伪狂犬病病毒鄂A株包膜糖蛋白gD基因的克隆与表达

CLONING AND EXPRESSION OF THE ENVELOPE GLYCOPROTEIN gD GENE OF PSEUDORABIES VIRUS EA STRAIN

克隆了伪狂犬病病毒鄂A株编码包膜糖蛋白gD的基因并进行了序列测定 ,与国外报道的Rice株相比 ,其核苷酸序列具有 98%的同源性 ,推导氨基酸序列同源性为 97%。将此基因克隆于具有全期启动子盒的杆状病毒转移载体pSX35A中 ,构建成重组转移质粒pSX35A gD ,与致死缺失型线性化苜蓿丫纹夜蛾核型多角体病毒 (AcMNPV OCC- )基因组DNA一起共转染粉纹夜蛾Hi5细胞 ,经同源重组 ,获得含gD基因的重组病毒AcMNPV OCC+ gD。重组病毒经空斑纯化后感染Hi5细胞进行表达分析 ,细胞裂解物的SDS PAGE及Western Blot ting均显示分子量约 47kD的gD蛋白得到了特异性表达 ,其表达量占细胞总蛋白的 6 2 % ,表达的gD蛋白具有免疫原性。

The envelope glycoprotein gD gene of pseudorabies virus Ea strain was cloned via PCR technique.Sequence analysis displayed 98% nucleotide sequence homology and 97% deduced amino acid sequence homology between our cloned gD gene and PRV Rice strain gD gene.The recombinant transfer plasmid pSX35A\|gD was obtained by inserting D gene into the baculovirus transfer vector pSX35A with whole\|phase promoter cassette,then transfected insect cell Hi5 with linearized AcMNPV\|OCC - virus DNA,and formed recombinant baculoviruses AcMNPV\|OCC + gD by homologous recombination in insect cell.Recombinant baculoviruses infected insect cell Hi5 after being purified by plaque assay.Both SDS\|PAGE and Western\|blotting showed glycoprotein gD with a molecular weight of about 47kD was expressed specificially,product was about 6 2% of total cellular protein,and expressed gD was of immunogenicity.