HPV11型L2E7融合蛋白的原核表达及其免疫效果观察

Prokaryotic expression and purification of human papillomavirus type 11 L2E7 fusion protein vaccine and its immunnogenicity

目的构建人乳头瘤病毒11型L2E7原核表达系统pET9aHPV11L2E7，并纯化蛋白进行小鼠免疫效果研究。方法从尖锐湿疣组织中扩增人乳头瘤病毒11型12、E7基因片段，构建DET9aHPV11L2E7原核表达重组质粒并测序，在大肠埃希菌宿主菌BL21（DE3＋）中经IPTG诱导表达融合蛋白L2E7（553个氨基酸），经SDS—PAGE电泳和Western Blot进行鉴定。CM离子交换介质纯化蛋白免疫BALB／c小鼠，进行细胞免疫水平和体液免疫水平的检测。结果成功构建了pET9 aHPV11L2E7原核表达系统，纯化获得的HPV11L2E7蛋白免疫BALB／c小鼠后能检测到针对HPV11E7特异性的细胞免疫，血清中能检测到高效价抗HPV11L2E7抗体。结论纯化的HPV11L2E7融合蛋白能够引发特异性细胞和体液免疫反应，能作为尖锐湿疣免疫治疗候选疫苗。

Objective To construct the Escherichia coli （E. coli） prokaryotic expression system pET9aHPV11L2E7, purify the fusion protein L2E7 and study the immunnogenicity of the protein. Methods The HPVll L2, E7 coding region was amplified from condyloma acuminata tissue specimen by PCR. The recombinant plazmid pET9aHPV11L2E7 was established and sequenced. Fusion protein L2E7 （553 amino acids） was expressed in host strain BL21 （DE3^＋） by IPTG inducing and identified by using SDS-PAGE and Western blotting. Then L2E7 protein purified with CM column was inoculated to BALB/c mice and its cell-mediated and humoral immunnogenicity was assessed by IFN-γ enzyme-linked immunospot （ELISPOT） and enzyme-linked immunosorbent assay （ELISA）. Results The E. coli prokaryotic expression system pET9aHPV11L2E7 was established and the purified fusion protein L2E7 was obtained successfully. The mice in vivo experiment indicated that the purified protein L2E7 could induce HPV11E7 specific cell-mediated immune responses and high level HPV L2E7 antibody was detected in serum. Conclusion The purified fusion protein L2E7 could induce specific cell-mediated and humoral immune responses. It can be used as a candidate of genital wart immune therapeutic vaccine.