鸡传染性支气管炎病毒S1基因在昆虫细胞中的表达

Preliminary Study on Expression of S1 Gene of Avian Infectious Bronchitis Virus in Insect Cells

将含有传染性支气管炎病毒（ＩＢＶ）广东地方分离株Ｄ４１的Ｓ１基因ｃＤＮＡ（从ＡＴＧ到Ｓ前体蛋白裂解位点）的片段克隆到具有多角体启动子（ＰＸＩＶ）和人工合成启动子（Ｐｓｙｎ）的杆状病毒转移载体质粒ｐＳＸ－ＩＶＶＩ＋Ｘ３中，构建了重组转基因载体质粒ｐＳＸＩＶＶＩ＋Ｘ３－Ｓ１．Ｄ４１。用该重组转移质粒与无包涵体的粉纹夜蛾核型多角体病毒ＴｎＮＰＶ－ＳＶＩ－ＧＤＮＡ（ｏｃｃ－，ｇａｌ＋）共转染草地夜蛾（Ｓｆ９）细胞，筛选并纯化出既能表达Ｓ１基因又能形成多角体的重组病毒ＴｎＮＰＶ－ＩＢＶＳ１－ｏｃｃ＋（ｏｃｃ＋，ｇａｌ－）。通过间接ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ等方法在感染了重组病毒的Ｓｆ９细胞中成功地检测到分子量约６２０００的Ｓ１基因表达产物。虽然该重组Ｓ１糖蛋白可能由于Ｎ－糖基化不完全而分子量小于天然蛋白，但它可以像正常病毒粒子一样，被鸡抗ＩＢＶＤ４１株血清特异识别。

A recombinant transfer vector plasmid pSXIVVI+X3S1.D41 was constructed by inserting the cDNA of gene (from ATG to the possible cleavage of spike glycoprotein) of IBV strain D41, an attenuated vaccine strain of an isolate from Guangdong province, into the transfer vector plasmid pSXIVVI+X3 containing synthetic and polyhedric promoters. After cotransfecting the cultured Sf9 cells by pSXIVVI+X3S1.D41 and the parent virus (TnNPVSVI-G) DNA, the recombinant virus-TnNPVIBVS1occ+ (occ+, gal-), which can express S1 gene and form polyhedra, was raised and purified by serial plaque assays. In the Sf9 cells infected with the recombinant virus, the expression product of the S1 glycoprotein with a molecular weight of 62 000 was successfully detected by indirectELISA and Western blot using chicken antiIBV D41 polyclonal sera. Maybe owing to incomplete Nglycosylation in the insect cells the recombinant S1 glycoprotein seems smaller than natural one.