钩体017株基因库pDJH_2与L．alstoni重组DNA探针同源性及pDJH_2在大肠杆菌中表达产物的分析  

Homology of the Recombinant DNA of Plasmid pDJH_2 with the Recombinant DNA Probe of L. alstoni and Analysis of Its Expression in Escherichia coli

作　　者：江南[1] 戴保民[1] 李胜富[1] 赵慧业 刘家福[1] 宋绍忠 杨耀 张云 屠云人[1] 杨洪彬[1] 黄自英[1] 梁莉[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》1995年第3期241-247,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：用地高辛标记含完整ＯｍｐＬ１结构基因之Ｌ．ａｌｓｔｏｎｉ２．５ｋｂＥｃｏＲ１重组ＤＮＡ探针与我们构建的赖型钩体０１７株基因库重组质粒ｐＤＪＨ＿２进行ＤＮＡ－ＤＮＡ杂交。结果显示：该重组ＤＮＡ探针与ｐＤＪＨ＿２１．９ｋｂ插入片段同源；使用ＩＰＴＧ诱导重组质粒ｐＤＪＨ＿２后，其在大肠杆菌中表达了６８ｋｄ产物。经蛋白酶Ｋ消化、免疫印迹及斑点一酶联免疫吸附试验，提示此６８ｋｂ产物是蛋内质抗原；用该６８ｋｂ蛋白质抗原主动免疫ＢＡＬＢ／ｃ小鼠，可抵抗强毒力赖型钩体０１７株的攻击，表现出对小鼠的免疫保护性，从而为赖型钩体０１７株基因工程疫苗的研制奠定了一定基础。此赖型０１７株钩体重组ＤＮＡ经ＩＰＴＧ诱导后能在大肠杆茵中表达，以及其表达产物的保护性动物试验结果，在所查国内外文献中尚属首次报道。Dlg-labeled recombinant DNA probe ofL.alstoni which contains the entire structural OmpL1gene was hybridized with the recombinant DNA of theplasmid named pDJH_2 of the gene library of L. interro-gans serovar lai strain 017.The result showed a highdegree of homology among them ; expression of recom-binant DNA of pDJH_2 was achieved by β-D-galactosi-dase(IPTG ) induction in E. coli. The molecularweight of this product is 68kd. Then they were treatedwith proteinase K and subjected to SDS-PAGE.The results showed it is a protein in nature. Using the spe- cific monoclonal antibody E_4B_8D_5 on immunoblottingand specific polyclonal antibedy on dot-ELISA assay,we investigated the immune reaction and noticed thatprotein 68kd might be an antigen in character.E. coli which contains the recombinant plasmidpmH_2 were injected into BALB/c mice,Then the micewere challenged by leptospires of the strong virulencestrain 0 1 7.but all the infected mice survived.In this paper,we first report the expression of re-combinant DNA of L.interrogans serovar lai strain 017in E. coli when injected with IPTG, and immunopro- tection of BALB/c mice which were injected with theexpression against the infection of L. inierrogansserovar lai strain 017.pDJH_2,may be the first recombi-nant for which the gene has been cloned and its ex-pression product 68kd may be the immunoprotectiveantigens.

关 键 词：钩端螺旋体 重组质粒 基因表达 pDJH2 

分 类 号：R377.5[医药卫生—病原生物学]