Preparation method and application of tilapia mossambica-derived streptococcus agalactiae recombinant CBP protein vaccine

A technology of streptococcus lactis and protein vaccines, applied in the field of molecular vaccinology, can solve the problems of ineffective Streptococcus iniae and achieve the effects of simple preparation method, low expression cost and safe preparation process

Inactive Publication Date: 2017-06-13
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 mossambica-derived streptococcus agalactiae recombinant CBP protein vaccine
  • Preparation method and application of tilapia mossambica-derived streptococcus agalactiae recombinant CBP protein vaccine
  • Preparation method and application of tilapia mossambica-derived streptococcus agalactiae recombinant CBP protein vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Preparation of tilapia-derived Streptococcus agalactiae recombinant CBP protein

[0051] 1. Primer design:

[0052] Using tilapia-derived Streptococcus agalactiae as a template, a pair of primers CBP-F and CBP-R containing restriction enzymes were designed, the sequences are as follows:

[0053] Primer CBP-F (sequence shown in sequence 3):

[0054] 5'- GGGGGGTCTCTAGTG GATCAAACTACATCGGTTCAA-3’

[0055] Primer CBP-R ((sequence is shown in sequence 4)):

[0056] 5'- GCCGGGTCTCGTGGG AATTTCAATATAGCGACGAAT-3’

[0057] 2. Cbp gene cloning

[0058] (1) PCR amplification of Cbp gene

[0059] The genomic DNA of Streptococcus agalactiae from tilapia was extracted, and the above-mentioned primers were used for PCR reaction of CBP-F and CBP-R with the genomic DNA as the template.

[0060] The PCR reaction system was: 25μl reaction system containing 50ng template DNA (genomic DNA), 2.5μl 10×PCRBuffer (Mg+), 2μl dNTP Mixture (2.5mM), 1μl CBP-F, 1μl CBP-R, 0.125μl r...

Embodiment 2

[0088] Example 2 Preparation of tilapia-derived Streptococcus agalactiae recombinant CBP protein vaccine

[0089] The purified recombinant CBP protein obtained in Example 1 was diluted to 1 mg / ml with sterilized PBS, and mixed with Freund's complete and incomplete adjuvant at 1:1 to prepare a subunit vaccine.

[0090] Take 1ml of the prepared vaccine and centrifuge at 3000×g for 5min. If the vaccine has no stratification, it can be used for subsequent immunization.

Embodiment 3

[0091] Example 3 Tilapia-derived Streptococcus agalactiae recombinant CBP protein vaccine immunization test

[0092] 1. Immune protection experiment

[0093] (1) Nile tilapia was purchased from Guangzhou Zhongxingou Aquaculture Development Co., Ltd., the size was 30±5.0g, and it was temporarily cultured for two weeks before the experiment.

[0094] (2) In the immunization experiment, tilapia were randomly divided into 3 groups, namely PBS group, PBS+adjuvant group and CBP vaccine group. There are 15 tails in each group, and three parallel groups are set up in each group.

[0095] The CBP group and the adjuvant group were injected intraperitoneally with 100 μl of recombinant antigen CBP and PBS mixed with an equal volume of Freund's adjuvant, and the PBS group was injected with 100 μl of sterile PBS.

[0096] 2. Immunization program

[0097] Tilapia were immunized twice, with Freund's complete adjuvant for the primary immunization and subunit vaccines for the booster immuniz...

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Abstract

The invention discloses a preparation method and application of a tilapia mossambica-derived streptococcus agalactiae recombinant CBP protein vaccine. The recombinant CBP protein vaccine is prepared from tilapia mossambica-derived streptococcus agalactiae recombinant CBP protein. The amino acid sequence of the tilapia mossambica-derived streptococcus agalactiae recombinant CBP protein is shown as a sequence 2. Tilapia mossambica-derived streptococcus agalactiae is used as a research object and is cloned into a Cbp gene from a tilapia mossambica-derived streptococcus agalactiae genome; the tilapia mossambica-derived streptococcus agalactiae is connected with a pET28a(+) carrier to construct an expression vector, and prokaryotic expression and purification are carried out in E.coliBL21(DE3), so as to obtain the recombinant CBP protein vaccine with high specificity and immune protective efficacy of 56.35 percent+ / -8.09 percent; a foundation is laid for immune prevention and treatment of tilapia mossambica streptococcus agalactiae diseases.

Description

technical field [0001] The present invention belongs to the technical field of molecular vaccinology. More specifically, it relates to a preparation method and application of a tilapia-derived Streptococcus agalactiae recombinant CBP protein vaccine. Background technique [0002] 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 marine and freshwater fish and cause death, causing significant economic losses to the aquaculture industry in my country and even the world. Therefore, it is of great practical significance to study the control technology related to Streptococcus agalactiae. [0003] At present, there are some patents related to streptococcal vaccine in China. Application number: 200580013779.X, name of invention: Streptococcus agalactiae vaccine A combination vaccine prepared from the complete inactivated cells of beta-h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/315C12N15/70A61K39/09A61P31/04C12R1/46
CPCA61K39/092C12N15/70C07K14/315
Inventor 李安兴李云
Owner SUN YAT SEN UNIV
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