see Table 2) showed that there was an amplified fragment of 1177 bp in all analyzed R. anatipestiferi type strains and reference serotype strains. Genomic DNA PCR analysis of the R. anatipestifer type strain and serotype reference strains and restriction enzyme analysis of the amplified DNA fragments showed that the OmpA gene is common to R. anatipestifer. However, restriction fragment length polymorphism (RFLP) analysis of PCR products using multiple cuts with the enzyme AluI revealed some heterogeneity in the OmpA gene. Three different groups using similar restriction enzymes are indicated (see Table 5). Although the gene showed some degree of variation between the different serotypes, these small intraspecific variations, mostly silent mutations, did not affect the phenotype. The variable region was found at the 3' end of ompA corresponding to the domain in which the nucleotide differences in the OmpA sequences of strains CVL110/89 and ATCC11845 were detected. Usually, the largest variation of the OmpA gene of different strains is also in this region.

Table 5 AluI restriction enzyme group of Rimmerella anatipestifer serotype

Group R. anatipestifer serotypes or strains 123 ATCC11845 serotypes 1, 2, 3, 5, 7, 17 serotypes 6, 13, 14, 16, 19 serotypes 9, 11, 15, 18

R. anatipestiferi serotype 15 strain CVL110 was studied by immunoblotting with monospecific polyclonal antiserum against 6xHis-OmpA and convalescent sera from ducks experimentally infected with R. anatipestiferi serotype 15 strain /89 total cell lysates and immunoreactions of purified recombinant 6xHis-OmpA-10xHis. Refer to Figure 2-4. The protein was boiled in the same volume of SDS loading buffer (62.2mM Tris-HCl, pH6.8, containing 2% SDS, 5% β-mercaptoethanol, 10% glycerol, and 0.005% bromophenol blue) for 10 minutes Separated by 10% SDS-PAGE, and then transferred to nitrocellulose membrane. The membrane was immersed in calcium binding buffer (60 mM KCl, 5 mM MgCl ₂ and 10 mM imidazole hydrochloride, pH 7.2) for 10 minutes. This was followed by incubation for 20 minutes in calcium-binding buffer supplemented with 1.0 μCi of ⁴⁵ Ca ⁺⁺ per milliliter (0.02 mCi per microgram of ⁴⁵ CaCl ₂ ; Amersham, Inc.); the membrane was then washed twice for 5 minutes with deionized water and incubated at room temperature. dry. ^{Incorporated45Ca} was visualized by autoradiography.

Monospecific polyclonal antisera against the OmpA protein were raised in mice and ducklings. Mice were immunized with 330 ug of purified recombinant polyhistidine-tailed fusion protein (6xHis-OmpA) mixed 1:1 with complete Freund's adjuvant (Difco Laboratoriss, Detroit, MI) in a total volume of 200 ul. Two weeks later a booster immunization containing 330 μg of purified recombinant protein in incomplete Freund's adjuvant was used. Sera from mice were collected seven days after the second immunization.

Convalescent duck serum was generated as follows. Eight-day-old ducklings were subcutaneously immunized with 1 ml of the inactivated antigen preparation. The inactivated antigen was prepared by heating R. anatipestifer serotype 15 strain CVL110/89 bacteria (105 cfu/ml) at 100° C. for 1 hour. A second immunization was performed 11 days later with an equal volume of the antigen preparation. Sera from ducklings were collected 10 days after the second immunization and combined. After these sera were diluted 1:2000, antigens were detected by SDS-polyacrylamide gel (SDS-PAGE) and immunoblotting according to known methods. Antibodies were detected chromogenically using phosphatase-conjugated goat anti-mouse antibody (IgG/IgM; KPL #0751806) or goat anti-duck antibody (IgG/IgM; KPL #052506, purchased from Kirkegaard and Perry Inc. Gaithersburg, MD, USA) combination.

The immunoblotting of the whole cell lysate of R. anatipestifer serotype 15 strain CVL110/89 showed that there were three bands at 55kDa, 53kDa and 51kDa, respectively, which were caused by the serum reaction with anti-6xHis-OmpA (Fig. 2, first lane). Immunoblot of the recombinant protein 6xHis-OmpA-10xHis showed three low molecular weight bands (46kDa, 44kDa and 42kDa) when reacted with the same serum (Figure 2, second lane). St represents a molecular weight standard. Molecular weights are expressed in kDa.

The immunoblotting of the whole cell lysate of R. anatipestifer and the convalescent duck serum revealed the same three bands of OmpA at 55kDa, 53kDa and 51kDa, and some other immunoreactive proteins (Figure 3, the first lane) . This duck recovery period mixed serum can also react with the recombinant protein 6xHis-OmpA-10xHis, showing that there are three characteristic bands at 46kDa, 44kDa and 42kDa (Figure 3, the second swimming lane), which is consistent with the polyclonal mouse anti-OmpA antibody The response is the same. Western blotting of sera from ducks immunized with whole-cell antigens of R. anatipestifer serotype 15 strain CVL110/89 gave the same results as convalescent sera (results not shown).

In immunoblots of whole-cell lysates of this strain of R. anatipestifer and reference strains of different serotypes, all polyclonal sera reacted with the recombinant protein 6xHis-OmpA at 55kDa, 53kDa, and 51kDa. Three characteristic bands were the same as the result of R. anatipestifer serotype 15 strain CVL110/89 ( FIG. 4 ).

In Fig. 4, T represents the type strain of R. anatipestifer ATCC11845, and C represents the purified recombinant 6xHis-OmpA-10xHis protein used as a control. Numbers indicate the serotype used. For serotype 11, ^a indicates strain CCUG25055-890822, ^b indicates strain DRL28020. St is the reference of pre-stained protein molecular weight standard in all figures.

The three distinct characteristic bands at 55kDa, 53kDa and 51kDa may be due to the detection at different stages of protein processing, just like OmpA in Escherichia coli and Bordetella avium, there can be multiple bands. These bands are OmpA precursors, which contain a signal peptide, located in the cytoplasm or associated with the plasma membrane (OmpA precursor), immature OmpA processed to remove the signal peptide, present in the periplasm or attached to the inner surface of the outer membrane (imp- OmpA), and mature OmpA. The molecular weight of the recombinant OmpA protein band was approximately 10 kDa smaller than that of OmpA from endogenous R. anatipestiferus and correlated better with the calculated molecular weight of OmpA. The difference in molecular weight may be due to further post-translational modifications of OmpA in R. anatipestifer, such as the addition of glycosaminoglycan chains, while the recombinant protein lacks glycosaminoglycan chains when expressed in E. coli.

The total antigen binding experiment of ⁴⁵ Ca ⁺⁺ to R. anatipestifer showed that one major band at 51-55 kDa corresponds to three bands at 55 kDa, 53 kDa and 51 kDa of OmpA, and three minor bands at 40 kDa, 32 kDa and 30 kDa, These three bands are bound to calcium ions (Fig. 5, first lane). However, the recombinant 6xHis-OmpA-10xHis protein binds calcium ions, showing a strong band between 42-46 kDa, corresponding to the three bands in 6xHis-OmpA-10xHis seen by western blot (Fig. 5 Compare the second lane in Fig. 3 with the second lane in Fig. 3). Each of the three bands seen in the western blot was indistinguishable in the calcium blot because of the low likelihood of separation on autoradiography.

It is known that amino acids 1-177 in the OmpA protein of Escherichia coli are transmembrane segments. In contrast, OmpA of R. anatipestifer does not have a similar region at the N-terminus, but contains a short inside-out transmembrane helix of 17 amino acids. A long stretch of hydrophilic amino acids indicates that a large portion of R. anatipestifer's OmpA is exposed on the surface. This coincides with its strong antigenicity.

The structural characteristics of this protein suggest that this protein is an important antigen in the process of preparing a vaccine against R. anatipestifer. The calcium-binding domain and PEST sequences on R. anatipestifer OmpA are important because calcium-binding proteins are involved in the development of many diseases, and proteins that produce immunogenic peptides preserved in MHC-I molecules are enriched in PEST sequence. Furthermore, hyperimmune serum against recombinant OmpA protein can interact with all serotypes of OmpA and OmpA precursors.

The high immunoactivity and conservation of this protein among different serotypes make OmpA protein can be used in specific R. anatipestifer diagnostic methods and vaccines, which has the advantage of applying one antigen for cross-protection against all bacterial serotypes . This is in contrast to previous reports that outer membrane protein vaccines have little effect on gut bacteria because potentially antigenic proteins cannot reach the surface of these cells. Clearly, the abundance and surface accessibility of this new major outer membrane protein to the antigen processing pathway is beneficial for its immunogenicity.

Poultry birds susceptible to infection with R. anatipestifer can be immunized against infection by this bacterium by administering the protein according to the invention in an immunogenic composition as a so-called subunit vaccine. The subunit vaccines of the present invention may contain OmpA protein from native bacterial cells, optionally using a pharmaceutically acceptable carrier.

Those skilled in the art will recognize that immunogenic subunits or fragments of the OmpA protein may also find use in vaccines and diagnostic methods. Likewise, recombinant proteins that bind one or more epitopes can be used.

Sometimes, the ability of these proteins to produce protective immunity may be low. Small fragments are preferably conjugated to carrier molecules to increase their immunogenicity. More suitable carriers are macromolecular substances, such as natural polymers (proteins, such as keyhole limpet hemocyanin, albumin, toxins), synthetic polymers, such as polyamino acids (polylysine, polyalanine) , or micelles of amphotropic compounds, such as saponins. Alternatively these segments may be polymers, preferably linear polymers.

According to the present invention, proteins and fragments used in vaccines can be modified in vitro or in vivo if necessary, such as glycosylation, amidation, carbonylation or phosphorylation.

In addition, the vaccine may also contain an aqueous medium or a suspension containing water, usually mixed with other components to enhance activity and/or extend shelf life. These ingredients can be salts, pH buffers, stabilizers (such as skim milk or casein hydrolyzed proteins), emulsifiers, adjuvants that can enhance the immune response (such as oils, muramyl dipeptides, aluminum hydroxide, polyanions and amphiphilic substances) and preservatives.

Another form of subunit vaccine is a live vaccine. Nucleic acid sequences encoding R. anatipestifer OmpA protein or immunogenic fragments can be introduced into microorganisms (such as bacteria or viruses) by DNA recombination technology, and the recombined microorganisms can also replicate simultaneously, so the encoded nucleic acid sequences can be expressed through the inserted nucleic acid sequences. peptides and elicit an immune response in the infected host.

One embodiment of the present invention is a recombinant vector virus comprising a nucleic acid encoding the R. anatipestifer OmpA protein or an immunogenic fragment thereof, as described above, which may be present in a host cell or in a host bird infected with the recombinant vector virus Express. The invention contains the host cell or cell culture infected by the recombinant vector virus, and can produce R. anatipestifer OmpA protein by expressing the nucleic acid sequence.

As in US Pat. No. 5,843,722, incorporated herein by reference, the nucleic acid sequence of the present invention can be introduced into the genome of a vector virus using well-known in vivo homologous recombination techniques.

First, the DNA fragment corresponding to the insertion region of the vector genome, that is, the region that can be used for the incorporation of heterologous sequences without affecting the essential functions of the vector, such as those necessary for infection or replication, can be obtained according to known DNA recombination techniques. Inserted into the cloning vector. Insertion regions are found in many kinds of microorganisms (such as EP 80806, EP110385, EP83286, EP314569, WO 88/02022, WO 88/07088, US Patent No. 4769330 and US Patent No 4722848).

Deletions can be introduced into the insertion region of the recombinant vector molecule obtained in the first step if necessary. For example, the recombinant vector molecule obtained in the first step can be appropriately digested or treated with restriction enzymes by exonuclease III.

Then insert the nucleic acid sequence of the gene or the immunogenic fragment encoding R. anatipestifer OmpA protein into the insertion region in the first step recombinant vector, or into the DNA deletion region in the above-mentioned vector. The length of the DNA sequence in the insertion region should be appropriate to facilitate homologous recombination with the vector genome. Appropriate cells can then be infected with wild-type vector virus, or transformed with vector genomic DNA in the presence of a recombinant vector containing insert sequences flanked by appropriate vector DNA sequences, thereby creating a gap between the corresponding region of the recombinant vector and the vector genome. recombination occurred. The progeny of the recombinant vector can now be prepared by cell culture and screened genotype or phenotype, such as by hybridization, or by detecting the expression of co-integrated marker genes, or the antigenic duck plague limer expressed by the recombinant vector Immunological detection of Bacillus OmpA polypeptide.

Subsequently, this recombinant vector can be used to immunize birds, which can then be maintained for a period of time, or can even be replicated in vaccinated animals, expressing R. anatipestifer OmpA protein or immunogenic fragments in vivo, and as a result can stimulate Vaccines the animal's immune system. Suitable vectors for incorporation of the nucleic acid sequences of the invention may be derived from viruses such as poxviruses, e.g. vaccine viruses (EP 110385, EP83286, US Patent No 4769330 and US Patent No 4722848) or fowl pox virus (WO 88/02022), herpes virus Such as HVT (WO 88/07088) or Marek's disease virus, adenovirus or influenza virus, or bacteria such as E. coli or some special Salmonella. Using such recombinant microorganisms, in order to expose the R. anatipestifer OmpA protein or immunogenic fragments as surface antigens, the protein or fragment can be expressed as a fusion protein with the host OMP protein or e.g. E. coli pili protein , or by artificially synthesizing a signal peptide or anchor sequence that can be recognized by microorganisms.

The preparation of the vector vaccine of the present invention can be by cultivating recombinant bacteria or host cells infected with the recombinant vector comprising the nucleic acid sequence of the present invention, and then the recombinant bacteria or cells containing the vector and/or recombinant vector viruses growing in the cells can be collected, optionally The ground is substantially purified and optionally lyophilized to make a vaccine.

The host cells transformed with the recombinant vector of the present invention can also be cultured under conditions favorable for the expression of nucleic acid sequences encoding R. anatipestifer OmpA protein or immunogenic fragments. Crude cultures, host cell lysates, or host cell extracts may be used to prepare vaccines, although in another embodiment, according to the invention, more pure polypeptides may be used to prepare vaccines according to specific needs. In order to purify the produced polypeptide, the host cells transformed with the recombinant vector of the present invention are cultured in an appropriate volume, and the produced polypeptide is isolated from the cells, if it is a secreted protein, it can also be isolated from the culture medium. The polypeptides secreted into the medium can be separated and purified by standard technical methods such as salt separation, centrifugation, ultrafiltration, chromatography, gel filtration or immunoaffinity chromatography, while the intracellular polypeptides can be purified by the following methods: Separation method: first collect the above cells, and then destroy these cells, such as by ultrasonic or other mechanical disruption methods such as French extrusion method, and then separate the polypeptide from other intracellular components, and form the polypeptide into a vaccine. Cell disruption can also be achieved by digestion with chemical methods (such as EDTA or detergents such as TritonX114) or enzymatic methods such as lysozyme.

Antibodies or antisera directed against the polypeptides of the present invention can be used in passive immunotherapy, diagnostic immunoassays and synthesis of anti-idiotypic antibodies.

As mentioned above, R. anatipestifer OmpA protein or immunogenic fragments can be used to prepare polyclonal, specific or monoclonal antibodies. The techniques used to prepare and process polyclonal antibody sera are well known to those skilled in the art (eg, Mayer and Walter, eds., Methods in Cell and Molecular Biology, Immunochemistry, Academic Press, London, 1987). Antibodies purified from polyspecific antisera have affinity for monospecific immunogens by a modification of the method of Hall et al. (Nature, 311, 379-387, 1984). As used herein, an antibody having monospecificity for an immunogen is defined as a single antibody type or a multiple antibody type having homogeneous binding properties for related antigens. Homospecific binding as used herein means that the antibody type can bind to a specific antigen or epitope.

The monoclonal antibody reactive with R. anatipestifer OmpA protein or its immunogenic fragments can be prepared by immunizing normal mice by techniques known in the art (Kohler and Milstein, Nature, 256, 495-497, 1975) .

Anti-idiotypic antibodies are immunoglobulins that carry the "endogenous image" of the pathogen antigen against which protective resistance is desired, and can be used as immunogens in vaccines (Dreesman et al., J. Infectious Diseases, 151, 761, 1985 ). Technical methods for generating anti-idiotypic antibodies are well known to those skilled in the art (MacNamara, et al., Science, 226, 1325, 1984).

The vaccine of the present invention can be used in the traditional activated immunization method: one or more times in an amount compatible with the dosage form that can play a prophylactic effect, that is, the amount of the immunizing antigen or the amount of the recombinant microorganism that can express the desired antigen will be Induction of poultry immunity to virulent Rimmerella anatipestifer. Herein, "immunity" refers to a level of protection against infection, ie higher than that of a non-vaccinated flock of birds.

For live viral vector vaccines, the dose may range between 10 ⁵ and 10 ⁹ CFU (colony forming units) per bird. A typical subunit vaccine of the invention contains 0.01 to 1 mg of immunogenic protein. These vaccines can be administered intradermally, subcutaneously, intramuscularly, peritoneally, intravenously, orally, or intraanally.

Novel R. anatipestifer OmpA proteins and immunogenic fragments, antibodies thereof and nucleic acids encoding such proteins and fragments can be used in diagnostic methods and kits for detecting the presence of R. anatipestifer in biological fluids. These diagnostic methods and kits can be in conventional formats, such as immunoassays for detection of antigens and antibodies, and optionally hybridization for nucleic acid detection after amplification.

Immunological detection techniques are based on the ability of antigenic substances to form complexes with one or more antibodies. One of the components of the complex can be labeled to facilitate the separation, detection and/or quantification of the labeled antigen or antibody complex from the uncomplexed labeled antigen or antibody.

In competitive immunoassays, antigenic material in a liquid sample, such as here the R. anatipestifer OmpA protein or an immunogenic fragment, competes with a known amount of labeled antigen for limited antibody-antigen binding sites. The amount of labeled antigen bound to the antibody is inversely related to the amount of antigen in the specimen.

In immunometric or noncompetitive assays, labeled antibody can be used in place of labeled antigen, and the amount of labeled antibody bound to the insoluble disaggregated complex is directly proportional to the amount of antigen in the fluid sample.

Competitive immunoassays and immunometric methods can be based on one of two basic approaches: heterogeneous assays and homogeneous assays. In competitive immunoassays, these two modalities involve the formation of a reaction mixture that includes a minimum of three reaction components: a known amount of analyte or analyte conjugate, an analyte-binding reagent, and a reagent suspected to contain the analyte. Sample liquid medium.

Heterogeneous or biphasic detection methods involving solid and liquid phases, involving the immobilization of one member of the analyte/analyte binder pair on a solid phase and the other member with a label and tracer such as an enzyme or radionuclide prime conjugated. The labeled analyte or conjugate competes with the analyte suspected of being present in the liquid medium of the sample for a limited number of analyte binding sites.

Homogeneous assays eliminate separation and pre-incubation steps by measuring the amount of enzyme activity in the analyte-enzyme conjugate, rather than the amount of analyte conjugate bound to the support. The presence of the analyte in the sample fluid can be determined by an increase in the activity of the enzyme conjugate (US Pat. Nos. 4,067,774 and 3,817,837).

When the added analyte conjugate or sample analyte binds to the analyte binding agent upon incubation, the analyte becomes insoluble. The liquid and insoluble phases are then separated and the analytes in each phase are quantified. After incubation, separation, the amount of analyte in the sample fluid medium can be determined from the amount of insoluble analyte conjugate. Since the amount of bound analyte conjugate is inversely proportional to the amount of analyte in the sample, the greater the amount of sample analyte in the sample fluid, the smaller the amount of analyte conjugate in an insoluble state.

Any of the various quantitative or qualitative immunoassays can be used to diagnose R. anatipestifer infection in birds.

Immunization kits for the immunological detection of R. anatipestifer in biological fluids generally comprise (I) antibodies to the R. anatipestifer OmpA protein or immunogenic fragments thereof, and (II) labeled R. anatipestiferi Bacterial OmpA protein or antigenic fragments (that is, fragments that can form immune complexes with antibodies). In such kits, the antibodies can be monoclonal, monospecific and polyclonal antibodies as described above. In the case of polyclonal antibodies, it is advantageous to select such antibodies to minimize concomitant interfering reactions and false positive results. The motifs used to label the R. anatipestifer OmpA protein or antigenic fragments can be any conventional markers used in heterogeneous or simultaneous assay methods. For example, it may be radioactive atoms, enzymes, fluorescent substances, ligands, luminescent substances or the like. The kit may optionally contain unlabeled R. anatipestifer OmpA protein or an antigenic fragment thereof as a control.

In another embodiment, the immunoassay kit for detection of R. anatipestifer in biological fluids may contain two or more antibodies against R. anatipestifer OmpA protein or antigenic fragments thereof. Wherein at least one antibody is labeled as above, alternatively, the kit may contain or be used in combination with a labeled reporter antibody that can immunoreact with an antibody to the R. anatipestifer OmpA protein or antigenic fragment. As mentioned above, the kit can optionally contain R. anatipestifer OmpA protein or antigenic fragment as a control.

The kit for detecting R. anatipestifer OmpA gene (or RNA) contains a detection nucleic acid, which can hybridize with the sequence in the R. anatipestifer OmpA gene under stringent hybridization conditions. "Stringent hybridization conditions" include: (1) washing at low ionic strength and high temperature, such as 0.015M NaCl/0.0015M sodium tartrate/0.1% sodium dodecyl sulfate, 50°C, or (2) during hybridization Use a denaturant such as formamide, such as 50% (v/v) formamide with 0.1% BSA/0.1% Ficoll/polyvinylpyrrolidone/50mM sodium phosphate buffer containing 750mM NaCl, 75mM sodium citrate, pH 6.5 , 42°C. Another example is the use of 50% formamide, 5X SC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon essence DNA (50mug/ml), 0.1% SDS, 10% dextran sulfate, 42°C, washed with 0.2XSSC and 0.1% SDS at 42°C. In another case, "stringent hybridization conditions" have more than 80% homology with the R. anatipestifer OmpA gene, preferably more than 90% homology, and most preferably more than 95% homology nucleic acid will be with The conditions under which the gene hybridizes.

The nucleic acid may be labeled, such as any of the labeling methods described above, or alternatively, a labeled reporter nucleic acid that binds to the detection nucleic acid may be used.

Preferably, the kit for detecting R. anatipestifer gene or RNA contains reagents that can amplify the target sequence in the gene before detection. These reagents include primers or probes for PCR, LCR or other well-known methods of nucleic acid amplification that flank the target sequence.

In any of the foregoing kits, one or more reagents may be immobilized on a solid support as is well known to those skilled in the art. These supports include, for example, the walls or bottoms of test tubes or the wells of microtiter plates, latex particles, test strips, water-absorbing materials, magnetic particles, slides, plates, and the like.

The invention includes a diagnostic method and a kit for detecting R. anatipestifer OmpA nucleic acid, protein and antibody.

The present invention is further illustrated by, but not limited to, the following examples.

Implementation column

Preparation and screening of embodiment 1 Rimmeria anatipestifer gene library

The gene DNA was extracted from R. anatipestifer serotype 15 strain CVL110/89 by rapid guanidine isothiocyanate method. This strain caused a severe outbreak with 25% mortality in duck farms in Singapore. Partially digested (Sau3A) genomic DNA was cloned into BamHI digested and dephosphorylated phage λZAP Express ^™ vector (Stratagene) and packaged with Gigapack II Gold Packaging Extract (Stratagene). The gene library was plated with the Escherichia coli XL1-Blue MRF strain according to standard method steps. The ligated products were transformed into XL1-Blue cells by the calcium chloride method. Following infection with helper phage M13, kanamycin-resistant clones selected from plaques were excised in vivo on the plasmid vector pBK-CMV following the manufacturer's instructions. A library of HindIII-digested fragments from genomic DNA of R. anatipestifer serotype 15 strain CVL110/89 can be constructed using the pBluescriptIISK plasmid.

The digoxin-labeled probe that is used to detect R. anatipestifer OmpA gene segment can be prepared by PCR, is template with plasmid pJFFRA6 DNA, and primer RA60MPA-L and RA60MPA-R (see Table 2) are from OmpA Clone fragment. This digoxin-labeled probe can be used to screen a HindIII-digested genomic DNA gene library based on a plasmid vector. Gene libraries can be screened with pooled sera from convalescent ducks infected with R. anatipestifer serotype 15, as described above.

For screening of recombinant E. coli clones, colonies were transferred from solid medium plates to nitrocellulose membranes (Schleicher-Schuell, Dasswl, Germany). Recombinants were lysed in situ and DNA was cross-linked to the membrane by drying the filter at 80°C for 2 hours. Then use hybridization buffer (750mM NaCl, 75mM trisodium citrate, 0.1% N-lauryl sarcosine, 0.02% SDS and 1% blocking agent (Bowringman, product number: #10961789), pH7.7, 68 ℃, pre-incubated for 2 hours. Then in hybridization buffer (750mM NaCl, 75mM trisodium citrate, 0.1% N-lauryl sarcosine, 0.02% SDS) containing 1 microgram of digoxin-labeled OmpA gene probe Hybridization at 68°C for 18 hours. The membrane was washed twice with a buffer solution containing 30mM NaCl, 3mM trisodium citrate, 0.1% SDS, pH7.7 at 68°C for 15 minutes each time. According to the instructions, label with phosphatase Digoxigenin-labeled DNA probes were detected with an anti-digoxigenin antibody (Bowringmans).

Clones with strong immune responses were collected and transformed into plasmid pJFFRA6. This clone contains a 859bp partial open reading frame which shows significant similarity to the outer membrane protein A of B. avium. The corresponding gene in the plasmid pJFFRA6 was therefore named OmpA. Plasmid pJFFRaOmpA15 containing the 2.2 kb HindIII fragment was further analyzed according to methods well known in the art including sequencing (see Table 3).

Example 2 Expression and antigen purification of OmpA fusion protein

In order to obtain purified recombinant R. anatipestifer OmpA antigen, in vitro with Taq/Pwo polymerase mixture, oligonucleotide primers RAOMPH1-L and RAOMPAH1-R (see Table 2), to R. anatipestifer serotype Genomic DNA of 15 strains CVL110/89 was used as template to amplify OmpA gene. The PCR product was purified and digested with NdeI and BamHI, and then cloned into the expression vector pETHIS-1 to obtain plasmid pJFFOMPA, which resulted in the in-frame fusion of 6 histidine codons at the 5' end of OmpA. The second plasmid, pJFFOMP13, was constructed in a similar manner using primers RAOMPAH1-L and RAOMPAH1A-R. The resulting sequence can generate an OmpA coding frame with 6 histidine codons fused at the 5' end of OmpA and 10 histidine codons fused at the 3' end. The cloned gene constructs in plasmids pJFFOMPA and pJFFOMP13 could be confirmed by DNA sequence analysis.

Plasmids pJFFOMPA and pJFFOMP13 were transformed into E. coli host strain BL21 (DE3), and E. coli carrying corresponding plasmids containing OmpA fusion constructs were induced by IPTG to express OmpA fusion proteins (6XHis-OmpA and 6XHis-OmpA-10XHis). After induction, cells were collected, washed with TES buffer (10 mM Tris, 1 mM EDTA, 0.8% NaCl, pH 8.0), and extracted with 50 mM phosphate buffer pH 8.0 containing 6M guanidine hydrochloride. Fusion proteins were purified from extracts of these cells by Ni ²⁺ -chelate affinity chromatography (Qiagen, GmbH, Hilden, Germany) following the manufacturer's instructions. The bound polyhistidine-tailed fusion protein was eluted by slowly lowering the pH of 50 mM phosphate buffer containing 300 mM NaCl and 6 M guanidine hydrochloride from 8.0 to 4.5. The fusion protein was eluted at pH 5.0. The fusion protein was then dialyzed against 50 mM phosphate buffer pH 8.0 containing 300 mM NaCl. A second plasmid, pJFFOMP17, was constructed separately as pJFFOMP13. Its gene product, 6XHis-OmpA-10XHis, exhibited the same characteristics as pJFFOMP13 obtained in this study.

sequence listing

<110> Joachim Frey

Sumati Subramaniam

School of Molecular Agricultural Biology

<120> OMPA gene encoding R. anatipestifer outer membrane protein and application method

<130>2577-113PCT

<140> pCT/SG99/00075

<141>1999-07-14

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gagatggaaa tgatagacgg aggtgcggaa gatatagact ctgatgaaac agaagttatg 1860

gtaactacgg cgtttgagga ttttgggtct ttatcacata agttagacga gctggggata 1920

gaggttaaga atgcagaact gcaaaggata cctaatatta gtaaatctgt atcagaagag 1980

caatttattg cgaatatgaa aatgttacaa aggtttgagg aagatgatga tgtacagaat 2040

gtatatcata acatggaaat tacagacgag ctaatgaaga aactataaaa tagaaaaaag 2100

gctacttaga ataggtagcc ttttttattt tttgtttacg aaaggagtaa gccattgaga 2160

taaacttgat aatcaatgcc gacattgggt tctaaagttt tggataccga acaatatttt 2220

tcaaaagaaa gttgagcagc cttcaaagct t 2251

<210>18

<211>388

<212>PRT

<213> Rimmerella anatipestifer

<400>18

Met Gly Lys Glu Phe Met Leu Met Thr Gly Leu Gly Leu Gln Leu Lys

1 5 5 10 15

Phe Ala Gly Leu Leu Phe Gly Asn Glu Asp Ala Trp Phe Asp Pro Tyr

20 25 30

Val Arg Val Gly Ala Asn Tyr Leu Arg His Asp Tyr Thr Gly Leu Thr

35 40 45

Phe Pro Val Thr Asp Ser Tyr Asn Asp Val Thr Tyr Ala Gly Tyr Ser

50 55 60

Glu Asn Lys Pro Tyr Thr Gln Gly Arg Ala Asp Hls Phe Ala Leu Ser

65 70 75 80

Thr Gly Ser Leu Gly Thr Asn Ile Trp Leu Thr Lys Asn Phe Gly Leu

85 90 95

Gly Ile Gln Gly Asp Tyr Val Ser Thr Pro Val Asp Lys Ser Arg Leu

100 105 110

Ala Asn Phe Trp Gln Ala Ser Ala Ser Leu Asn Phe Arg Phe Gly Asn

115 120 125

Arg Asp Lys Asp Lys Asp Gly Val Leu Asp Lys Asp Asp Leu Cys Ser

130 135 140

Glu Thr Pro Gly Leu Pro Glu Phe Gln Gly Cys Pro Asp Thr Asp Gly

145 150 155 160

Asp Gly Val Pro Asp Lys Asp Asp Asn Cys Pro Glu Val Ala Gly Pro

165 170 175

Val Glu Asn Asn Gly Cys Pro Trp Pro Asp Thr Asp Lys Asp Gly Val

180 185 190

Leu Asp Lys Asp Asp Ala Cys Val Asp Val Ala Gly Pro Ala Glu Asn

195 200 205

Asn Gly Cys Pro Trp Pro Asp Thr Asp Asn Asp Gly Val Leu Asp Lys

210 215 220

Asp Asp Lys Cys Pro Thr Val Pro Gly Leu Pro Gln Tyr Asp Gly Cys

225 230 235 240

Pro Lys Pro Gln Ser Ala Phe Ala Ala Glu Ala Thr Gly Ala Leu Gln

245 250 255

Gly Ile Phe Phe Asn Phe Asn Lys Ala Ser Ile Arg Ser Glu Ser Asn

260 265 270

Thr Lys Leu Asp Gln Ala Ala Glu Val Ile Lys Ser Ser Asn Gly Gly

275 280 285

Thr Phe Leu Val Val Gly His Thr Asp Val Lys Gly Asn Ala Asn Tyr

290 295 300

Asn Leu Lys Leu Ser Arg Glu Arg Ala Ala Ser Val Val Ala Ala Leu

305 310 315 320

Glu Ala Arg Gly Val Asn Pro Ser Gln Leu Lys Ser Lys Gly Val Gly

325 330 335

Ser Ala Glu Ala Thr Val Pro Ala Ser Ala Ser Asn Glu Glu Arg Met

340 345 350

Lys Asp Arg Lys Val Val Val Glu Ala Ile Ser Gly Ser Ala Trp Glu

355 360 365

Ala Leu Gln Lys Ser Asp Leu Pro Val Val Lys Lys Lys Val Val Lys

370 375 380

Lys Lys Arg Lys

385

<210>19

<211>6

<212>DNA

<213> Rimmerella anatipestifer

<400>19

taatat 6

<210>20

<211>6

<212>DNA

<213> Rimmerella anatipestifer

<400>20

ttgact 6

<210>21

<211>228

<212>PRT

<213> Rimmerella anatipestifer

<400>21

Met Ala Lys Thr Phe Ser Lys Ile Gly Lys Asp Ile Ala Leu Ala Val

1 5 10 15

Lys Ala Gly Gly Pro Asp Pro Asp Ser Asn Pro Ala Leu Arg Arg Cys

20 25 30

Ile Gln Asn Ala Lys Gly Ala Asn Met Pro Lys Asp Asn Val Glu Arg

35 40 45

Ala Ile Lys Lys Ala Ser Gly Ala Asp Ala Glu Asn Tyr Glu Glu Ile

50 55 60

Thr Tyr Glu Gly Tyr Gly Gln Gly Gly Val Ala Phe Phe Val Glu Cys

65 70 75 80

Thr Thr Asn Asn Ser Thr Arg Thr Val Ala Asn Val Arg Ala Ile Phe

85 90 95

Asn Lys Phe Asp Gly Asn Leu Gly Lys Asn Gly Glu Leu Ser Phe Leu

100 105 110

Phe Asp Arg Lys Gly Ile Phe Thr Leu Glu Lys Ser Leu Ile Asn Met

115 120 125

Asp Trp Glu Glu Phe Glu Met Glu Met Ile Asp Gly Gly Ala Glu Asp

130 135 140

Ile Asp Ser Asp Glu Thr Glu Val Met Val Thr Thr Ala Phe Glu Asp

145 150 155 160

Phe Gly Ser Leu Ser His Lys Leu Asp Glu Leu Gly Ile Glu Val Lys

165 170 175

Asn Ala Glu Leu Gln Arg Ile Pro Asn Ile Ser Lys Ser Val Ser Glu

180 185 190

Glu Gln Phe Ile Ala Asn Met Lys Met Leu Gln Arg Phe Glu Glu Asp

195 200 205

Asp Asp Val Gln Asn Val Tyr His Asn Met Glu Ile Thr Asp Glu Leu

210 215 220

Met Lys Lys Leu

225

Claims (14)

1. a nucleic acid, the Rimmerella anatipestifer OmpA albumen of its coding SEQ ID NO:18. 2. A nucleic acid consisting of nucleotide bases 82-1242 in SEQ ID No: 17. 3. A vector comprising the nucleic acid of claim 1 or 2 and a replicon capable of replicating in a host cell. 4. An expression vector comprising the nucleic acid of claim 1 or 2, wherein the coding sequence of the nucleic acid is operably linked to regulatory sequences that can direct the expression of the coding sequence in a host cell. 5. A host cell transformed with the vector of claim 3. 6. A host cell transformed with the vector of claim 4. 7. An isolated polypeptide consisting of SEQ ID No: 18. 8. A fusion protein comprising the polypeptide of claim 7. 9. A method of producing the polypeptide of claim 7, comprising: a) transforming the host cell with an expression vector encoding said polypeptide operably linked to a regulatory sequence directing expression of said polypeptide; b) cultivating the above-mentioned host cells under suitable conditions to produce the polypeptide; c) recovering said polypeptide; and optionally d) purifying said polypeptide. 10. An antibody capable of specifically binding to the polypeptide of claim 7. 11. A vaccine composition for stimulating an immune response against R. anatipestifer in members of avian species, comprising a peptide having the sequence of SEQ ID NO: 18, comprising bases 82-1242 of SEQ ID NO: 17 The nucleic acid of base, or the antibody that specifically binds to the peptide having the sequence of SEQ ID NO:18. 12. The vaccine composition according to claim 11, wherein the peptide is a synthetic peptide. 13. A vaccine composition for stimulating an immune response against R. anatipestifer in members of an avian species, comprising a viral vector that can replicate in an inoculated host and can be replicated in said host In vivo expression of nucleic acid encoding the R. anatipestifer OmpA protein of SEQ ID NO: 18. 14. Use of an antibody that specifically binds the fusion protein of claim 8 for the preparation of a composition for the diagnosis of R. anatipestifer infection in a member of an avian species.

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