Construction and Transduction of SARS-3CL Protease Gene in Baculovirus Vector
10.3969/j.issn.0253-9896.2009.07.008
- VernacularTitle:SARS-3CL蛋白酶基因在杆状病毒载体中的构建及其转染
- Author:
Qinghua HOU
;
Yingqi HOU
;
Nianci HANG
- Publication Type:Journal Article
- Keywords:
SARS virus;
viral proteins;
genes;
baculoviridae;
plasmids;
transfection
- From:
Tianjin Medical Journal
2009;37(7):550-552,后插3
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To construct severe acute respiratory syndrome (SARS)coronavirus 3CL protease gene into transforming vector prepare recombinant baculovirus and transduct it into infect insect cells to express SARS-3CL protease. Mothods: The 3cl-Teasy and pFastBac HTb bacmida were amplified. The 3cl gene was cloned into baculovirus transforming vector pFastBac HTb by enzyme-digest- and-ligase method,which was named recombinant pFB HTb-3cl. The pFB HTB-3cl was transformed into E.coli DH10Bac competent cells.The positive colonies were screened by three antibiotics and blue-white patch method. The bacmid-HTb-3cl recombinant baculovirus bacmid was obtained and purified to transfect St9 insect cells.The protease expressed in Sf9 insect cells were identified by SDS-PAGE. Results: Recombinant expression vector was obtained successfully. The 3CL protease expressed in insect cells were identified by SDS-PAGE. Conclusion: The expression of 3CL protease in insect cells provided foundation for detecting protein activities and screening inhibitor against SARS-3CL protease.
