重组人钙网蛋白的克隆与原核表达

Cloning and Prokaryotic Expression of Human Recombinant Calreticulin

目的:克隆人钙网蛋白(calreticulin,CRT)并进行原核表达和纯化。方法:采用RT-PCR法从人非小细胞肺腺癌A549细胞总RNA中克隆人钙网蛋白cDNA,构建CRT原核表达质粒(pE15b/CRT)并转化E.coli的Rossetta菌株。IPTG诱导后,表达蛋白在变性条件下经Ni-NTA树脂亲和层析纯化,然后透析复性。分别用SDS-PAGE和Western blotting鉴定CRT表达和纯化状态结果:从A549细胞总RNA中成功获得人CRTcDNA克隆,重组质粒pET-15b/CRT构建正确。转化pET-15b/CRT的E.coliRossetta诱导性表达重组人CRT蛋白,该蛋白可经Ni-NTA树脂亲和层析高度纯化。结论:成功建立了CRT原核表达和纯化的实验方法,该方法为后续的CRT蛋白功能研究奠定了基础。

Objective： Clone, express and purify human recombinant calreticulin （CRT）. Methods： Human CRT cDNA was amplified from total RNA of human lung cancer cell line A549 ceils by RT-PCR. Then, PCR product was subcloned into prokaryotic expression vector pET-15b. After sequencing, this recombinant plasmid was transformed into E. coli. Rossetta. Recombinant CRT was expressed in host ceils by IPTG induction. Resulted protein was purified by Ni-NTA resin under denature condition and dialyzed to recover its native structure. SDS- PAGE and Western blot method were used to identify the expression and purification of reconbinant CRT. Results： Human CRT cDNA was cloned from total RNA of A549 ceils. CRT prokaryotic expression vector pET-15b-crt was constructed. Reconbinant CRT was induced to express in E. coli and purified by Ni-NTA affinity chromatograph. Conclusion： A method for prokaryotic expression and purification of human recombinant CRT was successfully established. This method laid a foundation for the subsequent CRT research.