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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。
Objective To develop a monkey B virus ELISA test kit instead of the whole virus antigen. Methods The gB gene was synthesized from the reported sequence of gB protein of monkey B virus E2490 strain (GenBank accession number: AF533768). The gB gene was cloned into the insect baculovirus expression vector pFastBacHTA by Xba Ⅰ and EcoRI specific digestion. After PCR After digestion and sequencing, the recombinant plasmid pFastBac-HTa-gB carrying gB gene was successfully constructed. Results The recombinant plasmid was transformed into DH10BAC competent cells containing baculovirus shuttle vector. After antibiotic and PCR screening, bacmid-gB was obtained. Transfection of sf9 insect cells under liposome-mediated transfection led to enlargement of cells, enlargement of enlarged nuclei, harvest of recombinant baculovirus, re-infection of cells, and harvest of target proteins. By SDS-PAGE and Western blot analysis and indirect immunofluorescence assay, the results showed that the protein was expressed, and has good biological activity, the size of about 98kDa. The experimental results showed that the recombinant plasmid pFastBac-HTa-gB carrying the gene of interest and the bacmid-gB transposing bacmid were successfully constructed and expressed on sf9 insect cells after transfection. Conclusion The results of this study lay the foundation for the development of the ELISA kit for monkey B virus by using the expressed gB protein as the diagnostic antigen in the next step instead of the current stage.