全长和截短的δ内毒素cryIAc基因在昆虫杆状病毒表达系统中的表达

全长和截短的δ内毒素cryIAc基因在昆虫杆状病毒表达系统中的表达 EXPRESSION OF FULL-LENGTH AND TRUNCATED FORMS OF δ-ENDOTOXIN CRYIAC GENE IN INSECT BACULOVIRUS EXPRESSION SYSTEM 杨远征 1 2 3 first-author 杜全胜 1 2 3 朱反修 1 2 3 王福山 1 2 3 齐义鹏 1 2 3 黄永秀 1 2 3 武汉大学生命科学学院 武汉大学生命科学学院 School of life Science, Wuhan University School of life Science, Wuhan University 中国科学院水生生物学研究所 中国科学院水生生物学研究所 武汉大学生命科学学院中国科学院水生生物学研究所 武汉大学生命科学学院中国科学院水生生物学研究所 在昆虫病毒转移载体ＰＶＬ１３９３多角体基因启动子下游克隆了全长的苏芸金杆菌δ内毒素基因ｃｒｙＩＡｃ，得到了重组转移载体ｐＴｈＹ１，用ＫｐｎＩ将ｃｒｙＩＡｃ基因进行了截短，得到了重组转移载体ｐＶＬＹ２．随后在两种重组转移载体ｃｒｙ基因的下游引入了ＩＥ１启动于驱动的ｎｅｏ基因作为筛选标记和ＳＶ４０小ｔ抗原终止区作为ｃｒｙ基因的终止信号，得到了重组转移载体ｐＶＬＹｌｎｅｏ和ＰＶＬＹ２ｎｅｏ，分别用两种重组转移载体ＤＮＡ与野生型ＡｃＮＰＶＤＮＡ共转染昆虫Ｓｆ９细胞，用Ｇ４１８筛选后经有限稀释法纯化，得到了携带全长和截短。ｒｙＩＡｃ基因的两种重组病毒ｖＶＬＹ１ｎｅｏ和ｖＶＬＹ２ｎｅｏ，分别在昆虫细胞中表达了分子量为１３３３００和８２０００的ｃｒｙ蛋白，大小分别与ｃｒｙＩＡｃ晶体蛋白和预计的截短蛋白相同，并均具有与原晶体蛋白相同的免疫活性，生物测定表明两种表达产物均具有杀虫活性，和昆虫病毒一起产生协同增效毒性． Full-length and truncated forms of δ-endotoxin cryIAc gene, whichwere ligated with termination region of SV40 small t antigen gene as its terminalsignal and neo gene driven by IE1 promoter for selection marker, respectively,were cloned under the polyhedrin gene promoter located within baculovirus transfervector pVL1393 to derive two transfer vector PVLY1neo and pVLY2neo. TwoKinds of recombinant viruses vVLY1neo and vVLY2neo were constructed by cotransfection of transfer vector DNA with AcNPV DNA in insect sf9 cells, throughselection by G418 and purification by limited dilution. The recombinant virusesvVLYineo and vVLY2neo expressed 133 300 and 82 000 crystal protein in insectcells, the same molecular weight as normal CryIAc protein and putative protein,and the same immunogeniticity as the normal crystal protein. Bioassay indicatedthat two kinds of expression products have insecticidal activities’ synergism with’baculovirus. 基因表达 内毒素基因 重组杆状病毒 gene expression endotoxin gene recombinant baculovirus Q786 国家“八五”攻关项目资助 1995-04-01 2021-04-01 4