HPV16 L2肽段偶联AP205 VLP的原核表达及免疫原性分析

目的:将编码HPV16衣壳蛋白L2的65～71、112～120免疫优势表位连接到RNA噬菌体衣壳蛋白AP205氮端,组装形成病毒样颗粒,通过在大肠杆菌中实现表达及纯化,对其免疫原性进行研究。方法:合成编码AP205衣壳蛋白基因和HPV16 L2的65～71、112～120位氨基酸表位的基因序列,PCR连接并克隆至pET30a(+)原核表达载体,构建重组表达质粒pET30-AP205-HPV16ΔL2,转化大肠杆菌BL21(DH3)感受态细胞,IPTG诱导表达。表达蛋白经凝胶层析纯化及SDS-PAGE、Western blot等理化性质检测,免疫接种ICR小鼠,通过间接ELISA法检测其免疫原性。结果:成功构建重组表达质粒,重组蛋白在大肠杆菌中以可溶性表达,透射电镜观察可见典型病毒样颗粒,该VLP在动物实验中表现出较好的免疫原性。结论:成功将HPV16 L2表位偶联AP205以形成VLP,在大肠杆菌中实现可溶性表达。

Prokaryotic Expression and Immunogenicity Analysis of HPV16 L2 Peptide Coupled to AP205VLP

Objective: To connect the residues 65~71 and 112~120 of HPV minor capsid protein L2 to the coat protein of RNA bacteriophage AP205.The fusion protein was self-assembled into virus-like-particles(VLPs) after expressing in E.coli,then the protein was purified and the immunogenicity was analyzed.Methods: After being synthesized artificially,the AP205 coat protein gene and HPV16L2 gene were connected by PCR and cloned into vector pET-30a(+).The constructed plasmid pET30-AP205-HPV16ΔL2 was transformed to E.coli BL21(DH3),the recombinant protein was expressed under induction of IPTG and purified by Gel chromatography,then identified through SDS-PAGE and Western blot.The immunogenicity of purified product was measured by indirect ELISA after ICR mouse was immunized.Results: Recombinant plasmid pET30-AP205-HPV16ΔL2 was constructed correctly and the protein was expressed solubly in E.coli BL21(DH3).The typical VLPs can be observed through Transmission electron microscopy which Showed good immunogenicity in animal experiments.Conclusion: The HPV16L2 epitope was fused successfully to the N-terminus of AP205 coat protein,and The VLPs was self-assembled in E.coli.