用E.coli表达Canstatin-N及其表达条件优化

Expresstion of canstatin-N in E. coli and its expression condition optimization

以重组质粒pET-CN为模板设计引物CASN1和CASN2,PCR方法扩增约267bp的人血管能抑素N端1～89氨基酸基因片段,用EcoR I和Sal I双酶切将其克隆进pET-22b(+)载体获得重组表达质粒pET-22b(+)-CN,转化E.coli BL21(DE3),用IPTG诱导表达Canstatin-N,产物以包涵体形式存在。本文在摇瓶发酵条件下研究了诱导剂浓度、诱导培养时间对目标蛋白表达的影响,结果表明IPTG的最佳诱导浓度为0.1mmol/L;37℃下诱导培养2h时产物表达量最高。纯化获得的融合his6的重组Canstatin-N具有免疫和抑制鸡胚绒毛尿囊膜(CAM)新生血管生成活性。

The recombinant plasmid pET-CN was used as template to amplify the 267 bp fragment coding for canstatin N-terminal 1-89 amino acids（N domain） with CASN1N and CASN2 primers. The amplified N domain of canstatin （canstatin-N） gene was cloned into EcoR I and Sal I sites on pET-22b（ ＋ ） resulting in recombinant plasmid pET-22b （ ＋ ）-CN. The pET-22b（ ＋ ）-CN was then transformed into E. coli BL21（DE3） and expression of canstatin-N protein was induced with IPTG. The expressed product existed mainly as inclusion body. The influences of culture conditions such as concentration of the inducer, the time of induction culture on the expression of the recombinant protein of E. coli BL 21（DE3） were investigated in detail, The results showed that the best concentration of IPTG was 0.1 mmol/L, the expressing quantity of recombinant protein reached its maximum by culturing for two hours at 37℃. It inhibited the new vessel formation in a dose-dependent manner tested by CAM assay.