汉坦病毒H8205株G1P基因片段重组杆状病毒表达载体的构建及表达

将汉坦病毒H8205株G1P基因的保守序列(约1000bp)作为目的基因插入到BactoBac杆状病毒表达系统的pFastBacHTb供体质粒中,利用Tn7转座子同BacmidDNA同源重组,获得了含目的基因片段的重组杆状病毒DNA,并利用其转染Sf9昆虫细胞,72h后收集细胞悬液,再用该悬液侵染Sf9昆虫细胞,48h后收获病毒.采用IFA分析收获的产物,观察到了特异性的荧光,并且采用SDSPAGE和Western印迹也获得了与预期一致的结果.证明感染后的Sf9昆虫细胞所表达的蛋白中含有能与抗汉坦病毒H8205株多克隆抗体特异性结合目的蛋白.研究表明,采用杆状病毒表达系统可以成功表达出汉坦病毒H8205株包膜糖蛋白G1基因片段,为开发适合的以G1P为抗原的汉坦病毒诊断试剂进行了前期的探索.

The Construction of Recombination Baculovirus Expression Vector of Hantavirus H8205's G1P Gene Fragment and Its Expression

The conservative region (about 1 000 bp) of Hantavirus H8205 strain G1P gene was inserted into pFastBAc HTb donor plasmid of Bac-to-Bac baculovirus expression system as interest gene. Through the homologous recombination of donor plasmid with bacmid DNA at the site of Tn7. The recombinant baculovirus DNA containing the interest gene fragment was obtained, and then transfected into Sf9 insect cells. After 72 hours, the cells suspension was collected and used to infect Sf9 insect cells again, and the virus was harvested 48 hours postinfection. The final product was analyzed by IFA (Indirect immunofluorescence assay), and specific fluorescence was observed.The results of SDS-PAGE and western blotting were expected. It certified that the target proteins, which can react with Hantavirus H8205 strain polyclonal antibodies speci,ally are parts of the proteins expressed by infected Sf9 insect cells. The identity of the recombinant expression protein was further comfirmed by Western blotting showing a 36 kD specific band.The data indicated that Hantavirus H8205 strain G1P gene fragment could be expressed successfully by using baculovirus expression system, and the research took prophase exploration for developing apropos Hantavirus diagnostic reagents using G1P as their antigens.