CN108904792B

CN108904792B - Anti-nerve necrosis virus immersion vaccine using baculovirus as carrier and preparation method thereof

Info

Publication number: CN108904792B
Application number: CN201810797600.3A
Authority: CN
Inventors: 谢俊锋; 黄润庆; 张静; 何建国
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2018-07-19
Filing date: 2018-07-19
Publication date: 2021-10-08
Anticipated expiration: 2038-07-19
Also published as: CN108904792A

Abstract

The present invention provides an anti-neuronecrosis virus immersion vaccine using baculovirus as a carrier, which comprises a recombinant baculovirus expressing the outer convex domain of the neuronecrosis virus capsid protein on the outer membrane of the baculovirus. The anti-nerve necrosis virus immersion vaccine of the invention can effectively stimulate the innate immune system of fish, effectively prevent and cure nerve necrosis virus, and has high safety.

Description

Anti-nervous necrosis virus soaking vaccine using baculovirus as carrier and preparation method thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an anti-nervous necrosis virus immersion vaccine taking baculovirus as a vector and a preparation method thereof.

Background

The Nervous Necrosis Virus (NNV) mainly infects larval fish, juvenile fish and part of juvenile fish, and once outbreak, the lethality rate approaches 100%. However, due to the characteristics of small size, uneven feeding and the like of the larval fish, the vaccine for preventing and treating NNV at the stage cannot be injected and fed, so that a vaccine capable of immunizing by a soaking mode is urgently needed.

Disclosure of Invention

The invention aims to solve the technical problems and provides a soaking vaccine capable of effectively preventing and treating nervous necrosis virus for fish larvae.

Another object of the present invention is to provide a method for preparing the immersion vaccine.

In order to achieve the above objects, the present invention provides a baculovirus-derived anti-neurturin virus-impregnated vaccine, wherein the vaccine comprises a recombinant baculovirus in which a pro-domain (CP) of a Capsid Protein (CP) of a neurturin virus is displayed on an outer membrane of the baculovirus.

Preferably, the anti-neurturin virus immersion vaccine further comprises additives such as, but not limited to, vaccine adjuvants, preservatives, stabilizers, substances for adjusting the pH and/or isotonicity of the vaccine, or combinations thereof.

Preferably, the baculovirus is Autographa californica multiple nuclear polyhedrosis virus (AcMNPV).

Preferably, the anti-nervous necrosis virus soaking vaccine is suitable for various animals which can be infected with nervous necrosis virus, particularly fish, more particularly seawater fish, including but not limited to grouper or trachinotus ovatus.

The invention also provides a preparation method of the anti-nervous necrosis virus soaking vaccine, which comprises the following steps:

(1) taking a nervous necrosis virus genome as a template, amplifying in an RT-PCR mode to obtain an outer convex structural domain P domain gene coding sequence of a nervous necrosis virus capsid protein, carrying EcoR I and Hind III enzyme cutting sites, and cloning to a plasmid pFastBacHT A;

(2) amplifying gene coding sequence of N-terminal 40 amino acid residues NA40 of influenza virus neuraminidase and linker (GGGGS)₃The promoter P is obtained by inserting the plasmid pFactibacHT A into the restriction sites of Nco I and EcoR I_PHNA40 Gene coding sequence, linker (GGGGS)₃Recombinant baculovirus with coding sequence and P domain gene coding sequence.

Preferably, the preparation method of the anti-nervous necrosis virus soaking vaccine further comprises culturing and producing the recombinant baculovirus. More preferably, Sf9 cells are infected with recombinant baculovirus for production.

Preferably, the primers for amplifying the coding sequence of the evaginable domain P domain gene of the capsid protein of the nervous necrosis virus are as follows:

F1：GGAATTCACACCTGAAGAGACCACCGCTC；

R2：CCCAAGCTTTTAGTTTTCCGAGTCAACCCTG。

preferably, the primers for amplifying the gene coding sequence of the first 40 amino acid residues NA40 from the N-terminal of influenza virus neuraminidase are as follows:

F3：CATGCCATGGATATGAATCCAAATCAAAAAATAATAACCATTG；

R4：GGAATTCGCTGCCGCCACCGCCGCTTCC。

the invention also provides a method for immunizing fishes against the nervous necrosis viruses, wherein the nervous necrosis virus resistant immersion vaccine is used for immersing immune fish larvae at least twice for immersion immunization. Preferably, the final concentration of the anti-nervous necrosis virus immersion vaccine for immersion immunization of the fish larvae is 10⁴TCID₅₀and/mL. More specifically, at a final concentration of 10⁴TCID₅₀The anti-nervous necrosis virus soaking vaccine is used for soaking immune fish larvae for 2h, and after normal breeding for 48h, the same conditions (the final concentration is 10)⁴TCID₅₀/mL) soaking for the second time, and can effectively prevent the nervous necrosis virus infection under normal conditions (the nervous necrosis virus of natural infection is generally 10%³Below).

Preferably, the fish may be a variety of fish that can be infected with the neuro-necrosis virus, particularly marine fish, including but not limited to grouper or trachinotus ovatus.

As a preferred embodiment, if the culture conditions are not good, the culture medium can be soaked in the same conditions after 48 hours of the secondary immunization, and the immunization can be strengthened once.

The congenital immune system of the fish body is activated after the first immunization (the expression of related genes is up-regulated) by detecting the gene expression condition through RT-PCR (reverse transcription-polymerase chain reaction), and the immune system is more active and enough to resist the nervous necrosis virus infection under the natural condition after the second immunizationAnd (6) dyeing. Performing nervous necrosis virus injection to the young fish after twice immunization (10)⁴TCID₅₀mL), it was found that the immune protection rate was as high as 85.4%.

In addition, cell infection (cells of mammals and fishes) and fish body safety detection (long-time soaking and intraperitoneal injection) prove that the nervous necrosis virus resistant soaking vaccine can not generate toxic effect influence on vertebrates and cells. Therefore, the anti-nervous necrosis virus immersion vaccine is a novel anti-nervous necrosis virus immersion vaccine with simple preparation, convenient use, good effect and high safety.

The invention displays the evagination structure domain of the capsid protein of NNV on the outer membrane of baculovirus/Autographa californica nuclear polyhedrosis virus), and the baculovirus is known to be harmless to vertebrates but can effectively stimulate the immune system, so the invention uses the baculovirus as a carrier to display the excellent immunogen of the NNV on the outer membrane, and can effectively stimulate the congenital immune system of fish, thereby achieving the purpose of preventing and treating the NNV.

Through screening and optimizing a transmembrane region and a promoter, the transmembrane region of influenza virus neuraminidase is used, and P domain can be effectively displayed on the outer membrane with the outward end and the inward end; polyhedrin promoter (P) using AcMNPV_PH) The recombinant virus can efficiently express P domain, so that the recombinant virus has the highest P domain content, and the promoter is the original promoter of the baculovirus and is most clearly regulated. The recombinant baculovirus can produce 10 by infecting SF9 cell⁹TCID₅₀Viral load/mL, and the virus can be used directly without inactivation after simple purification.

Drawings

FIG. 1 shows the green fluorescent protein expression results of the recombinant baculovirus according to the present invention.

FIG. 2 shows the Western blot detection results of the recombinant baculovirus according to the present invention.

FIG. 3 is the death curve of the larvae after the nervous necrosis virus challenge immunity.

Detailed Description

The technical solutions of the present invention will be further described below with reference to the accompanying drawings and specific examples, but the present invention is not limited to the following examples.

Unless otherwise indicated, all reagents used in the present invention are commercially available reagents, and the operations involving, but not describing in detail, the parameters in the examples are well known to those skilled in the art.

Example 1 preparation of an anti-Neuronecrosis Virus immersion vaccine

1. P domain cloning of Capsid Protein (CP)

Taking a Nervous Necrosis Virus (NNV) genome as a template, obtaining 640-1017bp (SEQ NO.2) of a cp gene (SEQ ID NO.1) (the corresponding amino acid is 214-338aa) by RT-PCR amplification, carrying EcoR I and Hind III enzyme cutting sites (F1: GGAATTCACACCTGAAGAGACCACCGCTC (SEQ ID NO. 5); R2: CCCAAGCTTTTAGTTTTCCGAGTCAACCCTG (SEQ ID NO.6)), and cloning to a plasmid pFastBacHT A according to the instruction.

2. Transmembrane region screening

Since CP is C-terminal displayed outside the particle and N-terminal inside the particle, it is necessary to insert transmembrane signal of type II transmembrane protein at its N-terminal in order to display P domain of capsid protein outside the cell membrane.

Influenza virus Neuraminidase (NA) belongs to the II transmembrane protein (one transmembrane, N-terminal inside the cell membrane and C-terminal outside), TMHMM2.0 predicts the NA transmembrane region of Influenza A viruses (A/Sofia/418/2006(H1N1)) (GenBank: CY103786.1) at amino acid residues 7-29.

The first 40 amino acid residues from the N-terminus of NA (NA)₄₀) The gene coding sequence of (SEQ ID NO.3) and linker (GGGGS)₃The coding sequence of (SEQ ID NO.4) was amplified using the following primers (F3: CATGCCATGGATATGAATCCAAATCAAAAAATAATAACCATTG (SEQ ID NO. 7); R4: GGAATTCGCTGCCGCCACCGCCGCTTCC (SEQ ID NO.8)) and inserted into the plasmid pFactibacHT A via the Nco I and EcoR I cleavage sites.

3. Promoter screening

Multiple promoters (069/108/249, corresponding promoters of the corresponding genes, all of which have been confirmed to have high expression levels) and the polyhedrin promoter (P) of White Spot Syndrome Virus (WSSV) were selected_PHThe original plasmid carries itself) for expression screening. The WSSV promoter is amplified by the following primers, is connected to a pFastBacHTA vector through SnaB I and Rsr II enzyme cutting sites, and replaces P on the vector_PHA promoter. The GFP sequence was then ligated into the engineered vector via EcoR I and Hind III sites. The plasmids were transfected into Sf9 cells and the ability of each promoter to initiate gene transcription in Sf9 cells was assessed by observing GFP expression.

F-069：5‘GGTACGTATGAAAATGGCTGTT3’(SEQ ID NO.9)

R-069: 5 'GGCGGTCCGCTTGAGTGGAGAGAG 3' (SEQ ID No.10), product 984bp

F-249：5‘GGTACGTACTCGCCCACCACC3’(SEQ ID NO.11)

R-249: 5 'GGCGGTCCGGGCTGCGAGAATGGTTTG 3' (SEQ ID No.12), product 968bp

F-108：5‘GGTACGTAGAAAATATCCTCG3’(SEQ ID NO.13)

R-108: 5 'GGCGGTCCGCTTGATTTCTTGGTTG 3' (SEQ ID No.14), product 927bp

Based on the fluorescence quantity, 249 and P are selected_PHThe promoter is used for the next step, recombinant viruses Bac-249-NA-P and Bac-PH-NA-P are prepared under the condition that all the selected promoter, NA and P domain are recombined, and the Bac-249-NA-P recombinant viruses cannot well express P domain, but Bac-PH-NA-P can well display P domain on cell membrane (see figure 1), so the latter is named Bac-Noda. As shown in FIG. 1, P domain was localized to the cell membrane after Bac-Noda infected sf9 cells. Fresh sf9 cells are infected by P1 recombinant virus Bac-Noda, immunofluorescence detection is carried out by using a mouse anti-NNV polyclonal antibody and a green fluorescent secondary antibody after 48 hours, the upper left A is a P domain signal, a bright field (upper right B) is cell morphology under visible light, the lower left C is cell nucleus staining (Hoechst 33258), and the lower right D is the superposition effect of the front three pictures. The green fluorescence signal on the cell membrane can be seen, which proves that P domain can be expressed on the cell membrane.

4. Preparation of recombinant Virus Bac-Noda

Will contain the promoter P_PH、NA40、linker(GGGGS)₃And P domain transformed DH10B competent cells: mu.g of recombinant plasmid was added to 100. mu.l of DH10Bac competent cells, after heat shock, 900. mu.l LB medium was added, cultured at 37 ℃ for 4 hours, centrifuged at 4,000g for 4 minutes, 900. mu.l of the medium supernatant was aspirated, the cells were suspended in 100. mu.l of the medium, plated on LB solid plates containing 50. mu.g/ml kanamycin, 7. mu.g/ml gentamicin, 10. mu.g/ml tetracycline, 100. mu.g/ml Bluo-gal and 40. mu.g/ml IPTG, cultured at 37 ℃ for 48 hours, white plaques were picked, and PCR analysis and sequencing were carried out to determine positive clones.

Bacmid was extracted using a kit from OMEGA, but the procedure was slightly modified: (1) according to the following steps: 1000 inoculation of bacteria in fresh medium (containing 50 u g/ml kanamycin, 7 u g/ml gentamicin, 10 u g/ml tetracycline LB medium), overnight culture for about 16 hours, centrifugal collection of bacteria, adding 200 u l buffer T1/RNase A solution heavy suspension of bacteria. (2) Adding 200 mu l T2 Buffer, inverting 5-10 times, fully cracking the thallus, and standing for 5 minutes at room temperature. (3) 200. mu.l of ice-cold T3 Buffer was added, inverted 15-20 times, and left on ice for 5 minutes. (4)13,000g, centrifuged at 4 ℃ for 10 minutes, the supernatant was transferred to a 1.5ml centrifuge tube, and 0.1 volume ETR solution (endotoxin adsorption buffer by Omega, see the instructions of the Omega endotoxin free Kit E.Z.N.A.endo-free Plasmid Mini Kit I and E.Z.N.A.endo-free Plasmid Mini Kit I) was added, incubated on ice for 10 minutes, centrifuged at 42 ℃ for 5 minutes, 13,000g, and centrifuged for 3 minutes. (5) The supernatant was transferred to a 2.0ml centrifuge tube, 800. mu.l of ice-cold isopropanol was added, incubated on ice for 10 minutes, 13,000g, and centrifuged at 4 ℃ for 15 minutes. The white precipitate was rinsed twice with 75% ethanol solution, the residual liquid was blotted dry, dried at room temperature for 10 minutes, the precipitate was dissolved in 40. mu.l of sterile water and stored at 4 ℃.

Production of recombinant baculovirus Bac-Noda: (1) the 6-well plate was seeded with Sf9 cells at a density of about 70% effect. (2) The following morning, 3. mu.g bacmid were transfected with Fugene transfection reagent. (3) The supernatant was collected 72 hours after transfection, and was first generation virus P1.

5. Bac-Noda validation

Detecting whether the cell membrane of the infected cell has P domain by using an immunofluorescence technology: (1) recombinant P1 virus Bac-Noda (about 10)⁴TCID₅₀/mL) infected freshly cultured sf9 cells. (2) After 48h, the supernatant was removed and the cells were fixed with paraformaldehyde for 10min is the same as the formula (I). (3) After conventional antigen blocking, primary Antibody incubation (murine anti-NNV polyclonal Antibody) and incubation with green fluorescent Secondary Antibody (Donkey anti-Mouse IgG (H + L) Secondary Antibody, Alexa Fluor 488), nuclei were stained in contrast with Hoechst 33258 and observed with a fluorescent microscope to confirm the presence of fluorescent signals on the cell membrane (see fig. 1).

6. Bac-Noda mass production and purification

Mass production of recombinant viruses Bac-Noda: recombinant P1 virus Bac-Noda (about 10)²TCID₅₀/mL) infection 1X 10¹⁰Freshly cultured sf9 cells. And collecting the supernatant after 72h, namely P2. If larger amounts of recombinant virus are required, P2 can be used to infect fresh sf9 cells to culture P3 virus.

Density gradient centrifugation purification of recombinant virus: (1)1000g, centrifugation at 4 ℃ for 10min, supernatant was taken and repeated once to remove cells. (2) The supernatant was centrifuged through a 40% sucrose cushion at 100,000g for 60min, and the pellet was resuspended in PBS (phosphate buffered saline). (3) The resuspension was centrifuged on a 25% -56% sucrose density gradient, 100,000g, 90min, and a wide blue-white band was aspirated. (4) The virus solution was diluted in PBS, 100,000g was centrifuged for 60min, and the pellet was resuspended in an appropriate amount of PBS. -80 degree preservation. (5) The titer of the purified recombinant virus was determined by the conventional gradient dilution method, which required 10⁸TCID₅₀More than mL. (6) The purified virus particles were subjected to Western blot detection (using commercial anti-His monoclonal antibody and murine anti-NNV polyclonal antibody), and the corresponding bands could be detected, thus confirming that the particles had the designed His-NA-P protein sequence (see FIG. 2).

As shown in FIG. 2, purified Bac-Noda has His tag and P domain. And (3) carrying out Western blot on the purified Bac-Noda, and detecting the Bac-Noda with an anti-His monoclonal antibody and a mouse anti-NNV polyclonal antibody to prove that the Bac-Noda has a His label carried by the baculovirus and a recombinant cloned P domain. His tag is the N-terminus of the recombinant protein and P domain is the C-terminus of the recombinant protein, both were detected and were high (VH compared to VH, which is a His-tagged OGNNV VLP and thus both His and CP, which can be used as a positive control).

Example 2 soaking immunization

1. Immersion immunization procedure optimization

Separately diluted recombinationVirus Bac-Noda to a final concentration of 10³、10⁴、10⁵TCID₅₀Soaking grouper (body length below 3 cm) or trachinotus ovatus (body length below 5 cm) young fish for 1-2h, and soaking for the second time under the same conditions after 48 h. Another 3 groups of larvae were immunized in the same manner, and a third soaking immunization was performed. Performing OGNNV HN (Hainan strain of Epinephelus coioides Nees necrosis virus) challenge on 6 groups of young fishes with different concentrations and different immunity times to obtain a death rate similar to that of three times of immunity after twice immunization although 10 times of immunity⁵Group survival ratio of 10⁴Groups were low, but the differences were not significant, so a summary of the cost effective immunization protocol was 10⁴TCID₅₀Bac-Noda soaking immunization in/mL is carried out twice for 2 h. Under these conditions, the immunoprotection rate was 85.4% (see FIG. 3).

As shown in fig. 3, at 10⁴TCID₅₀After Bac-Noda soaking the immune grouper larvae twice, the concentration is 10⁴TCID₅₀/mL OGNNV HN injections were challenged for 11 consecutive days. Dead fish were collected daily and cumulative mortality was calculated. The mortality of the negative control (Bac-wt, i.e., wild-type baculovirus without insertion) was compared and gave an immunoprotection of 85.4%.

2. Detection of immune effects

And (3) detecting 15 genes of 8 organs of the grouper larvae after the two immunizations in an RT-qPCR mode, wherein a quantitative result shows that the expression of the genes related to the innate immunity is greatly increased, and the effects of the genes in a short period after soaking are comprehensively presumed to be mainly used for stimulating the innate immunity and the cellular immunity.

3. Security detection

Cell invasion performance: Bac-Noda was added at 10⁴TCID₅₀Fish cells (SB, SSN-1, FHM, ZF4 and CO) and mammalian cells (Hela, 293T and BHK) were soaked in/mL concentrations and found to partially enter the cells or attach to the cell surface. And the cells have no abnormal growth in the 48h soaking process. In addition, PCR or RT-PCR is carried out on the cells after 48h soaking to detect the gene sequences of baculovirus capsid protein and P domain, and the comparison of the 1h sample shows that the number of the two genes is not increased or is increased by only 1-2 times, and effective infection replication is not formed. Description of the inventionBac-Noda is highly safe to fish and mammalian cells.

The performance on fish bodies at high concentration: by 10⁷TCID₅₀the/mL Bac-Noda soaks grouper larvae for 10 days, and the death rate is 5%. By 10⁷TCID₅₀the/mL Bac-Noda intraperitoneal injection of grouper larvae is observed for-10 days, and the death rate is 7%. It can be seen that high concentrations of Bac-Noda have very low toxicity to fish larvae.

Sequence listing

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Claims

1. a kind of anti-neural necrosis virus soaked vaccine with baculovirus as carrier, it is characterized in that, described anti-neural necrosis virus soaked vaccine comprises the outer convex structure that expresses neuronecrosis virus capsid protein on the outer membrane of baculovirus Domain recombinant baculovirus containing the promoter P _PH , the NA40 gene coding sequence, the linker (GGGGS) ₃ coding sequence and the P domain gene coding sequence, and the recombinant baculovirus does not express neuronecrosis virus other parts of the capsid protein;

The NA40 gene coding sequence is the gene coding sequence of the first 40 amino acid residues NA40 of the N-terminal of influenza virus neuraminidase;

Described baculovirus is Spodoptera californica nuclear polyhedrosis virus;

The primers for amplifying the coding sequence of the outer convex domain P domain gene of the neuronecrosis virus capsid protein are as follows:

F1: GGAATTCACACCTGAAGAGACCACCGCTC;

R2: CCCAAGCTTTTAGTTTTCCGAGTCAACCCTG.

2 . The anti-nerve necrosis virus immersion vaccine according to claim 1 , further comprising an additive. 3 .

3. The anti-nerve necrosis virus immersion vaccine according to claim 1, wherein the anti-nerve necrosis virus immersion vaccine is suitable for fish.

4. the preparation method of the anti-nerve necrosis virus soaked vaccine described in any one of claim 1 to 3, is characterized in that comprising the following steps:

(1) Using the neuronecrosis virus genome as a template, the coding sequence of the outer convex domain P domain gene of the neuronecrosis virus capsid protein was obtained by RT-PCR amplification, and EcoR I and Hind III restriction sites were added. , cloned into plasmid pFastBacHT A;

(2) Amplify the gene coding sequence of the first 40 amino acid residues NA40 at the N-terminal end of influenza virus neuraminidase and the coding sequence of linker (GGGGS) ₃ , and insert into the plasmid pFactbacHT through Nco I and Eco R I restriction sites A, make a recombinant baculovirus containing promoter P _PH , NA40 gene coding sequence, linker (GGGGS) ₃ coding sequence and P domain gene coding sequence;

F1: GGAATTCACACCTGAAGAGACCACCGCTC;

R2: CCCAAGCTTTTAGTTTTCCGAGTCAACCCTG;

The primers for amplifying the gene coding sequence of the first 40 amino acid residues NA40 of the N-terminal of the influenza virus neuraminidase are as follows:

F3: CATGCCATGGATATGAATCCAAATCAAAAAATAATAACCATTG;

R4: GGAATTCGCTGCCGCCACCGCCGCTTCC.

5. The preparation method according to claim 4, further comprising culturing and producing recombinant baculovirus.