停乳链球菌isp基因的克隆与原核表达

Cloning and Prokaryotic Expression of Streptococcus dysgalactiae Gene isp

根据GenBank报道的停乳链球菌(Streptococcus dysgalactiae)isp基因(GenBank:CP002215.1)序列设计引物,PCR扩增得到isp基因。片段大小为1 500 bp,与从GenBank下载的序列进行同源性比对结果为98.57%。将isp基因克隆于表达载体pET-25b,成功构建了重组质粒pET-25b-isp,将重组质粒分别转化BL21(DE3),将BL21(pET-25b-isp)阳性重组菌株经IPTG诱导后,SDS-PAGE结果显示均有蛋白表达,大小分别为53 ku与目的蛋白大小相符;Western blot检测显示,表达的蛋白为目的蛋白。对成功构建的菌株BL21(pET-25b-isp)表达条件经优化后,在温度37℃,诱导时间12 h,IPTG浓度为1 mmol/L时,蛋白有较高的表达量。

According to the sequence of gene isp of Streptococcus dysgalactiae reported in the GenBank （ GenBank CP002215.1 ） a primer was designed and amplified by PCR. The fragment was 1 500 bp with 98.57% of homology as compared with the sequence downloaded from the GenBank. The /sp gene was cloned into expression vector pET-25b and successfully constructed a recombinant plasmid pET-25b-isp, and transformed into BL21 （DE3）. The positive recombinant strain of BL21 （pET-25b-isp） was induced with IPTG, the SDS-PAGE results showed that all had protein expression with the size of 53 ku and accord with the size of target protein. Western blot tests showed the expression protein was the target protein. After condition optimization the successful constructed strain of BI21 （pET-25b-isp） had fairly high protein expression volume at 37 ℃, induced for 12 h, and IPTG concentration at 1 mmol/L.