Screening Of Proteins Interacted With The SARS-CoV HEL By The Yeast Two-Hybrid System

The SARS-associated coronavirus (SARS-CoV) is positive-strand RNA genome viruse. The helicase (HEL) encoded were considered to be crucial for the proliferation and replication of the virus, yet the cellular cofactors sustaining the activity of HEL and the viral pathogenicity were not well elucidated. The aim of this study was to identify the cellular proteins interacted with HEL by yeast two-hybrid system through screening the eDNA library of the rat's pulmonary epithelial cells.The first part of this study was to examine the self-activation of SARS-CoV HEL. So pGBKT7-HEL (the recombinant bait vector pGBKT7 harboring HEL gene) was transformed into yeast AH109. The fusion protein was detected by Westem Blot to confirm the expression of HEL protein in AH109, and the intraeellularβ-galactosidase activity was qualitively evaluated by filter paper method to exclude the self-activation of HEL protein.In the second part of this study, pGBKT7-HEL and the prey plasmids were tranfomed sequentially to AH109, and the 133 preliminarily positive clonies were checked furtherly by phenotypes retesting, clonies sorting,β-galactosidase activity detecting, yeast mating and target DNA sequencing. Then the bioinformation of the screened proteins was analyzed by Blastp. Eventually, totally seven proteins were identified as Actn2(Actinin alpha 2), Boas2 (Breast carcinoma amplified sequence 2),Thap 11 (Thr-His-Ala-Pro domain containing 11),Ada (Adenosine deaminase),Ddx5 (Asp-Glu-Ala-Asp box polypeptide 5),Dars2(Aspartyl-tRNA synthetase 2) and Smarcb 1 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily b, member 1), respectively.In conclusion, seven kinds of the rat's pulmonary epithelial cell proteins were preliminarily confirmed that can interacted with SARS-CoV HEL. The ultimative identification of these proteins and the exact mechanism of their interactions with SARS-CoV HEL need further elucidation.