人乳头瘤病毒16型L1/E7融合蛋白在原核细胞中的表达及免疫效果观察

Expression of the human papillomavirus type 16L/E7 fusion protein in E.coli and observation of its immunogenicity in mice

目的构建能表达L1E7融合蛋白的原核表达菌株,纯化蛋白,并观察其免疫效果。方法用PCR方法分别扩增出C末端部分缺失的HPV16L1基因和HPV16E7编码基因N端部分序列。将上述基因连接,构建融合基因L1ΔCE7N并将其插到原核表达载体pGEX-2T中进行融合蛋白表达纯化,然后观察其免疫效果。结果L1ΔCE7N融合基因测序结果表明,序列与设计相符,读码框架正确。将其插入原核表达质粒在大肠埃希菌中获得高效表达;经Wester-Blot鉴定在相对分子质量约85×103处有特异性表达带,与预期相符。用亲和层析和分子筛可纯化L1ΔCE7N融合蛋白,将其免疫C57BL/6小鼠,结果表明融合蛋白能诱发高滴度L1、E7抗体,并能保护小鼠免受TC-1肿瘤细胞的攻击。结论本实验在原核系统中高效表达并纯化了L1ΔCE7N融合蛋白,该蛋白可作为预防和治疗HPV16感染以及相关肿瘤的候选疫苗株。为研制HPV16预防治疗性疫苗探索一条经济、易普及的途径。

Objective Many epidemiological and experimental evidences prove that cervical cancers are strongly associated with genital high-risk types of human papillomaviros （HPV）. HPV16 is present in 50% of the tumor specimens. Thus, it is important to develop vaccines against HPV16 and cervical cancer. The authors studied the expression of the HPV16 L1△CE7N fusion protein in E. coli and observed its immunogenicity. Method The fragment of HPV16 L1△C gene and the E7N gene were amplified by PCR separately; the fusion gene named L1△C E7N was generated by fusing E7N to the C terminal of L1△C then the chimeric gene was cloned into prokaryotic expression vector pGEX-2T and expressed in E. coli strain JM109. The L1△CE7N protein expressed were detected by Western-blot. Finally its immunogenicity was characterized in immunized mice. Result It was proved that the sequence and open reading frame of fussion gene L1△E7N was correct by sequencing; SDS-PAGE-gel analysis showed that HPV16 L1/E7 fusion protein was highly expressed in E. coli; the protein was expressed as soluble form and the molecular weight was about 85 × 10^3 . The fussion protein could be purified by affinity chromagraphy and gel filtration. The ELISA result indicated that L1/E7 could elicit specific antibodies against L1 and E7 in immunized mice. In vivo tumor protection test indicated that tumor formation was retarded or prevented in the mice after vaccination with L1/E7, when C57 BIJ6 mice were challenged by syngenetic HPV16E6 and E7 transformed tumor cells. Conclusion HPV16L1/E7 fusion protein was expressed in E. coli, it can be a candidate for prophylactic and therapeutic vaccine for HPV16-associated infection and tumors.