Streptococcus protective antigen C5a and preparation method thereof

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

Inactive Publication Date: 2012-10-24
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 pr

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 one, bacterial strain and growth condition

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

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

[0030] SEZ C55138 strain, Escherichia coli pET-28a vector and Escherichia coli BL21 competent cells are all commercial 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 primers were designed from the genome sequence of SEZ MGCS10565 (NCBI Accession: CP001129.1).

[0033] The preparation method of Streptococcus equi subspecies zooepidemic protective antigen GAPDH 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 restriction site of BamHI,

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

[0037] Ligation with the vector: the PCR product was digested with a restriction enzyme and then ligated with the pET-32a vector digested with...

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, 10 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 group 1 were immunized by intraperitoneal injection. After 14 days, the same antigen emulsified with 50 μg of Freund’s incomplete adjuvant was used in the same way. Secondary injection of immunized mice;

[0049] 3. Positive control group: Inject SEZ inactivated vaccine (use SEZ C55138 strain inactivated by Freund's complete adjuvant and Freund's incomplete adjuvant to emulsify and inactivate the mice in the second group, the bacterial concentration is 2×10 8 CFU / mL);

[0050] 4. Negative control group: inject PBS emulsified with adjuvant to the mice of the third group, and the adjuvant used is the same as that of the positive control group;

[0051] 5. Ten days after all mice...

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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 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 different cell wall polysaccharide antigens, it can be divided into 20 groups: A, B, C..., V (missing I and J). Among them, group A streptococci are mainly pathogenic to humans. (Group A Streptococci, GAS), other groups are pathogenic to animals. Streptococcus infections in livestock and poultry are mainly Group B Streptococci (GBS) and Group C Streptococci (Group C Streptococci, GCS) and other group hemolytic streptococci, such as bovine mammary gland infection caused by Group B Inflammation and swine impetigo; equine blight caused by group C and streptococcal infections in sheep and swine. At present, there are many studies on GAS and GBS. The C5a proteins (SCPA, SCPB) of GAS and GBS can inhibit neutrophil chemotaxis by cleav...

Claims

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Application Information

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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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