HPV16L1VLP纯化、鉴定及血凝抑制实验方法的建立

HPV16L1 VLP Purification and Identification and the Establishment of Hemagglutination Inhibition Assay

摘要通过昆虫细胞-杆状病毒表达系统表达HPV16L1 VLP,纯化鉴定后利用其建立血凝抑制实验,检测HPV16L1单克隆抗体的血凝抑制活性。利用重组杆状病毒感染悬浮培养的昆虫细胞表达HPV16L1蛋白,大量获得VLP;优化扩增蛋白的条件并用SDS-PAGE分析鉴定;经氯化铯密度梯度离心法纯化VLP;利用所得VLP建立血凝抑制试验,鉴定制备的HPV16L1单克隆抗体的血凝抑制活性。优化蛋白表达条件结果显示,当重组杆状病毒感染细胞的MOI=10时目的蛋白表达量最大;按此滴度感染悬浮培养的昆虫细胞,收获、纯化VLP并鉴定其特异性;利用所得VLP建立了血凝抑制实验,鉴定HPV16L1单克隆抗体的血凝抑制效价为1∶16。建立的血凝实验,可用于鉴定和检测HPV16L1相关抗体,为HPV诊断试剂的开发提供实验基础。

Through expression system of insect cells to express human papillomavirus （ HPV ） L1 virus-llke particle （VLP） hemagglutination inhibition assay was established and using it to test its hemagglutination activity of HPV16 L1 monoelonal antibody （MeAb） after purification and identification. Using insect cells suspensively cultured and infected with recombinant baculovirus to express HPV16 L1 proteir, VLP were obtained in great quantity, optimized the conditions of protein amplification analyzed and identified with SDS-PAGE, and then purified the VLP with CsCl gra- dient eentrifugation and established hemagglutination inhibition assay using obtained VLP to identify hemagglutination inhibition activity of the prepared HPV16 L1. The results of the optimized expression of protein showed that when MOI = 10 hours the infected cells with recombinant baeulovirus the expression amount of target protein was the largest. With this titer infected the suspensively cultured insect cells, harvested and purified VLP and identified its specificity. The valence of McAb identified with purified VLPs by hemagglutination inhibition assay was 1： 16. The established hemagglutination assay can be used to identify and test HPV16L1 correlated antibody and provide an assay foundation for HPV diagnosis kits and further development.