重组人钙网蛋白的克隆与原核表达

［摘要］目的: 克隆人钙网蛋白（calreticulin，CRT）并在E.coli中原核表达和纯化。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Western blotting法鉴定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。

Cloning and Prokaryotic Expression of Human Recombinant Calreticulin

Objective: Clone, express and purify human recombinant calreticulin (CRT). Methods: Human CRT cDNA was amplified by RT-PCR from total RNA of human lung cancer cell line A549 cells. 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 cells 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 cells. 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 succedent CRT research.