人乳头瘤病毒16型L1的表达、病毒样颗粒组装及其免疫原性研究

目的 在原核表达系统中表达HPV16 L1蛋白,纯化后在体外自组装成VLPs并鉴定。方法 优化GenBank中HPV16 L1基因序列并截短C末端25个氨基酸,构建至原核表达载体pET-28a上,获得重组表达载体pET28a-16L1△C25。采用镍亲和层析法纯化超声上清,于体外解组装-重组装HPV16 VLPs,采用动态光散射和透射电镜进行形貌分析,纯化后于第0、2和4周免疫小鼠,假病毒中和试验检测HPV16 VLPs免疫后血清中和抗体。结果 双酶切和测序结果表明成功构建pET28a-16L1△C25重组质粒,诱导表达后,经SDS-PAGE和Western blotting分析显示表达的L1蛋白大部分以可溶性形式存在,纯化后的蛋白样品于体外重新组装,动态光散射和透射电镜能够观察到形态与天然病毒颗粒相似的VLPs,第6周小鼠血清中和抗体滴度Log10平均值达到4.43。结论 利用原核表达系统成功表达了截短型HPV16 L1蛋白,并于体外组装成结构完整的VLPs,且具有较好的免疫原性,为低成本HPV预防性疫苗的研发奠定基础。

Expression of human papillomavirus type 16 L1 protein and assembly and immunization assessment of virus like particles

We expressed and purified the L1 major capsid protein of human papillomavirus type 16 (HPV16) in prokaryotic expression system and self-assemble virus like particles. Optimized HPV16 L1 gene according to the Escherichia coli cell codon in GenBank data base was truncated 25 mino acid residues at C-terminus and constructed pET28a-16L1△C25 expression vector. Ultrasound supernatant was purified by Ni-NTA affinity chromatography. Then the purified HPV16 L1 self-assemble into VLPs in vitro, we investigated the morphology of VLPs with dynamic light scattering and electronmicroscopy could assemble into VLPs, we used the purified VLPs to immunize BALB/c mice in 0, 2, 4 weeks and detected the titers of HPV16 specific neutralizing antibody in sera was detected by pseudovirus neutralization assay. HPV16 L1 we expressed in E. coli were major soluble forms analyzed by SDS-PAGE and western blotting. The soluble HPV16 L1 could self-assemble to VLPs in high efficiency, their shapes were similar to native HPV16 virions, The titer of HPV16 specific neutralizing antibody in sera was detected by pseudovirus neutralization assay at the sixth week,average value of Log10 was 4.43. The HPV16 VLPs prepared from prokaryotic expression system has the immunogenicity,which may provide a new way for research and development of low cost prophylactic HPV vaccine.