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Streptococcus adhesion antigen GAPDH and preparation method thereof

A streptococcus and adhesion technology, applied in the field of streptococcus adhesion antigen GAPDH and its preparation, can solve the problem of difficult to effectively control SEZ infection and other problems, and achieve the effect of weakening ability and weakening ability

Inactive Publication Date: 2012-10-24
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Effective control of SEZ infections has been difficult due to a lack of comprehensive understanding of SEZ virulence factors and protective antigens

Method used

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  • Streptococcus adhesion antigen GAPDH and preparation method thereof
  • Streptococcus adhesion antigen GAPDH and preparation method thereof
  • Streptococcus adhesion antigen GAPDH and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment one, bacterial strain and growth condition

[0026] 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.

[0027] The carrier is Escherichia coli pET-28a, and the competent cell is Escherichia coli BL21.

[0028] SEZ C55138 strain, pET-28a vector and Escherichia coli BL21 competent cells are all commercial products.

Embodiment 2

[0029] Embodiment two, preparation method

[0030] The GAPDH recombinant protein is encoded by the gapdh 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 the NCBI database (Genbank CP001129.1).

[0031] The preparation method of Streptococcus equi subspecies zooepidemic protective antigen GAPDH comprises the following steps:

[0032] PCR amplification: use the genome of C55138 as a template, and use the following primers for PCR amplification;

[0033] Forward primer (SEQ ID NO.3): 5'-TCTT GGATCC CGTCTTATCC-3' (the underlined part is the BamHI restriction site),

[0034] Reverse primer (SEQ ID NO.4): 5'-CATTTCGTT GAATTC CCAT-3' (the underlined part is the EcoRI restriction site);

[0035] Ligation with the vector: the PCR product was digested with a restriction enzyme and then ligated with the pET-28a vector digested wit...

Embodiment 3

[0046] Embodiment 3. Mice immunization and challenge test

[0047] 1. Twenty 4-week-old BALB / c female mice were randomly divided into 2 groups, 10 in each group;

[0048] 2. Experimental group: After 50 μg of purified rGAPDH was emulsified with Freund's complete adjuvant, the mice in group 1 were immunized by intraperitoneal injection. After 14 days, the same antigen was emulsified with Freund's incomplete adjuvant, and the second injection was immunized in the same way. mice;

[0049] 3. Control group: inject PBS emulsified with Freund's complete adjuvant and incomplete adjuvant to the mice of the second group as a control;

[0050] 4. After all mice were immunized for 10 days, blood was obtained from the tail vein to obtain serum, and then SEZ C55138 strain (2×10 5 CFU / mL) challenge;

[0051] 5. Observe and record the growth of all mice.

[0052] Experimental results: The mouse serum was collected 10 days after the second immunization with rGAPDH, and the antibody titer ...

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Abstract

The present invention relates to a streptococcus adhesion antigen GAPDH and a preparation method thereof. The GAPDH is an rGAPDH, which has high conservative property and sequence homology, consists of 319 amino acid residues, and has a molecular weight of 32.4kDa. The rGAPDH is encoded by a gapdh gene; and a forward primer and a reverse primer of the gapdh gene respectively have one restriction enzyme cutting site at 5' and 3'. The preparation method of the GAPDH comprises the following steps of: PCR amplification, connection to a carrier, transformation and induction, and purification. The rGAPDH and an SEZ (Streptococcus zooepidemicus) rehabilitation pig serum show good immune responses; the rGAPDH can protect SEZ infected mice with limitation; and the mice immunized by the rGAPDH show high level of antibody titer in serum. An anti-GAPDH antibody can induce high level of bactericidal capability; a gapdh gene has a transcription level in SEZ infected mice much higher than that of culture in vitro; and the rGAPDH can significantly weaken cell adhesion ability of the SEZ, and greatly reduce cell infection ability of the SEZ.

Description

technical field [0001] The invention relates to a streptococcus antigen, in particular to a streptococcus adhesive antigen GAPDH and a preparation method thereof. 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. [0003] Streptococcus zooepidemicus (SEZ) is a group C beta-hemolytic streptococcus in Lambert, which can cause sepsis, meningitis, su...

Claims

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Application Information

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Patent Type & Authority Applications(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 SUN YAT SEN UNIV
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