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WO2008037123A1 - Densovirus periplanata fuliginosa recombinant et utilisation correspondante - Google Patents

Densovirus periplanata fuliginosa recombinant et utilisation correspondante Download PDF

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Publication number
WO2008037123A1
WO2008037123A1 PCT/CN2006/002580 CN2006002580W WO2008037123A1 WO 2008037123 A1 WO2008037123 A1 WO 2008037123A1 CN 2006002580 W CN2006002580 W CN 2006002580W WO 2008037123 A1 WO2008037123 A1 WO 2008037123A1
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gene
black
virus
scorpion
breasted
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Chinese (zh)
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Yuanyang Hu
Hong Jiang
Jiamin Zhang
Xu Cao
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GREEN LIFE LABORATORY Ltd
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GREEN LIFE LABORATORY Ltd
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Priority to CN2006800559230A priority Critical patent/CN101522894B/zh
Priority to PCT/CN2006/002580 priority patent/WO2008037123A1/fr
Publication of WO2008037123A1 publication Critical patent/WO2008037123A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43522—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from scorpions
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/40—Viruses, e.g. bacteriophages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to biocide agents. Specifically, the present invention relates to the construction of a recombinant black-necked scorpion nucleus virus recombined with the prion insect toxin gene BmKIT1 and a cockroach insecticide containing the recombinant black-breasted scorpion scorpion nucleus virus.
  • Background of the Invention ⁇ is an important urban pest that is distributed throughout the world. Periplaneta fuliginosa is a dominant species of indoor carp in the Yangtze River and the southern provinces of China.
  • black-breasted cockroach It is also a common species in the southeastern United States, Japan, Southeast Asia and other countries and regions. There are three types of indoor cockroaches: black-breasted cockroach, German cockroach, and American cockroach. The black-breasted cockroach is the dominant species. In addition to stealing food, contaminating food, damaging clothing, books, and destroying computer and other sophisticated instruments, black-chested cockroaches are also the main cause of human asthma and allergies. Due to the harm to people, the Chinese government listed one of the four common habits in urban areas, including black-chested scorpion, in 1978, and called for the prevention and treatment of cockroaches throughout the year.
  • Periplaneta fuliginosa Densovirus was first isolated and identified by Professor Hu Yuanyang of Wuhan University in 1990. The morphological structure, nucleic acid properties, structural peptides, serum of the Institute of Insect Virology, School of Life Sciences, Wuhan University. Intensive research on learning, tissue toxicology, safety, virus recovery, in vitro fine-package culture, etc., and construction of the pfDNV whole genome cloning vector, complete genome-wide nucleotide sequence determination (GeneBank AFl 92260 black-chested big cockroach fDNV belongs to the subfamily of the virion virus of the parvovirus family, and the virion is a typical globular tetrahedron.
  • the viral genome is a single-stranded DNA molecule with a molecular weight of 5.5 kb.
  • domestic and foreign cockroaches rely mainly on chemical pesticides, lacking Environmentally compatible bio-killing agent. According to the statistics of the Ministry of Agriculture's Pesticide Control Institute in 2000, it obtained the Chinese Pesticide Registration Certificate, and there are 75 kinds of foreign pesticides with the control of cockroach.
  • domestic products only one of which is domestic. It is biologically derived, and the rest are chemical pesticides. So many chemical insecticides are used indoors. Agents must be harmful to human health.
  • Insect-specific scorpion neurotoxin is a type of toxin stored in the scorpion venom, which is a weapon for scorpion to prey and defend against enemies. Studies have shown that this indole toxin is a small molecule basic polypeptide consisting of 30-70 amino acid residues and containing 3 pairs or 4 pairs of double bonds. They can selectively act on Na + or K + ion channels on insect cell membranes, causing insects to die from nerve numbness and being harmless or less toxic to mammals and crustaceans.
  • AalT gene has been successfully expressed in BmNPV, AcMNPV, TnNPV and RoM PV, AaHT and LqhIT 2 genes in AcMNPV, and the expression products have insecticidal effects on various pests such as silkworm, cotton worm, cotton bollworm and rice borer. active.
  • One object of the present invention is to provide an expression vector for the recombinant R. striata genome.
  • the expression vector comprises inserting a genome of the black chestnut cockroach in a PUC119 plasmid to form a PUCA plasmid, and one or more exogenous genes inserted between the Blgll cleavage site of the PUCA plasmid and ITR2.
  • Another object of the present invention is to provide a recombinant concentrated nuclear virus. One or more exogenous genes are inserted into the genome of the recombinant nuclear virus.
  • Still another object of the present invention is to provide a recombinant black-breasted scorpion nucleus virus in which the scorpion insect toxin gene BmKIT1 is inserted. It is still another object of the present invention to provide a biological insecticide comprising a recombinant concentrated nuclear virus. It is still another object of the present invention to provide a biocide comprising a recombinant black-breasted scorpion nucleus virus. It is still another object of the present invention to provide a biocide comprising a recombinant black-breasted scorpion nucleus virus in which one or more exogenous genes are inserted.
  • FIG. 1 is a black-breasted scorpion-rich nuclear virus expression vector PfDNV-GFP.
  • the foreign gene is inserted between the Eco72I and Xhol sites and is regulated by the P97 promoter located in the ITR2 sequence.
  • the single line portion is the pUC119 sequence
  • the double line portion is the PfDNV sequence.
  • the nucleotide fragment of the PfDNV genome indicated by the arrow is replaced by the GFP gene, and X, E represent the 01, cleavage site, respectively.
  • the solid part is the terminal inverted repeat (ITR) of PfDNV.
  • ITR is a two-terminal regulatory sequence of the PfDNV genome, and its ITR2 contains a P97 promoter and a non-coding sequence to regulate the transcription of foreign genes.
  • the foreign gene was inserted at the 721 restriction site and fused to the 5' end of the GFP gene.
  • the GFP gene serves as an indicator gene for expression of a foreign gene.
  • Fig. 2 Recombinant plasmid of recombinant black-breasted scorpion scorpion nucleus virus containing pupae-specific neurotoxin-promoting gene pUCA + .
  • black-breasted scorpion nucleus virus refers to the representative species of the genus Pefudensovirus, a subfamily of the genus Parvodensovirus, named by the International Committee for Virology (ICTV) in 2004. Fuliginosa densovirus (PfDNV). The virus was isolated and identified by the School of Life Sciences of Wuhan University, and the genome sequence registration number: GeneBank Accession No. AB028936.
  • recombinant black-breasted scorpion nucleus virus refers to the use of genetic engineering methods such as insertion of foreign genes, deletion and modification of the virus's own sequence to engineer the viral genome to enhance the virus's insecticidal virulence and expand insecticidal activity.
  • Pu a black-breasted scorpion-rich nucleus virus containing such a modified genome is called a recombinant black-breasted scorpion nucleus virus.
  • the recombinant black-breasted scorpion scorpion nucleus virus is deposited in China's typical biological collection center is CCTCC.
  • wild-type black-breasted scorpion scorpion nucleus virus which refers to a gene sequence that is substantially identical to the black-breasted scorpion-rich nuclear virus gene sequence (GeneBank Accession No. AB028936) in a natural population.
  • PUC119 refers to a plasmid vector produced by Takara Bio, Japan, which is designed for single-strand, no longer than 7 kb DNA fragment clones. The vector sequence is named GeneBank Accession No. U07650 o
  • PUCA refers to the cloning vector constructed by cloning the whole genome of the wild-type P. striata nucleus virus into the multiple cloning site of PUC119.
  • PfDNV-GFP refers to an expression vector constructed by inserting a green fluorescent protein gene GFP into a PUCA plasmid.
  • the vector is deposited under the China National Collection of Microorganisms at CCTCC M 206092.
  • PUCA+ refers to the transformation of the genome of the black-breasted scorpion-rich nucleus virus in PUCA, the excision of the sequence between the Xhol and EcoR72I cleavage sites, the insertion of the scorpion insect toxin gene BmKITl and the green fluorescent protein gene GFP. Recombinant vector.
  • ITR refers to the terminal inverted repeats at both ends of the viral genome.
  • the ITR structure is one of the characteristics of members of the Parvoviridae family, which has various functions such as regulation of viral gene replication, transcription, and virus rescue.
  • ITR1 refers to the terminal inverted repeat sequence containing the P3 promoter at the forward end of the black-breasted scorpion;
  • ITR2 refers to the inverted end of the terminal containing the P97 promoter at the inverted end of the black-breasted scorpion sequence.
  • vector refers to an artificially constructed recombinant granule for cloning, expression, or transfection.
  • BmKIT 1 refers to Buthus from the East Asia, Butus martensi Karsch (Bm K a cDNA sequence isolated from the tail gland that encodes a signal peptide of 18 amino acid sequences and a mature indole-specific toxin, accession number GeneBank Accession No. AF057555.
  • GFP refers to the green fluorescent protein gene, which encodes a protein that emits green fluorescence upon excitation at 488 nm. Usually GFP The gene for the life science research was registered with GeneBank Accession No. 1155762.
  • ⁇ pheromone refers to a group of synthetic quinone pheromone analogs, including anthrone 3,11-dimercapto-2- undectanone and cis-9-nonatriene. Mainly to increase the lure of viral insecticides on cockroaches.
  • Ivermectin is a macrolide antibiotic-like insecticidal acaricide, the chemical name is 22,23-dihydroavermectin, the English name is ivemectin, and the structural formula is:
  • the expression vector comprises one or more exogenous genes inserted between the Blgll cleavage site of the PUCA plasmid and ITR2.
  • the nucleotide sequence of the whole genome of the black-breasted scorpion nucleus virus (PfDNV) is 5454 nucleotides, and the G + C content is 38.13%.
  • ITRs inverted repeats
  • the 3 palindrome can be used as a primer to initiate replication of the viral DNA, and the parent and progeny DNA strands are cleaved from the replication intermediate by specific site cleavage and packaged into the mature virus. particle.
  • the positive and negative strand coding ability of the PfDNV genome is comparable, and the open reading frames (ORFs) are concentrated at 5, half and half of each strand, and there is an overlap of coding genes.
  • the PfDNV genome has four large open reading frames (ORF1 - ORF4), and the encoded protein has high homology with other parvovirus NS proteins.
  • the ORF2 encoded protein is homologous to the GKRN conserved region of the parvovirus NS-1 protein.
  • the positive strand encodes a non-structural protein of PfDNV; the negative strand contains three large open reading frames (ORF5 ORF7), and the encoded protein has higher homology with other parvovirus VP proteins, among which the ORF5 encodes a protein and parvovirus.
  • the PG protein PGY conserved region has high homology, and it is speculated that the negative strand encodes the PfDNV structural protein.
  • the PfDNV genome functional promoter is located at map unit (mu)3 (positive strand) and mu97 (negative strand), and the Poly(A) site is located at mu69 (positive strand) and mu38 (negative strand).
  • the PfDNV genome will start to transcribe two large mRNA strands with positive and negative strands as templates, respectively.
  • the transcription process is similar to that of the deer venom butterfly nuclear virus (JcDNV) and the large waxy scorpion nucleus virus (GmDNV) 0 through 5, donor and 3, receptor search analysis found that PfDNV is positive
  • the negative strand genome contains a potential mRNA cleavage site, indicating a complex post-transcriptional process in the PfDNV genome.
  • the inventors of the present invention first constructed an expression vector for the recombinant BL-breasted scorpion genome.
  • the vector was constructed by inserting the genome of PUC119 into the PUC119 plasmid to construct a black-breasted sputum plasmid PUC A, and then using the PUC A as a material to the black-breasted scorpion genome in PUC A. Introduce foreign genes.
  • the inventors of the invention construct an expression vector for the recombinant Black-breasted cockroach genome.
  • the vector was constructed by inserting the genome of PUC119 into the PUC119 plasmid to construct a black-breasted sputum plasmid PUCA, and then using the PUCA as a material to introduce foreign sources into the black-breasted scorpion genome in PUCA.
  • the insertion site of the gene, the foreign gene, was selected between the Bgl II restriction site of the black chestnut genome and ITR2.
  • the Bgl II restriction site is at position 3514 of the genome of the black chest.
  • the inventors of the present invention constructed an expression vector for the recombinant Black-breasted cockroach genome.
  • the vector was constructed by inserting the genome of PUC119 into the PUC119 plasmid to construct a black-breasted sputum plasmid PUCA, and then using the PUCA as a material to introduce a foreign gene into the genome of the black-breasted scorpion in PUCA.
  • the insertion site of the foreign gene was selected between the Bgl II restriction site and ITR2 of the genome of the black chest.
  • Black-breasted scorpion gene The 3514th group, more specifically, was selected for enzymatic cleavage at positions 4034 to 4443 of the genome of the black-chested scorpion.
  • the selected foreign gene is the green fluorescent protein gene (GFP), and the selected green fluorescent protein gene is modified to have a Xhol cleavage site and an Eco72I cleavage site at both ends.
  • the modified green fluorescent protein gene is then inserted between positions 4034 to 4443 of the genome of the black chestnut.
  • the resulting recombinant expression vector PfDNV-GFP of the black-chested scorpion genome is as follows
  • the PfDNV genome contains Xhol and £co 72i cleavage sites at nucleotide positions 4034 and 4443, respectively.
  • the green fluorescent protein gene (genebank SEQ ID NO: U55762) was modified at the end, the ATG start codon was removed, the Eco721 cleavage site was added at the 5' end, and the restriction endonuclease was added at the 3' end.
  • the pUCA and the modified GFP gene were digested with Eco721 and _X3 ⁇ 4oI double enzymes, and the target fragment was recovered and ligated to construct the PfDNV-GFP of the black-breasted scorpion serovirus expression vector (Fig. 1).
  • Another aspect of the invention relates to a recombinant black-breasted scorpion-rich nucleus virus.
  • exogenously potentiating genes are introduced, and other exogenous genes can be introduced simultaneously or not.
  • the inventors of the present invention select the expression vector of the recombinant R. ruthenicum genome constructed by the present inventors, and insert the exogenous synergistic gene into the black breast of the vector by genetic engineering technology. Within the genome of the cockroach, a recombinant black-breasted scorpion-rich nucleus virus was obtained.
  • the expression vector of the recombinant Black-breasted cockroach genome is inserted into the cockroach insect-specific neurotoxin synergistic gene BmKIT1.
  • BmKIT1 is a cDNA sequence isolated from the tail gland of Buthus martensi Karsch (GeneBank SEQ ID NO: AF057555), which encodes an insect sodium ion channel-dependent toxin comprising an 18 amino acid signal peptide.
  • the end of BmKIT1 was modified, the 3, the terminal stop codon was removed, and the Eco721 restriction site was added at both ends.
  • the recombinant plasmid pfDNV-GFP and the modified BmKIT1 fragment were digested with Eco72 enzyme, and the target fragment was recovered and ligated to construct the recombinant plasmid pUCA+ (Fig. 2).
  • the results of restriction enzyme digestion, PCR and nucleic acid sequencing confirmed that the recombinant plasmid had the correct sequence of exogenous gene and the insertion direction.
  • the exogenous gene BmKIT1 and GFP marker were inserted into the structural gene (VP) reading frame (solid part) ), regulated by the P97 promoter and related non-coding sequences within the terminal ITR sequence.
  • Expression of a toxin-enhancing gene in sputum in insect-specific expression Another aspect of the invention relates to the expression of a scorpion-specific neurotoxin-promoting gene in sputum.
  • the cockroach insect-specific neurotoxin-promoting gene is inserted into the recombinant black-breasted scorpion nucleus virus, and then the inserted sputum-specific neurotoxin-promoting gene is expressed in sputum.
  • the recombinant black-breasted scorpion nucleus virus of the present invention co-transfects a third-instar nymph of the black-breasted cockroach with a wild-type black-breasted scorpion venom virus, and the reconstituted black chest is large. Both the sputum nucleus virus and the wild-type black-breasted scorpion scorpion virus were expressed in the nymphs of the transfected black-breasted cockroach.
  • the third instar larvae of the black-breasted cockroach were co-transfected with pUC A and pUC A+ by a peritoneal injection method at a dose of p ⁇ /nymph, containing 0.5 pUCA + , 0.5 ⁇ ⁇ pUCA, dissolved in 2 mg/ml of DEAE-dextran. in.
  • the transfected nymphs were reared at 28 ° C to provide rat feed and clean water, and the diseased nymphs were collected for further study.
  • the control group was transfected with the pUCA plasmid of ⁇ /nymph.
  • Infectious recombinant black-breasted scorpion nucleus virion containing scorpion insect-specific neurotoxin-promoting gene Another aspect of the invention relates to an infectious recombinant black-breasted scorpion nucleus containing a scorpion-specific insect-specific neurotoxin-promoting gene Virion preparation.
  • the present inventors selected a recombinant black-breasted scorpion nucleus virus of the present invention and a wild-type black-breasted scorpion-rich nucleus virus, as described above, for the third instar larva of the black-chested cockroach Co-transfection was performed to collect infectious recombinant black-breasted scorpion nucleus virions containing scorpion-specific insect-specific toxin-enhancing genes from co-transfected nymphs.
  • the method of collecting virions can be carried out by a conventional method well known to those skilled in the art.
  • the infected nymphal nymph is subjected to homogenization, centrifugation, etc., and the desired virions are collected, and the recombinant expression vector of the black-breasted scorpion genome constructed by the present inventor adopts genetic engineering technology.
  • the exogenous synergistic gene is inserted into the genome of the black-breasted cockroach in the vector to obtain a recombinant black-breasted scorpion scorpion virus.
  • Recombinant nuclear virions were prepared by co-transfecting pUCA+ and pUCA with the third instar nymph of the black-chested cockroach.
  • the transcription of BmKIT1 and GFP in the intestinal tissues of nymphs was detected by RT-PCR.
  • the nymphs in the treatment group showed obvious symptoms of viral infection, and RT-PCR was positive.
  • the nymphs of the treated group which were orally infected for 7 days, also detected green fluorescence in CFSM, and virions were observed by transmission electron microscopy.
  • the nucleus of the intestinal epithelial cells expands, and a large number of virus particles with a diameter of about 20 ⁇ m are accumulated in the nucleus and cytoplasm (Fig. 4).
  • the nymphs feed on the feed containing the supernatant of the co-transfected corpse, and after 7 days of oral infection, electron micrographs of the intestinal epithelial cells after infection with the nymph. It shows that the nucleus is enlarged, and a large amount of virions are accumulated in the nucleus.
  • Toxicity of Infectious Recombinant Black-breasted Large Scorpion Nucleus Virus Particles to Black-chested Scorpion Virus Another aspect of the present invention relates to the detection of infectious recombinant black-breasted scorpion nucleus virions containing black cockroach insect-specific neurotoxin-promoting gene The toxicity of the chest.
  • an infectious recombinant black-breasted scorpion nucleus virus particle containing a scorpion-containing insect-specific neurotoxin-promoting gene obtained as described above is subjected to toxicity test for measuring black-chested cockroach poisoning The post-mortem curve was used to assess the oral virulence of recombinant black-breasted scorpion venom virus.
  • the concentration of virions in each batch of samples can be calculated, so that the final virion concentration of the baits used in the virulence experiment is 6 ⁇ 10 2 , 6 ⁇ 10 3 5 6 10 4 respectively.
  • 6 ⁇ 10 5 , 6 ⁇ 10 6 viruses/g the same dose of wild-type virions were applied to the control group. After 3 days of attacking, the nymphs were transferred to clean feed and water. Another 60 nymphs were completely cleaned with water and water as a control. All risks are repeated three times in three months. The actual risk observation was 28 days, and the number of dead nymphs was recorded daily.
  • wt represents wild-type PfDNV
  • wt+re represents wild-type and recombinant PfDNV.
  • the dosage unit is the number of virions per gram. The experiment was observed for 28 days. F face f +p3 ⁇ 4 t
  • the cumulative mortality of the black nymphs nymphs at 6 ⁇ 10 4 viruses/g attack dose.
  • ( ⁇ ) indicates a mixed infection group of recombinant and wild type viruses;
  • ( ⁇ ) represents a wild type virus alone infection group;
  • ( ⁇ ) represents a clean feed only, water treatment group.
  • Each set of real faces was repeated 3 times, and the Log-rank test was used to compare the differences.
  • the dose of this real 3 ⁇ 4r bait is 6 ⁇ 10 4 toxic particles/g. Indole-specific neurotoxin-promoting gene
  • the recombinant black-breasted scorpion-rich nuclear virus insecticide containing a scorpion-specific insect-specific neurotoxin-promoting gene is prepared according to the principle of no harm to the environment, and has a strong specificity against cockroaches. A biological acaricide with a high rate of extinction.
  • the following insecticide formulations are formulations of conventional cockroach insecticides. These dosage forms are those commonly used in the art.
  • the insecticide of the present invention can also be prepared into other dosage forms depending on the needs of the particular situation. In these formulations, all except the virion dose, the weight percent, and all formulations were prepared in a conventional manner. It will be apparent to those skilled in the art that various dosage forms, various ingredients, amounts thereof, and the like can be variously changed without departing from the scope of the present invention, and that only conventional experimental means in the art can be used. Obtain the formula and ratio of ingredients required for a specific application.
  • urinary cheese resin (wall material) 80-40% Trichloromethane (heart solvent) 15-20% petroleum ether (filler) 0-25%
  • Example 1 Construction of PfDNV-GFP of the black-breasted scorpion serovirus expression vector PfDNV-GFP Construction material and method
  • the whole genome cloning plasmid UCA of the black-breasted scorpion scorpion virus was constructed by the School of Life Sciences of Wuhan University, at the China Center for Type Culture Collection (CCTCC).
  • the deposit number is: CCTCC M 205093.
  • the green fluorescent protein gene GFP accession number is GeneBank Accession Number: U55762.
  • the sequence is:
  • GATGAACT EcoR72I, XhoI, Taq, T 4 DNA ligase and DNA extraction kits were purchased from the company MBI, T vector kit was purchased from TaKaRa.
  • DH5 ⁇ Escherichia coli strain is from the College of Life Sciences of Wuhan University and is a commercially available strain.
  • DNA sequencing was completed by the professional sequencing company of Beijing Aoke Biotechnology Co., Ltd.
  • the gene was modified by PCR to remove the ATG start codon, and the EcoR72I and Xhol restriction sites were added at the 5 and 3 ends, respectively.
  • Primer sequences are: Forward primer: 5, -GCCACGTGGGTAAAGGAGAAGAAC-3' Reverse primer: 5, -GCCTCGAGAGTTCATCCCATGCCAT-3' Melting temperature 94. C, annealing temperature 50. C, extended temperature 72 °C, extension time 50 seconds.
  • the above PCR product was recovered, ligated with the T-vector, and the T-GFP cloning vector was purchased, and the DH5 a strain was transformed to select a positive single colony.
  • the constructed vector was subjected to PCR, identified by enzyme digestion, and sent to Beijing Aoke Biotechnology Co., Ltd. for sequencing.
  • pUCA and T-GFP were separately digested with EcoR72I and XhoI double enzymes, and the respective target fragments were recovered, ligated, transformed into DH5 a bacteria, and positive single colonies were selected.
  • the constructed vector was subjected to PCR, and after digestion and identification, the company was sent to determine the sequence of the inserted fragment.
  • the GFP sequence primers listed in the materials and methods were used, and the constructed vector was used as a template to identify by PCR; EcoR72I and XhoI digested the constructed vector; and the amplified specific sequence was sequenced.
  • the deposit number is CCTCC No. M206092.
  • Example 2 Expression of PfDNV-GFP in the scorpion of the black-breasted scorpion serovirus expression vector and method PfDNV-GFP was constructed by our laboratory, and DEAE-Dextran was purchased from bioengineering ( Shanghai Co., Ltd.
  • the mRNA extraction kit was TRIZOL Reagent Kit of Invitrogen, and the cDNA synthesis kit was purchased from TaKaRa (cDNA Synthesis Kit). Black-necked nymphs are kept by the actual danger room as usual.
  • the shield granule PfDNV-GFP was extracted according to conventional molecular biological methods, and the plasmid concentration was determined by Shimadzu UV-1601 ultraviolet spectrophotometer. .
  • Gen according to the measurement result was plasmid concentration with an aqueous solution of DEAE-Dextmn 2mg / ml of the formulated PfDNV-GFP plasmid 0.5X 10- 20 ⁇ g / ⁇ 1 , 0.5xl0 -10 ⁇ g / ⁇ 1, 0.5x10 g / ⁇ ⁇ gradient transfection solution.
  • the pUCA was also formulated into the above concentrations with a DEAE-Dextran aqueous solution. Take 420 black-necked nymphs of the third instar larvae and keep them for 24 hours to provide clean drinking water, but no feed.
  • the mRNA of the above midgut was extracted with TRIZOL Reagent Kit according to the instructions, at 42.
  • the first strand is synthesized under C.
  • the GFP gene transcription was detected by PCR using the primers and conditions in Example 1.
  • the above hindgut was washed three times with PBS solution (0.5 M NaCl, 0.1 M acid salt buffer, pH 7.2), and Beckman LCS-sp2-MP type laser confocal display: the mirror was detected at a wavelength of 488 nm.
  • Table 4 Table 4
  • Example 3 Infectious recombinant black-breasted concentrated nuclear virus vector containing cockroach insect-specific neurotoxin-enhancing gene Construction materials and methods Black-breasted cockroach
  • the concentrated nuclear virus expression vector PfDNV-GFP was constructed in Example 1.
  • the East Asian scorpion insect-specific neurotoxin gene BmKITl was presented by the Institute of Virology and Molecular Oncology, School of Life Sciences, Wuhan University. The gene registration number is:
  • DH5 ⁇ strain is preserved in the School of Life Sciences of Wuhan University. DNA sequencing by Beijing Aoke Biotechnology Engineering Co., Ltd. and other professional sequencing companies completed. Using the above BmKIT1 gene as a template, the gene was modified by PCR, and the TAA stop codon was removed, and the p EcoR72I restriction site was added at the ends of 5 and 3, respectively.
  • Primer sequences are: Forward primer: 5, -GC CACGTG ATG AAA TTT TTC CTT ATA TTT CTC G-3' Reverse primer: 5'-GC CACGTG ACC AAT TAT TTG GAC GTC ACAATAA-3' Melting temperature 94 °C The annealing temperature is 50 °C, the extension temperature is 72 °C, and the extension time is 30 seconds.
  • the above PCR product was recovered, ligated with the T-vector, and the T-BmKIT1 cloning vector was purchased, and the DH5a strain was transformed to select a positive single colony.
  • the constructed vector was subjected to PCR, identified by enzyme digestion, and sent to the company for sequencing.
  • pUCA-GFP and T-BmKIT1 were separately digested with EcoR72I enzyme, and the respective target fragments were recovered, ligated, transformed into DH5a bacteria, and positive single colonies were selected.
  • the constructed vector was subjected to PCR, and after digestion and identification, the company was sent to determine the sequence of the inserted fragment. The results were confirmed by PCR, restriction enzyme digestion and DNA sequencing.
  • the recombinant heterozygous neurotoxin-enhancing gene BmKIT1 was inserted into the recombinant black-breasted scorpion nucleus virus vector with correct orientation and named pUCA+.
  • Example 4 Infectious recombinant black-breasted scorpion nucleus virus particle containing cockroach insect-specific neurotoxin synergistic gene Preparation material and method Infectious recombinant black-breasted scorpion sputum containing scorpion insect-specific neurotoxin synergistic gene
  • the viral vector pUCA+ was constructed from Example 3, and the accession number of the China Type Culture Collection was CCTCC M206103.
  • plasmids pUCA+ and pUCA were formulated into a total concentration of lx10 _1 3 ⁇ 4 g/ ⁇ 1 transfection solution with 2 mg/ml of DEAE-Dextran aqueous solution.
  • the ratio of pUCA+ to pUCA was 2:8, 4:6, 5:5, 6:4, 8:2, respectively.
  • the detection was carried out by RT-PCR and laser confocal method, and the corresponding sequences were detected by RT-PCR using GFP and BmKIT1 specific primers, respectively.
  • the above-mentioned diseased intestines were homogenized in PBS solution (0.5 M NaCl, 0.1 M in acid buffer, pH 7.2), centrifuged at 8,000 g for 20 minutes, and the supernatant was taken, using Hitachi HITACHI H-8100 II transmission electron microscope. Initially check the virions in the supernatant. The supernatant was further centrifuged at 40,000 g for 2 hours, examined by electron microscopy, and counted to determine the concentration of virions in the solution.
  • the concentration of virions extracted from the intestines after transfection of each group was diluted to 3 ⁇ 10 4 and mixed with an equal amount of feed. Take 180 black-necked nymphs and keep them for 24 hours to provide clean drinking water, but no feed. Subsequently, it was divided into 6 groups, 30 in each group, and the virus was infected with the above-mentioned feed. After three days of infection, remove the virus-containing feed and give regular feed and clean water, 28 . C breeding. The infected nymphs were observed for 28 days, and no response to needle stimulation was the standard of death.
  • the scores in the table indicate the ratio of the positive number to the total number of samples in this treatment group. It can be found from Table 5.1 that pUCA and pUCA+ are mixed in the same amount, and the transcription and expression efficiency of Xibu source gene is the highest. Therefore, the following transfections were carried out by the same amount of pUCA and pUCA+ mixed transfection.
  • Table 5.2 shows that the infectious recombinant black-breasted scorpion-rich riboviral vector pUCA+ containing the scorpion-specific insect-specific neurotoxin-stimulating gene BmKIT1 is transfected with pUCA
  • the virions are infectious to the nymphs of the black-chested nymph, and the sputum of the infected sputum can detect the transcription of BmKIT1 and the expression of GFP, and verify the infectious recombinant black-breasted cockroach containing the insect-specific neurotoxin-promoting gene. Generation of dense nuclear virions.
  • Example 5 Toxicity test of infectious recombinant black-breasted scorpion nucleus virions containing cockroach insect-specific stimulating toxin-enhancing gene against black-breasted cockroach. Materials and methods ⁇ Insect-specific neurotoxin-promoting gene BmKITl Infectious recombinant black-breasted scorpion nucleus virions were transfected with pUCA+ and pUCA plasmids at a concentration of 0.5 x 10-1 () ⁇ ⁇ ⁇ 1 in the same manner as in Example 4, and isolated.
  • the prepared virions were mixed with A 1.2 ⁇ 10 s , 1.2 ⁇ 10 1.2 x 10 5 , 1.2 ⁇ 10 7 virus/liter aqueous solution, and mixed with an equal amount of feed to prepare 6 X 10 2 , 6 X 10 3, 6 X 10 4, 6 10 6 virus / gram oral bait.
  • the wild-type black-breasted scorpion nucleus virus particles were formulated into 6 ⁇ 10 4 virus/g scorpion scorpion bait as a control.
  • each calculation can be calculated
  • the concentration of virions in the batch is such that the prepared virions are formulated to the desired concentration for each dosage form.
  • the avermectin is purchased from Shijiazhuang Longhui Fine Chemical Co., Ltd., and the remaining reagents are conventional chemically pure reagents.
  • Recombinant virus insecticide bait, suspending agent, condensing capsules containing cockroach insect-specific neurotoxin synergistic gene,
  • the wettable powder is developed by the laboratory, and the relevant prescriptions can be found in the patent specification section.
  • the toxicological test of the poison bait is based on the national standard of the People's Republic of China.
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Abstract

La présente invention concerne un vecteur d'expression recombinant obtenu par génie génétique du génome du densovirus Periplaneta fuliginose (GeneBank Acc. N° AB028936), un densovirus Periplaneta fuliginose recombinant obtenu par génie génétique (PfDNV001, Acc. N° CCTCC V200606) associé par insertion à un gène de renforcement étranger tel que le gène BmKIT1 de la neurotoxine de Karsch Buthus martensi, et un pesticide du gardon comprenant le densovirus Periplaneta fuliginose recombinant obtenu par génie génétique.
PCT/CN2006/002580 2006-09-29 2006-09-29 Densovirus periplanata fuliginosa recombinant et utilisation correspondante Ceased WO2008037123A1 (fr)

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CN101871944A (zh) * 2010-06-02 2010-10-27 中国农业科学院茶叶研究所 一种茶尺蠖核型多角体病毒的定性检测方法

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CN103589807B (zh) * 2013-11-27 2015-09-30 武汉武大绿洲生物技术有限公司 一种黑胸大蠊浓核病毒实时荧光定量pcr试剂盒及检测方法和应用
CN117947099B (zh) * 2024-03-25 2024-07-09 中国林业科学研究院高原林业研究所 一种蜚蠊目昆虫的转基因方法

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WO2002000924A2 (fr) * 2000-06-28 2002-01-03 Institut National De La Recherche Scientifique Enzymes phospholipase a2 d'origine virale, agents antiviraux et procedes d'utilisation
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CN101871944A (zh) * 2010-06-02 2010-10-27 中国农业科学院茶叶研究所 一种茶尺蠖核型多角体病毒的定性检测方法
CN101871944B (zh) * 2010-06-02 2013-07-10 中国农业科学院茶叶研究所 一种茶尺蠖核型多角体病毒的定性检测方法

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