Improvement of the cytotoxity of Autographa californica Nuclear Polyhedrosis Virus--Cloning the Bacillus thuringiensis subsp. ai -zawai 7.29 delta-endotoxin gene

• Main Authors: Hu,Yaochung, 胡耀中
• Other Authors: Shih,Cheng-Jen;Lo,Chu-Fang
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/72758228171864185030

碩士 === 國立臺灣大學 === 植物病蟲害學系 === 81 === The delta-endotoxin gene from Bacillus thuringiensis susbp. aizaw ai 7.29 was insered into Autographa californica Nuclear Polyhedr osis Virus (AcNPV). Three transfer vector, T7, pVL1393 and pAcUW1 , were used to clone the endotoxin gene After a series of constru ction, three recombinant transfer vectors, pIBL-T7, pIBL-UW1 and pIBL-1393 were obtained and then cotransfected with modified AcN PV DNA(Baculogold kit) into IPLB-SF21-AE cell. The recombinant vi ruses, Acendo-T7,Acendo- UW1 and Acendo-1393,were purified by end point dilution and identified by dot-blot hybridization, PCR meth od and southern blotting. The mRNA isolated from putative recomb inant virus infected cells was for detecting the transcription le vel of endotoxin by dot-blot hybridization method. The result con firmed that the mRNA of endotoxin had been expressed by these r ecombinant viruses. The production of endotoxin protein was dete cted by Western blotting method and by indirect immunofluorescenc e assay.Trypan blue and MTT method were used toassay the viabilit y of infected cells. Cytotoxity of recombinant virus had shown mu ch higher than that of wild type virus.