人乳头瘤病毒11型DNA中CpG基序甲基化状态分析及生物活性的研究  

Methylation and biological activity of CpG motifs in human papillomavirus 11 DNA

作　　者：艾军华[1] 吕凤林[1] 左国伟[1] 金丹[1] 李元朝[1] 胡承香[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所第一研究室创伤烧伤复合伤国家重点实验室,重庆400042

出　　处：《免疫学杂志》2005年第1期29-32,36,共5页Immunological Journal

基　　金：国家自然科学基金 (30 4 0 0 386);重庆市科技攻关课题(0 1 6647)资助项目

摘　　要：目的　对人乳头瘤病毒 11型全核苷酸序列的CpG基序 (CpGmotifs)进行分析 ,为阐明尖锐湿疣的发病机制和提高DNA疫苗的效能奠定理论基础。方法　对从pBR32 2 HPV11重组质粒上酶切下来的HPV11全长DNA进行计算机分析 ,包括分类、计数和定位 ;并用限制性内切酶PCR法分析HPV11DNA中CpG基序的甲基化状态 ,即分别用甲基化敏感的限制性内切酶AccⅡ、HaeⅡ和HpaⅡ对HPV11DNA进行酶切消化 ,再以HpaⅡ切割位点侧翼序列为引物 ,以HpaⅡ酶切产物为模板 ,用聚合酶链反应 (PCR)扩增HPV11DNA片断 ,并用HPV11DNA刺激表达有pCIneo TLR9的HEK2 93细胞 ,ELISA法检测TNF al pha、IFN alpha和IL 12的分泌。结果　CpG的“C”的侧翼为两个嘌呤 ,“G”的侧翼为两个嘧啶 ,在HPV11中共 14个。HPV11全基因组被AccⅡ切成 2个片断、被HaeⅡ切成 3个片断、被HpaⅡ切成 5个片断 ,而且以HpaⅡ酶切产物为模板进行PCR扩增 ,未能得到相应的片断。HPV 11DNA刺激有人TLR9表达的HEK2 93细胞后能检测到TNF alpha、IFN alpha和IL 12的分泌。结论　乳头瘤病毒 11型DNA中含有GACGTT等非甲基化的CpG基序 。Objective To analyze the methylation of CpG motifs in human papillomavirus 11 nucleic acid for elucidating the mechanism of condyloma acuminata (CA) and optimizing the efficacy of the DNA vaccine. Methods The computer-assisted analysis was used to analyze the total number, classification, and localizations of CpG motifs in HPV11 total nucleic acids from the recombination plasmid pBR322-HPV11. Then the status of methylation of CpG motifs in HPV11 sequences was analyzed by restriction endonuclease polymerase chain reaction (PCR). The HPV11 total DNA was digested by restriction endonucleases Acc Ⅱ, Hae Ⅱ, and Hpa Ⅱ, respectively, then the HPV11 fraction was amplified by PCR with the flanked sequence of Hpa Ⅱ digest sites as primers and HpaⅡ digested products as template. The HEK293 cells with expression of pCIneo-TLR9 were stimulated with the HPV11 DNA, and the secretion of TNF-alpha, INF-alpha, and IL-12 were measured by ELISA kit. Results The CpG motifs was flanked by 5'purines and 3'pyimidines. The HPV11 contained 14 sequences. The HPV11 total genome was digested into two fractions by Acc Ⅱ, three fractions by Hae Ⅱ, and five fractions by Hpa Ⅱ, respectively, and had no corresponding fractions after PCR. The HPV11 DNA could induce the secretion of TNF-alpha, INF-alpha, and IL-12. Conclusion There are unmethylated CpG motifs with immunogenicity in the HPV11 entire sequences, such as GACGTT.

关 键 词：人乳头瘤病毒11型 CPG基序 核苷酸序列分析 限制性内切酶PCR法 

分 类 号：R346[医药卫生—基础医学]