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HtpsA-gene-knock-out mutant strain of Streptococcus suis serotype 2 and application thereof

A Streptococcus suis, gene knockout technology, applied in the field of genetic engineering, can solve problems such as no research

Inactive Publication Date: 2013-10-16
中国人民解放军南京军区军事医学研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Reports in recent years have shown that members of the Htp family protein have been found in group A, B, C, and G streptococci, and related studies on the functions of this family member in Streptococcus pyogenes and Streptococcus agalactiae have also been reported, but there is no report yet. Research on the function of the gene in S. suis and its molecular mechanism involved in bacterial pathogenicity

Method used

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  • HtpsA-gene-knock-out mutant strain of Streptococcus suis serotype 2 and application thereof
  • HtpsA-gene-knock-out mutant strain of Streptococcus suis serotype 2 and application thereof
  • HtpsA-gene-knock-out mutant strain of Streptococcus suis serotype 2 and application thereof

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Experimental program
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Effect test

Embodiment 1

[0049] Embodiment 1: Construction of gene knockout vector

[0050] (1) According to the upstream and downstream DNA sequences of the htpsA coding gene of the S. suis2 wild strain 05ZYH33 genome, design PCR-specific primers, the base sequence of which is as follows:

[0051] LA1: 5′-CG GCATGC GAAAGTGATAAGAGG-3' (SEQ ID NO.5, the underline is the introduced Sph I restriction site)

[0052] LA2: 5′-C GTC GAC TACGGAGCCAACAACT-3' (SEQ ID NO.6, the underline is the introduced Sal I restriction site)

[0053] RA1: 5′- GGATCC GCGCACAAGCAAGT-3' (SEQ ID NO.7, the underline is the introduced BamH I restriction site)

[0054] RA2: 5′-AC GGTACC TCAGGATGTTGCATGA-3' (SEQ ID NO.8, the underline is the introduced Kpn I restriction site)

[0055] According to the pSET2 plasmid sequence, a pair of specific primers spc-F / spc-R was designed to amplify the entire spectinomycin resistance gene cassette with the pSET2 plasmid as a template. The primer sequence is:

[0056] Spc-F: 5'-C GTC ...

Embodiment 2

[0073] Embodiment 2: Screening and identification of mutant strains

[0074] (1) The gene knockout vector pUC::htpsA was electrotransformed into 05ZYH33 competent

[0075] ①Preparation of S. suis2 wild strain 05ZYH33 competent cells: Pick a single colony of 05ZYH33 and inoculate it with 3ml THB medium, cultivate overnight on a shaking table at 37°C, transfer to THY containing DL-threonine at 1:50 the next day at 37°C Shaker shake culture to OD 600 0.3-0.4, collect the bacteria by centrifugation at low speed at 4°C, wash the bacteria with pre-cooled 10% glycerol 4 times, each time not less than 25ml, and finally resuspend the bacterial pellet with 0.5ml of 0.3M sucrose containing 15% glycerol, and Aliquot 50μl / tube and store at -80°C for later use.

[0076] ② Electroporation: Add 10ul of pUC::htpsA plasmid to 50μl of the competent state prepared by the above method, and then add it to the electroporation cup (operated on ice). After electroporation at 22.5kV / cm, 200Ω and 25μF...

Embodiment 3

[0085] Embodiment 3: in vitro experiment

[0086] (1) Gram staining

[0087] According to the instructions of the Gram staining solution produced by Beijing Suolaibao Technology Co., Ltd., Gram staining was performed on 05ZYH33 and 05ZYH33ΔhtpsA (CGMCC No.7375, the same below), and the chain arrangement of the mutant strain 05ZYH33ΔhtpsA compared with the wild strain was found More scattered, and the length of the chain was significantly shorter than that of the wild strain, indicating that the chain-forming ability of the mutant strain 05ZYH33ΔhtpsA was weakened.

[0088](2) Growth characteristics

[0089] Under the same culture conditions, single colonies of 05ZYH33ΔhtpsA and wild strain 05ZYH33 were picked and inoculated in 3 mL of THB medium containing spectinomycin (100 mg / mL) and without spectinomycin, respectively, and cultured overnight at 37 °C with shaking. The next day, the overnight cultured bacteria were taken out, the absorbance value at 600nm was measured, and...

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Abstract

The invention relates to an htpsA-gene-knock-out mutant strain of Streptococcus suis serotype 2 and application thereof, belonging to the field of genetic engineering. The mutant strain is 05ZYH33 delta htpsA with an accession number of CGMCC No. 7375. A preparation method for the mutant strain comprises the following steps: carrying out gene knockout on an htpsA gene by using a homologous recombination gene knockout technology, wherein the htpsA gene is located on chromosome 05ZYH33 of a wild S. suis 2 strain and codes histidine triad protein Htp; and determining that an obtained strain is a gene-knocked-out mutant strain through combined PCR product electrophoresis, RT-PCR and DNA sequencing identification and naming the obtained gene as 05ZYH33 delta htpsA. Pathogenicity of the mutant strain provided by the invention is researched through animal experiments, and results show that virulence of the mutant strain to a tested animal is substantially reduced; so the mutant strain can be used for developing an attenuated vaccine of Streptococcus suis serotype 2.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a htpsA gene knockout mutant strain of Streptococcus suis type 2 and an application thereof. Background technique [0002] Streptococcus suis type 2 (S. suis2) is a zoonotic pathogen that endangers the world. The pathogen can not only cause pig meningitis, septicemia, arthritis, pneumonia and endocarditis, but also infect humans, posing a serious threat to the lives of relevant practitioners and the general public. According to the antigenicity of its capsular polysaccharide, it can be divided into 35 serotypes, of which type 2 (SS2) is the most virulent and has the highest clinical detection rate. In recent years, the prevalence of S.suis2 in swine herds in southern provinces of my country has become increasingly serious, and large-scale outbreaks have occurred from time to time, causing economic losses of billions of yuan each year. In 1998 and 2005, large-scale SS2 infection ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/09C12N15/63A61K39/09A61P31/04C12R1/46
Inventor 潘秀珍李敏王晶王长军邵珠卿李先富高基民
Owner 中国人民解放军南京军区军事医学研究所
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