【摘要】 目的为研究UQCRB(ubiquinol-cytochrome c reductase binding protein)与中药的作用机制,本实验室进行了UQCRB基因的克隆、原核表达和纯化。方法提取人肝癌SMMC-7721细胞总RNA,反转录后采用特异引物进行PCR扩增获取UQCRB基因全长,产物纯化后连入T载体测定正确,克隆经NheI、XhoI双酶切后连入PET28a(+)载体,构建PET28a(+)-UQCRB原核表达质粒。在BL21(DE3)宿主菌中表达超声处理后,应用Amicon○R Pro Affinity Concentration Kit-Ni-NTA纯化,表达及纯化产物用westernblot验证。结果构建了PET28a(+)-UQCRB表达质粒,经过酶切鉴定和测序鉴定正确。重组UQCRB在BL21(DE3)中的最优表达条件为IPTG 1mM诱导5h。Westernblot证实了表达和纯化结果。结论重组UQCRB的原核表达和纯化成功,为进一步研究UQCRB的作用奠定了基础。更多还原

【Abstract】 Objective To study the interaction between UQCRB and traditional Chinese medicine,cloning,prokaryotic expression and purification of UQCRB(ubiquinol-cytochrome c reductase binding protein)was done in our laboratory.Methods Total RNA was extracted from SMMC-7721 cells of human liver cancer.The UQCRB full length gene was obtained by PCR amplification after reverse transcription using specific primers and the PCR product was purified and cloned into T vector which was sequenced to be correct.After being digested by Restriction Enzymer NheI and XhoI,it was connected to PET28a(+)to construct prokaryotic expression plasmid of PET28a(+)-UQCRB.UQCRB recombinant protein was expressed in BL21(DE3)host bacteria and purified Using Amicon○R Pro Affinity Concentration Kit-Ni-NTA.Both the products of expression and purification was verified using Westernblot.Results Expression plasmid of PET28a(+)-UQCRB was constructed and measured by restriction enzyme digestion identification and sequencing analysis.It was optimal that the expression of recombinant UQCRB in BL21(DE3)was induced 5hours with1 mM IPTG.The product of expression and purification was confirmed to be UQCRB recombinant protein by Westernblot.Conclusion Success of prokaryotic expression and purification of recombinant UQCRB establishes the foundation of further study for the function of UQCRB.更多还原