Establishment and application of Streptococcus suis type 2 cell wall protein antibody detection method

A technology of Streptococcus suis and Streptococcus, which is applied in the establishment and application field of detection of Streptococcus suis cell wall protein antibody, can solve the problem of inability to determine the infection antibody or immune antibody of Streptococcus suis, and achieve low detection cost, Good sensitivity and specificity effect

Inactive Publication Date: 2015-07-08
广西壮族自治区动物疫病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The evaluation methods of serological antibodies against Streptococcus suis include agar diffusion test and indirect ELISA based on the whole bacteria and capsular polysaccharide as antigen. Patented CN201210245250.2, which can be used to detect serum antibodies to Streptococcus suis. This method can detect antibodies produced by inactivated vaccines and wild virus infections, and cannot determine antibodies to Streptococcus suis infection or immune antibodies

Method used

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  • Establishment and application of Streptococcus suis type 2 cell wall protein antibody detection method
  • Establishment and application of Streptococcus suis type 2 cell wall protein antibody detection method
  • Establishment and application of Streptococcus suis type 2 cell wall protein antibody detection method

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Embodiment 1

[0055] Embodiment 1: the extraction of swine type 2 Streptococcus genomic DNA

[0056] The Streptococcus suis type 2 preserved in our laboratory was inoculated on TSA medium containing 10% inactivated newborn bovine serum, and a single colony was picked and inoculated in TSB liquid medium, placed on a shaker at 150 r / min, 37°C Incubate overnight with shaking. Take 1.0 ml of bacterial liquid in a 1.5 ml centrifuge tube, and extract streptococcal DNA according to the instructions of the Bacterial Genomic DNA Extraction Kit from TIANGEN Company.

Embodiment 2

[0057] Example 2: Amplification of the target gene

[0058] According to GenBank published Streptococcus suis type 2 strains sspA The sequence of the gene (GenBank accession number: CP000407.1, specifically as figure 1 shown) , using Primer Premier 5.0 software to design the fragment as sspA (328-3228 bp) primers, the underlined part is the introduced enzyme cutting site Eco RI and Sal I.

[0059] Primers are as follows:

[0060] SspA upstream primer U 5'–GC GAATTC tggcattggtatatgcgcttccgat–3'

[0061] Downstream Primer D 5'–C GTC GAC cctacggtcaacttagaattgaagC–3'

[0062] The upper and lower primer restriction sites contain restriction restriction bases, which are GC and C bases, respectively. Primers were synthesized by Shanghai Sangon Biotechnology Company. The extracted DNA was amplified by PCR in a 50 μl system. PCR products were detected by 1.2% agarose gel electrophoresis. see results image 3 , the size of the amplified band is consistent ...

Embodiment 3

[0063] Example 3: Construction and Screening of Expression Recombinant Plasmids

[0064]The PCR amplified product of the target gene fragment was digested with the corresponding endonuclease, and the pET28a vector was digested with the same double enzymes, recovered and purified separately, and transformed into Escherichia coli DH5α after T4 DNA ligase ligation. The positive clones were screened with bacterial liquid, and the plasmids of the positive clones were sequenced. The recombinant plasmid was heat-shock transformed into E. coli BL21 competent, and finally the prokaryotic expression vector carrying the target fragment was obtained. pET28a- sspA for cloning Eco RI and Sal Ⅰ The double enzyme digestion identification is consistent with the expected size, which further indicates that the target gene has been successfully cloned into the expression vector. Plasmid double enzyme digestion identification after cloning of the target fragment, see Figure 4 .

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Abstract

Belonging to the technical field of veterinary microbiology and animal immunology, the invention discloses cloning of Streptococcus suis type 2 cell wall protein gene main functional domain fragment and application of the recombinant protein expressed by the fragment in Streptococcus suis type 2 antibody detection. The invention prepares an expression strain Escherichia coli BL21 / pET28a-sspA able to secrete Streptococcus suis type 2 cell wall protein SspA, and the preservation number is CCTCC NO: M2014344. The invention also discloses establishment of an SspA recombinant protein based ELISA detection method and application in detection of Streptococcus suis type 2 antibodies. A large number of SSPA antibodies exist in swine infected serum, and the antibody of the protein cannot be detected in immune serum. The method provided by the invention can be employed to detect swine serum antibody and can distinguish Streptococcus suis type 2 infection and immune animals.

Description

technical field [0001] The invention relates to the technical fields of veterinary microbiology and animal immunology, in particular to a recombinant Escherichia coli strain based on cell wall protein SspA (Subtilisin-like serine protease, SspA, subtilisin-like serine protease) Escherichia coli BL21 / pET28a-SspA, and the recombinant protein of Streptococcus suis type 2 expressed by the strain, were used to establish an ELISA detection method, which is suitable for the detection of antibodies infected with Streptococcus suis type 2. Background technique [0002] Streptococcus suis is an important pathogen that can cause infection and disease in humans, pigs and other animals. According to capsular polysaccharide typing, there are 35 serotypes of Streptococcus suis, namely type 1 / 2 and types 1-34. Among them, Streptococcus suis type 2 is the most virulent and most prevalent serotype. Streptococcus suis type 2 mainly causes meningitis, bronchitis, endocarditis, arthritis, abs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21G01N33/68C12R1/19
CPCC12N9/54C12Y304/21062G01N33/569
Inventor 胡巧云熊毅袁小宁黄胜斌何其松杨荣李军郭建刚
Owner 广西壮族自治区动物疫病预防控制中心
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