摘要
目的利用昆虫细胞表达体系制备人乳头瘤病毒(HPV)31 L1病毒样颗粒(VLP)疫苗。方法化学合成法获得昆虫Sf9细胞偏性密码子优化的、C端删除27个氨基酸编码序列的HPV31 L1截短基因(HPV31 L1MΔC),构建重组杆状病毒,感染Sf9细胞进行表达;分别采用超声破碎、含离子型表面活性剂(1%SDS等)或含非离子型表面活性剂(0.5%TritonX-100)的裂解缓冲液,制备细胞裂解上清;氯化铯超速离心法纯化后经动态光散射及透射电子显微镜鉴定;0.1μg的HPV31 L1MΔC VLP于第0和2周肌肉免疫BALB/c小鼠,第4周采集血清分析中和活性。结果 3种裂解方法制备的上清均显示HPV31 L1MΔC蛋白的特异表达,其表达量约占裂解上清总蛋白的10%;采用非离子型表面活性剂获得的裂解上清纯化后,目的蛋白纯度高,产量达11.5 mg/L;纯化蛋白的水化分子直径为103.3 nm,均一性好;电镜分析为直径50~60 nm的VLP。免疫血清中HPV31中和抗体滴度为1 440。结论 HPV31 L1MΔC在昆虫细胞表达体系制备的VLP产量高、免疫原性好,可用于生产含HPV31 L1 VLP的多价疫苗。
Objective To produce human papillomavirus(HPV) type 31 L1 virus-like particle(VLP) vaccine in baculovirus system. Methods A C-terminal 27 residues truncated HPV31 L1 gene (HPV31 L1MΔC) which optimized with Sf9 cell bias condons, was synthesized and expressed in baculovirus system. Cells infected with HPV31 L1MΔC recombinant baculovirus were lysed by sonification, or incubation with either ionic surfactant-containing buffer or nonionic surfactant-containing buffer, and the supernatants were harvested respectively for expression assays. The supernatants prepared by incubation with nonionic surfactant-containing buffer were purified by CsCl ultracentrifugation and HPV31 L1MΔC protein was identified by dynamic light scatter (DLS) and transmission electronic microscopy (TEM). BALB/c mice were immunized with 0.1 μg HPV31 L1MΔC VLP intramuscularly at weeks 0, 2 and sera at week 4 were taken for neutralization assay against HPV31 pseudovirus. Results HPV31 L1MΔC protein was expressed in all three kinds of supernatants prepared by either sonification or incubation with two different surfactant-containing buffer, and amounted for 10% of the total proteins. The yield of purified HPV31 L1MΔC was up to 11.5 mg per liter suspension medium. HPV31 L1MΔC protein showed an average hydrodynamic diameter of 103.3 nm with a mean polydispersity index of 0.190 by DLS, and presented VLP with an average diameter of 50~60 nm which is similar to native virions shown by TEM. HPV31 L1MΔC VLP induced high titer of HPV31 neutralizing antibody (1 440) in mice. Conclusions The VLP produced with HPV31 L1MΔC truncated gene in baculovirus system is a promising candidate for development of multivalent HPV L1VLP vaccine for its high yield and potent immunogenicity.
作者
郝亚茹
张婷
刘洪洋
王志荣
周艳
夏百成
许雪梅
HAO Ya-ru;ZHANG Ting;LIU Hong-yang;WANG Zhi-rong;ZHOU Yan;XIA Bai-cheng;XU Xue-mei(Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC, Beijing 100005,China)
出处
《基础医学与临床》
CSCD
2019年第3期343-347,共5页
Basic and Clinical Medicine
基金
中国医学科学院医学与健康科技创新工程项目基金(2016-I2M-3-026)
北京市自然科学基金(5162025)
国家国际科技合作项目(2013DFA32430)