恶性疟原虫信号肽肽酶原核表达载体的构建及融合表达蛋白的鉴定

目的构建恶性疟原虫PfSPP基因pMAL-p2x的原核表达载体,并鉴定表达,为其功能研究奠定基础。方法体外培养恶性疟原虫（3D7株和FCR3株）,提取虫体总RNA,进行反转录后,采用RT-PCR扩增PfSPP的全编码区基因,将其克隆入原核表达载体pMAL-p2x,PCR、双酶切鉴定重组质粒,并进行序列测定,将测序正确的重组质粒转化入大肠杆菌进行表达获得MBP-PfSPP融合蛋白,采用Western blotting对表达产物进行鉴定。结果成功构建pMAL-PfSPP,转化菌经诱导表达出分子量约为77 000Mr的MBP-PfSPP融合蛋白,抗MBP多克隆抗体可特异性地识别表达的融合蛋白。结论成功表达具有多个跨膜结构的膜蛋白PfSPP,从而为深入研究PfSPP的酶活性及生物学功能奠定基础。

Cloning,prokaryotic expression and identification of Plasmodium falciparum signal peptide peptidase

Objective To clone and prokaryotic express Plasmodium falciparum signal peptide peptidase in E.coli. Methods Plasmodium falciparum（3D7 strain and FCR3 strain） total RNA was extracted from cultured malaria parasite. PfSPP gene was amplified by RT-PCR,and then cloned into prokaryotic expression vector pMAL-p2 x.The correct recombinant constructs were identified by PCR screening,double enzymes digestion and DNA sequencing. PfSPP / pMAL-p2 x constructs were transformed to E.coli BL21（DE3）,and expressed in E.coli by IPTG induction.The MBP-PfSPP fusion protein expression was identified by SDS-PAGE and Western blotting using anti-MBP polyclonal antibody. Results PfSPP / pMALp2 x recombinant vectors（3D7 strain and FCR3 strain）were constructed successfully,PfSPP gene sequences were confirmed by DNA sequencing.After IPTG induction,MBP-PfSPP fusion proteins were expressed in E. coli. The recombinant MBPPfSPP was identified by anti-MBP polyclonal antibody showing a molecular mass of 77 000 kD. Conclusions Multitransmembrane protein SPP of P.falciparum（3D7 and FCR3） was successfully expressed in E.coli as soluble form,which laid the foundation for the further analysis of its enzyme activity and biological function.