Tilapia streptococcus agalactiae vaccine and preparation method thereof

A technology for the preparation of Streptococcus lactis and vaccines, which is applied in the direction of vaccines, veterinary vaccines, chemical instruments and methods, etc., can solve the problems of difficulty in prevention and control, lack of safe and effective vaccines, and great hazards in tilapia production, so as to improve pro- Water and dispersibility, increased durability, high expression effect

Inactive Publication Date: 2020-05-08
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the problems that Streptococcus agalactiae is harmful to tilapia production, causing serious economic losses, lack of safe and effective vaccines and great difficulty in prevention and control, the purpose of the present invention is to provide

Method used

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  • Tilapia streptococcus agalactiae vaccine and preparation method thereof
  • Tilapia streptococcus agalactiae vaccine and preparation method thereof
  • Tilapia streptococcus agalactiae vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Screening of target polypeptides

[0043] There are a large number of cell surface proteins, which are not only related to the virulence and pathogenic mechanism of pathogenic microorganisms, but also can be assembled into heterogeneous complexes containing different receptors and effector proteins, which have a conduction function between cells, and the surface proteins have strong mobility , are more easily agglutinated by the corresponding antibody. Therefore, cell surface proteins with good immunogenicity will surely provide more and better choices for the development of new vaccines. This application uses bioinformatics methods to analyze the functional structural sites and antigenic epitope regions of multiple surface protein gene sequences of Streptococcus agalactiae predicted to have immune function published on GenBank, including http: / / tools .immunepitope.org / bcell / Online analysis of Bepipred linear epitopes, antigenicity, β-Turn, surface accessibi...

Embodiment 2

[0047] Example 2: Preparation of target polypeptide by genetic engineering method

[0048] 2.1 Preparation of genomic DNA

[0049] After the tilapia-derived Streptococcus agalactiae was amplified and cultivated, the genomic DNA was extracted according to the instructions of the commercial bacterial genome extraction kit.

[0050] 2.2 PCR primer design

[0051] According to the prediction results of the epitope region of the gene sequence, specific primers were designed with primer5.0, and the primer sequences were:

[0052] Forward primer: 5'-GGGGGATCCACAAGTGATAAGAATACTGACACGA-3' (SEQ ID NO: 2).

[0053] Reverse primer: 5'-GGGGTCGACACTTA ATGATAACAGAATAATCCC-3' (SEQ ID NO: 3).

[0054] 2.3 Recovery of amplified products and construction of recombinant expression vectors

[0055] After PCR amplification, the PCR product was separated, and the target fragment was recovered using an agarose gel recovery kit. The PCR-amplified gene was double-digested with restriction endonucl...

Embodiment 3

[0061] Example 3: Functional modification of single-walled carbon nanotubes

[0062] Heat the single-walled carbon nanotube sample at 450°C for 30-40 minutes to convert the amorphous carbon in the sample into CO 2 . Using the mixed acid oxidation method, the carbon nanotubes processed by the above air oxidation method were put into the mixed acid (concentrated H 2 S0 4 : concentrated HNO 3 =3:1 (V / V)) at 60°C for heating and acidification for 12h. The carbon nanotube mixed acid mixture is filtered through a microporous membrane, washed with pure water until the pH does not change, and then dried at 80°C to constant weight to obtain oxidized single-walled carbon nanotubes.

[0063] Disperse the low-temperature defatted soybean meal powder in deionized water at a ratio of 1:15, adjust the pH to 8.0 with 2M NaOH, stir at a low speed for 2 hours, and then centrifuge (8000g, 20min, 40°C) to remove insoluble substances, take the supernatant and wash with 2M Adjust the pH to 4.5...

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Abstract

The invention relates to a tilapia streptococcus agalactiae vaccine and a preparation method thereof. According to bioinformatics principles, a part of polypeptide sequences of a surface anchoring protein of streptococcus agalactiae are analyzed and screened as an antigen for preparing the vaccine, in addition, a carbon nanotube carried surface protein system is constructed by using a chemical synthesis technology, and the selected polypeptide antigen is in nano combination with functionalized single-wall carbon nanotubes, so that a tilapia streptococcus agalactiae subunit vaccine is prepared.The polypeptide antigen screened by the invention is capable of remarkably increasing the antibody level of inoculated tilapia when being compared with an inactivated vaccine, and is capable of resisting attack of streptococcus agalactiae of high toxicity. In addition, the polypeptide antigen screened by the invention can be tightly and uniformly connected with surfaces of functionalization modified carbon nanotubes, and compared with independent polypeptide antigen groups, the polypeptide antigen is capable of remarkably improving the immunity of the tilapia.

Description

technical field [0001] The invention relates to the field of aquatic vaccines, in particular to a tilapia streptococcus agalactiae vaccine and a preparation method thereof. Background technique [0002] Streptococcus agalactiae (S. agalactiae) is a Gram-positive coccus, which originally caused mastitis in dairy cows and caused a decrease in milk production. Subsequently, Streptococcus agalactiae was isolated from pregnant women and newborns in many countries, often causing neonatal pneumonia, meningitis and sepsis, etc., leading to serious death. With the development of the world's aquaculture industry, Streptococcus agalactiae has been frequently isolated from aquatic animals, especially since 2007, the bacteria have been breaking out every year in major tilapia breeding areas such as Guangdong, Hainan, Fujian, and Guangxi in my country It is prevalent, with a morbidity rate as high as 10%-30%, and a mortality rate as high as 25%-80%, which seriously threatens the healthy d...

Claims

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Application Information

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IPC IPC(8): A61K39/09A61K39/385A61P31/04C07K14/00C12N15/70
CPCA61K39/092A61K39/385A61K2039/54A61K2039/552A61K2039/60A61P31/04C07K14/00C12N15/70
Inventor 梁芳叶剑敏郭政
Owner SOUTH CHINA NORMAL UNIVERSITY
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