幽门螺杆菌HspA乳酸菌疫苗构建及免疫反应性鉴定  

Construction and immunological identification of lactic acid bacteria vaccine to Helicobacter pylori HspA

作　　者：张小娟[1] 王新彩[1] 段广才[2] 张荣光[3] 

机构地区：[1]河南科技大学医学院病原生物学教研室,河南洛阳471003 [2]郑州大学公共卫生学院流行病学教研室 [3]郑州大学基础医学院寄生虫学教研室

出　　处：《中国公共卫生》2013年第7期1070-1073,共4页Chinese Journal of Public Health

基　　金：中国博士后科学基金(200801273)

摘　　要：目的探讨幽门螺杆菌(Hp)热休克蛋白A(HspA)在食品级乳酸乳球菌NZ3900菌株中的克隆表达及免疫反应性。方法利用基因重组技术构建乳酸乳球菌重组子NZ3900/pNZ8110-hspA;绘制生长曲线,观察hs-pA插入对乳酸乳球菌重组子生长影响及乳酸链球菌素(Nisin)对重组子生长影响;采用钠十二烷基硫酸盐-聚丙烯酰胺凝胶电泳法(SDS-PAGE)检测HspA在乳酸乳球菌中的表达;应用western-blot鉴定重组子表达HspA的免疫反应性。结果成功扩增出幽门螺杆菌河南分离株(MEL-Hp27)的hspA基因,构建了hspA基因的乳酸乳球菌原核表达系统(NICE);细菌生长曲线显示hspA的插入未对乳酸乳球菌重组子的生长产生影响,除10 ng/mL Nisin对重组子生长影响较小外,20～100 ng/mL nisin对重组子的生长均有明显抑制;SDS-PAGE和Tricine SDS-PAGE检测均未观察到HspA条带;western-blot鉴定结果显示乳酸乳球菌表达的HspA抗原蛋白具有良好免疫反应性。结论HspA在乳酸乳球菌中的少量表达影响重组菌生长。Objective To explore the expression and immune activity of Helicobacter pylori（Hp） heat shock protein A（HspA） in Lactococcus lactis（L.lactis）NZ3900,a food-grade delivery vehicle.Methods NZ3900/pNZ8110-hspA was constructed using gene recombination technique.Growth curves were plotted to study whether the growth of recombinant bacteria was affected after exogenous gene hspA cloned into L.lactis and whether the growth of empty bacteria,empty plasmid bacteria and recombinant bacteria was affected after treatment of nisin at different concentrations.Sodium dodecylsulfate polyacrglaide gel electrophoreses（SDS-PAGE） and tricine SDS-PAGE were used to detect HspA expressed in recombinant bacteria.Western blot was adopted to confirm whether the HspA expressed by transformant bacteria having immunoreactivity.Results The hspA gene was amplified from genome DNA of Hp MEL-Hp27 and then cloned in the E.coli-L.lactis shuttle vector pNZ8110 and transformed into E.coli MC1061.The positive recombinant plasmid was electro-transformed into L.lactis NZ3900.There was no obvious affection observed from the growth curve after exogenous gene hspA cloned into L.lactis NZ3900.The growth of recombinant bacteria was affected after Nisin treatment at different concentrations except for 10 ng/ml Nisin treatment,while the growth of empty bacteria,empty plasmid bacteria was not affected by different concentrations of nisin.No HspA strip was observed in SDS-PAGE and tricine SDS-PAGE analyses.Western blot analysis showed that HspA expressed by recombinant bacteria had good immunoreactivity.Conclusion The growth of recombinant bacteria was affected by the expressioin of HspA.

关 键 词：幽门螺杆菌(Hp) 乳酸乳球菌 原核表达系统(NICE) 热休克蛋白A(HspA) 

分 类 号：R37[医药卫生—病原生物学]