摘要
为了制备针对鹿源BVDV E2蛋白的单克隆抗体,本试验通过RT-PCR技术扩增出鹿源BVDV E2的全长基因,并将其插入杆状病毒bac to bac表达系统的供体质粒pFast BacHTA中,构建含有BVDV E2基因的重组供体质粒pFast-E2,转化至含穿梭载体Bacmid的受体菌DH10Bac E.coli感受态中,经半乳糖苷酶的蓝白斑筛选,获得重组杆粒rBacmid-E2,然后将其转染sf9细胞后得到重组杆状病毒vBac-E2,E2基因会随着杆状病毒的增殖而获得表达。表达产物经直接免疫荧光检测,可见特异性荧光;Western-Blot定性分析,在大约38 000处出现1条特异性的蛋白印迹与预期大小相符,表明BVDV E2基因在该系统中得到了表达。本研究为研制针对鹿源BVDV E2蛋白的单克隆抗体及胶体金试纸条奠定了基础。
To prepare for the sika BVDV E2 protein monoclonal antibodies,full-length gene sika BVDV E2 was amplified by RT-PCR,and was inserted into the donor plasmid pFastBacHTA baculovirus expression system,to construct recombinant plasmid pFast-E2 containing BVDV E2 gene,then transformed to the DH10 Bac E.coli competent cells,the recombinant bacmid rBacmid-E2 cells were obtained by the method of blue-white screening,transfected recombinant rod sf9 like virus to get vBac-E2,and the E2 gene was amplified with the virus proliferation.The direct immunofluorescence showed specific fluorescence;Western blot analysis showed a specificity of about38 000 protein bands.Experimental results showed that BVDV E2 gene was expressed in insect cells.This experiment developed sika BVDV E2 protein monoclonal antibodies and laid a foundation for the colloidal gold test strip.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2014年第12期1899-1905,共7页
Chinese Journal of Veterinary Science
基金
吉林省科技支撑计划重点攻关资助项目(20130206021NY)
吉林大学2013年度创新训练国家级资助项目(2013A81275)
