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Streptococcus equi subsp. zooepidemicus protective antigen HP0623 and preparation method thereof

A protective antigen, Streptococcus equi technology, applied in bacterial antigen components, botanical equipment and methods, biochemical equipment and methods, etc., to achieve good passive immune protection and high protective efficacy

Inactive Publication Date: 2017-09-05
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since inactivated whole-bacteria vaccines often cause side effects such as immune syndrome, it is necessary to develop new subunit protein vaccines to prevent the occurrence of such diseases

Method used

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  • Streptococcus equi subsp. zooepidemicus protective antigen HP0623 and preparation method thereof
  • Streptococcus equi subsp. zooepidemicus protective antigen HP0623 and preparation method thereof
  • Streptococcus equi subsp. zooepidemicus protective antigen HP0623 and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment one, bacterial strain and growth condition

[0036] The bacterial strain is SEZ C55138; the culture condition is TSB medium or TSA medium with 5% fetal bovine serum; shake culture at 37°C for about 8 hours, or the OD600 reaches 0.6-1.0. The carrier is Escherichia coli pET-32a, and the competent cells are Escherichia coli BL21.

[0037] The SEZ C55138 strain, Escherichia coli pET-32a, fetal bovine serum and Escherichia coli BL21 competent cells are all commercially available products.

Embodiment 2

[0038] Embodiment two, preparation method

[0039] The HP0623 protein is encoded by the sez_0623 gene, the forward primer of the HP0623 gene has a BamH I restriction site, and the reverse primer has a Hind III restriction site. The forward and reverse primers were designed from the SEZ genome sequence and synthesized by a primer synthesis company.

[0040] The preparation method of Streptococcus equi zooepidemic subspecies protective antigen HP0623 comprises the following steps:

[0041] 1) PCR amplification: use the genome of the C55138 strain as a template, and perform PCR amplification using the following primers:

[0042] Forward primer (SEQ ID NO.3): 5'-CCC GGATCC GCTTGCCTGCTAGTG-3', the underlined part is the BamH I restriction site;

[0043] Reverse primer (SEQ ID NO.4): 5'-CTC AAGCTT GAGGGGAAGATCGTATC-3', the underlined part is the HindIII restriction site;

[0044] 2) Ligation with the vector: the PCR product was digested with a restriction enzyme and then liga...

Embodiment 3

[0053] Embodiment three, antibody titer determination

[0054] Mouse serum antibody IgG was measured by enzyme-linked immunosorbent assay. After the purified recombinant protein HP0623 was coated overnight at 4°C, blood was collected from mice 10 days after the second immunization to separate the serum. After gradient dilution, all dilutions were equal to Take 100 μL and add it to the enzyme-linked plate. After reacting at 37°C, add horseradish-enzyme-labeled goat anti-mouse IgG. After the color development is completed, the reaction is terminated. Read the OD value with a microplate reader. The OD value is greater than the average OD value of the control serum +3 The maximum dilution factor of the serum with the variance SD (standard deviation) was used as the serum antibody. To infer the immune type, IgG1 and IgG2a were further tested to determine Th2 and Th1 immune responses.

[0055] Experimental results:

[0056] The IgG titer level of the immune group was significantly...

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Abstract

The invention relates to a streptococcus equi subsp. zooepidemicus (SEZ) protective antigen HP0623 and A preparation method thereof. The SEZ protective antigen HP0623 is a SEZ HP0623 recombinant protein, is composed of 429 amino acid residues, and has a molecular weight of 47.04 kDa. The preparation method comprises steps of gene clone, enzyme cutting and enzyme linking, inducible expression, and protein purification. The HP0623 can be used to prepare vaccines. The provided SEZ protective antigen HP0623 expresses good immunogenicity in mouse experiments; the generated antibody can provide a strong protective force and induce a high level sterilizing performance; the SEZ protective antigen HP0623 is located on the surface of SEZ, and rHP0623 can inhibit the adhesion of SEZ on Hep-2 cells.

Description

technical field [0001] The invention relates to a streptococcus protective antigen, in particular to a Streptococcus equi subspecies zooepidemic protective antigen HP0623 and a preparation method thereof. Background technique [0002] Streptococcus is a very common animal pathogen, which can be divided into 20 groups according to group-specific antigens, from A to V, lacking I and J. Animal pathogens are mostly Group B Streptococci (GBS) and C Group Streptococci (Group C Streptococci, GCS), while human pathogens are mostly Group A Streptococci (GroupAS Streptococci, GAS). [0003] Streptococcus zooepidemicus (SEZ) belongs to Group C Streptococcus of Langer's subgroup. It has no host-specific tropism and is prone to mutation. The antigenicity of strains from different sources may be different. The bacterium can infect animals through digestive tract, surgery, trauma, vaccination and trauma, etc., thereby causing animal diseases such as sepsis, arthritis, mastitis, meningitis...

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
CPCA61K39/092A61K2039/552C07K14/315C12N15/70
Inventor 魏子贡赵鹏鹏梁辉煌唐斌
Owner HUBEI UNIV
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