Streptococcus zooepidemicus protective antigen Sec_205 and preparation method thereof

A protective antigen, Streptococcus equi technology, applied in the direction of bacterial antigen components, chemical instruments and methods, antibacterial drugs, etc., can solve the problem that it is difficult to develop subunit vaccine infection, and achieve a good immune response effect

Inactive Publication Date: 2017-11-17
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there are few studies on SEZ virulence factors and protective antigens, making it difficult to develop effective subunit vaccines to control SEZ infection in animals

Method used

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  • Streptococcus zooepidemicus protective antigen Sec_205 and preparation method thereof
  • Streptococcus zooepidemicus protective antigen Sec_205 and preparation method thereof
  • Streptococcus zooepidemicus protective antigen Sec_205 and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] This embodiment provides a protective antigen Sec_205 of Streptococcus equi subspecies zooepidemicus, which is a recombinant protein of SEZ Sec_205, encoded by the sesec_205 gene (SEQ ID NO.1), consisting of 650 amino acid residues, with a molecular weight of 70.9 kDa, amino acid The sequence is shown in SEQ ID NO.2.

[0037] The forward primer of the sesec_205 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 genome sequence of the NCBI database (Genbank CP001129.1).

[0038] Further, this embodiment also provides a method for preparing the above-mentioned Streptococcus equi subspecies zooepidemic protective antigen Sec_205, the specific steps comprising:

[0039] PCR amplification: use the genome of C55138 as a template, and use the following primers to perform PCR amplification and clone the sesec_205 gene under conventional conditions;

[0040] Forward primer (SEQ ID N...

Embodiment 2

[0051] Embodiment 2 Mice immunization and virus challenge test

[0052] In this example, the SEZ Sec_205 recombinant protein obtained in Example 1 was tested for mouse immunization and challenge, and the test steps included:

[0053] 1. Thirty 6-week-old KM female mice were randomly divided into 3 groups, 10 in each group;

[0054] 2. Experimental group: After 50 μg of purified rSec_205 was emulsified with Montanide Gel 01PR (SEPPIC, France) adjuvant, the first group of mice was immunized by intraperitoneal injection, and 14 days later, the mice were immunized for the second time in the same way;

[0055] 3. Positive control group: C55138 was inactivated and emulsified with Montanide Gel 01 PR (SEPPIC, France) adjuvant, and mixed with 1×10 9 The mice were immunized with CFU / mL by intraperitoneal injection, and after an interval of 14 days, the second immunization was carried out in the same way;

[0056] 4. Negative control group: the mice in the second group were intraperit...

Embodiment 3

[0062] Embodiment three, passive protection test analysis

[0063] In order to confirm that the protection is due to Sec_205-specific stimulation of the immune response, mice were passively immunized with anti-rSec_205 mouse serum, and then challenged.

[0064] 1. Thirty 6-week-old KM female mice were randomly divided into 3 groups, 10 in each group;

[0065] 2. Experimental group: immunize the first group of mice with 100 μL anti-rSec_205 second-immune mouse serum intravenously;

[0066] 3. Positive control group: Inject the second group of mice intravenously with SEZ inactivated vaccine second-immune mouse serum;

[0067] 4. Negative control group: Inject the third group of mice intravenously with the serum of normal mice;

[0068] 5. After 24 hours of immunization, all mice were challenged with SEZ C55138 (2×10 5 CFU / mL);

[0069] 6. Observe and record the growth of all mice.

[0070] Experimental results: 1) All mice in the positive control group survived the challeng...

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Abstract

The invention relates to Streptococcus zooepidemicus protective antigen Sec_205 and a preparation method thereof. The Streptococcus zooepidemicus protective antigen Sec_205 is rSec_205. The antigen preparation method comprises cloning, conversion, inducible expression and purifying. The rSec_205 and an SEZ (Streptococcus zooepidemicus) rehabilitation pig serum show good immune responses; and the mice immunized by the recombinant protein detects high level of antibody titer in mice serum. mice second immune serum has obvious passive immune protection to mice, and an anti-rSec_205 antibody can induce high-level sterilization capability. The immunity protective tests show that after the mice is immunized by the rSec_205, high protection effectiveness is provided. The transcription level of a gene for coding the Sec_205 protein in SEZ-infected mice body is obviously higher than the transcription level of in vitro culture, a flow cytometry result shows that the Sec_205 is distributed on the surface of SEZ cell, which is cell surface protein, in a SEZ adhesion test, Sec_205 can obviously reduce the SEZ adhesion capability for cells, and the inhibition rate is 30.3%.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention relates to a protective antigen Sec_205 of Streptococcus equi subspecies zooepidemicus and a preparation method thereof. Background technique [0002] Streptococcus is a common pathogen. According to different cell wall polysaccharide antigens, it can be divided into 20 groups: A, B, C..., V (missing I and J), among which Group A Streptococci (GAS) It is mainly pathogenic to humans, and other groups are mainly pathogenic to animals. Group B Streptococci (GBS) and C Streptococci (Group C Streptococci, GCS) and other group hemolytic streptococci mainly infect livestock and poultry, such as GBS mainly causing bovine mastitis and swine impetigo; GCS is Mainly cause equine blight and sheep and swine sepsis. [0003] Streptococcus zooepidemicus (SEZ) is a kind of GCS, which is a β-hemolytic streptococcus. At present, the research on SEZ in China still needs to be further deepe...

Claims

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Application Information

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IPC IPC(8): C07K14/315C12N15/70A61K39/09A61P31/04
Inventor 魏子贡梁辉煌唐斌
Owner HUBEI UNIV
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