Xu Yu-fei, Wang Qing-yong, Zhang Hong-tao, Han Ye-hua, Song Guo-xing, Xu Xue-mei
Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China.
Chin Med J (Engl). 2007 Mar 20;120(6):503-8.
Human papillomaviruses (HPVs) can infect squamous or mucosal epithelia and cause cervical cancer or genital warts. Coinfection with multiple HPV types is a common finding of many epidemiological studies. Therefore, it is necessary to develop a vaccine, which can eradicate established HPV infections and prevent other HPV infections. In this study, we generated chimeric virus like particles (cVLPs) composed of HPV-6b L1, HPV-6b L2 and one artificial HPV-16 mE7 proteins.
The artificial HPV-16 mE7 gene was designed by codon modification, point mutation and gene shuffling then chemically synthesized and subcloned behind HPV-6b L2. HPV-6b L1 and L2-mE7 were expressed in insect cells by using Bac-to-Bac system. The generated cVLPs were purified by CsCl gradient ultracentrifuge and analyzed by immunoblot, electron microscope and haemagglutination assay.
The HPV-6b L1 and L2-mE7 proteins were well expressed in insect cells and could selfassemble into cVLPs, whose diameter was about 55 nm and similar to that of HPV-6b L1/L2 VLPs. Intact cVLPs could be recognized by H6.M48 neutralizing monoclonal antibody and HPV-6b L2 polyclonal antibody, while the denatured cVLPs, but not the intact cVLPs, were reactive to HPV-16 E7 polyclonal antibody. HPV-6b L1/L2-mE7 cVLPs haemagglutinated mouse erythrocytes as efficiently as HPV-6b L1/L2 VLPs did.
The insertion of the 158 amino acid HPV-16 mE7 protein behind L2 did not disrupt the correct assembling of cVLPs. The morphological characteristics and haemagglutinating activity of cVLPs were similar to those of HPV-6b L1/L2 VLPs. The cVLPs retained conformational B cell epitopes of HPV-6 VLPs and HPV-16 mE7 protein had an internal location in the cVLPs. Therefore, large modified E7 protein with higher immunogenicity could be incorporated into cVLPs by fusing to the C-terminus of L2, which would help to improve the therapeutic effects of L1/L2-E7 cVLPs.
人乳头瘤病毒（HPV）可感染鳞状上皮或黏膜上皮，引发宫颈癌或尖锐湿疣。多种HPV类型的合并感染是许多流行病学研究中的常见发现。因此，有必要研发一种能够根除已有的HPV感染并预防其他HPV感染的疫苗。在本研究中，我们构建了由HPV-6b L1、HPV-6b L2和一种人工合成的HPV-16 mE7蛋白组成的嵌合病毒样颗粒（cVLP）。
通过密码子修饰、点突变和基因改组设计人工合成的HPV-16 mE7基因，然后进行化学合成并亚克隆至HPV-6b L2基因的下游。利用杆状病毒表达系统在昆虫细胞中表达HPV-6b L1和L2-mE7。通过氯化铯梯度超速离心法纯化所产生的cVLP，并采用免疫印迹、电子显微镜和血凝试验进行分析。
HPV-6b L1和L2-mE7蛋白在昆虫细胞中得到良好表达，并能自组装成cVLP，其直径约为55 nm，与HPV-6b L1/L2 VLP相似。完整的cVLP可被H6.M48中和单克隆抗体和HPV-6b L2多克隆抗体识别，而变性的cVLP（而非完整的cVLP）可与HPV-16 E7多克隆抗体发生反应。HPV-6b L1/L2-mE7 cVLP凝集小鼠红细胞的效率与HPV-6b L1/L2 VLP相同。
在L2蛋白的下游插入158个氨基酸的HPV-16 mE7蛋白并不影响cVLP的正确组装。cVLP的形态特征和血凝活性与HPV-6b L1/L2 VLP相似。cVLP保留了HPV-6 VLP的构象性B细胞表位，且HPV-16 mE7蛋白位于cVLP的内部。因此，通过与L2的C末端融合，具有更高免疫原性的大型修饰E7蛋白可被整合到cVLP中，这将有助于提高L1/L2-E7 cVLP的治疗效果。
