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(pET-15b/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Westem 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CRT cDNA克隆,重组质粒pET-15b/CRT构建正确。转化pET-15b/CRT的E.coli Rossetta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.