HPV6b L1蛋白在昆虫细胞中的表达及其作为特异性诊断抗原的初步研究

Expression of HPV6b L1 protein in insect cells and its application to the diagnosis of specific antigen

目的 :研究人乳头瘤病毒 6b结构蛋白 (HPV6bL1)在昆虫细胞系统的表达及其作为特异性诊断抗原的可行性 ,为HPV感染的血清学诊断及基因工程疫苗研究作准备。方法 :采用HPV6bL1重组杆状病毒感染sf 9昆虫细胞制备HPV6bL1,经氯化铯梯度离心纯化 ,Westernblot分析及电镜观察鉴定 ;用 1μg、5 μg和2 0 μg三种剂量免疫小鼠 ,以检测小鼠血清抗体反应 ;并用HPV6bL1作为诊断抗原检测HPV感染者 (尖锐湿疣及宫颈癌 )和健康对照者共 112例血清特异性抗体。结果 :HPV6bL1在昆虫细胞中得到表达 ,并可自行组装形成VLPs,且予 1μg剂量免疫小鼠就能产生阳性血清抗体反应 ,HPV6bL1VLPs特异性抗体对HPV6感染的敏感性和特异性分别为 78.6 %和 97.1%。结论 :HPV6bL1VLPs具较强的免疫原性和抗原性 ,血清中检测到的HPV6bL1抗体对HPV6感染具较高的特异性 ,可进一步用于HPV感染的血清学诊断及防治尖锐湿疣的基因工程疫苗研究。

Objective:To develop a diagnostic antigen for HPV infection and genetic engineering vaccine against condyloma acuminatum(CA),the HPV6b L1 protein expressed in sf-9 insect cells with baculovirus express vector system and used as a special diagnostic antigen for HPV infection was studied.Methods:HPV6b L1 VLPs were expressed with HPV6b L1 recombinant baculovirus in sf-9 insect cells,purified on CsCl density gradients,identified by Western blot analysis and examined by electron microscopy. Mice were immunized with HPV6b L1 VLPs without adjuvant in 1μg,5μg,20μg doses respectively and sera were prepared.By ELISA analysis the HPV6b L1 VLPs were also used to screen human serum antibody from 40 patients with HPV infection(CA and cervical cancer) and 72 control subjects.Results:The HPV6b L1 protein could be expressed in sf-9 insect cells and assembled into virus-like particles(VLPs). Specific serum antibody against HPV6b L1 VLPs could be measured in the mice immunized with only 1μg HPV6b L1 VLPs.It showed that the sensitivity and specificity were 78.6% and 97.1% respectively,by using ELISA to detected the serum specific antibody used for diagnosing HPV6 infection. Conclusion:The HPV6b L1 VLPs possessed strong antigenicity and could be used to study a specific diagnostic antigen for HPV infection and to develop genetic engineering vaccine against CA.