[Cloning of ACE-2 gene encoding the functional receptor for the SARS coronavirus and its expression in eukaryotic cells].
OBJECTIVE
Angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor of severe acute respiratory syndrome coronavirus (SARS-CoV), so its gene was cloned and eukaryotic expressed for further insight into mechanisms in SARS-CoV entry and pathogenesis, as well as development of a safe and reliable neutralization assay for SARS-CoV.


METHODS
Total RNA was extracted from right atrial tissue of a patient with right heart failure resected during a valvular replacement surgery by Trizol one-step method, and the full-length ACE-2 encoding gene was acquired by RT-nested-PCR. The ACE-2 encoding gene was then cloned into pcDNA4/HisMax-TOPO eukaryotic expression vector to construct the recombinant plasmid pcDNA4/ ACE-2, which was then transfected into 293 T cell and ACE-2 eukaryotic transient expression was detected by Western Blot. Syncytia inhibition assay was established to detect SARS-CoV neutralizing antibody, and compared parallelly with SARS pseudovirus neutralization assay.


RESULTS
The recombinant plasmid pcDNA4/ ACE-2 could express ACE-2 protein in eukaryotic cells and induce cell-cell fusion between S protein- and ACE2-expressing cells. This cell-cell fusion assay could be used to detect SARS-CoV neutralizing antibody.


CONCLUSIONS
SARS-CoV receptor ACE-2 gene was successfully cloned and eukaryotic expressed, and used to establish syncytia inhibition assay for SARS-CoV neutralizing antibody assay.
Lu
Jian
J
Shenzhen Donghu Hospital, Shenzhen 518020, China. szlujian@163.net
Zhou
Bai-Ping
BP
Wen
Li-Xia
LX
Jiang
Xiao-Ling
XL
D000085962:Angiotensin-Converting Enzyme 2; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D009500:Neutralization Tests; D007703:Peptidyl-Dipeptidase A / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011991:Receptors, Virus / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology / Q000378:metabolism*; D014162:Transfection; D014709:Vero Cells
D011991:Receptors, Virus; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; D000085962:Angiotensin-Converting Enzyme 2