含人类乳头瘤病毒16E7-L1的嵌合型病毒样颗粒的构建、表达和纯化鉴定

Construction, Expression, Purification and Identification of Chimeric HPV16 E7-L1 Virus Like Particles

目的研究人类乳头瘤病毒(HPV)16型的早期基因E7 ,构建、表达并纯化鉴定了HPV16E7与BPVL1重组的嵌合型病毒样颗粒VLPs。方法HPV16E7基因分3段经PCR扩增后分别克隆入连有BPVL1的质粒PUC形成BPVL1 HPV16E7 ;以质粒PVL1393为载体将BPVL1 HPV16E7基因转染杆状病毒并在昆虫细胞中进行表达 ;用超声粉碎和蔗糖超离、氯化铯梯度离心等方法纯化以及免疫印迹、ECL、透射电镜等方法鉴定表达产物。结果和结论成功地获得表达BPVL1 HPV16E7重组体 ,形态和结构与野生型HPV几乎一致的嵌合型病毒样颗粒VLPs。将为进一步的HPV16E7的疫苗研究等奠定基础。

Objective To study the human papillomavirus (HPV) type 16 E7, the chimeric HPV16 E7 L1 virus like particles (VLPs) were constructed, produced, purified and identified. Methods The HPV16E7 gene was cut to three parts, amplified by PCR, and connected individually with BPVL1 on plasmid PUC. With PVL1393 as a transfect vector, the BPVL1/HPV16E7 recombinants were transfected to baculovirus which subsequently expressed the chimeric BPVL1/HPV16E7 protein in the SF 9 cells. The recombinant proteins were then purified by centrifuge, sonication, sucrose and CsCl ultracentrifuge, and were identified by SDS PAGE, Western blot, ECL and TEM. Results and Conclusion The chimeric BPVL1/HPV16E7 recombinants were constructed, expressed and purified successfully, and they were also identified as VLPs which were similar to the wild type HPV particles in terms of their biological and structural characteristics. Our results had laid the foundation of further study of the vaccine of HPV16E7.