CAJ | 학술논문

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Western blot检测鉴定生物学功能,并与HCV NS3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.Western blot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HCV NS3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HCV NS3蛋白存在相互作用.
목적 구건포함HCV핵심단백전장기인적중조원핵표체질립,경전화표체형대장간균후자유도배양이획취가용성중조핵심단백,병검측기생물학공능. 방법 이H/FL질립위모판,통과PCR확증전장HCV핵심단백DNA.PCR산물경쌍매절후정향극륭도pET28a원핵표체재체중.장중조적원핵표체재체전화표체형대장간균BL21 (DE3) pLysS,병통과자유도배양적방식사기재고농도상태하표체중조핵심단백.중조핵심단백경Western blot검측감정생물학공능,병여HCV NS3단백진행상호결합후공동순화실험. 결과 중조핵심단백대량존재우표체균파균상청액중.Western blot검측결과현시기구유핵심단백항원성.중조핵심단백불능단독통과Ni-NTA친화층석주순화,단가이여HCV NS3단백결합후공동순화. 결론 성공표체가용성적중조핵심단백,증명료기생물학활성.중조핵심단백여HCV NS3단백존재상호작용.
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 ceil-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 BL21 (DE3)pLysS E.coli to achieve high-concentration expression of the recombinant C protein by auto-induction.The recombinant protein C was purified by NiNTA 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 supernatant 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.