新疆南疆地区奶牛乳房链球菌gapC基因原核表达载体的构建

Construction of Recombinant Expression Plasmid of gapC Gene of Streptococcus uberis Isolated from the Southern Xinjiang

为获得乳房链球菌(Streptococcus uberis)gapC基因的原核表达载体,通过PCR方法扩增出新疆南疆地区奶牛乳房链球菌临床分离株的gapC基因,并克隆到原核表达载体pET32a(+)中,构建了重组质粒pET32a(+)-gapC,将重组质粒转化宿主菌E.coli BL21进行重组蛋白的表达。实验结果表明,IPTG诱导5～9 h均可获得大量重组蛋白。重组蛋白分子量约为54.0 kD,介于43 kD～66.2 kD之间,与预期大小一致,说明表达载体构建成功。通过优化最佳诱导条件,获得乳房链球菌gapC基因重组蛋白的最佳诱导条件为0.5 mmol/L IPTG诱导6 h即可获得大量重组蛋白,为进一步确定蛋白的免疫保护性和制备疫苗奠定了基础。

To obtain expression vector of gapC gene of Streptococcus uberis , the gapC gene of S. uberis field strain from the southern Xinjiang dairy herds was amplified by PCR and was cloned into the prokaryotic expression vector pET32a （ ＋ ）. The recombinant plasmid was transformed into E. coli BL21 cells. The recombinant protein was detected with the induction of 0.5 mmol/L and 1 mmol/L IPTG at different inducible time, respectively. The results showed that the fusion protein （ His - GapC） was massly expressed after induced for 5～9 h with 0. 5 mmol/L and 1 mmol/L IPTG and the optimum condition for GapC expression was 0. 5 mmol/L IPTG for 6 h. The molecular weight of expressed protein was about 54. 0 kD, between 43 kD ～ 66. 2 kD, which was expected. This study would supply a basis for the detection of GapG immunogenicity and the preparation of vaccine.