Wen Kun, Qiu Li-Wen, Wang Ya-di, Che Xiao-Yan
Central Laboratory of Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2007 Jan;27(1):20-3.
To clone and express avian influenza A virus [A/Hong Kong/482/97(H5N1)] H5 subtype hemagglutinin in baculovirus-insect cell expression system and investigate the antigenicity and bioactivity of the recombinant protein.
H5 gene of influenza A virus was amplified by PCR. The recombinant bacmid was obtained by cloning the gene to the donor plasmid of pFastBacHTB and transformed into DH10Bac competent cells. The recombinant baculovirus stock was prepared by transfecting the recombinant bacmid DNA into the insect cell line for protein expression after amplification. Immunofluorescene assay (IFA) and Western blotting were performed to identify the antigenicity of the recombinant protein, and hemagglutination assay was used to identify its bioactivity.
The recombinant his-H5 protein was expressed in the insect cells with a relative molecular mass of 64,000, which showed erythrocyte-agglutinating activities with the red blood cells of guinea pig. Western blotting and IFA demonstrated that the recombinant his-H5 could be recognized and bound by standard anti-H5 sera.
The recombinant his-H5 with a post-translation modification is successfully obtained in insect cells, which may provide a potential source for further study of the antigen's biological function and for production of the subunit vaccine or monoclonal antibodies.
在杆状病毒-昆虫细胞表达系统中克隆并表达甲型禽流感病毒[A/香港/482/97(H5N1)]H5亚型血凝素，研究重组蛋白的抗原性和生物活性。
通过PCR扩增甲型流感病毒的H5基因。将该基因克隆到pFastBacHTB供体质粒中，获得重组杆粒，然后转化到DH10Bac感受态细胞中。扩增后将重组杆粒DNA转染到昆虫细胞系中制备重组杆状病毒储备液用于蛋白表达。进行免疫荧光分析(IFA)和蛋白质印迹法鉴定重组蛋白的抗原性，并用血凝试验鉴定其生物活性。
重组his-H5蛋白在昆虫细胞中表达，相对分子质量为64000，对豚鼠红细胞显示红细胞凝集活性。蛋白质印迹法和IFA表明重组his-H5能被标准抗H5血清识别并结合。
在昆虫细胞中成功获得了具有翻译后修饰的重组his-H5，这可能为进一步研究该抗原的生物学功能以及生产亚单位疫苗或单克隆抗体提供潜在来源。
