赖型钩体内鞭毛基因的扩增、克隆、核苷酸序列测定及其在大肠杆菌中的表达  

The PCR Amplification,Cloning, Sequencing,Expression in E;coli of Gene encoding Endoflagella Subunit Protein(fla B)from Leptospira interrogans serovar lai

作　　者：陈庄[1] 戴保民[1] 阎和平[1] 李胜富[1] 赵慧业 刘家福[1] 宋绍忠 杨耀 张云 刘富先 屠云人[1] 杨洪斌[1] 黄自英[1] 梁莉[1] 胡利贞[1] 赵慕愚[1] 

机构地区：[1]华西医科大学,成都生物制品研究所,四川省卫生防疫站

出　　处：《华西医科大学学报》1996年第1期10-16,共7页Journal of West China University of Medical Sciences

基　　金：高等学校博士学科点专项基金;卫生部科研基金

摘　　要：根据钩体内鞭毛蛋白（Ｅｎｄｏｆｌａｇｅｌｌｉｎ）ｆｌａＢ基因核苷酸序列自行设计一对引物，以赖型钩体ＤＮＡ为模板，用ＰＣＲ扩增到约８４０ｂｐ的片段。扩增片段经酶切（ＢａｍＨＩ、ＰｓｔＩ）定向克隆入ｐＵＣ８。重组质粒ｐＬＦ１插入片段与哈勒焦型钩体ｆｌａβ基因相比，核苷酸序列和推测的氨基酸序列同源性很高。ＳＤＳ－ＰＡＧＥ表明有一约３３ｋｄ的特异蛋白带，表达量占菌体蛋白１１％。免疫印迹试验表明此区带能被抗赖型钧体内鞭毛（轴丝）抗血清识别。首次以ＢＡＬＢ／ｃ小鼠钧体病模型为基础，用含重组质粒ｐＬＦ１的大肠杆菌作免疫原进行免疫保护试验，实验组存活率高子对照组，表现了一定程度的保护作用。本研究结果在所查国内外文献中尚属首次报道。A pair of oligonucleotide primers weredesigned by ourselves to amplify the endoflagella geneof L.interrogans serovar lai.Afragment about 840 bpwas generated with PCR and inserted into plasmidpUC8 after the fragrnent and pUC8 were digested re-spectively with Bam HI and Pst I.Arecombinant plas-mid(designated as pLF1) was obtained. SDS-PAGEanalysis indicated that a 33 kd was expressed in E, coliJM103 harboring pLF1 and the expression level of theprotcin was 11%of total bacterial soluble proteins.Western blot analysis showed that the protein bandcould be recognized by the antiserum against theendoflagella ( Axiall filament ) of Leptospira inter-rogans serovar lai.Nucleotide seguence data showed anopen reading frame encoding 282 aminoacids residues,corresponding to a protein of molecular weight 33.6kd.The G+C content of endoflagella subunit proteingene was 48 mol%, Therefore, the G+C content ofthe leptospiral fla B Gene is significantly higher thanthe reported 39 mol%G+C content of leptospiralgenorne of L. interrogans serovar lai but similar to theG+C of the Treponema pallidum genome. Comparisonof the deduced endoflagellar subunit protein (fla B )amino acid sequence with flagellins from other bacteriarevealed a high level of identity with the Trebonemapallidum, fla B proteins. Immunization/protection ex-periment was performed on the rnodel of BALB/c miceand showed that the survival rate in the group JM103-pLF1 was higher than that in the group JM103-pUC8,but statistically the difference between them wassignificant (P<0. 05 )and pLF1 did not induce signifi-cant levels of agglutinating antibodies against L.inter-rogans serovar lai.

关 键 词：钩端螺旋体 内鞭毛 基因扩增 聚合酶链反应 克隆 

分 类 号：R377.5[医药卫生—病原生物学]