摘要
根据马立克氏病病毒 (MDV)强毒株GA的基因序列 ,设计和合成一对引物 ,以特超强毒 6 48株基因组DNA为模板 ,通过PCR技术 ,扩增其囊膜糖蛋白gI基因阅读框 (ORF)中 ,除去其N_端编码疏水区的 16 5个碱基对 (bp)以外的其余部分 ;将PCR产物按正确的阅读框架定向克隆到表性载体pGEX_6P_1中谷胱甘肽转移酶 (GST)基因的下游 ;将重组质粒转化进大肠杆菌BL2 1株 ,在 1.0mMIPTG浓度和 30℃的条件下诱导 ,gI_GST基因融合蛋白获得了理想的表达 ;经聚丙烯酰胺凝脉电泳 ,West ern_blot试验 ,验证其表达的融合蛋白产物大小为预期的 6 3kD。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 6
Glycoprotein I(gI) gene was amplified from genomic DNA of MDV 648 with the dismission of 165bps at the N-terminal of ORF by polymerase chain reaction (PCR) technique. 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 Westernblotting, and found to be 63kD in size as a fusion protein with glutathione stransferase(GST) 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 stains were found in the MDV plaques and localized on the cytomembrane of the infected cells. The results of the study showed that the vitro expressed protein of gI gene via recombinant plasmid vector maintains some antigenicity of MDV.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2001年第1期7-10,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家"86 3计划资助项目! (课题号 :10 1_5 2_0 3_0 2 )
