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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。
Abstract：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 59 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 GapC immunogenicity and the preparation of vaccine.
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基本信息:
中图分类号:S852.611
引用信息:
[1]张辉,焦海宏,陈伟.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[J].塔里木大学学报,2012,24(02):7-13.
基金信息:
国家自然科学基金项目(30860018);; 教育部“新世纪优秀人才支持计划”(NCET-11-1071)
2012-06-15
2012-06-15