心肌钙蛋白I在杆状病毒表达系统中的表达

Expression of cardiac troponin I in baculovirus system

目的在杆状病毒表达系统中表达心肌钙蛋白(Icardiac troponin I,cTnI)。方法将带有6×His标签的cTnI基因亚克隆至杆状病毒转移载体pFastBac-dua(lpFBd)中,构建重组转移质粒pFBd-cTnI,转化大肠埃希菌DH10-Bac,筛选获得重组杆粒rbacmid-cTnI,转染昆虫细胞Sf9,SDS-PAGE和Western blot检测转染细胞中cTnI蛋白的表达。结果重组杆粒rbacmid-cTnI经PCR鉴定证明构建正确;重组杆粒rbacmid-cTnI转染的Sf9细胞(Invitrogen细胞株)可表达相对分子质量约25 000的cTnI蛋白条带,表达的cTnI蛋白可被鼠抗His标签单抗和兔抗cTnI多抗特异性识别。结论成功在Sf9细胞中表达了cTnI蛋白,为下一步大规模制备cTnI蛋白以及建立cTnI相关检测方法奠定了基础。

Objective To express cardiac troponin I （cTnI） in baculovirus system. Methods The cTnI gene with 6 x His tag was subcloned to baculovirus transfer vector pFastBac-dual （pFBd）. The constructed recombinant transfer plasmid pFBd-cTnI was transformed to competent E. coli DHIOBac, based on which recombinant bacmid rbacmid-cTnI was screened and transfected to Sf9 cells. The expression of cTnI protein was determined by SDS-PAGE and Western blot. Results PCR proved that rbacmid-cTnI was constructed correctly. The cTnI protein, with a relative molecular mass of about 25 000, was expressed in Invitrogen straion of Six） cells transfeeted with rbacmid-cTnI, and was recognized by mouse monoclonal antibody against His tag and rabbit polyelonal antibody against cTnI. Conclusion The cTnI protein was successfully expressed in Sf9 cells, which laid a foundation of further preparation of cTnI protein and development of test method for cTnI.