CN109136200A

CN109136200A - A kind of recombination infectious hematopoietic necrosis poison and its construction method and application

Info

Publication number: CN109136200A
Application number: CN201811100029.1A
Authority: CN
Inventors: 赵景壮; 徐黎明; 卢彤岩
Original assignee: Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
Current assignee: Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
Priority date: 2018-09-20
Filing date: 2018-09-20
Publication date: 2019-01-04
Anticipated expiration: 2038-09-20
Also published as: CN109136200B

Abstract

本发明公开了一种重组传染性造血器官坏死病毒及其构建方法与应用。本发明所提供的重组传染性造血器官坏死病毒与改造前的传染性造血器官坏死病毒相比，差别仅在于在所述改造前的传染性造血器官坏死病毒的基因组RNA中的P基因和M基因之间还含有目的蛋白的编码基因(如传染性胰脏坏死病毒的VP2基因)。本发明以IHNV为亲本病毒，利用反向遗传操作技术体外拯救成功获得了能够表达传染性胰脏坏死病毒VP2基因的重组病毒rIHNV‑VP2。本发明所构建得到的重组病毒rIHNV‑VP2，通过系列试验证明了该重组病毒能够起到同时防治IHNV和IPNV的作用，本发明为进一步开展IHNV和IPNV的防治研究奠定了坚实的基础。The invention discloses a recombinant infectious hematopoietic organ necrosis virus and a construction method and application thereof. Comparing the recombinant infectious hematopoietic necrosis virus provided by the present invention with the infectious hematopoietic organ necrosis virus before the transformation, the difference only lies in the P gene and the M gene in the genomic RNA of the infectious hematopoietic organ necrosis virus before the transformation. It also contains the gene encoding the target protein (such as the VP2 gene of the infectious pancreatic necrosis virus). In the present invention, IHNV is used as the parent virus, and the recombinant virus rIHNV-VP2 capable of expressing the VP2 gene of the infectious pancreatic necrosis virus is successfully obtained by using the reverse genetic manipulation technology to rescue in vitro. The recombinant virus rIHNV-VP2 constructed by the present invention has been proved through a series of tests that the recombinant virus can play the role of preventing and treating IHNV and IPNV at the same time, and the present invention has laid a solid foundation for further research on the prevention and treatment of IHNV and IPNV.

Description

A kind of recombination infectious hematopoietic necrosis poison and its construction method and application

Technical field

The present invention relates to field of biotechnology, and in particular to a kind of recombination infectious hematopoietic necrosis is malicious and its constructs Method and application.

Background technique

Infectious hematopoietic necrosis (Infectious hematopoietic necrosis, IHN) and infectiousness pancreas Dirty necrosis sick (Infectious pancreatic necrosis, IPN) is disease that is most common, seriously endangering salmon trout health Toxoinfection disease, is the most important two kinds of diseases for causing salmon trout industry heavy economic losses in world wide at present.It infects Property hematopoietic necrosis virus (Infectious haematopoietic necrosis virus, IHNV), belong to play shape disease Malicious section (Rhabdoviridae), Nola's Rhabdovirus (Novirhabdovirus) are sub-thread minus-stranded rna virus, virus Full-length genome is about 11kb, include six genes, be separately encoded virus nucleoprotein (N), phosphoprotein (P), stromatin (M), Glycoprotein (G), non-structural protein (NV) and polymerase protein (L).It can be by worldwide IHNV according to G-protein gene order It is divided into five kinds of genotype of U, M, L, E and J.Wherein, for U, M, L genotype Major Epidemic in North America, E genotype is popular in Europe, J base Because type is popular in Asia.According to pathogenic strain, environmental factor, the difference of the age of a fish, IHNV can cause salmon trout be up to 100% it is dead Rate is died, is the number one killer of serious obstruction salmon trout sustainable health development at present.Infectious pancreas necrosis virus (Infectious pancreatic necrosis virus, IPNV) belongs to the aquatic birnavirus of biplate section RNA virus section Belong to.IPNV genome is made of (A segment and B segment) two double-stranded RNAs, A segment overall length 3092bp, encodes 106kDa poly egg White (NH2-VP2-VP4-VP3-COOH), wherein VP2 and VP3 is two kinds of major structural proteins of the virus, and VP2 is induction protection The main immunogens of property neutralizing antibody.B segment overall length 2777bp encodes albumen pVP1 (94kDa), is that virion relies on RNA polymerase.Due to the difference of strain, host and environmental factor, IPNV can cause 10%~90% death rate.IHN and IPN master Endanger fry and the rainbow trout (Onchorhyncus mykiss) of juvenile fish stage, brook trout (Salvelinus Fontinalis), brown trout (Salmo trutta), Atlantic salmon (Salmo salar) and hemp, which breathe out, belongs to (Oncorhynchus Spp. fry and juvenile fish) are the main viral infectious of serious obstruction salmon trout sustainable health development at present.

With classics slave phenotypic alternation to the thinking of progress gene expression characteristics research on the contrary, reverse genetics manipulation technology (reverse genetics) refers to the infective molecule cloning by constructing RNA virus, in viral cDNA molecules level on pair It carries out external manual operation (such as progress point mutation, missing, insertion, transversion, indexing and complementation transformation), changes virus Certain features (virulence as lowered virus, improve the infecting potential etc. of virus), the cDNA molecule of virus is connected into expression and is carried Body, transfection cell obtain the infection clones of virus, also referred to as " virus rescue " (the rescue of virus).Pass through Reverse genetics manipulation technology can study the gene duplication of RNA virus and expression regulation mechanism, rna editing and spontaneous recombination and lure Interaction relationship, the viral resistant strategies, gene therapy research between recombination, virus and host are led, and building new virus carries Body carrys out expression alien gene and carries out the development etc. of vaccine.

Summary of the invention

The object of the present invention is to provide a kind of recombination infectious hematopoietic necrosis poison and its construction method and applications.

In a first aspect, a kind of claimed recombination infectious hematopoietic necrosis poison.

Infectious hematopoietic organ necrosis before recombination infectious hematopoietic necrosis's poison provided by the present invention and transformation Virus is compared, difference be only that P gene in the geneome RNA of infectious hematopoietic necrosis's poison before the transformation and Also containing the encoding gene of destination protein between M gene.

Recombination infectious hematopoietic necrosis's poison provided by the present invention is in infectious hematopoietic necrosis's poison (IHNV) recombinant virus obtained after the encoding gene of destination protein is inserted between the P gene in geneome RNA and M gene.

Wherein, the phosphoprotein (P) of the P gene coding infectious hematopoietic necrosis malicious (IHNV)；The M gene is compiled The stromatin (M) of code infectious hematopoietic necrosis malicious (IHNV).

Further, the encoding gene of the destination protein can be the antigen gene of target viral or the coding of labelled protein Gene.

Further, the target viral can be other viruses outside infectious hematopoietic necrosis's poison, such as infect Property pancreas necrosis virus (IPNV) etc.；Correspondingly, the antigen gene of the target viral concretely infectious pancreatic necrosis The VP2 gene etc. of malicious (IPNV).The labelled protein can be green fluorescent protein etc..

In a specific embodiment of the invention, the infectious hematopoietic necrosis before the transformation malicious (IHNV) is specific It is BLk94 plants of infectious hematopoietic necrosis's poison (IHNV-BLk94).The infectious pancreas necrosis virus (IPNV) is specific It is ChRtm213 plants of infectious pancreas necrosis virus (IPNV-ChRtm213).

Further, the P in the geneome RNA of the infectious hematopoietic necrosis before the transformation malicious (IHNV) Gene encodes phosphoprotein (P) shown in SEQ ID No.1；Infectious hematopoietic necrosis's before the transformation malicious (IHNV) Stromatin (M) shown in M gene coding SEQ ID No.2 in geneome RNA.

Corresponding to gene level, in the geneome RNA of the infectious hematopoietic necrosis malicious (IHNV) before the transformation The P gene coding region nucleotide sequence as shown in SEQ ID No.4；Infectious hematopoietic organ necrosis before the transformation The coding region nucleotide sequence of the M gene in the geneome RNA of viral (IHNV) is as shown in SEQ ID No.5.

Further, shown in the VP2 gene coding SEQ ID No.3 of the infectious pancreas necrosis virus (IPNV) VP2 albumen.

Corresponding to gene level, the nucleotide sequence such as SEQ of the VP2 gene of the infectious pancreas necrosis virus (IPNV) Shown in ID No.6.

Further, recombination infectious hematopoietic necrosis's poison and the infectious hematopoietic organ necrosis before transformation Virus is compared, and difference is only that the P gene in the geneome RNA of infectious hematopoietic necrosis's poison before the transformation A segment (" P GE-M GS " in Fig. 3 in A) between code area and the code area of the M gene is replaced for segment B (Fig. 3 " P GE-M GS-VP2gene-P GE-M GS " in middle B).Wherein, the sequence of the segment A is SEQ ID No.7；It is described The sequence of segment B is SEQ ID No.8 (i.e. SEQ ID No.7+SEQ ID No.6+SEQ ID No.7).

More specifically, the recombination infectious hematopoietic necrosis poison can be according to method described in following second aspect It prepares.

Second aspect, it is claimed a kind of to prepare recombination infectious hematopoietic organ described in first aspect above The method of necrosis virus.

Preparation provided by the present invention recombinates the side of infectious hematopoietic necrosis's poison above described in first aspect Method, it may include following steps: by the corresponding cDNA sequence of geneome RNA containing recombination infectious hematopoietic necrosis's poison The recombinant plasmid and helper plasmid cotransfection EPC cell of column, to obtain the recombination infectious hematopoietic necrosis poison.

Wherein, described " the corresponding cDNA sequence of geneome RNA containing recombination infectious hematopoietic necrosis's poison Recombinant plasmid " in the corresponding cDNA sequence of geneome RNA of starting recombination infectious hematopoietic necrosis's poison express Promoter be T7 promoter, it is described recombination infectious hematopoietic necrosis's poison the corresponding cDNA sequence of geneome RNA Coded sequence and T7 termination sequence (being denoted as pIHNV-VP2) also containing fourth hepatovirus ribozyme afterwards.The helper plasmid shares 4 It is a: N gene (the coding base of nucleoprotein N in the geneome RNA containing infectious hematopoietic necrosis's poison before the transformation Cause) the corresponding cDNA sequence in code area helper plasmid 1 (being denoted as pHelp-N)；Contain the infectious hematopoietic before the transformation The auxiliary of the corresponding cDNA sequence in code area of P gene (encoding gene of phosphoprotein P) in the geneome RNA of organ necrosis virus Plasmid 2 (is denoted as pHelp-P)；NV gene in geneome RNA containing infectious hematopoietic necrosis's poison before the transformation The helper plasmid 3 (being denoted as pHelp-Nv) of the corresponding cDNA sequence in code area of (encoding gene of non-structural protein NV)；Contain L gene (encoding gene of polymerase protein L) in the geneome RNA of infectious hematopoietic necrosis's poison before the transformation The helper plasmid 4 (being denoted as pHelp-L) of the corresponding cDNA sequence in code area.

In the method, when carrying out the cotransfection, the pIHNV-VP2, the pHelp-N, the pHelp-P, The quality proportioning of the pHelp-Nv and the pHelp-L are 2.0:1.0:0.5:0.25:0.5.

In the method, when CPE occurs in 60% or more cell, the recombination infectious hematopoietic organ necrosis is harvested Virus.

The third aspect, the biomaterial of claimed following (a1) or (a2) or (a3):

(a1) in vitro zooblast or recombinant bacterium containing previously described recombination infectious hematopoietic necrosis's poison；

(a2) carrier of geneome RNA or cDNA containing previously described recombination infectious hematopoietic necrosis's poison；

(a3) vaccine containing previously described recombination infectious hematopoietic necrosis's poison.

Since the IHNV-BLk94 virus is low virulent strain, direct injection can carry out immune effect detection, nothing after 60d is immunized Need inactivation treatment.

Fourth aspect, claimed following any application:

(b1) previously described recombination infectious hematopoietic necrosis poison or the biomaterial are used for pre- in preparation Anti- and/or treatment disease due to caused by infectious hematopoietic necrosis malicious (IHNV) and/or the infection of previously described target viral Application in the product of disease；

(b2) previously described recombination infectious hematopoietic necrosis poison or the biomaterial are being prepared for pressing down Application in the product of infectious hematopoietic necrosis processed malicious (IHNV) and/or the infection of previously described target viral；

(b3) previously described recombination infectious hematopoietic necrosis poison or the biomaterial are preventing and/or are controlling It treats due to answering in infectious hematopoietic necrosis malicious (IHNV) and/or previously described target viral infection associated diseases With；

(b4) previously described recombination infectious hematopoietic necrosis poison or the biomaterial are inhibiting infectiousness Hematopoietic necrosis virus (IHNV) and/or previously described target viral infection in application.

In a specific embodiment of the invention, the target viral is specially infectious pancreas necrosis virus (IPNV). The infectious hematopoietic necrosis malicious (IHNV) and/or infectious pancreas necrosis virus (IPNV) infection are infection rainbow trout Fish.

The present invention is saved in vitro using reverse genetics manipulation technology and is successfully obtained using IHNV strain BLk94 as parental virus RIHNV-BLk94.The Viral structural protein VP2 gene of IPNV is inserted into P and the M gene of rIHNV-BLk94 genome by the present invention Between, building has obtained recombinant virus rIHNV-VP2.Demonstrating the recombinant virus by campaign can play while preventing and treating The effect of IHNV and IPNV, the present invention are that the study on prevention of further development IHNV and IPNV has established solid foundation.

Detailed description of the invention

Fig. 1 is rIHNV-BLk94 strain full length cDNA sequence plasmid construction policy map.

Fig. 2 is the construction strategy figure of recombinant plasmid pIHNV-GFP-N/P.

Fig. 3 is interleaving for the P and M gene of the invention that the VP2 gene of IPNV strain is inserted into rIHNV-BLk94 genome Enter position view.A is rIHNV-BLk94 genome relevant range schematic diagram；B is recombinant virus rIHNV-VP2 genome phase Close area schematic.

Fig. 4 is rIHNV-VP2 strain full length cDNA sequence plasmid construction policy map.

Fig. 5 is the cDNA clones of IHNV-BLk94 whole genome sequence.H1, H2, H3 are IHNV-BLk94 full-length genome sequence Column cDNA clones product；M is DL15000DNA molecular weight standard；The amplification of pFLC-LaSota carrier for expression of eukaryon skeleton.

Fig. 6 is each recombinant plasmid PCR qualification result for expressing GFP.M is DL2000DNA molecular weight standard；pIHNV-GFP- N/P, pIHNV-GFP-P/M, pIHNV-GFP-M/G, pIHNV-GFP-G/NV, pIHNV-GFP-NV/L are respectively each recombinant plasmid PCR qualification result.

Fig. 7 is the clone of IHNV-BLk94 helper plasmid.N, P, Nv and L are IHNV-BLk94 genetic fragment；M₁For DL15000DNA molecular weight standard；M₂For DL2000DNA molecular weight standard.

Fig. 8 is the TCID of each recombinant virus₅₀Detection.

Fig. 9 is the growth curve of each recombinant virus.

Figure 10 is the GFP mRNA expression of each recombinant virus.

Figure 11 is the GFP protein expression level of each recombinant virus.

Figure 12 is the cytopathy of recombinant virus rIHNV-VP2.

Figure 13 is that protective rate detects after IHNV attacks poison.

Figure 14 is that IPNV attacks virus load detection in rainbow trout body after poison.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The genome of infectious hematopoietic necrosis's poison BLk94 plants (IHNV-BLk94) involved in following embodiments The coding region nucleotide sequence of P gene in RNA encodes phosphoprotein shown in SEQ ID No.1 as shown in SEQ ID No.4 (P).The nucleotide sequence of the code area of M gene in the geneome RNA of IHNV-BLk94 is compiled as shown in SEQ ID No.5 Stromatin (M) shown in code SEQ ID No.2.It is located at the code area of the P gene in the geneome RNA of IHNV-BLk94 The sequence such as SEQ ID of RNA segment (" P GE-M GS " in corresponding diagram 3 in A) between the code area of the M gene Shown in No.7.

The VP2 of infectious pancreas necrosis virus ChRtm213 plants (IPNV-ChRtm213) involved in following embodiments The nucleotide sequence of the code area of gene encodes VP2 albumen shown in SEQ ID No.3 as shown in SEQ ID No.6.

The building and application of embodiment 1, recombinant virus rIHNV-VP2

One, materials and methods BLk94

(1) experimental material and reagent

Trizol is Invitrogen company (10296028) product；(the letter of BLk94 plants of infectious hematopoietic necrosis's poison Claim IHNV-BLk94) (Genbank accession number: DQ164100) and ChRtm213 plants of (abbreviation IPNV- of infectious pancreas necrosis virus ChRtm213) (Genbank accession number: KX234591) is saved by this laboratory；EPC cell ATCC CRL-2872；Contain fourth liver Carrier pFLC-LaSota is transformed in the pBluescript II SK of virus ribozymal, and helper plasmid carrier pTM-NP is protected by this laboratory Deposit (bibliography " foundation of the novel recombinant Newcastle disease virus of Zhang Zhenyu (NDV) expression system and best exogenous gene expression position The Heilungkiang determination [D] set: Northeast Agricultural University, 2015:16-19. ").Cell culture fluid MEM, trypsase, Hank ' s liquid For Hyclone biotech firm product.Fetal calf serum is Gibco biotech firm product.RT-PCR one-step method kit (12574035), liposome 2000 is Invitrogen biotech firm product.In-Fusion PCR Cloning Kit kit For Clontech company (600670) product.Anti- IHNV antibody is prepared by this laboratory and is saved, which is the IHNV disease of expression The surface glycoprotein of poison is immunogene, and (bibliography is " Zhao Jingzhuan, Xu to the polyclonal antibody that immune new zealand white rabbit obtains Dawn, Liu Miao wait the truncation expression of the fishes infectious Hematopoietic Necrosis's disease viral glycoprotein of and immunogenicity detection [J] thin Born of the same parents and molecular immunology magazine, 2014,30 (12): 1238-1242. "), fluorescent marker secondary antibody is that Ebioscience biology is public Department.

(2) design of primers and synthesis

According to IHNV-BLk94 whole genome sequence and pFLC-LaSota carrier for expression of eukaryon frame sequence, Prime is utilized 5.0 software design specific primer of primer, for constructing the true of recombinant virus rIHNV-BLk94 full-length genome cDNA sequence Consideration convey records plasmid, and primer sequence is as shown in table 1.

1 rIHNV-BLk94 full-length genome cDNA sequence cloning primer of table

Primer	Sequence (5 ' → 3 ')
		IH1-F	5’-CGACTCACTATAGGGGTATAAAAAAAGTAACTTGACTA-3’
IH1-R	5’-AATCCAATCATACAGGCCCGATGCAGT-3’
		IH2-F	5’-CTGTATGATTGGATTCTGTGGGGGGCAGTGGATAC-3’
IH2-R	5’-ACTTTGTTGTTGACGCGCTCT-3’
		IH3-F	5’-TGTCAACAACAAAGTCGGGGTGCATCTCTTT-3’
IH3-R	5’-GGGACCATGCCGGCCGTATAAAAAAAGTAACAGAGAGA-3’
		pB-F	5’-GGCCGGCATGGTCCCAGCCTCCTCG-3’
pB-R	5’-CCCTATAGTGAGTCGTATTAGCGGC-3’

It is soft using Prime primer 5.0 according to IHNV-BLk94 whole genome sequence and helper plasmid carrier pTM-NP Part designs specific primer, is respectively used to building recombinant virus rIHNV-BLk94 nucleoprotein (N), phosphoprotein (P), non-structural protein The eukaryon helper plasmid of white (NV) and polymerase protein (L) cDNA sequence, primer sequence are as shown in table 2.

2 rIHNV-BLk94 helper plasmid cDNA sequence cloning primer of table

According to IHNV-BLk94 whole genome sequence and GFP gene order, 5.0 software design of Prime primer is utilized Specific primer, for constructing rIHNV-BLk94 carrier expression GFP recombinant plasmid, primer sequence is as shown in table 3.

Table 3 constructs rIHNV-BLk94 carrier and expresses GFP plasmid cloning primer

According to the VP2 gene order of IPNV strain, it is used for using 5.0 software design specific primer of Prime primer It is as shown in table 4 to construct rIHNV-VP2 carrier expression VP2 recombinant plasmid primer sequence.

Table 4 constructs rIHNV-VP2 recombinant plasmid clone primer

Primer	Sequence (5 ' → 3 ')
		ZT-F	5’-ATGTCCATTTTCAAGAGAGCAAAGA-3’
ZT-R	5’-GCTCTCGTTTGAACTGACTCTTGGACTT-3’
		VP2-F	5’-AGTTCAAACGAGAGCATGAACACATCCAAGGCAACCGCAA-3’
VP2-R	5’-CTTGAAAATGGACATTCATGCCTTTGAGGTTGGTAGGTCA-3’

(3) viral amplification and RNA are extracted

The viral suspension of IHNV-BLk94 and IPNV-ChRtm213 is used into the cell maintenance medium (MEM containing 2%FBS respectively Culture medium) dilution 10⁵Times, it is inoculated in the EPC cell that single layer converges, is cultivated in 15 DEG C.When cytopathy occurs in 80% or more cell Become (cytopathic effect, CPE) when, collect cell culture fluid (i.e. viral suspension), take 250 μ L viral suspensions (adjust to Contain 10⁵Virion) in the centrifuge tube of no RNA enzyme.12 000g are centrifuged 5min, removal precipitating.750 μ L Trizol are added Lysate concussion mixes, and 200 μ L phenol chloroforms are added and mix, and 12 000g are centrifuged 15min after 10min, and Aspirate supernatant is added to newly Centrifuge tube in, isometric isopropanol is added, is mixed by inversion repeatedly, 12 000g be centrifuged 15min, abandon to the greatest extent supernatant, be added 75% Ethyl alcohol 1mL, overturn washing, 12 000g be centrifuged 10min, abandon to the greatest extent supernatant, 4000 centrifugation 10s, blot the liquid of tube bottom, room temperature Dry 3min；Be added 100 μ L without RNA enzyme water, RNA precipitate is completely dissolved, be sub-packed in -80 DEG C it is spare.

(4) building of rIHNV-BLk94 strain full length cDNA sequence plasmid and eukaryon helper plasmid

1, the building of rIHNV-BLk94 strain full length cDNA sequence plasmid

The construction strategy of rIHNV-BLk94 strain full length cDNA sequence plasmid is as shown in Figure 1.

The IHNV-BLk94RNA extracted using step (3) is primer as the sequence of template, table 1, anti-using mono- step of RT-PCR Kit amplification is answered to cover IH1, IH2, IH3 segment of IHNV-BLk94 strain full-length genome.It expands IH1 segment and uses primer IH1-F and IH1-R；It expands IH2 segment and uses primer I H2-F and IH2-R；It expands IH3 segment and uses primer I H3-F and IH3-R. Then, according to Pfu Ultra^TMII Fusion HS archaeal dna polymerase specification, the pFLC-LaSota eukaryon saved with laboratory Expression vector is template, and the sequence (pB-F and pB-R) using table 1 is primer, expands pFLC-LaSota carrier for expression of eukaryon bone Frame segment.Object above genetic fragment is recycled using plastic recovery kit, is said according to In-Fusion PCR Cloning Kit Bright book connects IH1, IH2, IH3 and pFLC-LaSota carrier for expression of eukaryon skeleton segment, constructs containing rIHNV-BLk94 poison The plasmid pIHNV-BLk94 of strain full length cDNA sequence, and it is correct through sequence verification.

2, the building of eukaryon helper plasmid

The IHNV-BLk94RNA extracted using step (3) is primer as the sequence of template, table 2, anti-using mono- step of RT-PCR Kit is answered to expand IHNV-BLk94 strain N, P, NV and L genetic fragment respectively.Expand N genetic fragment using primer I HN-F and IHN-R；It expands P genetic fragment and uses primer I HP-F and IHP-R；It expands NV genetic fragment and uses primer I HNv-F and IHNv-R； It expands L genetic fragment and uses primer I HL-F and IHL-R.According to Pfu Ultra^TMII Fusion HS archaeal dna polymerase specification, For the helper plasmid carrier pTM-NP saved using laboratory as template, the sequence (phelp-F and phelp-R) using table 2 is primer, Expand helper plasmid carrier pTM-NP skeleton segment.Object above genetic fragment is recycled using plastic recovery kit, according to In- Fusion PCR Cloning Kit specification is respectively by N, P, NV and L genetic fragment and helper plasmid carrier pTM-NP matrix tablet Section connection, constructs Revive virus helper plasmid pHelp-N, pHelp-P, pHelp-Nv and pHelp-L.And through sequence verification Correctly.

(5) building of GFP subclone

Using the pIHNV-BLk94 full-length cDNA constructed in step 41 as template, NF and NR in table 3 are primer, by IHNV N gene and M gene between sequence be cloned into pcDNA3.1 carrier, obtain IHNV P gene subclone pcDNA3.1-NM/ P⁺。

It will be template using p519gfp plasmid (ATCC:87452), the GFP insert F and GFP insert R in table 3 For primer, amplification obtains GFP segment；Utilize pcDNA3.1-NM/P⁺For template, the GFP Vet F and GFP Vet R in table 3 is Primer, amplification obtain the carrier segments pcDNA3.1-NM/P of removal P gene open reading frame^-；By GFP segment and pcDNA3.1- NM/P^-Carrier connection after, obtain include Gene end-Gene start-GFP GFP subclone (GFP template), and It is correct through sequence verification, it is convenient to the building of later period recombinant plasmid.

(6) recombinant plasmid pIHNV-GFP-N/P, pIHNV-GFP-P/M, pIHNV-GFP-M/G, pIHNV-GFP-G/NV, The building of pIHNV-GFP-NV/L and the rescue of virus

In order to determine the optimum position of IHNV expression alien gene, indicator protein GFP is successively inserted by the present invention Between N the and P gene of IHNV genome, between P and M, between M and G, between G and NV and between NV and L, a system is constructed Arrange the different pIHNV-GFP plasmid in insertion position.

Below by taking external source GFP gene is inserted between N and P gene as an example, be described in detail the building of serial recombinant plasmid with Rescue strategy, specific strategy are as shown in Figure 2.

1, the building of recombinant plasmid pIHNV-GFP-N/P

PIHNV-BLk94 plasmid obtained in the GFP template and step 41 obtained respectively using step 5 as template, Utilize the primer amplification GFP N/P segment and pIHNV-GFP-N/P Vet segment in table 3.It is used when expanding GFP N/P segment Primer is N/P insert F and N/P insert R；The primer used when expanding pIHNV-GFP-N/P Vet segment is N/P Vet F and N/P Vet R.The segment that PCR is obtained is attached after glue recycles respectively, connection product converts bacillus coli DH 5 alpha Competent cell, picking single bacterium colony identify that correct positive plasmid is named as pIHNV-GFP-N/P plasmid (and through sequence verification Correctly).

2, the rescue of recombinant virus rIHNV-GFP-N/P

Take 2.0 μ g of pIHNV-GFP-N/P plasmid, 1.0 μ g of helper plasmid pHelp-N, 0.5 pHelp-P μ g, pHelp-Nv 0.25 0.5 μ g cotransfection EPC cell of μ g and pHelp-L, concrete operation method is according to Invitrogen company 2000 transfection procedure specification of Lipofectamine carries out.Cell after transfection is placed in 15 DEG C of incubator cultures, is seen daily Cell state is examined, when CPE occurs in 60% or more cell, harvest viral suspension is placed in -80 DEG C of packing and saves backup.

3, recombinant plasmid pIHNV-GFP-P/M, pIHNV-GFP-M/G, pIHNV-GFP-G/NV, pIHNV-GFP-NV/L Building and the rescue of virus

The structure of recombinant plasmid pIHNV-GFP-P/M, pIHNV-GFP-M/G, pIHNV-GFP-G/NV, pIHNV-GFP-NV/L Build with the rescue of virus when in addition to GFP Insert Fragment and vector amplification that the primer is different, other steps are and pIHNV- The building of GFP-N/P and virus rescue are identical.The primer used when expanding GFP P/M segment is the P/M insert F in table 3 With P/M insert R, the primer used when expanding pIHNV-GFP-P/M Vet segment is P/M the Vet F and P/M in table 3 Vet R；The primer used when expanding GFP M/G segment is M/G insert F and M/G the insert R in table 3, amplification The primer used when pIHNV-GFP-M/G Vet segment is the M/G Vet F and M/G Vet R in table 3；Expand GFP G/NV piece The primer used when section expands pIHNV-GFP-G/NV Vet piece for G/NV insert F and G/NV the insert R in table 3 The primer used when section is the G/NV Vet F and G/NV Vet R in table 3；When expanding GFP NV/L segment the primer that uses for NV/L insert F and NV/L insert R in table 3, when expanding pIHNV-GFP-NV/L Vet segment the primer that uses for NV/L Vet F and NV/L Vet R in table 3.

(7) Revive virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV And the TCID of rIHNV-GFP-NV/L₅₀Measurement

The good EPC cell of growth conditions is taken, according to 2 × 10 after digestion⁴A/100 μ L are sub-packed in 96 orifice plates, are inoculated with each point Viral suspension from strain difference dilution, each 8 hole of dilution, every 100 μ L of hole set up blank control group simultaneously, and 15 DEG C are cultivated, After observation 7 days, TCID is calculated using Reed-Muench method₅₀。

(8) Revive virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV And the growth curve of rIHNV-GFP-NV/L

The good EPC cell of growth conditions is taken, according to 2 × 10 after digestion⁴A/100 μ L are sub-packed in 96 orifice plates, are inoculated with each point Viral suspension from strain difference dilution, each 8 hole of dilution, every 100 μ L of hole set up blank control group simultaneously, and 15 DEG C are cultivated, Every 12h collects primary virus, calculates TCID using Reed-Muench method₅₀Draw viral growth curves.

(9) transcriptional level detection Revive virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, The expression quantity of rIHNV-GFP-G/NV and rIHNV-GFP-NV/L GFP mRNA

Inoculation EPC cell monolayer in be changed in 6 porocyte culture plates, before virus inoculation containing 10% serum MEM training Support base.It is inoculated with each Revive virus respectively in 6 orifice plates with 0.1MOI (Multiplicity of infection), 15 DEG C are cultivated The maintaining liquid of 2% serum is changed to after 1h.15 DEG C, 0.5%CO₂Under the conditions of cultivate 48h after extract viral RNA, utilize One Step SYBR PrimeScript PLUS RT-PCR Kit (RR096A, Takara, Japan) is to recombinant virus GFP mRNA Expression quantity detected, the detection primer used is RT-GFP F:5 '-CGAGGTGGTGTACATGAACGA-3 ', RT-GFP R:5 '-GCTGTAGAACTTGCCGCTGTT-3 ', reference gene are the N protein gene of virus, and internal control primer is RT-N F:5 '- AGGAGAGGGAACGAGAAGG-3 ', RT-N R:5 '-TGTTGGGATCTGCGAAAGTG-3 '.

(10) recombinant virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV And the detection of rIHNV-GFP-NV/L GFP expressing quantity

Inoculation EPC cell monolayer in be changed in 6 porocyte culture plates, before virus inoculation containing 10% serum MEM training Support base.It is inoculated with each Revive virus respectively in 6 orifice plates with 0.1MOI (Multiplicity of infection), 15 DEG C are cultivated The maintaining liquid of 2% serum is changed to after 1h.15 DEG C, 0.5%CO₂Under the conditions of cultivate 48h after in using pancreatin will be under cell dissociation Come, PBS utilizes the expression of each recombinant virus green fluorescent protein GFP of flow cytomery after washing 2 times.

(11) rIHNV-VP2 strain full length cDNA sequence plasmid construction

Early-stage study result demonstrates IHNV mRNA transcription amount and successively successively decreases according to the sequence of genome 3 ' to 5 ', and Compared with other insertion points, when between P the and M gene that GFP is inserted into rIHNV-BLk94 carrier, the expression quantity of GFP albumen Highest.This explanation, the transcription properties and foreign gene for comprehensively considering IHNV genome mRNA on influence caused by virus replication, Noncoding region between rIHNV-BLk94 genome P and M gene is the optimum position of expression alien gene.Therefore the present invention will The VP2 gene of IPNV strain is inserted between P the and M gene of rIHNV-BLk94 genome, is constructed containing IPNV strain VP2 The recombinant virus rIHNV-VP2 (Fig. 3) of gene.

The RNA of the IPNV-ChRtm213 extracted using step (3) is primer as template, the VP2-F of table 4 and VP2-R, is utilized RT-PCR single step reaction kit expands the target fragment for covering IPNV strain VP2 gene.With the pIHNV- constructed in step 63 GFP-P/M recombinant plasmid is template, and the ZT-F and ZT-R using table 4 are primer, the carrier sequence of amplification removal GFP gene.Benefit With Pfu Ultra^TMII Fusion HS archaeal dna polymerase, connection VP2 gene target fragment and the carrier sequence for removing GFP gene Column, construct the recombinant plasmid pIHNV-VP2 containing VP2 cDNA sequence, construction strategy is as shown in Figure 4.

The rescue of (12) recombinant virus rIHNV-VP2 strain

Take 2.0 μ g of pIHNV-VP2 plasmid, 1.0 μ g of helper plasmid pHelp-N, 0.5 pHelp-P μ g, pHelp-Nv 0.25 0.5 μ g cotransfection EPC cell of μ g and pHelp-L, concrete operation method is according to Invitrogen company 2000 transfection procedure specification of Lipofectamine carries out.Cell after transfection is placed in 15 DEG C of incubator cultures, is seen daily Cell state is examined, when CPE occurs in 60% or more cell, harvest viral suspension is placed in -80 DEG C of packing and saves backup.

The application of (13) recombinant virus rIHNV-VP2

By the recombinant virus rIHNV-VP2 injecting immune SPF rainbow trout (5 ± 1g) of harvest, every group of immune 50 tails, immunizing dose For every tail 50pfu/ tail, control group carries out inoculation using PBS.60d after immune utilizes IHNV (10²Pfu/ tail) and IPNV(10⁶Pfu/ tail) virus infect respectively, carry out immunoprotection detection.

Two, result and analysis

1, rIHNV-BLk94 strain full-length cDNA plasmid construction

The long 11132bp of IHNV-BLk94 whole genome sequence, the present invention are classified as tri- sections of genetic fragments of IH1, IH2, IH3 It is cloned, size is followed successively by 3756bp, 3680bp, 3726bp, the product after the amplification of RT-PCR single step reaction kit Electrophoretic analysis result shows that 3 sections of genetic fragment sizes are consistent with expected results (see Fig. 5).Application specific primer pair pFLC- LaSota carrier for expression of eukaryon frame sequence is expanded, and electrophoresis result shows that carrier size is consistent with expected results 3140bp (see Fig. 5).IH1, IH2, IH3 and pFLC-LaSota eukaryon table are connected according to In-Fusion PCR Cloning Kit specification Up to vector backbone segment, the plasmid pIHNV-BLk94 containing IHNV-BLk94 strain full length cDNA sequence is constructed.

2, recombinant plasmid pIHNV-GFP-N/P, pIHNV-GFP-P/M, pIHNV-GFP-M/G, pIHNV-GFP-G/NV, The building of pIHNV-GFP-NV/L

GFP N/P segment, GFP P/M segment, GFP M/G segment, GFP G/NV are expanded respectively using the primer of specificity Segment, GFP NV/L segment and pIHNV-GFP-N/P Vet carrier segments, pIHNV-GFP-P/M Vet carrier segments, PIHNV-GFP-M/G Vet carrier segments, pIHNV-GFP-G/NV Vet carrier segments, pIHNV-GFP-NV/L Vet carrier-pellet Target fragment, connect to obtain recombinant plasmid pIHNV-GFP-N/P, pIHNV-GFP-P/M, pIHNV-GFP- respectively with carrier by section It is as shown in Figure 6 to construct successful recombinant plasmid PCR qualification result by M/G, pIHNV-GFP-G/NV and pIHNV-GFP-NV/L.

3, the building of rIHNV-GFP strain eukaryon helper plasmid

Using the RNA of IHNV-BLk94 strain as template, expanded respectively using specific primer IHNV-BLk94 strain N, P, Nv and L genetic fragment, size are respectively 1176bp, 693bp, 336bp and 5961bp (see Fig. 7).Specific primer is used simultaneously Expand pFLC-LaSota vector backbone segment, according to In-Fusion PCR Cloning Kit specification respectively by N, P, Nv and L genetic fragment is connect with pFLC-LaSota vector backbone segment, constructs rescue rIHNV-GFP strain helper plasmid phelp- N, phelp-P, phelp-NV and phelp-L.

4, Revive virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and The TCID of rIHNV-GFP-NV/L₅₀Measurement

After determining different loci insertion GFP gene, the situation of change of recombinant virus infection ability, the present invention is right respectively The TCID of each recombinant virus₅₀It is detected.The result shows that the TCID of each Revive virus₅₀Titre and parental virus rIHNV- BLk94 is similar, illustrates Revive virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/ NV and rIHNV-GFP-NV/L maintains the infection ability (Fig. 8) of its parental virus.

5, Revive virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and The growth curve of rIHNV-GFP-NV/L

In order to study the duplication characteristic and its growth kinetics characteristic of Revive virus, the present invention is by viral dilution 10^-5After times EPC cell is infected respectively, later every primary virus of 12h harvest, and to the TCID of different infection time point viruses₅₀It is detected, Draw the growth curve (Fig. 9) of virus infection.As shown, each recombinant virus of expression GFP albumen is equal on same time point Even rIHNV-BLk94 parental virus maintains similar map, shows duplication similar with rIHNV-BLk94 parental virus Characteristic and growth kinetics characteristic.But the speed of growth of rIHNV-GFP-N/P is slightly compared with the recombinant virus of other expression GFP Aobvious lag about falls behind other viral 0.5-1 titres.Although rIHNV-GFP-P/M virus is slightly above on same time point RIHNV-GFP-N/P, but still it is significantly lower than other Revive virus.The titre of same time point virus is from high to low successively are as follows: rIHNV-GFP-N/P,rIHNV-GFP-P/M,rIHNV-GFP-M/G,rIHNV-GFP-G/NV,rIHNV-GFP-NV/L.This says Bright, the insertion of foreign protein GFP has a great impact for the duplication of virus, and is inserted into 3 ' ends of the gene closer to genome The duplicating efficiency of virus is influenced bigger.

6, transcriptional level detection Revive virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, The expression quantity of rIHNV-GFP-G/NV and rIHNV-GFP-NV/L

Using specific Real-time PCR primer, using the N protein gene of virus as internal reference, in transcriptional level to each The expression quantity of Revive virus expression alien gene GFP is detected (Figure 10).The results show that each Revive virus GFP mRNA RIHNV-GFP-N/P > rIHNV-GFP-P/M > rIHNV-GFP-M/G > rIHNV-GFP-G/NV > rIHNV-GFP- is presented in expression quantity NV/L。

7, recombinant virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and The detection of rIHNV-GFP-NV/L GFP expressing quantity

Real-time PCR is the results show that foreign protein about holds the transcription amount of its mRNA to get over close to virus genomic 3 ' It is more；But the growth curve of virus but shows that foreign gene gets over the influence of the duplicating efficiency of virus closer to 3 ' ends of genome Greatly.In order to determine the optimum expression position of foreign protein, this research has carried out streaming to the GFP expressing quantity of each Revive virus Cell instrument detects (Figure 11).It can be seen from the results that the GFP expression quantity of Revive virus rIHNV-GFP-P/M is maximum, specific order For rIHNV-GFP-P/M > rIHNV-GFP-M/G > rIHNV-GFP-N/P > rIHNV-GFP-G/NV > rIHNV-GFP-NV/L.By Result above can be seen that the transcription properties for comprehensively considering IHNV genome mRNA and foreign gene to caused by virus replication It influences, foreign gene is inserted between the P gene of IHNV-BLk94 genome and M gene to the highest that can obtain foreign gene Expression quantity.

8, rIHNV-VP2 strain full-length cDNA plasmid construction and virus rescue

Using the RNA of IPNV-ChRtm213 strain as template, using primer amplified IPNV strain VP2 genetic fragment, Its size is 1329bp.Application specific primer is according to In-Fusion PCR Cloning Kit specification by VP2 gene replacement The GFP gene for falling pIHNV-GFP-P/M plasmid constructs the pIHNV-VP2 recombination matter containing IPNV strain VP2 genetic fragment Grain.After 1.0 μ g of pIHNV-VP2, pHelp-N, pHelp-P, pHelp-Nv and pHelp-L cotransfection EPC cell, save out Recombinant virus rIHNV-VP2, Cytopathic effect are as shown in figure 12.

In addition, genome sequence determination is carried out to recombinant virus rIHNV-VP2, the results show that recombinant virus rIHNV-VP2 Compared with IHNV-BLk94, difference, which is only that, to be located at the gene coding region P in IHNV-BLk94 geneome RNA (in Fig. 3 in A " P cds ") and the gene coding region M (" M CDS " in Fig. 3 in A) between A segment (" P GE-M GS " in Fig. 3 in A) replace It has been changed to segment B (" P GE-M GS-VP2gene-P GE-M GS " in Fig. 3 in B).Wherein, the sequence of the segment A is SEQ ID No.7；The sequence of the segment B is SEQ ID No.8 (i.e. SEQ ID No.7+SEQ ID No.6+SEQ ID No.7)。

9, the application of recombinant virus rIHNV-VP2

After recombinant virus rIHNV-VP2 dilution, SPF rainbow trout is immunized, the 60d after immune utilizes IHNV and IPNV virus Infection, respectively detects IPNV virus carrying capacity in the immune protective rate of IHNV and tissue.It can be seen from the results that through recombinating Viral rIHNV-VP2 immune rainbow trout (substitutes recombinant virus rIHNV- with control group in IHNV virus infection with PBS It VP2 is about) 85% (Figure 13) compared to the immune protective rate of recombinant virus；In IPNV virus infection, recombinate compared with the control group The virus load of the immunoprotection group IPNV of virus is substantially reduced (Figure 14).These results suggest that rIHNV-VP2 pairs of recombinant virus Preventing rainbow trout IHNV and IPNV virus infection has good immune protective effect, this result is the strong of China rainbow trout aquaculture Kang Fazhan provides technical guarantee.

<110>Heilongjiang Inst. of Aquatic Products, Chinese Academy of Aquatic Products Scie

<120>a kind of recombination infectious hematopoietic necrosis poison and its construction method and application

<130> GNCLN181321

<160> 8

<170> PatentIn version 3.5

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Claims

1. Recombinant infectious hematopoietic organ necrosis virus, characterized in that: the recombinant infectious hematopoietic organ necrosis virus is compared with the infectious hematopoietic organ necrosis virus before the transformation, and the difference is only in the infectious hematopoietic organ necrosis before the transformation. The gene encoding the target protein is also contained between the P gene and the M gene in the genomic RNA of the virus.

2. The recombinant infectious hematopoietic organ necrosis virus according to claim 1, wherein the encoding gene of the target protein is the antigen gene of the target virus or the encoding gene of the marker protein;

Further, the antigen gene of the target virus is the VP2 gene of infectious pancreatic necrosis virus; the marker protein is green fluorescent protein;

Furthermore, the infectious pancreatic necrosis virus is the infectious pancreatic necrosis virus ChRtm213 strain.

3. The recombinant infectious hematopoietic organ necrosis virus according to claim 1 or 2, characterized in that: the P gene encoding SEQ ID No.1 in the genomic RNA of the infectious hematopoietic organ necrosis virus before the transformation. phosphoproteins shown; and/or

The M gene in the genomic RNA of the pre-transformed infectious hematopoietic necrosis virus encodes the matrix protein shown in SEQ ID No. 2; and/or

The VP2 gene of the infectious pancreatic necrosis virus encodes the VP2 protein shown in SEQ ID No.3.

4. The recombinant infectious hematopoietic necrosis virus according to claim 3, wherein: the nucleotide sequence of the coding region of the P gene in the genomic RNA of the infectious hematopoietic necrosis virus before the transformation is as follows: shown in SEQ ID No. 4; and/or

The nucleotide sequence of the coding region of the M gene in the genomic RNA of the pre-transformed infectious hematopoietic necrosis virus is shown in SEQ ID No. 5; and/or

The nucleotide sequence of the coding region of the VP2 gene of the infectious pancreatic necrosis virus is shown in SEQ ID No.6.

5. The recombinant infectious hematopoietic organ necrosis virus according to any one of claims 1-4, wherein the recombinant infectious hematopoietic organ necrosis virus is compared with the infectious hematopoietic organ necrosis virus before the transformation, The only difference is that the A segment between the coding region of the P gene and the coding region of the M gene in the genomic RNA of the pre-transformed infectious hematopoietic necrosis virus is replaced by segment B; The sequence is SEQ ID No.7; the sequence of the fragment B is SEQ ID No.8.

6 . The recombinant infectious hematopoietic organ necrosis virus according to any one of claims 1 to 5 , wherein the recombinant infectious hematopoietic organ necrosis virus is prepared according to the method of claim 7 or 8 .

7. A method for preparing the recombinant infectious hematopoietic organ necrosis virus described in any one of claims 1-5, comprising the steps of: recombining the cDNA sequence corresponding to the genomic RNA containing the recombinant infectious hematopoietic organ necrosis virus The plasmid and the helper plasmid were co-transfected into EPC cells to obtain the recombinant infectious hematopoietic necrosis virus.

8. The method according to claim 7, wherein the recombinant infectious hematopoietic organ necrosis virus is activated in the "recombinant plasmid containing the cDNA sequence corresponding to the genomic RNA of the recombinant infectious hematopoietic organ necrosis virus" The promoter expressed by the cDNA sequence corresponding to the genomic RNA is the T7 promoter;

and / or

There are 4 helper plasmids in total: helper plasmid 1 containing the cDNA sequence corresponding to the coding region of the N gene in the genomic RNA of the pre-transformed infectious hematopoietic necrosis virus; Auxiliary plasmid 2 containing the cDNA sequence corresponding to the coding region of the P gene in the genomic RNA of The helper plasmid 4 of the cDNA sequence corresponding to the coding region of the L gene in the genomic RNA of the infectious hematopoietic necrosis virus before the transformation.

9. Biological material of the following (a1) or (a2) or (a3):

(a1) an isolated animal cell or a recombinant bacteria containing the recombinant infectious hematopoietic necrosis virus described in any one of claims 1-6;

(a2) a vector containing the genomic RNA or cDNA of the recombinant infectious hematopoietic necrosis virus according to any one of claims 1-6;

(a3) A vaccine comprising the recombinant infectious hematopoietic necrosis virus of any one of claims 1-6.

10. Any of the following applications:

(b1) The recombinant infectious hematopoietic necrosis virus described in any one of claims 1-6 or the biological material according to claim 9 is prepared for the prevention and/or treatment of infectious hematopoietic organ necrosis virus and/or rights Application in the product of the disease caused by the target virus infection described in requirement 2;

(b2) The recombinant infectious hematopoietic necrosis virus described in any one of claims 1-6 or the biological material according to claim 9 is prepared for inhibiting infectious hematopoietic organ necrosis virus and/or described in claim 2 the application of target virus-infected products;

(b3) the recombinant infectious hematopoietic organ necrosis virus described in any one of claim 1-6 or the biological material described in claim 9 in the prevention and/or treatment of infectious hematopoietic organ necrosis virus and/or claim 2 The application in the disease caused by the target virus infection;

(b4) The recombinant infectious hematopoietic necrosis virus described in any one of claims 1-6 or the biological material described in claim 9 inhibits the infectious hematopoietic organ necrosis virus and/or the target virus described in claim 2 application in infection;

Further, the target virus is infectious pancreatic necrosis virus.