重组人乳头瘤病毒16型L1 VLP的初步纯化、鉴定及免疫效果的初步研究

目的 对昆虫细胞-杆状病毒系统表达的人乳头瘤病毒(human papillomavirus,HPV)16型L1蛋白病毒样颗粒(virus-like particle,VLP)进行初步纯化研究,并在小鼠中检测纯化VLP的免疫原性.方法 采用蔗糖-氯化铯密度梯度超速离心纯化方法纯化HPV16 LI VLP.对纯化得到的VLP分别进行蛋白印迹法、十二烷基硫酸钠一聚丙烯酰胺凝胶电泳(SDS-PAGE)及电镜鉴定.然后用VLP免疫6周龄BALB/c小鼠.将小鼠随机分成6组(3个剂量组、2个佐剂组和1个对照组),间隔2周免疫3次,3次剂量相同.取血分离血清,采用间接ELISA法检测抗HPV16 L1 VLP抗体滴度.结果 经蔗糖-氯化铯密度梯度超速离心纯化后,用SDS-PAGE鉴定纯化的HPV16 LI VLP,纯度达到90%.电镜观察可见完整的VLP颗粒.间接EUSA检测结果显示,剂量组及佐剂组小鼠血清中均可检测到特异性抗HPV16 L1 VLP抗体,且抗体滴度随针次和剂量的增加而增高.结论 采用蔗糖-氯化铯密度梯度超速离心纯化法得到的HPV16 L1 VLP可用于免疫学初步研究.通过昆虫细胞表达产生的HPV16 LI VLP 具有较强的免疫原性,能诱导小鼠产生强的体液免疫应答.

A preliminary study on purification, identification and immunogenicity of HPV16 L1 VLPs

Objective To study a preliminary purification technique of human papillomavims 16 (HPVl6)L1 protein virus-like particles(VLPs) expressed in insect cell-baculovirus system and the immunogenicity of the purified VLPs in mice.Methods HPV16 L1 VLPs were purified by CsC1 and sucrose density gradient ultracentrifugation,and the purified VLPs were identified by Western blot,sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and electron microscopy.Six-week-old BALB/c mice were randomly divided into 6 groups,including 3 dose groups,2 adjuvant groups and a control group,and vaccinated with the purified VLPs 3 times at intervals of 2 weeks.Sera were taken and determined for anti-L1 antibodies by indirect ELISA.Results The purity of HPV16 L1 VLPs was up to 90% by SDS-PAGE identification.Intact VLPs were observed under the electron microscope.Anti-L1 antibodies were detected by ELISA in all dose groups and adjuvant groups.The titers of antibodies increased with vaccination times and dosage.Conclusion The HPV16 L1 VLPs expressed in insect cell-baculovirus system and purified by CsC1 and sucrose density gradient ultracentrifugation have immunogenicity and can induce strong humoral immune responses in mice.