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 g
Glycoprotein 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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
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 )
