人乳头瘤病毒6b型L1基因的克隆及表达  

Cloning and expression of human papilloma virus type 6b-L1 gene

作　　者：邓列华[1] 计雄飞[2] 胡云峰[1] 殷董[3] 田静 郭秀枝[1] LIU Jie FAN Hong-tao 林泽[1] 赵永铿[1] 

机构地区：[1]暨南大学附属第一医院皮肤科,广州510630 [2]福建医科大学附属第一医院皮肤科,福州350005 [3]陕西省人民医院皮肤科,西安710068 [4]广州市天河区慢性病防治中心皮肤性病科,广州510000 [5]Program in Genetics and Genomic Medicine University of Maryland, Baltimore, USA [6]Medical Department of Johnson,Radnor PA, USA

出　　处：《解放军医学杂志》2011年第3期235-236,240,共3页Medical Journal of Chinese People's Liberation Army

基　　金：广东省自然科学基金(04021019);暨南大学自然科学基金团队项目(620026)

摘　　要：目的研究尖锐湿疣患者人乳头瘤病毒(HPV)6b型晚期基因L1(HPV6bL1)在真核细胞内的表达。方法 PCR技术扩增原核重组质粒PQE40-HPV6bL1中L1基因,酶切后与真核表达质粒PEGFP-C1连接,转化入感受态大肠埃希菌DH5α,经扩增后酶切鉴定,挑选阳性克隆进行测序。以重组质粒PEGFP-HPV6bL1转染COS-7细胞,荧光显微镜下观察融合蛋白的表达,RT-PCR检测HPV6bL1 mRNA的表达。结果双酶切及测序鉴定显示,重组质粒中插入的目的基因片段及载体DNA大小、方向和插入位点均正确,PEGFP-HPV6bL1构建成功。重组体成功转染进COS-7细胞,在荧光倒置显微镜下可观察到细胞内有绿色荧光蛋白表达,RT-PCR检测到HPV6bL1 mRNA的表达。结论建立了HPV6bL1绿色荧光真核表达系统,为研究该蛋白质的功能奠定了基础。Objective To investigate the expression of green fluorescent protein plasmid of human papilloma virus 6b L1 gene（HPV6bL1） in eukaryotic cells.Methods The L1 gene of PQE40-HPV6bL1 was amplified by PCR,purified by restriction enzyme digestion,and then connected to eukaryotic expression plasmid PEGFP-C1.The recombinant expression vector was then transformed into E.coli DH5a,which was identified by BamH Ⅰ and Hand Ⅲ digestion and the positive vector was selected.The recombinant plasmid PEGFP-HPV6bL1 was transfected into COS-7 cells by liposomal transfection technique and the expression of fusion protein was observed under fluorescence microscope.The generation of HPV6bL1 mRNA was detected by RT-PCR.Results Identification of PEGFP-HPV6bL1 by enzyme digestion and sequencing showed that the length,direction and inserted location of target,which was inserted into the recombinant,was correct and the expression of EGFP in transfected cell was observed.Conclusions A new type of green fluorescent HPV6bL1 eukaryotic expression system has been established.It may provide a research foundation for the study of the protein.

关 键 词：人乳头瘤病毒6 绿色荧光蛋白类 转染 基因表达 

分 类 号：R373[医药卫生—病原生物学]