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Preparation method and application of tilapia source streptococcus agalactiae recombinant GroEL protein vaccine

A technology for streptococcus niles and protein vaccine, which is applied in the field of molecular vaccinology, can solve the problem of ineffectiveness of streptococcus iniae and other problems, and achieves the effects of simple preparation method, high yield and safe preparation process

Active Publication Date: 2016-08-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the Streptococcus agalactiae vaccines used above are all single or double-unit inactivated vaccines, and the immune efficacy of the prepared vaccines is directly related to the immunogenicity of the vaccine strains, and is only effective against Streptococcus agalactiae with the same serotype. Effective against cocci, not against Streptococcus iniae

Method used

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  • Preparation method and application of tilapia source streptococcus agalactiae recombinant GroEL protein vaccine
  • Preparation method and application of tilapia source streptococcus agalactiae recombinant GroEL protein vaccine
  • Preparation method and application of tilapia source streptococcus agalactiae recombinant GroEL protein vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of Streptococcus agalactiae Recombinant from Tilapia GroEL protein

[0052] 1. Primer design:

[0053] Using Streptococcus agalactiae from tilapia as a template, a pair of primers GroEL-F and GroEL-R containing restriction enzymes were designed with the following sequences:

[0054] Primer GroEL-F (sequence shown in SEQ ID NO.3):

[0055] 5'-G GAATTC ATGGCAAAAGATATTAAATTTTC-3'

[0056] Primer GroEL-R ((sequence shown in SEQ ID NO.4)):

[0057] 5'-GGG TTCGAA GAAGCCACCCATCATAGATGG-3'

[0058] The underlined part of the upstream primer GroEL-F is EcoR I Restriction site, the underlined part of the downstream primer GroEL-R is Hind III Restriction sites.

[0059] 2, GroEL gene cloning

[0060] (1) PCR amplification GroEL Gene

[0061] The genomic DNA of Streptococcus agalactiae derived from tilapia was extracted, and the genomic DNA was used as a template to carry out PCR reaction on GroEL-F and GroEL-R with the above primers...

Embodiment 2

[0092] Example 2 Preparation of Streptococcus agalactiae Recombinant from Tilapia GroEL protein vaccine

[0093] The purified recombinant GroEL protein obtained in Example 1 was diluted to 1 mg / ml with sterilized PBS, mixed evenly with Freund's complete and incomplete adjuvant at a ratio of 1:1, and prepared into a subunit vaccine.

[0094] Take 1ml of the prepared vaccine and centrifuge at 3000×g for 5 minutes. If there is no stratification of the vaccine, it can be used for subsequent immunization.

Embodiment 3

[0095] Example 3 Streptococcus agalactiae recombinant from tilapia GroEL Protein Vaccine Immunization Test

[0096] 1. Immune protection experiment

[0097] (1) Nile tilapia was purchased from Guangzhou Chengyi Aquatic Technology Co., Ltd., with a size of 50±5.0g, and was kept for two weeks before the experiment.

[0098] (2) During the immunization experiment, the tilapia were randomly divided into 3 groups, namely PBS group, PBS+adjuvant group and GroEL vaccine group. There are 14 tails in each group, and each group is set up in three parallels.

[0099] The GroEL group and the adjuvant group were injected intraperitoneally with 100 µl of recombinant antigen GroEL mixed with Freund's adjuvant and PBS, respectively, and the PBS group was injected with 100 µl of sterilized PBS.

[0100] 2. Immunization procedure

[0101] The tilapia were immunized twice, the initial immunization with Freund's incomplete adjuvant, and the booster immunization with Freund's complete ...

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Abstract

The invention discloses a preparation method and application of a tilapia source streptococcus agalactiae recombinant GroEL protein vaccine. The recombinant GroEL protein vaccine is prepared by tilapia source streptococcus agalactiae recombinant GroEL protein. An amino acid sequence of the tilapia source streptococcus agalactiae recombinant GroEL protein is as shown in SEQ ID NO.2. In the invention, tilapia source streptococcus agalactiae is taken as a research object, a GroEL gene is obtained from a tilapia source streptococcus agalactiae genome by cloning, the cloned GroEL gene is connected with a pET28a(+) carrier to construct an expression vector, and the vector is subjected to prokaryotic expression and purification in E.coli BL21(DE3) to obtain the recombinant GroEL protein vaccine with strong specificity and an immune protective efficacy of 68.61+ / -7.39 percent, so that a foundation is laid for immune prevention and treatment of a tilapia streptococcus agalactiae disease.

Description

technical field [0001] The invention belongs to the technical field of molecular vaccinology. More specifically, it relates to a preparation method and application of tilapia-derived Streptococcus agalactiae recombinant GroEL protein vaccine. Background technique [0002] Streptococcus agalactiae ( Streptococcus agalactiae ) is a zoonotic Gram-positive bacterium that can cause neonatal meningitis, bovine mastitis and fish meningoencephalitis. Streptococcus agalactiae can infect a variety of seawater and freshwater fish and cause death, causing significant economic losses to the aquaculture industry in my country and the world. Therefore, it is of great practical significance to study the control technology related to Streptococcus agalactiae. [0003] At present, there are already some patents related to streptococcal vaccines in China. Application No.: 200580013779.X, Invention Name: Streptococcus agalactiae Vaccine discloses a combination vaccine prepared from the comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C07K14/315A61K39/09A61P31/04
CPCA61K39/092C07K14/315A61K2039/545A61K2039/552A61K2039/54
Inventor 李安兴李薇李云
Owner SUN YAT SEN UNIV
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