人乳头瘤病毒52型L1蛋白病毒样颗粒在毕赤酵母中的优化表达、纯化及其免疫原性

Optimized expression of virus-like particles of human papillomavirus 52 L1 protein in Pichia pastoris and purification and immunogenicity of expressed product

目的 利用毕赤酵母系统表达人乳头瘤病毒52型(human papillomaviruses 52,HPV52)L1蛋白,并检测其病毒样颗粒(virus-like particle,VLP)的免疫原性。方法 采用同义密码子替换的方法对HPV52 L1蛋白的野生型基因进行密码子优化,并在体外构建多拷贝表达质粒,经转化和筛选获得在毕赤酵母系统中高表达的HPV52 L1 VLP菌种。采用15 L发酵罐大规模培养,菌液破碎上清经阳离子交换层析和分子筛排阻层析两步法纯化,获得HPV52 L1VLP。动态光散射和透射电子显微镜观察HPV52 L1 VLP的大小和形态,假病毒中和试验检测HPV52 L1 VLP在小鼠及大鼠体内的免疫原性。结果 HPV52 L1 VLP在溶液中呈较均一的单一组分,水合粒径为91. 37 nm;镜下观察呈均一的、直径约50 nm的球状空心颗粒,大小与HPV52天然病毒颗粒相近。HPV52 L1 VLP相对分子质量约56 000,纯度达95%以上,产量为3. 6 mg/L,且可与小鼠抗HPV L1多克隆单抗发生特异性结合。HPV52 L1 VLP在小鼠体内的半数有效剂量(ED50)为0. 010μg,在大鼠体内诱导产生的中和抗体滴度高达106。结论 于毕赤酵母系统成功表达了HPV52 L1 VLP,且具有良好的免疫原性,为相关预防性疫苗的研发奠定了基础。

Objective To express human papillomavirus 52(HPV52)L1 protein in Pichia pastoris,purify the expressed product and evaluate its immunogenicity.Methods The codon of wild-type gene of HPV52 L1 was optimized by synonymous codon substitution,then the multi-copy expression vector was constructed and transformed to P.pastoris.The clones for high expression of virus-like particles(VLPs)of HPV52 L1 protein were obtained through screening for largescale fermentation culture in 15 L fermenter.The culture supernatant was purified by cation-exchange chromatography and size-exclusion chromatography.The obtained VLPs were observed for size and shape by dynamic light scattering and transmission electronic microscopy,and evaluated for immunogenicity by pseudovirus neutralization test in mice and rats.Results HPV52 L1 VLPs showed a homogeneous single component in solution,at a hydrated size of 91.37 nm,which were homogeneous spherical empty particles at a size of about 50 nm under microscope,similar to those of natural virus particles of HPV52.The relative molecular mass of HPV52 L1 VLPs was about 56000,while the purity was more than 95%and the yield was 3.6 mg/L.The VLPs showed specific binding to mouse polyclonal antibody against HPV L1,of which the ED50 was 0.010μg in mice.However,the VLPs induced a neutralizing antibody titer of 106 in rats.Conclusion HPV52 L1 VLPs were successfully expressed in P.pastoris,which showed good immunogenicity.It laid a foundation of development of relevant prophylactic vaccines.