甲型H5N1禽流感病毒血凝素在sf9昆虫细胞中的表达及其鉴定

Expression of H5N1 avian influenza A virus hemagglutinin in sf9 insect cells and its identification

目的利用杆状病毒-昆虫细胞表达系统表达甲型H5N1禽流感病毒血凝素(hemagglutinin,HA)并对其进行鉴定。方法根据sf9昆虫细胞密码子偏好性,对A/Goose/Guangdong/1/96(H5N1)禽流感病毒的HA基因进行序列优化并全长合成。构建重组供体质粒pFastHA,经PCR及测序鉴定后,转化DH10Bac感受态细胞,获得重组穿梭质粒BacmidHA,用M13引物进行PCR鉴定。采用脂质体法转染sf9细胞,获得重组杆状病毒rBacHA。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)、蛋白质印迹法以及间接免疫荧光法对rBacHA表达产物进行分析。结果供体质粒pFastHA经双酶切后产生了1条与预期大小相符的1 707 bp条带,序列测定证实目的基因无突变。穿梭质粒BacmidHA经PCR扩增,得到1条与预期大小相符的3 180 bp条带。SDS-PAGE结果显示,rBacHA表达产物的相对分子质量约为65 000。蛋白质印迹法分析表明,表达产物能与禽流感病毒阳性血清结合。在荧光显微镜下,感染rBacHA的sf9细胞呈现绿色荧光,提示HA基因得到表达。结论利用杆状病毒-昆虫细胞表达系统成功制备了具有良好抗原性的重组HA蛋白。

Objective To express hemagglutinin (HA) of H5N1 avian influenza A virus using a baculovirus-insect cell expression system and identify the expression product.Methods The HA gene of A/Goose/Guangdong/1/96 (H5N1) avian influenza A virus was optimized according to the sf9 insect cell codon preference and full-length synthesized. The recombinant donor plasmid pFastHA was constructed, and then transformed into DH10Bac competent cells after PCR and sequence identification. The recombinant shuttle plasmid BacmidHA was constructed, identified by PCR with M13 primer, and transfected into sf9 cells with liposome method to obtain recombinant baculovirus rBacHA. The expression product of rBacHA was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and indirect immunofluorescence assay.Results A band with the expected size of 1 707 bp was observed after the donor plasmid pFastHA was double-enzyme digested. No mutation in the target gene was found by gene sequencing. A band with the excepted size of 3 180 bp was obtained from shuttle plasmid BacmidHA by PCR amplification. SDS-PAGE showed that the relative molecular weight of the rBacHA expression product was about 65 000. Western blotting analysis revealed that the expression product was able to react with avian influenza A virus positive serum. The rBacHA-infected sf9 cells emitted green fluorescence under a fluorescence microscope, indicating the correct expression of HA gene.Conclusion The recombinant HA protein with good antigenicity is expressed successfully by the baculovirus-insect cell expression system.