摘要
分析广东分离株HPV16型的L2和E7基因结构,并对E7的4个功能区重新排列组合形成失去致癌作用但其抗原表位不改变,其命名为rE7,构建pET-28a-L2、pET-28a-rE7、pET-28a-L2-rE7、pET-28a-rE7-L24个原核表达质粒.采用PCR技术从广东分离株HPV16基因组中扩增L2基因;用重叠PCR技术人工合成rE7基因和L2-rE7、rE7-L2融合基因,并构建到原核表达载体pET-28a(+)上.成功扩增广东分离株HPV16型L2基因,经重叠PCR技术成功得到rE7、L2-rE7和rE7-L2基因并成功构建了原核表达的4个载体:pET-28a-L2、pET-28a-rE7、pET-28a-L2-rE7、pET-28a-rE7-L2.广东分离株HPV16型L2基因与中国标准株有9处不同,同源性为99.37%,其编码氨基酸序列有8处突变.成功合成失去致癌作用的基因rE7及构建4个原核表达载体.
This study was aimed to investigate the structures of HPV16L2 and E7 gene.We have generated a recombinant E7 gene（rE7） in which four domains were rearranged and,in order to maintain all possible T cell epitopes,certain sequences were duplicated.Four prokaryotic expression vectors pET-28a-L2,pET-28a-rE7,pET-28a-L2-rE7,pET-28a-rE7-L2 were constructed.The HPV16L2 gene was amplified from human cervical carcinoma tissues by PCR;The HPV16rE7 gene was synthsized by using overlapping multi-step PCR.HPV16L2 and HPV16rE7 were linked together by using overlapping PCR technology and then integrated into L2-rE7 and rE7-L2.L2,rE7,L2-rE7 and rE7-L2,which were cloned into prokaryotic expression vector pET28a.Gene HPV16L2 and HPV16rE7 were successfully amplified by using overlapping PCR,and prokaryotic expression vectors pET-28a-L2,pET-28a-rE7,pET-28a-L2-rE7,pET-28a-rE7-L2 were constructed.The Sequence of HPV16L2 gene was obtained in which there were 9 nucleotides different between Guangdong strain and Chinese standard strain,but the homology was 99.37%.Subsequently,the differences lead to change in 8 amino acids.The rE7 gene that lost the effects of carcinogenicity was synthesized.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2010年第3期312-317,共6页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
国家自然科学基金重点项目(30730087)
教育部留学回国人员科研启动基金项目
广东省科技攻关项目(2007B020709003)
The Project-sponsored by SRF for ROCS
SEM~~
