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Construction method and application of vector vaccine for resisting infectious spleen and kidney necrosis viruses

A spleen and kidney necrosis virus, vector vaccine technology, applied in antiviral agent, virus/phage, application and other directions, can solve the problems of large amount of vaccine, poor immune effect, time-consuming and labor-intensive, etc., and achieve high biological safety and good immune effect. , easy to use effect

Inactive Publication Date: 2021-10-08
苏州培恩特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Injection immunization is the most common immunization method, which can ensure the amount of antigen and stimulate the fish to produce long-term immune protection, but it is easy to cause damage to the fish, time-consuming and laborious, and is not suitable for smaller fish
Oral immunization is to feed the antigen coated and mixed with the feed, which can avoid the fish stress response, and can immunize a large number of fish, with little damage, simple method and easy operation, but the vaccine is easily destroyed and degraded in the digestive tract, resulting in immune protection effect Poor; immersion immunization is not easy to cause damage to fish, and there is no requirement for the size of immunized fish. It is the most convenient way of immunization, but it requires a large amount of vaccine and the immunization effect is relatively poor

Method used

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  • Construction method and application of vector vaccine for resisting infectious spleen and kidney necrosis viruses
  • Construction method and application of vector vaccine for resisting infectious spleen and kidney necrosis viruses
  • Construction method and application of vector vaccine for resisting infectious spleen and kidney necrosis viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Construction of the carrier vaccine against infectious spleen and kidney necrosis virus and the antibody produced by perch induced by injection immunization

[0063] (1) Synthesize the infectious spleen and kidney necrosis virus main capsid protein gene expression cassette cmv-mcp (1997bp) controlled by the cytomegalovirus promoter as shown in SEQ ID NO: 1, clone it into the T-vector, carry out sequencing verification, and Verify that the correct plasmid is named pMD-CMV-MCP;

[0064] (2) Digest the pMD-CMV-MCP plasmid with BamHI and XbaI, recover the cmv-mcp fragment, and clone it into the same digested pFSATBac TM In Dual, the recombinant plasmid pFAST was obtained TM Dual-cmv-mcp;

[0065] (3)pFAST TMDual-cmv-mcp transformed DH10 / Bac competent cells, coated with tetracycline (10μg / ml), kanamycin (50μg / ml), gentamicin (7μg / ml), IPTG (40μg / ml) , X-gal (100μg / ml) on the LB agar culture plate; cultured at 37°C for 48 hours, picked white colonies for culture...

Embodiment 2

[0092] Example 2 The carrier vaccine BmNPV-MCP against infectious spleen and kidney necrosis virus can be transduced into the tissues of mandarin fish and express the main capsid protein of infectious spleen and kidney necrosis virus

[0093] Use 8×10 9 The copied recombinant virus BmNPV-MCP was injected into mandarin fish (body length 7-13cm, weight 35-60g). One week later, a total of 0.1g of spleen and kidney tissue was taken, and the genome was extracted. Primers MCP-1 (SEQ ID NO: 4) and MCP -2 (SEQ ID NO:5) was amplified. The PCR amplification system and amplification conditions are the same as in step (4) of Example 1.

[0094] Such as Figure 6 The results shown showed that the main capsid protein gene of infectious spleen-kidney necrosis virus could be amplified by PCR from the DNA of the spleen and kidney tissues of mandarin fish injected with BmNPV-MCP, indicating that BmNPV-MCP had entered the spleen and kidney tissues of mandarin fish.

Embodiment 3

[0095] Example 3 Immunoprotective effect of immunization injection of vector vaccine against infectious spleen and kidney necrosis virus to infectious spleen and kidney necrosis virus infection

[0096] (1) Select healthy and disease-free largemouth bass from the same batch (8-12cm in length, 35-55g in weight), and raise them with oxygen at a water temperature of 19-22°C for 2 weeks.

[0097] (2) Take 4×10 7 copy / μL of BmNPV-MVP, inject 200 μL / tail from the base of the pectoral fin, and set wild BmNPV (4×10 7 copy / μL) as the negative control, and the non-immunized blank control group (200 μl of PBS per fish, 20 sea bass in total).

[0098] (3) Take 5 g of the kidney of mandarin mandarin fish infected with infectious spleen-kidney necrosis virus, add 5 mL of phosphate buffer to fully grind, centrifuge at 8000 r / min for 30 min, take the supernatant, repeat this step 4 times, and use 0.22 μm Membrane filtration, the filtrate is infectious spleen and kidney necrosis virus liquid...

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Abstract

The invention discloses a construction method and application of a vector vaccine for resisting infectious spleen and kidney necrosis viruses, and belongs to the technical field of virus genetic engineering. The vector vaccine comprises an antigen protein MCP expressed by a bombyx mori nuclear polyhedrosis virus and shown as SEQ ID NO: 10, and the antigen protein is encoded by a cmv-mcp expression cassette shown as SEQ ID NO: 1. According to the construction method, the mcp expression cassette cmv-mcp controlled by a cytomegalovirus promoter cmv is cloned to a transfer plasmid to construct a recombinant plasmid, then competent cells are transformed to obtain Bacmid-MCP, the Bacmid-MCP DNA transfects bombyx mori culture cells to obtain BmNPV-MCP, then the BmNPV-MCP is inoculated to larvae or primary pupae of bombyx mori at the age of 4-5, and homogenate is carried out after the disease is attacked to obtain the vector vaccine. The vaccine is used for immunizing siniperca chuatsi / weever through an injection, oral administration or soaking method, and the occurrence of infectious spleen and kidney necrosis diseases can be reduced.

Description

technical field [0001] The invention relates to the technical field of virus genetic engineering, in particular to a construction method and application of a carrier vaccine against infectious spleen and kidney necrosis virus. Background technique [0002] Infectious spleen and kidney necrosis virus (ISKNV) is a member of the swollen cell virus genus and can infect Siniperca chuatsi and Largemouth bass (Micropterus salmoides). Development of the sea bass farming industry. Immunization technology has increasingly become a safe, effective and environmentally friendly new prevention and control technology in the prevention and control of fish diseases. China has successfully developed more than 50 kinds of aquatic vaccines, involving nearly 30 kinds of pathogens. At present, common vaccines mainly include: inactivated vaccines (killed vaccines), attenuated vaccines, subunit vaccines, synthetic peptide vaccines, recombinant live vector vaccines, DNA vaccines, and mRNA vaccines...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61P31/20C12N15/34C12N15/866C07K14/01
CPCA61K39/12A61P31/20C07K14/005C12N15/86A61K2039/552A61K2039/542A61K2039/541A61K2039/54A61K2039/5252A61K2039/575C12N2710/00022C12N2710/00034C12N2710/14043
Inventor 贡成良蒋军孟爱军宋学宏
Owner 苏州培恩特生物科技有限公司
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