SARS病毒受体ACE2的克隆、原核表达及其功能区鉴定

ACE2(angiotensin-converting enzyme 2,ACE2)是SARS冠状病毒(severe acute respiratory syndrome associatedcoronavirus,SARS-CoV)的主要受体。此研究旨在鉴定ACE2的SARS-CoV受体功能区,为进一步阐明SARS-CoV与细胞间的相互作用机制及研制抗病毒药物等提供理论依据。利用RT-PCR从Vero-E6细胞的mRNA中分两段扩增ACE2基因,其中N端片段ACE2A1-367(102~1 210nt)不包括ACE2的酶活性位点(1 223~1 237nt,或374~378aa),而C端片段ACE2B335-805(1 101~2 524nt)包括ACE2的酶活性位点。扩增片段克隆入pMD-18T,并进行测序鉴定。进一步构建与GST基因融合表达的原核表达质粒pGEX-ACE2A与pGEX-ACE2B,IPTG诱导表达。表达的融合蛋白分子量为65kD和77kD,主要以包涵体形式存在。Western blot证实表达产物具有免疫学活性。将纯化的包涵体蛋白质复性后进行Western blot分析,证实pGEX-ACE2A表达的蛋白(~65kD)能与SARS-CoV S1蛋白特异结合,而pGEX-ACE2B表达的蛋白(~77kD)不能与S1蛋白结合。结果表明,ACE2的受体活性与酶活性位点无关,受体功能区在ACE2 N端367个氨基酸内。

Prokaryotic Expression of SARS-CoV Receptor ACE2 in E. coli and Identification of Its Functional Domain

Two fragments of angiotensin-converting enzyme 2(ACE2) gene,ACE2A(102-1210 nt)and ACE2B(1101-2524 nt),were amplified with RT-PCR and cloned into pMD-18T.The insertion was confirmed by sequencing and restriction enzyme digestion.The gene fragments were sub-cloned into pGEX-KG separately and expressed as GST fusion proteins after IPTG induction in E.coli BL21(DE3).The expression was confirmed by SDS-PAGE and Western blot with polyclonal antibody to human ACE2.The molecular masses of the two fusion proteins were respectively 65kD and 77kD.Western blot analysis demonstrated that the recombinant proteins had immunological activity.Furthermore,only ACE2A(65kD),which excludes the enzyme active site HEXXH(374-378aa),could bind with S1 protein of SARS-CoV.These results showed that the receptor activity of ACE2 was not dependent on the enzyme activity,and the receptor functional domain was within N terminal 367 amino acids.