Cloning, expression and sequence analysis and tissue distribution of angiotensin-converting enzyme 2 (ACE2) gene in adult mice / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>o clone angiotensin-converting enzyme 2(ACE2) gene, to analyze its amino acids and nucleotides sequence and to investigate tissue distribution of ACE2 in adult mice.</p><p><b>METHODS</b>The full-length ACE2 encoding sequence was amplified from the RNA of mice kidney tissue by RT-PCR technique, cloned into plasmid pGEM-T easy, then subcloned into plasmid pcDNA3.1+. After identification of DNA sequence, the recombinant plasmid pmACE2 was transfected into Cos7 cells with lipofectin reagent. The transient expression of ACE2 molecule was detected by SDS-PAGE. Sequence analysis was conducted with CLUSTALX program. Tissue distribution of ACE2 in mice was detected by RT-PCR.</p><p><b>RESULTS</b>A fragment about 2.6 kb was amplified and the recombinant plasmid pmACE2 was confirmed by two-enzyme digesting and DNA sequencing. The cloned DNA sequence was consistent with that previously reported, except for 3 variations A701G, T1102C and T1330C. SDS-PAGE proved that expression of a soluble, truncated products form of ACE2 was a glycoprotein of approximately 80 kD in Cos7 cells. The predicted mice ACE2 sequence contained an N-terminal signal sequence (amino acid residues 1-18), a single HHEMGHIQ zinc-binding domain (amino acid residues 373-380) and C-terminal membrane anchor (amino acid residues 738-765). Mice ACE2 showed 84 % identity with that of human, and 90 % identity with that of rat. Expression of ACE2 was the greatest in lungs, hearts and kidneys, and moderate levels were also detected in testes and livers.</p><p><b>CONCLUSION</b>Mice ACE2 gene has been cloned and successfully expressed in vitro. The tissue-specific expression of ACE2 in different species is not identical.</p>