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Streptococcus protective antigen C5a and preparation method thereof

A protective antigen, streptococcus technology, applied in the direction of bacterial antigen components, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as SEZ difficulties, and achieve a good immune response effect

Inactive Publication Date: 2014-06-25
广东艾佩克科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of this, it is necessary to provide a streptococcal protective antigen C5a for the problem that SEZ is difficult to control

Method used

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  • Streptococcus protective antigen C5a and preparation method thereof
  • Streptococcus protective antigen C5a and preparation method thereof
  • Streptococcus protective antigen C5a and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, bacterial strain and growth condition

[0028] The strain adopts SEZ C55138 strain; the medium is TSB medium or TSA medium containing 5% fetal bovine serum; shake cultured at 37° C. for about 8 hours, or OD600 reaches 0.6-1.0.

[0029] The vector is Escherichia coli pET-28a, and the competent cell is Escherichia coli BL21.

[0030] SEZ C55138 strain, E. coli pET-28a vector and E. coli BL21 competent cells are all commercially available products.

Embodiment 2

[0031] Embodiment two, preparation method

[0032] The SCPZ protein is encoded by the scpZ gene, the forward primer of the gapdh gene has a BamHI restriction site, and the reverse primer has an EcoRI restriction site. The forward and reverse swim primers were designed from the SEZ MGCS10565 genomic sequence (NCBI Accession: CP001129.1).

[0033] The preparation method of the protective antigen GAPDH of Streptococcus equi subsp. zooepidemicus comprises the following steps:

[0034] PCR amplification: use the genome of the C55138 strain as a template, and use the following primers for PCR amplification;

[0035] Forward primer (SEQ ID NO.3): 5'-CAGAGGGC GGATCC TTT-3', the underlined part is the BamHI restriction site,

[0036] Reverse primer (SEQ ID NO. 4): 5'-CTGCTG GAATTC TGAGATAT-3', the underlined part is the EcoRI restriction site;

[0037] Linking with the vector: The PCR product is digested with restriction enzymes and then linked with the pET-32a vector digested by...

Embodiment 3

[0046] Embodiment 3. Mice immunization and challenge test

[0047] 1. Thirty 4-week-old BALB / c female mice were randomly divided into 3 groups with 10 mice in each group;

[0048] 2. Experimental group: After 50 μg of purified recombinant SCPZ protein was emulsified with Freund's complete adjuvant, the mice in the first group were immunized by intraperitoneal injection. The second injection of immunized mice;

[0049] 3. Positive control group: inject SEZ inactivated vaccine (using Freund's complete adjuvant and Freund's incomplete adjuvant emulsification inactivated SEZ C55138 strain, the bacterial concentration is 2 × 10) into the second group of mice. 8 CFU / mL);

[0050] 4. Negative control group: mice in group 3 were injected with PBS emulsified with adjuvant, and the adjuvant used was the same as that of the positive control group;

[0051] 5. After 10 days of immunization of all mice (10 days after the second immunization injection in the experimental group), blood wa...

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Abstract

The present invention relates to a streptococcus antigen C5a and a preparation method thereof. The streptococcus protective antigen C5a is an SEZC5a recombinant protein, which consists of 571 amino acids and has a molecular weight of 60.3kDa; a forward primer has one BamHI restriction enzyme cutting site, and a reverse primer has one EcoRI restriction enzyme cutting site. According to the preparation method of the streptococcus protective antigen C5a, the SEZ C5a recombinant protein is treated with cloning, expression and purification; and a series of biological engineering technologies and experiments on mice are applied to conduct system analysis on an rSCPZ. After vaccination, the rSCPZ can provide high protective efficacy; an anti-rSCPZ mice double-immunized serum has significant passive immune protection on mice; and the mice immunized by the rSCPZ show high level of antibody titer in serum. The anti-rSCPZ antibody can induce high level of bactericidal capability; an scpZ gene has a transcription level in SEZ (Streptococcus zooepidemicus) infected mice higher than that of culture in vitro; and the rSCPZ can adhere to hep-2 cells and inhibit cell infection ability of SEZ.

Description

technical field [0001] The present invention relates to a streptococcus protective antigen C5a, and also relates to a preparation method of the protective antigen C5a. Background technique [0002] Streptococcus is a common pathogenic bacteria. According to the different cell wall polysaccharide antigens, it can be divided into 20 groups A, B, C..., V (lack of I and J), among which group A Streptococcus is mainly pathogenic to humans. (Group A Streptococci, GAS), other groups are pathogenic to animals. Streptococcus infections in livestock and poultry, mainly group B Streptococcus (Group B Streptococci, GBS) and Group C Streptococci (Group C Streptococci, GCS) and other group hemolytic streptococci, such as the bovine mammary gland caused by group B Inflammation and swine pustulosis; equine bubonic plague caused by group C and Streptococcus septicaemia in sheep and swine. At present, there are many studies on GAS and GBS. The C5a proteins (SCPA, SCPB) of GAS and GBS can in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/315C12N15/31C12N15/70A61K39/09A61P31/04
Inventor 陈瑶生魏子贡付强刘小红莫德林
Owner 广东艾佩克科技有限公司
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