Kirnbauer R, Taub J, Greenstone H, Roden R, Dürst M, Gissmann L, Lowy D R, Schiller J T
Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892.
J Virol. 1993 Dec;67(12):6929-36. doi: 10.1128/JVI.67.12.6929-6936.1993.
The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection.
来自尖锐湿疣的两株人乳头瘤病毒16型（HPV16）的L1基因，通过重组杆状病毒用于在昆虫细胞中表达L1主要衣壳蛋白。两种L1主要衣壳蛋白都能高效自组装成病毒样颗粒（VLP），并可通过密度梯度进行大量纯化。VLP的产量比以前使用源自原型HPV16的L1所获得的产量高3个数量级。DNA序列比较确定了一个单一的非保守氨基酸变化是原型L1低效自组装的原因。通过表达HPV6、HPV11和棉尾兔乳头瘤病毒的L1也获得了VLP，这表明来自多种乳头瘤病毒的L1具有自组装成VLP的内在能力。使用杆状病毒双表达载体共表达HPV16 L1和L2也导致VLP的高效自组装，与仅表达L1相比，平均颗粒产量增加了约四倍。L1和L2的共免疫沉淀以及两种蛋白在蔗糖梯度中的共沉降表明L2被整合到颗粒中。能够大量制备HPV16 L1和L1-L2 VLP可能对开发用于检测针对HPV16病毒体构象表位的抗HPV16病毒免疫反应的血清学检测以及针对HPV16感染的免疫预防具有重要意义。
