摘要
目的研发猴B病毒ELISA检测试剂盒中替代全病毒的抗原。方法根据已报道的猴B病毒E2490株gB蛋白基因序列(GenBank登录号:AF533768)人工合成gB基因,通过XbaⅠ和EcoRⅠ特异性酶切,将gB基因克隆于昆虫杆状病毒表达载体pFastBacHTA,经PCR、酶切、测序鉴定后,成功构建了携带gB基因的重组质粒pFastBac-HTa-gB。结果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、PCR筛选,获得转座的杆粒bacmid-gB。在脂质体介导下转染sf9昆虫细胞,细胞变大变圆细胞核扩大,收获重组杆状病毒,再感染细胞,收获目的蛋白。通过SDS-PAGE和Western blot分析及间接免疫荧光检测,结果表明该蛋白得到表达,且具有良好的生物活性,大小约为98kDa。实验结果表明已成功构建了携带目的基因的重组质粒pFastBac-HTa-gB和转座的杆粒bacmid-gB,转染后在sf9昆虫细胞上得到表达。结论研究结果为下一步以表达的gB蛋白为诊断抗原,替代现阶段以病毒作为抗原的猴B病毒ELISA检测试剂盒的研制奠定了基础。
In order to obtain antigenic properties that used to construct a tLISA kit to test BV antibody and take the place of the BV, gB gene of simian B virusE2490 was synthesized according to the reported BV virus gB gene （GenBank acces- sion number： AF533768）. The gB gene fragment was than cloned in the vector pFastBac HTa. The recombinant plasmid pFastBac-HTa-gB was constructed and identified by PCR and enzyme digestion and sequenced. Then the plasmid pFast-HTa- gB was transformed into DH10Bac complement cells. It was identified by antibiotics and PCR, indicating that we have con- structed baculovirus expression vector to gB gene. On this basis, transposition bacmid DNA was extracted to transfect sf9 in sect cells. After transfected for 96 h, the sf9 insect cells showed expansion, round and cell nucleus turned to great, and baculo- virus was harvested. Then the SDS-PAGE and Western blot and indirect immunofluorescence assay results showed the expres sion of gig protein in insect ceils. The gB protein has a good biological activity and was approximate 98kDa. It was a base to construct ELISA kit to test BV antibody. The result showed that we have constructed the recombinant plasmid pFastBac-HTa gB and baculovirus expression vector to gB gene, and the gB gene was expressed in insect cells.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2012年第7期674-678,共5页
Chinese Journal of Zoonoses
基金
国家质检总局科技计划项目(2009IK009)资助
云南省高端科技人才引进项目(2009C1125)资助~~
