Construction of eukaryotic expression system of wild type and A294G mutant human angiotensin-converting enzyme 2

Objectives To construct the eukaryotic expression system of human wild type and mutant A294G angiotensin-converting enzyme 2(ACE2).Methods Human ACE2 gene was amplified by reverse transcriptase polymerase chain reaction(RT-PCR),linkaged with pcDNA3.1 eukaryotic expression vector to construct recombinant pcDNA3.1-hACE2,and then identified the recombinant plasmid by enzyme digestion and sequencing.Using the wild-type ACE2 expression vector as a templet,the mutant recombinant plasmids was amplified by PCR with pyrobest DNA polymerase.The template DNA was removed by digestion with Dpn I,and the amplified DNA was directly transformed into E.coli DH5α.After plasmid DNA was recovered from a colony,the sequence was confirmed.Results The wild-type and mutant ACE2 genes were subcloned into eukaryotic expression vector pcDNA3.1+,and the recombinant plasmids were validated by sequencing.The sequence of the wild-type ACE2 showed the same sequence as that in the NCBI(RefSeq: NM_021804.2),and the sequence of mutant ACE2 was concordant with that of the wild-type ACE2 except that the number 294 base A was replaced by G.Conclusions The eukaryotic expression plasmids containing human wild and mutative ACE2 were successfully constructed,which will enable us to explore the biological function of the mutative ACE2 gene and the role of ACE2 in the pathogenesis of disease futher.