人乳头瘤病毒16型L1-E7c嵌合基因的构建表达及免疫学效应的研究

Construction of a chimeric gene of human papillomavirus type 16 L1-E7 and immunogenicity of expressed fusion protein

目的构建人乳头瘤病毒16型(HPV16) L1-E7c嵌合基因并表达融合蛋白,以期获得防治HPV16感染及相关肿瘤的疫苗。方法以HPV16型的中国人野毒株为模板,利用PCR克隆技术制备HPV16 L1-E7c嵌合基因,并转入原核表达载体pET28a(+),获得pET28a(+)-L1-E7c表达质粒。以Western blot方法鉴定融合蛋白与HPV16L1和E7抗体的特异性结合。应用蛋白纯化仪纯化HPV16 L1-E7c融合蛋白,经过复性后,电镜观察病毒样颗粒(cVLP)的形态结构。纯化的蛋白免疫动物,测定疫苗抗肿瘤的细胞免疫及抗体产生情况。结果经过序列分析和酶切鉴定表明成功构建了HPV16 L1-E7c嵌合基因,并在大肠杆菌中可高效表达L1-E7c融合蛋白。此融合蛋白具有HPV16 L1和E7的抗原性,纯化的蛋白复性后可形成嵌合病毒样颗粒(cVLP),纯化的蛋白免疫动物后,产生特异性细胞和体液免疫反应,具有抗肿瘤活性。结论构建的嵌合基因HPV16 L1-E7c可有效表达HPV16 L1-E7c重组蛋白并形成cVLP。此蛋白在动物实验中具有疫苗的免疫保护作用,为HPV16疫苗的研究奠定了实验基础。

Objective To develop a combined prophylactic and therapeutic vaccine by using a chimeric protein consisting of a C-terminally truncated HPV 16 E7 portion fused to a LI capsid protein. Methods The HPV16 L1 and E7 genes were obtained from Chinese patients infected with HPVI6 using polymerase chain reaction （PCR）. The plasmid pET28a（ ＋ ）-L1-E7c contained L1 ORF and C-terminal portion of E7 ORF was constructed. The expression of the chimeric protein was identified using both HPV16 L1 and E7 specific antibodies by Western blot assay, while the chimeric virus-like particle （cVLP） was observed by the electron microscopy. The immunogenicity of the fusion protein was studied through HPV16 E7 CTL and L1 antibody in HPV16 E7 tumor-carried mouse. Results The plasmid pET28a（ ＋ ）-L1-E7c was constructed and checked by DNA sequencing and enzyme digestion. The fusion protein of HPV16 L1-E7c was expressed at high level in E. coli. The purified protein was re-natured and able to self-assemble into chimeric virus-like particle （cVLP） in vitro, which is morphologically indistinguishable from the native virion under the electron microscope. Vaccination with HPV16 L1-E7c induced regression of established tumors expressing HPV16 E7. And the fusion protein was a potent inducer of HPV16 E7 CTL responses and HPV16 L1 antibody. Conclusion The HPV16 L1-E7c fusion protein can be expressed in high level and self-assembled into the chimeric virus-like particles in vitro, which indicates that cVLP are suitable for prevention and therapy of HPV16 infection and related tumors.