摘要
目的构建包含HCV核心蛋白全长基因的重组原核表达质粒，经转化表达型大肠杆菌后自诱导培养以获取可溶性重组核心蛋白，并检测其生物学功能。方法以H／FL质粒为模板，通过PCR扩增全长HCV核心蛋白DNA。PCR产物经双酶切后定向克隆到pET28a原核表达载体中。将重组的原核表达载体转化表达型大肠杆菌BL21（DE3）pLysS，并通过自诱导培养的方式使其在高浓度状态下表达重组核心蛋白。重组核心蛋白经Westernblot检测鉴定生物学功能，并与HCVNS3蛋白进行相互结合后共同纯化实验。结果重组核心蛋白大量存在于表达菌破菌上清液中。Westernblot检测结果显示其具有核心蛋白抗原性。重组核心蛋白不能单独通过Ni-NTA亲和层析柱纯化，但可以与HCVNS3蛋白结合后共同纯化。结论成功表达可溶性的重组核心蛋白，证明了其生物学活性。重组核心蛋白与HCVNS3蛋白存在相互作用。
Objective To investigate the biological role of auto-induced expression of hepatitis C virus （HCV） core protein （protein C） using a recombinant protein in an in vitro cell-based system. Methods The PCR-amplified full-length HCV protein C gene （573 bp） was inserted into the pET28a prokaryotic expression vector. The recombinant plasmid was transformed into B L2 I（DE3）pLysS E. coli to achieve high-concentration expression of the recombinant C protein by auto-induction. The recombinant protein C was purified by Ni- NTA affinity chromatography, and tested in a protein binding assay for its ability to bind the HCV NS3 protein. Results The transformed E. coli produced a large amount of recombinant protein C, as detected in the sonicated supematant of the bacteria culture. The antigenic reactivity of the recombinant protein C was confirmed by western blotting. However, the recombinant protein C could not be purified by Ni-NTA affinity chromatography, but co-precipitated with the HCV NS3 protein. Conclusion Soluble recombinant protein C was successfully expressed by auto-induction, and shown to interact with the HCV NS3 protein, which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes. Future studies of this recombinant HCV protein C＇s crystal structure and antigenicity may provide further clues to its biological function（s） and potential for clinical applications.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2013年第8期565-569,共5页
Chinese Journal of Hepatology
