A kind of perch rhabdovirus g2-2m subunit vaccine and its preparation method

A technology of subunit vaccines and rhabdoviruses, applied in biochemical equipment and methods, viruses, antiviral agents, etc., can solve the problems of MSRV-free subunit vaccines on the market, improve immunogenicity and protective efficacy, and improve the production process Effects of simplification and production cost reduction

Active Publication Date: 2022-02-22
深圳万可森生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As mentioned above, vaccines have been reported as the most effective way to prevent viral infection in the prevention of MSRV, but there is no MSRV subunit vaccine available on the market

Method used

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  • A kind of perch rhabdovirus g2-2m subunit vaccine and its preparation method
  • A kind of perch rhabdovirus g2-2m subunit vaccine and its preparation method
  • A kind of perch rhabdovirus g2-2m subunit vaccine and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] This example illustrates recombinant Escherichia coli E. coli Construction and identification of BL21 / pET32a-G2-2M strain.

[0028] Amplification of gene fragments and M gene fragments

[0029] On the basis of previous research and bioinformatics analysis, combined with the research of other rhabdoviruses, the G2 gene fragment with the nucleotide sequence of SEQ ID No: 1 and the M gene with the nucleotide sequence of SEQ ID No: 2 were selected fragments, primers were designed, and the corresponding gene fragments were respectively amplified, purified and ligated with pMD19-T vector, and the recombinant products were named pMD19-T-G2 and pMD19-T-M. The recombinant plasmids were transformed into competent Escherichia coli DH5α, and positive strains were obtained by blue-white screening, and the plasmids were extracted for PCR amplification detection and sequencing identification.

[0030]Construction, Transformation and Screening of Recombinant Plasmid pET32a-G2-2M

...

Embodiment 2

[0036] This example illustrates the expression and purification of MSRV-G2-2M recombinant protein.

[0037] Recombinant bacteria E. coli Induced expression of BL21 / pET32a-G2-2M

[0038] strains for production E. coli BL21 / pET32a-G2-2M Use an inoculation loop to pick up a small amount of bacterial liquid, streak and inoculate it on an LB solid medium plate, and after standing at 37°C for 16-18 hours, pick a single colony and inoculate it in the LB liquid medium. Cultivate at 37°C and 160-180r / min for 12-16 hours as primary seeds. The primary seeds were inoculated in LB liquid medium at 1% (V / V), placed at 37°C and cultured at 160-180r / min for 14-16 hours as the secondary seeds. Secondary seeds were inoculated into the modified LB medium at 1% (V / V), and ampicillin was added to a final concentration of 100 μg / mL. 37 ℃ aerated fermentation culture for 5 to 7 hours, dissolved oxygen 30% to 40%, to the OD of the bacterial liquid 600 When the value is 1.1-1.3, add IPTG to a ...

Embodiment 3

[0049] This example illustrates the evaluation of the immune efficacy of recombinant proteins.

[0050] Recombinant protein bath immunoassay

[0051] Take 300 healthy perch of 3-5g and divide them into immune group 1, immune group 2 and control group, with 100 fish in each group. Immunization 1 and immunization 2 groups were treated with G2 recombinant protein solution (20mg / L, prepared based on ZL202111066165.5) and G2-2M recombinant protein group solution (20mg / L) respectively, and the control group was treated with normal saline instead of vaccine , after soaking for 6 hours, transfer to normal breeding water.

[0052] Determination of serum neutralizing antibody titer

[0053] On the 3rd, 7th, 14th, 21st, and 28th day after immunization, 3 perch were randomly selected from each group, blood was drawn and serum was separated, mixed and stored at -80°C for the determination of antibody neutralization titer.

[0054] Determination method of serum neutralizing antibody tite...

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Abstract

The present invention constructs the G2‑2M recombinant protein subunit vaccine based on carbon nanotubes. After bathing and immunizing, the protection rate of the fish school reaches about 94%, which is higher than that of the G2 protein fragment. Based on the introduction of the M protein fragment, the recombinant protein Compared with previous studies, it does not require mannosaccharification modification, the production process is greatly simplified, and the production cost is also greatly reduced, which is of great significance to the sustainable development of fisheries and the safe production of aquatic products .

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a perch rhabdovirus G2-2M subunit vaccine and a preparation method thereof. Background technique [0002] largemouth bass ( Micropterus salmoides ), commonly known as California perch and black perch, is native to the Mississippi River Basin in North America. Because the breeding of largemouth bass has good economic benefits in China, it has rapidly developed into the main economically farmed fish species in my country in the past ten years, and has been recognized as one of the famous and high-quality species, which has important economic value. Bass is susceptible to many diseases, such as gill rot, canker, nocardiosis, trichotillobia and so on. Among them, perch rhabdovirus ( Micropterus salmoides rhabdovirus, MSRV ) caused rhabdovirus disease in perch ( Micropterus salmoides rhabdovirus disease ) are the most dangerous. It is endemic in a wide area, has a lon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/205A61K39/385A61P31/14B82Y5/00
CPCA61K39/12A61K39/385A61P31/14B82Y5/00C12N2760/20034
Inventor 焦铁军孟强马瑞王文博
Owner 深圳万可森生物科技有限公司
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