表达幽门螺杆菌NAP蛋白的减毒沙门疫苗菌株预防幽门螺杆菌感染  

Prevention of the Helicobacter pylori infection in mice with recombinant attenuated Salmonella typhimurium vaccine expressing the neutrophil-activating protein

作　　者：焦志勇[1] 陈旻湖[1] 朱森林[1] 陈洁[1] 李国庆[1] 陈为[1] 胡品津[1] 

机构地区：[1]中山大学附属第一医院,广州510080

出　　处：《中国人兽共患病学报》2006年第7期629-632,共4页Chinese Journal of Zoonoses

基　　金：教育部2004年度新世纪优秀人才支持计划(NCET-04-0785);2002年广东省自然科学基金重点项目资助(021898);2002年广东省医学科研项目(B-2002027);美国CMB基金(098-677)

摘　　要：目的建立表达幽门螺杆菌(Helicobacterpylori,Hp)中性粒细胞活化蛋白(Hp-NAP)的减毒沙门疫苗菌,并利用人类幽门螺杆菌感染的小鼠模型研究疫苗菌对Hp感染的免疫保护作用。方法利用分子克隆技术构建携带Hp-NAP基因的重组原核表达质粒pTrc99A-NAP,经PCR及酶切鉴定后测定其基因序列,并与美国国立医学图书馆基因文库中相关序列的同源性进行比较。重组质粒pTrc99A-NAP转化减毒伤寒沙门菌SL3261,培养后筛选阳性菌落,抽提质粒进行PCR及酶切鉴定。表达的Hp-NAP蛋白用SDS-PAGE和West-blotting进行鉴定,用薄层扫描分析蛋白含量。C57BL/6小鼠随机分成4组,分别灌胃给予生理盐水(A组)、减毒鼠伤寒沙门菌SL3261(B组)、携带pTrc99A的SL326组(C组)、携带pTrc99A-NAP的SL326组重(D组)。免疫后4周,予Hp攻击,攻击菌量107CFU/只,灌胃2次,每日1次。攻击4周后处死动物,取胃组织分别行改良Giemsa染色及定量细菌培养,观察Hp定植情况。结果核苷酸序列测定及同源性分析证实,克隆的Hp-NAP基因与GenBank中相关序列的同源性为98%(397/402),氨基酸序列的同源性为98%(131/133)。pTrc99A-NAP转化的减毒沙门菌,可表达Mr约15000的Hp-NAP蛋白,表达量约占菌体蛋白量的37·5%;表达的新生蛋白能与小鼠抗Hp血清特异性反应,具有良好的免疫原性。同空白对照组、SL3261组和SL3261(pTrc99A)组相比,表达Hp-NAP的疫苗株组实验动物的胃组织Hp定植密度明显下降(P<0·05)。结论成功构建表达Hp-NAP的减毒沙门疫苗菌;重组疫苗菌对小鼠Hp感染具有一定免疫预防作用,可作为未来多组分重组减毒沙门疫苗菌的侯选菌株之一。To construct the attenuated Salmonella typhimurium strain SL3261 vaccine expressing the neutrophil-activating protein of Helicobacter pylori （Hp-NAP） and to investigate its immune protection in mouse model with human H. pylori infection, the recombinant prokaryotic expression plasmid pTrc99A-NAP was constructed with molecular clone techniques, and this expression vector was identified successively by PCR, enzyme digestion analysis and sequencing. The homology was compared with the cloned Hp-NAP gene and the related genes in GenBank. Meanwhile, the vaccine strain SL3261 was transformed by pTre99A-NAP. After cultivation, the positive colonies were picked out and identified with PCR and enzyme digestion analysis again. The expression of the Hp-HAP protein in SL3621 strain of bacteria was identified by SDS-PAGE, thin layer scanning and Western blotting analysis. BALB/c mice were randomly divided into 4 groups and immunized orally with normal saline （group A） , attenuated S. typhimurium SL3216 vaccine（group B）, SN3216 vaccine bearing pTrc99A（group C） and SN3216 vaccine bearing pTrc99A-NAP （group D）, respectively. Four weeks after the last immunization, all the mice were challenged with H. priori （1 × 10^7 CFU per mouse） twice through oral route, and then were sacrificed 4 weeks after challenging with H. pylori. The gastric tissue specimens were taken for examination with modified Giemsa staining and for quantitative cultivation of bacteria, to observe the colonization of H. priori. The experimental results showed that the homology between the cloned Hp-NAP gene and the related genes in GenBank could reach up to 98% of their nucleotide and the deduced amino acid sequences,and the expression of Hp-NAP protein could be detected in the cultural fluids of the cultivated SL3261 strain of bacteria. The relative molecular mass （Mr） of the expressed protein was about 150 000 and the quantity of expression accounted for 37.5% of the total bacterial protein. The expressed newly generated protein coul

关 键 词：幽门螺杆菌 Hp-NAP 减毒伤寒沙门菌 疫苗 免疫 

分 类 号：R378.2[医药卫生—病原生物学]