马立克氏病病毒广西株G2囊膜糖蛋白gI基因的克隆和表达  

Cloning and Expression of Glycoprotein I Gene of Marek′s Disease Virus Strain G2

作　　者：丁家波[1] 崔治中[2] 韦平[3] 卢银华[1] 赵文明[1] 韩凌霞[1] 吉荣[1] 

机构地区：[1]扬州大学牧医学院,江苏扬州225009 [2]山东农业大学动物科技学院,山东泰安271018 [3]广西大学动物科技学院,广西南宁530005

出　　处：《中国兽医学报》2001年第2期109-112,共4页Chinese Journal of Veterinary Science

基　　金：国家"8 6 3"计划资助项目 !(10 1-5 2 -0 3-0 2 )

摘　　要：根据马立克氏病病毒 ( MDV)强毒株 GA的基因序列 ,设计并合成了 1对引物 ,以强毒株 G2基因组DNA为模板 ,通过 PCR技术 ,扩增其囊膜糖蛋白 g I基因阅读框 ( ORF)中 ,除去其 N-端编码疏水区 1 6 5个碱基对 ( bps)以外的部分 ;将 PCR产物按正确的阅读框架定向克隆到表性载体 p GEX-6 P-1中谷胱甘肽转移酶( GST)基因的下游 ;将重组质粒转化入大肠杆菌 BL2 1 株 ,在 1 .0 mmol/L IPTG浓度和 30℃的条件下诱导 ,g I-GST基因融合蛋白获了理想的表达 ;经聚丙烯酰胺凝胶电泳和 Western-blotting试验 ,验证其表达的融合蛋白产物大小为预期的 6 30 0 0。将表达产物回收后免疫小鼠 ,所得抗血清可与 MDV感染的鸡胚成纤维细胞 ( CEF)在免疫荧光试验 ( FA)中 ,呈细胞膜阳性染色。试验结果表明 ,在大肠杆菌中表达的 G2株 MDV gGlycoprotein I(gI) gene was amplified from genomic DNA of MDV G2 with the dismission of 165 bp at the N-terminal of ORF by polymerase chain reaction (PCR) technigue. PCR product was cloned into the expressing plasmid vector pGEX-6p-1 via restriction endonucleases BamHⅠ and SmaⅠ. The recombinant was verified by restriction endonuclease analysis and nucleotide sequencing. Then it was transformed into its host E.coli strain BL 21 for gI expression. The expression of gI gene was indentified by SDS-PAGE and Western-blotting, and found to be 63 000 in size as a fussion protein with glutathione transferase protein. The specific bands of expression was excised from the gel and injected into the mice once a week and for 5 weeks. The antisera was collected from the immunized mice and used for fluorescence assay(FA) with CEF monolayers infected by RB1B and GA of MDV. The positive stainings were found in the MDV plaques and localized on the cytomembrane of the infected cells. The results showed that the in vitro expressed protein of gI gene via recombinant plasmid vector maintains some antigenicity of MDV.

关 键 词：马立克氏病病毒 GI基因 抗原性 广西株 囊膜糖蛋白 基因重组 基因表达 疫苗 

分 类 号：S852.652[农业科学—基础兽医学] Q78[农业科学—兽医学]