人乳头瘤病毒16型晚期基因L1在原核细胞的表达及免疫效果观察  

Expression of human papillomavirus type16 late gene (L1) in prokaryotic cells and its immune effict observation

作　　者：刘朝奇[1] 许雪梅[2] 徐建青[2] 李昆[2] 司静懿[2] 刘世德[2] 

机构地区：[1]三峡大学分子生物学研究所,湖北宜昌443003 [2]中国医学科学院中国协和医科大学基础医学研究所

出　　处：《中国妇幼保健》2007年第35期5028-5032,共5页Maternal and Child Health Care of China

基　　金：国家自然科学基金资助(39680011)

摘　　要：目的：人乳头瘤病毒16型（HPV16）疫苗的研制可望防治宫颈癌的发生、发展，本实验从中国感染人群中克隆L1基因，期望制备中国人群的HPV16的预防性疫苗。方法：从HPV16阳性的尖锐湿疣临床标本中，PCR扩增HPV16L1基因，克隆、测序，并在原核细胞表达L1蛋白，纯化蛋白经过复性后，电镜观察病毒样颗粒（VLP），VLP免疫动物观察抗体产生情况。结果：克隆的HPV16L1，测序结果发现有4个特异性突变位点是nt6240的C-G、nt6432 A→G、nt6901-03及nt6951-53。将HPV16L1基因克隆到原核表达载体pET-28a（＋），高表达的蛋白应用蛋白转印证实是HPV16L1蛋白，纯化的蛋白复性后可以自组装VLP，免疫动物产生高滴度的抗体。结论：从临床标本克隆表达了HPVl6L1基因，可以在原核细胞高表达及体外组装VLP，并且具有良好的免疫原性。为研制HPV16预防性疫苗探索一条易于推广而经济的制备途径。Objective： Human papillomavirus typel6 vaccine is expected to prevent and cure cervical cancer. In the research, expeer to prepare the preventive vaccine of HPV16 of Chinese population by cloning L1 gene from infected crowd in China. Methods： The HPV 16 L1 gene was amplified by PCR from HPV16 positive condyloma acuminata clinical samples, then cloned, sequenced and expressed L1 protein in prokaryotic cells. The recombinant protein was purified and re - natured. VLP was identified with electronic microscopy. Finally immunized animals and observed the production of antibody. Results： The LI gene sequencing was as following： Four of the point mutations were C-G, nt6432 A→G, nt6901-03 and nt6951-53 in nt6240. The L1 gene was inserted into prokaryote expression vector pET- 28a （ ＋ ）. The protein was identified by Western blot. Purified protein after reannealing could self- assemble VLP , and could elicit high titers specific antibody in immunized animals. Conclusion： HPV16 L1 gene has been cloned from clinical samples, sequenced, expressed and self- assembled into VLP, which has a good immunogenicity. It makes a foundation for prophylactic vaccine of HPV16.

关 键 词：HPVL6 L1基因 蛋白表达 免疫 

分 类 号：R737.33[医药卫生—肿瘤]