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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。
OBJECTIVE: To construct a virus-like particle by ligating 65-171,112-120 immunodominant epitopes encoding HPV16 capsid protein L2 to the N-terminus of the RNA bacteriophage capsid protein AP205 and expressing them in Escherichia coli Immunogenicity was studied. Methods: The gene sequences of AP205 capsid protein gene and HPV16 L2 amino acid epitopes 65-71,112-120 were synthesized and cloned into prokaryotic expression vector pET30a (+) by PCR. The recombinant plasmid pET30-AP205-HPV16ΔL2 , Transformed into E. coli BL21 (DH3) competent cells, induced by IPTG. The expressed protein was purified by gel chromatography and detected by SDS-PAGE, Western blot and other physical and chemical properties, immunized ICR mice, and its immunogenicity was detected by indirect ELISA. Results: The recombinant plasmid was constructed successfully. The recombinant protein was expressed in Escherichia coli in soluble form. The typical virus-like particles were observed by transmission electron microscopy. The VLP showed good immunogenicity in animal experiments. CONCLUSIONS: AP205 was successfully coupled to HPV16 L2 epitopes to form VLPs, achieving soluble expression in E. coli.