摘要
目的应用杆状病毒-昆虫细胞表达体系克隆及表达禽流感病毒H5N1血凝素H5抗原,鉴定其抗原性和生物活性。方法PCR扩增禽流感病毒H5全长基因序列,克隆、转化制备重组杆状病毒Bacmid质粒,通过转染昆虫细胞制备重组杆状病毒进行蛋白表达。采用细胞免疫荧光、Westernblotting和血凝试验分别对表达的蛋白进行抗原性和生物活性分析。结果在昆虫细胞中成功表达禽流感病毒重组his-H5蛋白,其相对分子质量约为64000,与豚鼠红细胞能够发生凝集反应,免疫荧光与Westernblotting结果提示该重组蛋白能够与禽流感病毒H5标准血清中的抗体特异性结合。结论经杆状病毒-昆虫细胞表达系统获得的重组his-H5抗原具备天然的生物活性和抗原性,该方法为进一步研究禽流感病毒血凝素抗原生物学特性以及制备亚单位疫苗、相关抗体奠定了基础。
Objective To clone and express avian influenza A virus [A/Hong Kong/482/97（HSN 1）] H5 subtype hemagglutinin in baculovirus-insect cell expression system and investigate the antigcnicity and bioactivity of the recombinant protein. Methods H5 gene of influenza A vires was amplified by PCR. The recombinant bacmid was obtained by cloning the gene to the donor plasmid ofpFastBacHTB and transformed into DHIOBac 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. Results 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. Conclusion 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.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2007年第1期20-23,共4页
Journal of Southern Medical University
基金
广东省科技计划项目(2005B31201007)
