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KMID : 0578319930030030309
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Molecules and Cells
1993 Volume.3 No. 3 p.309 ~ p.313
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Transient Expression of the E. coli β-galactosidase Linked next to the Polyhedrin Promoter of the Autographa california Nuclear Polyhedrosis Virus in Insect Cells
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Um, Wan-suk
Yang, Ji-Won/Kang, Suk-kwon/Chung, In-Shik/Yoon, Tae-Young/Yang, Jai-Myung
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Abstract
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Optimum conditions for transient expression of the p-galactosidase inserted at N-terminal co-ding region of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin were examined. The plasmid pHBG-1 carrying (3-galactosidase was transfected into the Sf21 cells and the polyhedrin promoter was trans-activated either by infecting wild type AcNPV or by cotransfecting with wild type viral DNA. Analysis of the level of the Q-galactosidase activity indicated that the polyhedrin promoter of pHBG-1 was not efficiently trans-activated by the cotransfected viral DNA nor by the virus infected before the transfection. The most efficient way of trans-activating polyhedrin promoter of pHBG-1 was by infecting AcNPV into Sf21 cells previously transfected with the plasmid. To find out optimum conditions for obtaining high level of transient expression, transfection was performed at number of different conditions. The crucial factors for obtaining efficient expression were as follows; 1) 2 X 106 cells per dish 2) 10 pg of plasmid DNA per dish 3) 4 h of absorption period in DNA-calcium phosphate suspension 4) virus infection at 24 h of post-transfection 5) virus titer of 6 X 105
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