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Clone and Expression of Carbamate Kinase and Ornithine Carbamoyl Transferase Genes of Streptococcus Suis Serotype 2

Author: WanJun
Tutor: HuJianCheng
School: Lanzhou University
Course: Biochemistry and Molecular Biology
Keywords: Streptococcus suis Carbamate kinase Ornithine carbamoyl transfer enzyme Clone The prokaryotic expression
CLC: S852.61
Type: Master's thesis
Year: 2009
Downloads: 57
Quote: 0
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Abstract


Swine streptococcus (Streptococcus suis) is an important zoonotic disease pathogens can cause a variety of diseases, including meningitis, septicemia, pneumonia, endocarditis and arthritis. The 33 serotypes bacteria most type 2 bacteria virulence. It recognized the virulence factor capsular polysaccharide, muramidase released protein and extracellular factor, hemolysin, but recently found that some strains does not have the above-mentioned one or several virulence factors, but has a strong pathogenicity, indicating that Streptococcus suis type 2 there is a more complex pathogenic mechanism. This study according to the pigs in GenBank Streptococcus 2 type (SS2) 98HAH33 strains of amino formate kinase (carbamate kinase, CK) sequence design and synthesis of a pair of primers, the primer 5 'ends respectively join containing EcoR Ⅰ and Xho I restriction enzyme digestion locus sequence. SS2 Qinghai strain genome as a template, CK complete coding genes were amplified by PCR and cloned into pMD18-T simple vector and further subcloned into pET32a (), was transformed into E.coli BL21 (DE3), screening positive transformants express the target protein was induced by IPTG. The sequencing results showed that the gene length 948bp, strain ck announced the 98HAH33 of nucleotide sequences in GenBank sequences are identical. SDS-PAGE analysis showed that expression of a molecular weight of approximately 53 kD fusion protein after nickel column affinity chromatography, Western blot analysis showed that this fusion protein and SS2 Qinghai strain whole cell preparation of rabbit antiserum specifically reacted. That CK fusion protein has been successfully expressed. Analysis of the characteristics of the carbamate kinase function to lay the foundation, helps to further study the molecular mechanisms of Streptococcus suis type 2 infection of host. Streptococcus suis type 2 (SS2) 98HAH33 strains ornithine carbamoyl transfer enzyme (OrnithineCarbamoyl transferase, OCT) sequence design Nde Ⅰ and Hind Ⅲ containing primers amplified OCT full gene sequence SS2 Qinghai strain genome as a template, cloned into pET28a () prokaryotic expression vector was transformed into E.coli BL21 (DE3) competent cells, through IPTG induction OCT fusion protein. Nucleotide sequencing showed that the the oct gene is 1014bp, with the the oct logged strains 98HAH33 sequence homology to 100%. SDS-PAGE OCT detected 40 kD fusion protein, enzyme activity analysis test showed that the fusion protein OCT activity optimum reaction temperature of 37 ° C, optimal PH of about 6.5. Western blot analysis showed that the OCT fusion protein with rabbit anti-SS2 Qinghai strain bacteria positive sera react specifically with good reactogenicity.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Pathogenic bacteria
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