CN109136200B

CN109136200B - Recombinant infectious hematopoietic necrosis virus and construction method and application thereof

Info

Publication number: CN109136200B
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: 2022-02-18
Anticipated expiration: 2038-09-20
Also published as: CN109136200A

Abstract

The invention discloses a recombinant infectious hematopoietic necrosis virus and a construction method and application thereof. Compared with the infectious haematopoietic necrosis virus before modification, the recombinant infectious haematopoietic necrosis virus provided by the invention only has the difference that a coding gene of a target protein (such as VP2gene of the infectious pancreatic necrosis virus) is also contained between a P gene and an M gene in the genomic RNA of the infectious haematopoietic necrosis virus before modification. The invention uses IHNV as a parent virus, and successfully obtains the recombinant virus rIHNV-VP2 capable of expressing the infectious pancreatic necrosis virus VP2gene by utilizing the reverse genetic manipulation technology for in vitro rescue. The recombinant virus rIHNV-VP2 constructed by the invention is proved to play a role in preventing and treating IHNV and IPNV simultaneously through series of tests, and the invention lays a solid foundation for further research on preventing and treating IHNV and IPNV.

Description

Recombinant infectious hematopoietic necrosis virus and construction method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a recombinant infectious hematopoietic necrosis virus and a construction method and application thereof.

Background

Infectious Hematopoietic Necrosis (IHN) and Infectious Pancreatic Necrosis (IPN) are the most common viral infections that seriously harm the health of salmon and trout, and are the two most important diseases causing significant economic loss in the salmon and trout industry worldwide. Infectious Hematopoietic Necrosis Virus (IHNV), belonging to Rhabdoviridae (Rhabdoviridae), norrhabdovirus (Novirhabdovirus), is a mononega RNA virus whose viral genome is approximately 11kb in length and contains six genes encoding viral nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), nonstructural protein (NV), and polymerase protein (L), respectively. IHNV worldwide can be divided into U, M, L, E and J five genotypes according to the G protein gene sequence. Among them, U, M, L genotype prevails mainly in north america, E genotype in europe, and J genotype in asia. According to different pathogenic strains, environmental factors and fish ages, IHNV can cause the mortality rate of salmon and trout to be as high as 100 percent, and is the number one killer which seriously hinders the healthy and sustainable development of the salmon and trout at present. Infectious Pancreatic Necrosis Virus (IPNV) belongs to the genus waterborne birnavirus of the family of double-fragment RNA viruses. The IPNV genome consists of two double-stranded RNAs (A-and B-segments), the A-segment has a full length of 3092bp and encodes a 106kDa polyprotein (NH2-VP2-VP4-VP3-COOH), where VP2 and VP3 are the two major structural proteins of the virus and VP2 is the major immunogen for inducing protective neutralizing antibodies. The B fragment is 2777bp in length, encodes protein pVP1(94kDa), and is a virion-dependent RNA polymerase. IPNV can cause 10% to 90% mortality due to differences in strains, hosts, and environmental factors. IHN and IPN mainly harm fry and fry of rainbow trout (Onchorhynchus mykiss), American redspot salmon (Salvelinus fontinalis), brown trout (Salmo truta), Atlantic salmon (Salmo salar) and Cannabis (Oncorhynchus spp.) in fry and parr stage, and are the main viral infectious diseases which seriously obstruct the healthy sustainable development of the salmon and trout at present.

Contrary to the classical thinking of changing phenotype to gene characteristics, reverse genetic manipulation (reverse genetics) refers to the construction of infectious molecular clones of RNA viruses, which are artificially manipulated in vitro at the viral cDNA molecular level (e.g., genetic point mutation, deletion, insertion, inversion, transposition, complementation, etc.) to change certain characteristics of the viruses (e.g., reducing the virulence of the viruses, increasing the infectivity of the viruses, etc.), and the introduction of the cDNA molecules of the viruses into an expression vector to transfect cells to obtain infectious clones of the viruses, which is also called "viral rescue" (the rescue of viruses). By reverse genetic manipulation technology, the gene replication and expression regulation mechanism, RNA editing and spontaneous recombination and induced recombination, the interaction relation between virus and host, antiviral strategy and gene therapy research of RNA virus can be researched, and a novel virus vector is constructed to express foreign genes and develop vaccines.

Disclosure of Invention

The invention aims to provide a recombinant infectious haematopoietic necrosis virus and a construction method and application thereof.

In a first aspect, the invention claims a recombinant infectious hematopoietic necrosis virus.

Compared with the infectious haematopoietic necrosis virus before modification, the recombinant infectious haematopoietic necrosis virus provided by the invention only has the difference that a coding gene of a target protein is also contained between a P gene and an M gene in a genome RNA of the infectious haematopoietic necrosis virus before modification.

The recombinant infectious haematopoietic necrosis virus provided by the invention is obtained by inserting a coding gene of a target protein between a P gene and an M gene in a genome RNA of the Infectious Haematopoietic Necrosis Virus (IHNV).

Wherein the P gene encodes phosphoprotein (P) of Infectious Hematopoietic Necrosis Virus (IHNV); the M gene encodes the matrix protein (M) of Infectious Hematopoietic Necrosis Virus (IHNV).

Further, the encoding gene of the target protein may be an antigen gene of a target virus or an encoding gene of a marker protein.

Further, the target virus may be other viruses than the infectious hematopoietic necrosis virus, such as Infectious Pancreatic Necrosis Virus (IPNV), etc.; accordingly, the antigen gene of the target virus can be specifically the VP2gene of the Infectious Pancreatic Necrosis Virus (IPNV) and the like. The marker protein can be green fluorescent protein and the like.

In a specific embodiment of the invention, the Infectious Hematopoietic Necrosis Virus (IHNV) before modification is specifically infectious hematopoietic necrosis virus BLk94 strain (IHNV-BLk 94). The Infectious Pancreatic Necrosis Virus (IPNV) is specifically infectious pancreatic necrosis virus ChRtm213 strain (IPNV-ChRtm 213).

Further, the P gene in the genome RNA of the Infectious Hematopoietic Necrosis Virus (IHNV) before modification encodes phosphoprotein (P) shown in SEQ ID No. 1; the M gene in the genome RNA of the Infectious Haematopoietic Necrosis Virus (IHNV) before modification encodes matrix protein (M) shown in SEQ ID No. 2.

The nucleotide sequence of the coding region of the P gene in the genome RNA of the Infectious Hematopoietic Necrosis Virus (IHNV) before modification is shown as SEQ ID No.4 corresponding to the gene level; the nucleotide sequence of the coding region of the M gene in the genome RNA of the Infectious Hematopoietic Necrosis Virus (IHNV) before modification is shown as SEQ ID No. 5.

Further, the VP2gene of the Infectious Pancreatic Necrosis Virus (IPNV) encodes VP2 protein shown in SEQ ID No. 3.

The nucleotide sequence of the VP2gene of the Infectious Pancreatic Necrosis Virus (IPNV) is shown in SEQ ID No.6, corresponding to the gene level.

Still further, the recombinant infectious hematopoietic necrosis virus differs from the infectious hematopoietic necrosis virus before modification only in that the a fragment ("P GE-M GS" in fig. 3 a) between the coding region of the P gene and the coding region of the M gene in the genomic RNA of the infectious hematopoietic necrosis virus before modification is replaced with a fragment B ("P GE-M GS-VP2gene-P GE-M GS" in fig. 3B). Wherein the sequence of the fragment A is SEQ ID No. 7; the sequence of the fragment B is SEQ ID No.8 (namely SEQ ID No.7+ SEQ ID No.6+ SEQ ID No. 7).

More specifically, the recombinant infectious hematopoietic necrosis virus may be prepared as described in the second aspect below.

In a second aspect, the invention claims a method for preparing a recombinant infectious haematopoietic necrosis virus as described in the first aspect hereinbefore.

The present invention provides a method for preparing a recombinant infectious haematopoietic necrosis virus as described in the first aspect hereinbefore, comprising the steps of: co-transfecting an EPC cell with a recombinant plasmid containing a cDNA sequence corresponding to the genomic RNA of the recombinant infectious hematopoietic necrosis virus and a helper plasmid, thereby obtaining the recombinant infectious hematopoietic necrosis virus.

Wherein, the promoter for starting the expression of the cDNA sequence corresponding to the genome RNA of the recombinant infectious hematopoietic necrosis virus in the recombinant plasmid containing the cDNA sequence corresponding to the genome RNA of the recombinant infectious hematopoietic necrosis virus is a T7 promoter, and the recombinant infectious hematopoietic necrosis virus further contains a coding sequence of hepatitis D virus ribozyme and a T7 termination sequence (marked as pIHNV-VP2) after the cDNA sequence corresponding to the genome RNA of the recombinant infectious hematopoietic necrosis virus. The helper plasmids had a total of 4: helper plasmid 1 (designated as pHelp-N) containing a cDNA sequence corresponding to the coding region of the N gene (the coding gene for nucleoprotein N) in the genomic RNA of the infectious hematopoietic necrosis virus before modification; helper plasmid 2 (designated as pHelp-P) containing a cDNA sequence corresponding to the coding region of the P gene (coding gene for phosphoprotein P) in the genomic RNA of the infectious hematopoietic necrosis virus before modification; helper plasmid 3 (designated as pHelp-NV) containing a cDNA sequence corresponding to the coding region of the NV gene (the coding gene of the non-structural protein NV) in the genomic RNA of the infectious hematopoietic necrosis virus before modification; and a helper plasmid 4 (pHelp-L) containing a cDNA sequence corresponding to the coding region of the L gene (the gene encoding the polymerase protein L) in the genomic RNA of the infectious hematopoietic necrosis virus before transformation.

In the method, the mass ratio of pIHV-VP 2, pHelp-N, pHelp-P, pHelp-Nv and pHelp-L is 2.0: 1.0: 0.5: 0.25: 0.5.

in the method, the recombinant infectious hematopoietic necrosis virus is harvested when more than 60% of the cells present CPE.

In a third aspect, the invention claims the following (a1) or (a2) or (a3) biomaterials:

(a1) an isolated animal cell or recombinant bacterium comprising a recombinant infectious hematopoietic necrosis virus as described above;

(a2) a vector comprising the genomic RNA or cDNA of the recombinant infectious hematopoietic necrosis virus described above;

(a3) a vaccine comprising the recombinant infectious haematopoietic necrosis virus as described hereinbefore.

The IHNV-BLk94 virus is a low virulent strain, so that the immune effect can be detected after the virus is directly injected for 60 days without inactivation.

In a fourth aspect, the invention claims any of the following applications:

(b1) use of a recombinant infectious haematopoietic necrosis virus as described hereinbefore or a biological material as described hereinbefore in the manufacture of a product for the prevention and/or treatment of a disease caused by infection with an Infectious Haematopoietic Necrosis Virus (IHNV) and/or a target virus as described hereinbefore;

(b2) use of a recombinant infectious haematopoietic necrosis virus as described hereinbefore or a biological material as described hereinbefore in the manufacture of a product for inhibiting infection by an Infectious Haematopoietic Necrosis Virus (IHNV) and/or a target virus as described hereinbefore;

(b3) use of a recombinant infectious haematopoietic necrosis virus as described hereinbefore or of a biological material as described hereinbefore for the prevention and/or treatment of a disease caused by infection with an Infectious Haematopoietic Necrosis Virus (IHNV) and/or a target virus as described hereinbefore;

(b4) use of a recombinant infectious haematopoietic necrosis virus as hereinbefore described or a biomaterial as hereinbefore described for inhibiting infection by an Infectious Haematopoietic Necrosis Virus (IHNV) and/or a target virus as hereinbefore described.

In a particular embodiment of the invention, the target virus is in particular Infectious Pancreatic Necrosis Virus (IPNV). The infection of Infectious Hematopoietic Necrosis Virus (IHNV) and/or Infectious Pancreatic Necrosis Virus (IPNV) is infection of rainbow trout.

The invention takes IHNV strain BLk94 as a parental virus, and obtains rIHNV-BLk94 successfully by in vitro rescue by utilizing a reverse genetic manipulation technology. The invention inserts the IPNV structural protein VP2gene between the P and M genes of the rIHNV-BLk94 genome to construct a recombinant virus rIHNV-VP 2. Series of experiments prove that the recombinant virus can play a role in preventing and treating IHNV and IPNV simultaneously, and the invention lays a solid foundation for further developing the prevention and treatment research of IHNV and IPNV.

Drawings

FIG. 1 is a schematic diagram of plasmid construction of the full-length cDNA sequence of rIHNV-BLk94 strain.

FIG. 2 is a schematic diagram of the construction of recombinant plasmid pIHNV-GFP-N/P.

FIG. 3 is a schematic diagram showing the insertion position of VP2gene of IPNV strain between P and M genes in rIHNV-BLk94 genome according to the present invention. A is a schematic diagram of the relevant region of the rIHNV-BLk94 genome; b is a schematic diagram of the relevant region of the genome of the recombinant virus rIHNV-VP 2.

FIG. 4 is a schematic diagram of plasmid construction of the full-length cDNA sequence of rIHNV-VP2 strain.

FIG. 5 is a fragmented clone of the whole genome sequence of IHNV-BLk 94. H1, H2 and H3 are segmented clone products of IHNV-BLk94 whole genome sequence; m is DL15000DNA molecular weight standard; and (3) amplifying the skeleton of the pFLC-LaSota eukaryotic expression vector.

FIG. 6 shows the results of PCR identification of each recombinant plasmid expressing GFP. M is DL2000DNA molecular weight standard; pIHV-GFP-N/P, pIHNV-GFP-P/M, pIHNV-GFP-M/G, pIHNV-GFP-G/NV and pIHV-GFP-NV/L are respectively the PCR identification results of each recombinant plasmid.

FIG. 7 shows the cloning of the helper plasmid IHNV-BLk 94. N, P, Nv and L is IHNV-BLk94 gene fragment; m₁Is DL15000DNA molecular weight standard; m₂Is DL2000DNA molecular weight standard.

FIG. 8 shows TCID of each recombinant virus₅₀And (6) detecting.

FIG. 9 is a growth curve of each recombinant virus.

FIG. 10 shows the expression level of GFP mRNA of each recombinant virus.

FIG. 11 shows the expression level of GFP in each recombinant virus.

FIG. 12 shows cytopathic effect of recombinant virus rIHNV-VP 2.

FIG. 13 shows the protection rate measurements after IHNV challenge.

FIG. 14 shows the in vivo viral load assay of rainbow trout after IPNV challenge.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The nucleotide sequence of the coding region of the P gene in the genomic RNA of the infectious hematopoietic necrosis virus strain BLk94 (IHNV-BLk94) involved in the following examples is shown in SEQ ID No.4, which encodes phosphoprotein (P) shown in SEQ ID No. 1. The nucleotide sequence of the coding region of the M gene in the genomic RNA of IHNV-BLk94 is shown in SEQ ID No.5, which codes for the matrix protein (M) shown in SEQ ID No. 2. The sequence of the RNA fragment located between the coding region of the P gene and the coding region of the M gene (corresponding to "P GE-M GS" in A in FIG. 3) in the genomic RNA of IHNV-BLk94 is shown in SEQ ID No. 7.

The nucleotide sequence of the coding region of the VP2gene of the infectious pancreatic necrosis virus ChRtm213 strain (IPNV-ChRtm213) involved in the following examples is shown in SEQ ID No.6, which encodes the VP2 protein shown in SEQ ID No. 3.

Example 1 construction and application of recombinant virus rIHNV-VP2

A, material and method BLk94

(I) test materials and reagents

Trizol is a product of Invitrogen corporation (10296028); infectious hematopoietic necrosis virus strain BLk94 (IHNV-BLk94 for short) (Genbank accession number: DQ164100) and infectious pancreatic necrosis virus ChRtm213 strain (IPNV-ChRtm213 for short) (Genbank accession number: KX234591) were stored in the laboratory; EPC cells ATCC CRL-2872; pBluescript II SK modified vector pFLC-LaSota containing hepatitis D virus ribozyme, and helper plasmid vector pTM-NP were stored in the laboratory (reference "Zhang Zheng. establishment of novel recombinant Newcastle Disease Virus (NDV) expression system and determination of optimal foreign gene expression position [ D ]. Heilongjiang: northeast agriculture university, 2015: 16-19."). The cell culture solution MEM, trypsin and Hank's solution are products of Hyclone organism company. Fetal bovine serum is a product of Gibco Biochemical company. RT-PCR one-step kit (12574035) and liposome 2000 are Invitrogen Biotech products. The In-Fusion PCR Cloning Kit is a product of Clontech (600670). An anti-IHNV antibody is prepared and stored in the laboratory, the antibody is a surface glycoprotein of an expressed IHNV virus as an immunogen, and a polyclonal antibody obtained by immunizing a New Zealand white rabbit (the reference is 'Zhaoqiang, Xudawn, Liu \ 2815634, and the like; detection of truncated expression and immunogenicity of glycoprotein of fish infectious hematopoietic necrosis virus [ J ]. journal of cell and molecular immunology, 2014,30(12):1238 + 1242.'), and a fluorescence-labeled secondary antibody is Ebioscience biology company.

(II) primer design and Synthesis

Specific primers are designed by using Prime primer 5.0 software according to IHNV-BLk94 whole genome sequence and pFLC-LaSota eukaryotic expression vector framework sequence, and are used for constructing eukaryotic transcription plasmids of recombinant virus rIHNV-BLk94 whole genome cDNA sequence, and the primer sequences are shown in Table 1.

TABLE 1 rIHNV-BLk94 Whole genome cDNA sequence cloning primers

Primer and method for producing the same	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’

Specific primers are designed by using Prime primer 5.0 software according to the IHNV-BLk94 whole genome sequence and an auxiliary plasmid vector pTM-NP, and are respectively used for constructing eukaryotic auxiliary plasmids of recombinant virus rIHNV-BLk94 nucleoprotein (N), phosphoprotein (P), non-structural protein (NV) and polymerase protein (L) cDNA sequences, and the primer sequences are shown in Table 2.

TABLE 2 cloning primers for the rIHNV-BLk94 helper plasmid cDNA sequence

Specific primers are designed by using Prime primer 5.0 software according to the whole genome sequence of IHNV-BLk94 and the GFP gene sequence, and are used for constructing rIHNV-BLk94 vector expression GFP recombinant plasmids, and the primer sequences are shown in Table 3.

TABLE 3 construction of rIHNV-BLk94 vector expression GFP plasmid cloning primers

Specific primers were designed using Prime primer 5.0 software to construct rIHNV-VP2 vector expression VP2 recombinant plasmid primer sequences based on the VP2gene sequence of the IPNV strain are shown in Table 4.

TABLE 4 construction of rIHNV-VP2 recombinant plasmid cloning primers

Primer and method for producing the same	Sequence (5 '→ 3')
		ZT-F	5’-ATGTCCATTTTCAAGAGAGCAAAGA-3’
ZT-R	5’-GCTCTCGTTTGAACTGACTCTTGGACTT-3’
		VP2-F	5’-AGTTCAAACGAGAGCATGAACACATCCAAGGCAACCGCAA-3’
VP2-R	5’-CTTGAAAATGGACATTCATGCCTTTGAGGTTGGTAGGTCA-3’

(III) amplification and RNA extraction of viruses

Viral suspensions of IHNV-BLk94 and IPNV-ChRtm213 were diluted 10 times with cell maintenance medium (2% FBS in MEM medium)⁵Double, inoculated into monolayer confluent EPC cells, at 15 degrees C culture. Collecting cells and culturing when more than 80% of cells have cytopathic effect (CPE)The nutrient solution (i.e., virus suspension) was sampled to obtain 250. mu.L of virus suspension (adjusted to 10%⁵Viral particles) in rnase-free centrifuge tubes. Centrifuge at 12000 g for 5min to remove the precipitate. Adding 750 mu L of Trizol lysate, shaking and mixing uniformly, adding 200 mu L of phenol chloroform, mixing uniformly, centrifuging 12000 g for 15min after 10min, sucking supernatant fluid, adding into a new centrifuge tube, adding isopropanol with the same volume, repeatedly reversing and mixing uniformly, centrifuging 12000 g for 15min, discarding the supernatant fluid, adding 1mL of 75% ethanol, reversing and washing, centrifuging 12000 g for 10min, discarding the supernatant fluid, centrifuging 4000 for 10s, sucking liquid at the bottom of the tube, and drying at room temperature for 3 min; adding 100 μ L of RNase-free water, dissolving the RNA precipitate completely, and packaging at-80 deg.C.

Construction of full-length cDNA sequence plasmid and eukaryotic auxiliary plasmid of (tetra) rIHNV-BLk94 strain

1. Construction of full-length cDNA sequence plasmid of rIHNV-BLk94 strain

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

And (3) amplifying IH1, IH2 and IH3 fragments covering the full-length genome of the IHNV-BLk94 strain by using the IHNV-BLk94RNA extracted in the step (three) as a template and the sequences in the table 1 as primers by using an RT-PCR one-step reaction kit. Amplifying the IH1 fragment by using primers IH1-F and IH 1-R; amplifying the IH2 fragment by using primers IH2-F and IH 2-R; the IH3 fragment is amplified by using primers IH3-F and IH 3-R. Then, according to Pfu Ultra^TMII Fusion HS DNA polymerase instruction, using laboratory preserved pFLC-LaSota eukaryotic expression vector as template, using the sequence (pB-F and pB-R) of Table 1 as primer, amplifying pFLC-LaSota eukaryotic expression vector skeleton fragment. The target gene fragments are recovered by using a gel recovery Kit, IH1, IH2, IH3 and pFLC-LaSota eukaryotic expression vector framework fragments are connected according to the In-Fusion PCR Cloning Kit specification, a plasmid pIHV-BLk 94 containing the full-length cDNA sequence of the rIHNV-BLk94 strain is constructed, and the plasmid pIHV-BLk 94 is verified to be correct by sequencing.

2. Construction of eukaryotic helper plasmids

And (3) respectively amplifying the N, P, NV and L gene fragments of the IHNV-BLk94 strain by using the IHNV-BLk94RNA extracted in the step (three) as a template and the sequences in the table 2 as primers and using an RT-PCR one-step reaction kit. The primer IHN is adopted for amplifying the N gene segmentF and IHN-R; primers IHP-F and IHP-R are adopted for amplifying the P gene segment; primers IHNv-F and IHNv-R are adopted for amplifying the NV gene segment; primers IHL-F and IHL-R are adopted for amplifying the L gene segment. According to Pfu Ultra^TMII Fusion HS DNA polymerase instruction, laboratory preservation of the auxiliary plasmid vector pTM-NP as template, using Table 2 sequences (phelp-F and phelp-R) as primers, amplification of auxiliary plasmid vector pTM-NP framework fragment. The above target gene fragments were recovered using a gel recovery Kit, and N, P, NV and the L gene fragment were ligated to the helper plasmid vector pTM-NP backbone fragment according to the In-Fusion PCR Cloning Kit instructions, respectively, to construct helper plasmids pHelp-N, pHelp-P, pHelp-Nv and pHelp-L for virus rescue. And verified to be correct by sequencing.

(V) construction of GFP subclones

Cloning the sequence between the N gene and the M gene of IHNV into pcDNA3.1 vector by using the pIHHNV-BLk 94 full-length cDNA constructed in the step four 1 as a template and NF and NR in the table 3 as primers to obtain the IHNV P gene subcloned pcDNA3.1-NM/P⁺。

The GFP fragment was amplified using p519GFP plasmid (ATCC: 87452) as a template and GFP insert F and GFP insert R in Table 3 as primers; using pcDNA3.1-NM/P⁺Using GFP Vet F and GFP Vet R in Table 3 as primers as template, and amplifying to obtain vector fragment pcDNA3.1-NM/P with P gene open reading frame removed^-(ii) a The GFP fragment was mixed with pcDNA3.1-NM/P^-After vector ligation, GFP subclone (GFP template) containing Gene end-Gene start-GFP was obtained and verified by sequencing to be correct, facilitating the construction of later-stage recombinant plasmids.

(VI) construction of recombinant plasmid pIHV-GFP-N/P, pIHNV-GFP-P/M, pIHNV-GFP-M/G, pIHNV-GFP-G/NV and pIHV-GFP-NV/L and rescue of virus

In order to determine the optimal position of IHNV expression foreign gene, the invention inserts the indicator protein GFP between N and P genes, between P and M, between M and G, between G and NV and between NV and L of IHNV genome in sequence, and constructs a series of pIHNV-GFP plasmids with different insertion positions.

The following describes the construction and rescue strategy of a series of recombinant plasmids in detail by taking the insertion of an exogenous GFP gene between N and P genes as an example, and the specific strategy is shown in FIG. 2.

1. Construction of recombinant plasmid pIHV-GFP-N/P

The GFP template obtained in step five and pIHV-BLk 94 plasmid obtained in step four 1 were used as templates, respectively, to amplify the GFP N/P fragment and pIHV-GFP-N/P Vet fragment using the primers shown in Table 3. Primers adopted when GFP N/P fragments are amplified are N/P insert F and N/P insert R; primers used for amplifying pIHV-GFP-N/P Vet fragments are N/P Vet F and N/P Vet R. And respectively recovering the fragments obtained by PCR, connecting, transforming the connecting product into escherichia coli DH5 alpha competent cells, selecting a single colony, and identifying the correct positive plasmid, namely the pIHV-GFP-N/P plasmid (and verifying the correctness by sequencing).

2. Rescue of recombinant virus rIHNV-GFP-N/P

pIHNV-GFP-N/P plasmid 2.0. mu.g, helper plasmid pHelp-N1.0. mu.g, pHelp-P0.5. mu.g, pHelp-Nv 0.25. mu.g and pHelp-L0.5. mu.g were co-transfected into EPC cells, and the procedure was carried out according to the Invitrogen Lipofectamine 2000 transfection protocol. Culturing the transfected cells in an incubator at 15 ℃, observing the cell state every day, and when more than 60% of the cells have CPE, harvesting the virus suspension and storing the virus suspension at-80 ℃ for later use.

3. Construction of recombinant plasmid pIHV-GFP-P/M, pIHNV-GFP-M/G, pIHNV-GFP-G/NV and pIHV-GFP-NV/L and virus rescue

The construction of recombinant plasmids pIHV-GFP-P/M, pIHNV-GFP-M/G, pIHNV-GFP-G/NV and pIHV-GFP-NV/L is different from that of virus rescue except for the GFP insert and primers used in vector amplification, and other steps are the same as the construction of pIHV-GFP-N/P and the virus rescue. The primers adopted during the amplification of the GFP P/M fragment are P/M insert F and P/M insert R in the table 3, and the primers adopted during the amplification of the pIHV-GFP-P/M Vet fragment are P/M Vet F and P/M Vet R in the table 3; the primers adopted during the amplification of the GFP M/G fragment are M/G insert F and M/G insert R in the table 3, and the primers adopted during the amplification of the pIHV-GFP-M/G Vet fragment are M/G Vet F and M/G Vet R in the table 3; the primers adopted during the amplification of the GFP G/NV fragment are G/NV insert F and G/NV insert R in the table 3, and the primers adopted during the amplification of the pIHV-GFP-G/NV Vet fragment are G/NV Vet F and G/NV Vet R in the table 3; primers used for amplifying the GFP NV/L fragment are NV/L insert F and NV/L insert R in Table 3, and primers used for amplifying the pIHV-GFP-NV/L Vet fragment are NV/L Vet F and NV/L Vet R in Table 3.

(VII) rescue virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and rIHNV-GFP-NV/L TCID₅₀Measurement of

Taking EPC cells with good growth state, digesting, and then obtaining the EPC cells according to the 2 x 10⁴ Subpackaging 100 μ L/100 μ L in 96-well plate, inoculating virus suspension of each isolate with different dilutions, each dilution having 8 wells, setting blank control group for 100 μ L each, culturing at 15 deg.C, observing for 7 days, and calculating TCID by Reed-Muench method₅₀。

(eight) growth curves of rescued viruses rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and rIHNV-GFP-NV/L

Taking EPC cells with good growth state, digesting, and then obtaining the EPC cells according to the 2 x 10⁴ Subpackaging 100 μ L/100 μ L in 96-well plate, inoculating virus suspension of each isolate with different dilutions, each dilution having 8 wells, setting blank control group for 100 μ L each well, culturing at 15 deg.C, collecting virus every 12h, and calculating TCID by Reed-Muench method₅₀And (5) drawing a virus growth curve.

(nine) detecting the expression quantity of mRNA of rescued virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and rIHNV-GFP-NV/L GFP by transcription level

EPC monolayers were seeded in 6-well cell culture plates and replaced with MEM medium containing 10% serum before virus inoculation. Each rescued virus was inoculated into 6-well plates at 0.1MOI (multiplex of infection), cultured at 15 ℃ for 1 hour, and replaced with a 2% serum maintenance solution. 0.5% CO at 15 ℃₂After culturing for 48 hours under the conditions, viral RNA was extracted, and the expression level of recombinant viral GFP mRNA was detected using One Step SYBR PrimeScript PLUS RT-PCR Kit (RR096A, Takara, Japan) using RT-GFP F: 5'-CGAGGTGGTGTACATGAACGA-3', RT-GFP R: 5'-GCTGTAGAACTTGCCGCTGTT-3', the internal reference gene is the N protein gene of the virus, the internal reference primer is RT-N F: 5'-AGGAGAGGGAACGAGAAGG-3' the flow of the air in the air conditioner,RT-N R：5′-TGTTGGGATCTGCGAAAGTG-3′。

(ten) detection of recombinant virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and rIHNV-GFP-NV/L GFP protein expression level

EPC monolayers were seeded in 6-well cell culture plates and replaced with MEM medium containing 10% serum before virus inoculation. Each rescued virus was inoculated into 6-well plates at 0.1MOI (multiplex of infection), cultured at 15 ℃ for 1 hour, and replaced with a 2% serum maintenance solution. 0.5% CO at 15 ℃₂After culturing for 48h under the condition, cells are digested by pancreatin, washed by PBS for 2 times, and then the expression condition of the green fluorescent protein GFP of each recombinant virus is detected by a flow cytometer.

Construction of full-Length cDNA sequence plasmid of (eleven) rIHNV-VP2 Strain

The results of the previous studies demonstrated that the amount of IHNV mRNA transcribed decreases from 3 'to 5' of the genome and that the expression level of GFP protein is highest when GFP is inserted between the P and M genes of the rIHNV-BLk94 vector compared to other insertion sites. This indicates that the noncoding region between the P and M genes of the rIHNV-BLk94 genome is the best location for expressing the foreign gene, considering the transcription characteristics of the mRNA of the IHNV genome together with the effect of the foreign gene on viral replication. Therefore, the invention inserts VP2gene of IPNV strain between P and M genes of rIHNV-BLk94 genome to construct recombinant virus rIHNV-VP2 (figure 3) containing VP2gene of IPNV strain.

And (3) amplifying a target fragment covering the IPNV strain VP2gene by using the RNA of the IPNV-ChRtm213 extracted in the step (three) as a template and the VP2-F and VP2-R in the table 4 as primers through an RT-PCR one-step reaction kit. And (3) taking the pIHNV-GFP-P/M recombinant plasmid constructed in the step six 3 as a template, and using ZT-F and ZT-R in the table 4 as primers to amplify and remove the vector sequence of the GFP gene. By means of Pfu Ultra^TMII Fusion HS DNA polymerase, connecting VP2gene target fragment and vector sequence of removing GFP gene, constructing recombinant plasmid pIHV-VP 2 containing VP2 cDNA sequence, the construction strategy is shown in figure 4.

Rescue of (twelve) recombinant virus rIHNV-VP2 strain

pIHNV-VP2 plasmid 2.0. mu.g, helper plasmids pHelp-N1.0. mu.g, pHelp-P0.5. mu.g, pHelp-Nv 0.25. mu.g and pHelp-L0.5. mu.g were co-transfected into EPC cells, and the procedure was carried out according to the Invitrogen Lipofectamine 2000 transfection protocol. Culturing the transfected cells in an incubator at 15 ℃, observing the cell state every day, and when more than 60% of the cells have CPE, harvesting the virus suspension and storing the virus suspension at-80 ℃ for later use.

Application of (thirteen) recombinant virus rIHNV-VP2

The harvested recombinant virus rIHNV-VP2 was injected into groups of SPF rainbow trout (5 + -1 g) immunized with 50 tails per group at an immunization dose of 50 pfu/tail per tail, and control groups were immunized with PBS. IHNV (10) was used 60d after immunization²pfu/tail) and IPNV (10)⁶pfu/tail) virus were infected separately and immunoprotection assays were performed.

Second, results and analysis

1. Construction of full-Length cDNA plasmid of rIHNV-BLk94 Strain

The IHNV-BLk94 whole genome sequence is 11132bp in length, the invention divides the IH1, IH2 and IH3 three segments of gene fragments into 3756bp, 3680bp and 3726bp in sequence for cloning, and the electrophoretic analysis result of products amplified by the RT-PCR one-step reaction kit shows that the size of the 3 segments of gene fragments is consistent with the expected result (see figure 5). Specific primers are used for amplifying the pFLC-LaSota eukaryotic expression vector skeleton sequence, and the electrophoresis result shows that the vector size is 3140bp consistent with the expected result (see figure 5). The IH1, IH2, IH3 and pFLC-LaSota eukaryotic expression vector skeleton fragments were ligated according to the In-Fusion PCR Cloning Kit instructions to construct a plasmid pIHNV-BLk94 containing the full-length cDNA sequence of IHNV-BLk94 strain.

2. Construction of recombinant plasmid pIHV-GFP-N/P, pIHNV-GFP-P/M, pIHNV-GFP-M/G, pIHNV-GFP-G/NV and pIHV-GFP-NV/L

Respectively amplifying a GFP N/P fragment, a GFP P/M fragment, a GFP M/G fragment, a GFP G/NV fragment, a GFP NV/L fragment, a pIHV-GFP-N/P Vet carrier fragment, a pIHV-GFP-P/M Vet carrier fragment, a pIHV-GFP-M/G Vet carrier fragment, a pIHV-GFP-G/NV Vet carrier fragment and a pIHV-GFP-NV/L Vet carrier fragment by using specific primers, the target fragment is respectively connected with the vector to obtain recombinant plasmids pIHNV-GFP-N/P, pIHNV-GFP-P/M, pIHNV-GFP-M/G, pIHNV-GFP-G/NV and pIHNV-GFP-NV/L, and the PCR identification result of the successfully constructed recombinant plasmids is shown in figure 6.

3. Construction of eukaryotic helper plasmid of rIHNV-GFP strain

The RNA of IHNV-BLk94 strain is used as a template, and specific primers are used for respectively amplifying N, P, Nv and L gene segments of IHNV-BLk94 strain, wherein the sizes of the gene segments are 1176bp, 693bp, 336bp and 5961bp respectively (see figure 7). Meanwhile, a pFLC-LaSota vector framework fragment is amplified by using a specific primer, and N, P, Nv and an L gene fragment are respectively connected with the pFLC-LaSota vector framework fragment according to the In-Fusion PCR Cloning Kit specification to construct helper plasmids phelp-N, phelp-P, phelp-NV and phelp-L for rescuing the rIHNV-GFP strain.

4. TCID for rescuing virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and rIHNV-GFP-NV/L₅₀Measurement of

In order to determine the change of the infection capacity of the recombinant viruses after inserting GFP genes at different sites, the invention respectively treats the TCID of each recombinant virus₅₀Detection was performed. The results showed that the TCID of each rescued virus₅₀The titers were similar to those of the parental virus rIHNV-BLk94, indicating that the rescued viruses rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and rIHNV-GFP-NV/L retained the infectivity of their parental viruses (FIG. 8).

5. Growth curves of rescued viruses rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and rIHNV-GFP-NV/L

To study the replication and growth kinetics of rescued viruses, the virus was diluted 10 in the present invention^-5After doubling, EPC cells were infected separately, and then the virus was harvested every 12h and TCID of the virus was determined at different infection time points₅₀Detection was performed and growth curves of the infecting virus were plotted (FIG. 9). As shown, the homogeneous rIHNV-BLk94 parental virus at the same time point for each recombinant virus expressing GFP protein maintains a similar map, showing similar replication and growth kinetics as the rIHNV-BLk94 parental virus. However, the growth rate of rIHNV-GFP-N/P is slightly delayed compared to other recombinant viruses expressing GFP, about0.5-1 titer of other viruses laggard. Although the rIHNV-GFP-P/M virus was slightly higher than the rIHNV-GFP-N/P virus at the same time point, it was still significantly lower than other rescued viruses. The titer of the virus at the same time point is sequentially from high to low: rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV, rIHNV-GFP-NV/L. This indicates that the insertion of the foreign protein GFP has a great influence on the replication of the virus, and that the closer the inserted gene is to the 3' end of the genome, the greater the influence on the replication efficiency of the virus.

6. Transcript level detection for expression level of rescued virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and rIHNV-GFP-NV/L

The expression level of the foreign gene GFP expressed by each rescued virus was detected at the transcriptional level using specific Real-time PCR primers and the viral N protein gene as an internal control (FIG. 10). The results show that the expression level of GFP mRNA of each rescue virus presents rIHNV-GFP-N/P > rIHNV-GFP-P/M > rIHNV-GFP-M/G > rIHNV-GFP-G/NV > rIHNV-GFP-NV/L.

7. Detection of recombinant virus rIHNV-GFP-N/P, rIHNV-GFP-P/M, rIHNV-GFP-M/G, rIHNV-GFP-G/NV and rIHNV-GFP-NV/L GFP protein expression quantity

The Real-time PCR result shows that the more the foreign protein is transcribed near the 3' end of the viral genome, the more the mRNA is transcribed; however, the growth curve of the virus shows that the closer the foreign gene is to the 3' end of the genome, the greater the influence on the replication efficiency of the virus. In order to determine the optimal expression site of the foreign protein, the expression level of GFP protein of each rescued virus was examined by flow cytometry in this study (fig. 11). As can be seen from the results, the rescued virus rIHNV-GFP-P/M has the largest GFP expression amount, and the specific sequence is rIHNV-GFP-P/M > rIHNV-GFP-M/G > rIHNV-GFP-N/P > rIHNV-GFP-G/NV > rIHNV-GFP-NV/L. From the above results, it was found that the highest expression level of the foreign gene was obtained by inserting the foreign gene between the P gene and M gene of IHNV-BLk94 genome, taking into consideration the transcription characteristics of mRNA in IHNV genome and the influence of the foreign gene on viral replication.

8. Full-length cDNA plasmid construction and virus rescue of rIHNV-VP2 strain

The RNA of the IPNV-ChRtm213 strain is used as a template, and the specific primer is used for amplifying the gene segment of the VP2gene of the IPNV strain, wherein the size of the gene segment is 1329 bp. The VP2gene is replaced by the GFP gene of the pIHNV-GFP-P/M plasmid by using specific primers according to the In-Fusion PCR Cloning Kit instruction, and the pIHNV-VP2 recombinant plasmid containing the IPNV strain VP2gene fragment is constructed. pIHV-VP 2, pHelp-N1.0 μ g, pHelp-P, pHelp-Nv, and pHelp-L were co-transfected with EPC cells, and recombinant virus rIHNV-VP2 was rescued, and the cytopathic results are shown in FIG. 12.

In addition, genome sequencing of recombinant virus rIHNV-VP2 revealed that recombinant virus rIHNV-VP2 differed from IHNV-BLk94 only in replacing the fragment A ("P GE-M GS" in A in FIG. 3) between the P gene coding region ("P CDS" in A in FIG. 3) and the M gene coding region ("M CDS" in A in FIG. 3) in the genomic RNA of IHNV-BLk94 with the fragment B ("P GE-M VP2gene-P GE-M GS" in B in FIG. 3). Wherein the sequence of the fragment A is SEQ ID No. 7; the sequence of the fragment B is SEQ ID No.8 (namely SEQ ID No.7+ SEQ ID No.6+ SEQ ID No. 7).

9. Application of recombinant virus rIHNV-VP2

The recombinant virus rIHNV-VP2 is diluted to immunize SPF rainbow trout, and the immune protection rate of IHNV and the IPNV virus load in tissues are respectively detected by infecting IHNV and IPNV viruses at 60d after immunization. As can be seen from the results, the rainbow trout immunized with the recombinant virus rIHNV-VP2 showed an immunoprotection rate of about 85% compared to the control group (replacing the recombinant virus rIHNV-VP2 with PBS) when infected with IHNV virus (FIG. 13); upon IPNV virus infection, the viral load of IPNV was significantly reduced in the immunoprotected group of recombinant viruses compared to the control group (fig. 14). The results show that the recombinant virus rIHNV-VP2 has good immune protection effect on preventing infection of the rainbow trout IHNV and IPNV viruses, and the result provides technical support for the healthy development of the rainbow trout breeding industry in China.

<110> institute of aquatic products of Heilongjiang, China institute of aquatic science

<120> recombinant infectious hematopoietic necrosis virus, and construction method and application thereof

<130> GNCLN181321

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Claims

1. A recombinant infectious hematopoietic necrosis virus characterized by: compared with the infectious haematopoietic necrosis virus before modification, the recombinant infectious haematopoietic necrosis virus only has the difference that a coding gene of a target protein is also contained between a P gene and an M gene in the genome RNA of the recombinant infectious haematopoietic necrosis virus;

the coding gene of the target protein is an antigen gene of a target virus;

the antigen gene of the target virus is VP2gene of the infectious pancreatic necrosis virus;

the infectious hematopoietic necrosis virus before modification is infectious hematopoietic necrosis virus strain BLk 94.

2. The recombinant infectious hematopoietic necrosis virus of claim 1, wherein: the infectious pancreatic necrosis virus is an infectious pancreatic necrosis virus ChRtm213 strain.

3. The recombinant infectious hematopoietic necrosis virus of claim 1, wherein: the P gene in the genome RNA of the infectious haematopoietic necrosis virus before modification encodes the phosphoprotein shown in SEQ ID No. 1.

4. The recombinant infectious hematopoietic necrosis virus of claim 1, wherein: the M gene in the genome RNA of the infectious haematopoietic necrosis virus before modification encodes the matrix protein shown in SEQ ID No. 2.

5. The recombinant infectious hematopoietic necrosis virus of claim 2, wherein: the gene VP2 of the infectious pancreatic necrosis virus encodes VP2 protein shown in SEQ ID No. 3.

6. The recombinant infectious hematopoietic necrosis virus of claim 3, wherein: the nucleotide sequence of the coding region of the P gene in the genome RNA of the infectious haematopoietic necrosis virus before modification is shown as SEQ ID No. 4.

7. The recombinant infectious hematopoietic necrosis virus of claim 4, wherein: the nucleotide sequence of the coding region of the M gene in the genome RNA of the infectious haematopoietic necrosis virus before modification is shown as SEQ ID No. 5.

8. The recombinant infectious hematopoietic necrosis virus of claim 5, wherein: the nucleotide sequence of the coding region of the VP2gene of the infectious pancreatic necrosis virus is shown in SEQ ID No. 6.

9. The recombinant infectious hematopoietic necrosis virus of claim 1, wherein: the recombinant infectious hematopoietic necrosis virus differs from the infectious hematopoietic necrosis virus before the alteration only in that a fragment a located between the coding region of the P gene and the coding region of the M gene in the genomic RNA of the infectious hematopoietic necrosis virus before the alteration is replaced with a fragment B; the sequence of the fragment A is SEQ ID No. 7; the sequence of the fragment B is SEQ ID No. 8.

10. A method of preparing the recombinant infectious hematopoietic necrosis virus of any one of claims 1-9, comprising the steps of: co-transfecting an EPC cell with a recombinant plasmid containing a cDNA sequence corresponding to the genomic RNA of the recombinant infectious hematopoietic necrosis virus and a helper plasmid, thereby obtaining the recombinant infectious hematopoietic necrosis virus.

11. The method of claim 10, wherein: the promoter for promoting the expression of the cDNA sequence corresponding to the genome RNA of the recombinant infectious haematopoietic necrosis virus in the recombinant plasmid containing the cDNA sequence corresponding to the genome RNA of the recombinant infectious haematopoietic necrosis virus is a T7 promoter.

12. The method of claim 10, wherein: the helper plasmids had a total of 4: a helper plasmid 1 containing a cDNA sequence corresponding to the coding region of the N gene in the genomic RNA of the infectious hematopoietic necrosis virus before modification; a helper plasmid 2 containing a cDNA sequence corresponding to the coding region of the P gene in the genomic RNA of the infectious hematopoietic necrosis virus before modification; a helper plasmid 3 containing a cDNA sequence corresponding to the coding region of the NV gene in the genomic RNA of the infectious hematopoietic necrosis virus before modification; and a helper plasmid 4 containing a cDNA sequence corresponding to the coding region of the L gene in the genomic RNA of the infectious hematopoietic necrosis virus before modification.

13. The following (a1) or (a2) or (a3) biomaterials:

(a1) an isolated animal cell or recombinant bacterium comprising the recombinant infectious hematopoietic necrosis virus of any one of claims 1 to 9;

(a2) a vector comprising the genomic RNA or cDNA of the recombinant infectious hematopoietic necrosis virus of any one of claims 1 to 9;

(a3) a vaccine comprising the recombinant infectious hematopoietic necrosis virus of any one of claims 1-9.

14. Any of the following applications:

(b1) use of the recombinant infectious haematopoietic necrosis virus of any one of claims 1 to 9 or the biomaterial of claim 13 in the manufacture of a product for the prevention and/or treatment of a disease caused by infection with an infectious haematopoietic necrosis virus and/or an infectious pancreatic necrosis virus;

(b2) use of a recombinant infectious haematopoietic necrosis virus of any one of claims 1 to 9 or a biomaterial according to claim 13 in the manufacture of a product for use in inhibiting infection by an infectious haematopoietic necrosis virus and/or an infectious pancreatic necrosis virus.