禽分枝杆菌副结核亚种hsp65与鹅副黏病毒HN融合基因真核表达载体的构建与表达

Construction of an eukaryotic expression vector containing hsp65 of Mycobacterium avium subsp.Paratuberculosis and HN of Goose paramyxovirus and expression in Marc-145 cells

关键词:  鹅副黏病毒  HN  禽分枝杆菌副结核亚种  hsp65  真核表达载体

Key words:  Goose paramyxovirus  HN  Mycobacterium avium subsp. Paratuberculosis  hsp65  eukaryotic expression vector

摘要:

为探索禽分枝杆菌副结核亚种（Mycobacterium avium subsp. Paratuberculosis,MAP）的热休克蛋白65（Heat Shock Protein 65,Hsp65）对鹅副黏病毒（goose paramyxovirus,GPMV）血凝素神经氨酸酶（hemagglutinin neuraminidase,HN）的分子佐剂作用及构建GPMV DNA疫苗，针对GPMV 的HN基因和MAP的 hsp65基因设计引物，经PCR克隆扩增两个基因；分别将二者先后与真核表达载体pVAX1连接，构建了pVAX1-HN、pVAX1-hsp65-HN，并通过PCR、酶切和序列鉴定所获重组质粒；应用脂质体法将其转染至Marc-145细胞，经RT-PCR和间接免疫荧光试验证实hsp65和HN在Marc-145细胞中获得了表达。本研究为制备新型GPMV DNA疫苗及探索Hsp65在DNA疫苗中的应用奠定了基础。

Abstract:

To explore the molecular adjuvant role of heat shock protein 65 (Hsp65) in Mycobacterium avium subsp.Paratuberculosis(MAP) on hemagglutinin neuraminidase (HN) of goose paramyxovirus (GPMV) and construct GPMV DNA vaccine, the primers for HN and MAP hsp65 gene were designed . and the two genes were amplified by polymerase chain reaction then linked to the eukaryotic expression vector pVAX1. Correct pVAX1-hsp65-HN and pVAX1-HN vectors was identified by PCR, enzyme digestion, sequence identification and then transfected to Marc-145 cells with liposome mediated transfection method. Successful expression in Marc-145 cells was confirmed by RT-PCR and indirect immunofluorescence test. The research laid the foundation for new GPMV DNA vaccines and application of Hsp65 on DNA vaccines.