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Position: Home > Articles > Expression of VP7 gene of bovine rotavirus in Sf9 cells Chinese Veterinary Science 2013 (2) 159-163

牛轮状病毒VP7基因在昆虫细胞中的表达

作  者:
范晴;谢芝勋;刘加波;庞耀珊;邓显文;谢志勤;谢丽基;罗思思
单  位:
广西兽医研究所广西动物疫苗和新技术重点实验室
关键词:
牛轮状病毒;VP7蛋白;杆状病毒表达体系;昆虫细胞
摘  要:
利用PCR方法扩增出牛轮状病毒外衣壳蛋白VP7基因,经BamHⅠ+PstⅠ特异性酶切后,连接于昆虫杆状病毒表达载体pFastBacHTA中,成功构建了重组质粒pFastBacHTA-VP7。该重组质粒转化含有杆状病毒穿梭载体的DH10BAC感受态细胞,经抗生素筛选和PCR筛选,获得转座的杆粒Bacmid-VP7。在脂质体介导下转染Sf9昆虫细胞,获得重组杆状病毒,再感染细胞,收获目的蛋白,对表达的蛋白进行SDS-PAGE分析和Western-blot分析。结果显示,成功扩增了VP7基因,大小为981bp,所表达的重组蛋白大小为40.4ku,与预期值相符,该蛋白能与牛轮状病毒阳性血清发生特异性反应,具有较好的反应原性。此研究结果为进一步研发牛轮状病毒ELISA检测试剂盒奠定了物质基础。
译  名:
Expression of VP7 gene of bovine rotavirus in Sf9 cells
作  者:
FAN Qing,XIE Zhi-xun,LIU Jia-bo,PANG Yao-shan,DENG Xian-wen,XIE Zhi-qin,XIE Li-ji,LUO Si-si(Guangxi Key Laboratory for Animal Vaccine and New Technologies,Guangxi Veterinary Institute,Nanning 530001,China)
关键词:
bovine rotavirus;VP7 protein;baculovirus expression system;insect cell
摘  要:
The outer capsid glycoprotein VP7 gene of bovine rotavirus was amplified by PCR.The PCR product was digested by BamHⅠ+PstⅠ,and then was cloned into the vector pFastBacHTA to construct the recombinant plasmid pFastBacHTA-VP7.Then the plasmid pFastHTA-VP7 was transformed into DH10Bac complement cells.The constructed baculovirus expression vector with VP7 gene was identified by PCR.On this basis,transposition bacmid DNA was extracted to transfect Sf9 insect cells.After re-transfection,the baculovirus was harvested.SDS-PAGE and Western-blot analyses showed that the expressed VP7 protein with 40.4 ku in size has good biological activity and could react with BRV positive serum.The result is a base for development of an ELISA kit for testing BRV antibody.

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