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目的在毕赤酵母中表达人乳头瘤病毒18型(Human papilomavirus 18,HPV18)L1蛋白,纯化病毒样颗粒(Virus-like particles,VLPs),并检测其免疫原性。方法将含HPV18 L1基因的酵母表达质粒pHPV18 L1电转化毕赤酵母X-33,甲醇诱导表达,重组HPV18 L1蛋白经SDS-PAGE和Western blot分析后,经离子交换层析、分子筛层析纯化,采用SEC-HPLC分析纯度;动态光散射和透射电镜分析粒径分布和结构;并通过小鼠半数有效剂量(ED50)和大鼠抗体滴度测定分析其免疫原性。结果 HPV18 L1蛋白在毕赤酵母中获得了有效表达,相对分子质量约为55 000,可与小鼠抗HPV18 L1单抗特异性结合;经纯化的HPV18 L1 VLPs纯度为99%,且颗粒大小均一,直径约50 nm;经吸附铝佐剂后,免疫小鼠的ED50为0.006 68μg;并可诱导大鼠产生较高的抗体滴度。结论 HPV18 L1可在毕赤酵母中高效表达并自动形成VLPs,该VLPs经纯化、吸附铝佐剂后具有良好的免疫原性,为工业化生产HPV18 L1疫苗奠定了基础。
Objective To express human papilomavirus 18 (HPV18) L1 protein in Pichia pastoris and purify the virus-like particles (VLPs) and test its immunogenicity. Methods The HPV18 L1 gene expression plasmid pHPV18 L1 was transformed into Pichia pastoris X-33 and induced by methanol. The recombinant HPV18 L1 protein was purified by ion-exchange chromatography and molecular sieve chromatography after SDS-PAGE and Western blot. The purity was analyzed by SEC-HPLC. The particle size distribution and structure were analyzed by dynamic light scattering and transmission electron microscopy. The immunogenicity was analyzed by the half-effective dose (ED50) and rat antibody titers. Results HPV18 L1 protein was efficiently expressed in Pichia pastoris. The relative molecular mass of HPV18 L1 protein was about 55 000, which could specifically bind to mouse anti-HPV18 L1 monoclonal antibody. The purified HPV18 L1 VLPs had a purity of 99% with uniform particle size , The diameter of about 50 nm; adsorbed aluminum adjuvant, the ED50 of mice immunized 0.006 68μg; and can induce high antibody titers in rats. Conclusion HPV18 L1 can express efficiently in Pichia pastoris and form VLPs automatically. The purified VLPs have good immunogenicity after adsorbed aluminum adjuvant, which lays the foundation for the industrial production of HPV18 L1 vaccine.