临床淋病奈瑟菌外膜蛋白PⅠ基因重组质粒的构建与生物信息分析  

CONSTRUCTION OF NEISSERIA GONORRHOEAE PⅠ GENE RECOMBINANTS FROM CLINICAL ISOLATES AND THE BIOINFORMATICS ANALYSIS

作　　者：滕毅[1] 汪东篱 雍刚[3] 谢志梅[1] 陈恒[1] 叶盛[1] 裴晓方[1] 

机构地区：[1]四川大学华西公共卫生学院医学检验教研室,成都610041 [2]深圳市宝安区公明镇防保所 [3]四川省医学科学院.四川省人民医院皮肤病性病研究所

出　　处：《现代预防医学》2007年第19期3621-3624,共4页Modern Preventive Medicine

基　　金：四川大学科技创新基金项目(2004CF02)

摘　　要：[目的]研究淋病奈瑟菌临床分离株外膜蛋白PⅠ基因的结构特点和变异情况,为淋球菌疫苗靶位点的选取提供参考。[方法]提取9株成都地区临床分离淋球菌的基因组,PCR扩增PⅠ基因,与表达载体pET-30b(+)构建重组质粒pET-30b(+)-PⅠ,重组子经双酶切鉴定后测序,利用一系列生物软件对序列进行生物信息学分析。[结果]重组子经双酶切后,在5400 bp和900 bp左右分别得到条带,与预期片断的大小相符合;各PⅠ序列在GeneBank中均搜索到相似性大于98.9%的相似序列,根据碱基数目及序列同源性比较结果,4条属于PⅠA亚型,5条属于PⅠB亚型;PⅠA亚型较PⅠB亚型(除2-7外)含有相对较高的α-螺旋和β-折叠结构,具有单一筒状的三级结构,而PⅠB亚型含有相对较高的环或其他结构,其中的7-2具有相连接的两筒状结构。[结论]成功构建9个pET-30b(+)-PⅠ重组子;PⅠA和PⅠB两亚型核苷酸数目、对应蛋白的二级结构和三级结构均有差异,两亚型内各核苷酸和氨基酸序列的变异程度也不同,本研究结果将为PⅠ蛋白抗原靶位的筛选提供参考。[Objective]To study the variation of Neisseria gonorrhoeae PⅠGene obtained from clinical isolates for the purpose of potential vaccine component protein selections.[Methods]The different PⅠ gene fragments were obtained with PCR amplification of Neisseria gonorrhoeae genomic DNA extracted from 9 clinial strains isolated in Chengdu.They were ligated with prokaryotic exprssion vector pET-30b.The resulting recombinants which designed as pET-30b（＋）-PⅠ plus the strain number were confirmed by EcoR Ⅰand Xho Ⅰ digestion and sequencing analysis.Different software were applied to analyze the DNA and resulting protein structures.[Results]After digesting with EcoR Ⅰ and Xho Ⅰ,two bands were observed at about 900 bp and 5 400 bp position respectively,which were in accordance with the size of the insert and the vector.Similar sequences（homology≥98.9%）with each PⅠ sequence obtained have been found in GeneBank after homologous comparison.By comparing these nine PⅠ DNA sequences,four of them were PⅠA subtype and the remaining were fallen into the subtype PⅠB.Moreα-helix and β-sheets have been found in subtype PⅠA（except 2-7 strain）with the predicted single cylinder tertiary structure,while subtype PⅠB demonstrated more loop and other structures with a predicted two connective cylinder for strain 7-2 PⅠ.[Conclution]Nine pET-30b（＋）-PⅠs were constructed successfully.The number of the nucleotides,the predicted protein secondary and tertiary structures between PⅠA and PⅠB subtype were different.The mutation degree of nucleotide and amino acid sequences within the same subtype were also different.These results will be very helpful for the further study of PⅠ antigenic domains as the potential vaccine components.

关 键 词：淋球菌 外膜蛋白PⅠ 重组质粒 生物信息分析 

分 类 号：R378.16[医药卫生—病原生物学]