HPV 18 L1病毒样颗粒的表达纯化及其豚鼠抗血清制备

Expression and purification of HPV 18 L1 virus like particles and preparation of guinea pig antiserum against HPV 18 L1 VLP

目的优化人乳头瘤病毒(HPV)18型晚期基因L1并在昆虫细胞中表达,获得HPV18L1病毒样颗粒(VLP),制备豚鼠抗血清。方法联合利用昆虫细胞偏性密码子、C末端截短32个氨基酸、降低mRNA二级结构等策略优化HPV18L1基因,合成后克隆入pFastBac1载体,构建重组病毒,感染sf9昆虫细胞72h后进行Western blot表达鉴定,密度梯度离心法纯化后进行纯度鉴定及形态学鉴定,获得HPV18L1VLP,联合弗氏佐剂免疫豚鼠制备抗血清。结果HPV18L1优化基因在昆虫细胞中可有效表达,纯化的HPV18L1蛋白纯度达90%以上,电镜观察可见直径约为50nm的VLP,豚鼠免疫血清中HPV18L1VLP特异性抗体滴度达5.12×105。结论HPV18L1优化基因可在昆虫细胞中高效表达并组装成VLP,制备的高滴度抗血清为HPV18L1VLP疫苗的进一步研究打下基础。

Objective To obtain human papillomavirus type 18 virus-like particles using an optimized HPV 18 L1 gene and to prepare antiserum with guinea pig. Methods HPV 18 L1 gene was optimized by combined strategy including codon replacement, truncation of 32 amino acids at C terminal and reduction of secondary structure of mRNA. The optimized HPV 18 L1 gene was synthesized and cloned into pFastBael vector. The recombinant baculovirus was obtained and then used to infect sf9 cells. Seventy-two hours later, the cell lysate was analyzed by Western blot. HPV 18 L1 VLP was purified by gradient centrifugation, analyzed by SDS-PAGE and transmission electron microscopy. The guinea pig was immunized with purified HPV 18 L1 VLPs. Then antiserum against HPV 18 L1 VLP was produced. Results Optimized HPV 18 L1 gene was efficiently expressed in si9 cells. The HPV 18 L1 VLP could be purified by gradient centrifugation and the purity was over 90%. Electron microscopy showed the VLP particles with diameter of 50 nm. The serum antibody titer of guinea pig was 5.12×10^5. Conduslon HPV 18 L1 VLP could be expressed efficiently in sf9 cells using optimized HPV 18 L1 gene . And guinea pig antisera could be used for future research of HPV 18 L1 VLP based vaccine.