幽门螺杆菌alpA基因在乳酸菌中的表达及免疫原性分析

发布时间：2018-01-14 08:05

本文关键词：幽门螺杆菌alpA基因在乳酸菌中的表达及免疫原性分析　出处：《细胞与分子免疫学杂志》2010年03期 　论文类型：期刊论文

【摘要】：目的:在乳酸菌中表达幽门螺杆菌(Hp)黏附素al-pA基因,口服免疫小鼠后检测其免疫原性。方法:PCR方法从基因组中扩增alpA基因,将其克隆至表达载体pNICE:sec,将重组质粒转化乳酸菌NZ9000株,筛选鉴定阳性菌落,诱导表达的alpA蛋白用SDS-PAGE和Western blot进行鉴定。将雌性ICR(CV级)小鼠随机分为4组,分别用PBS、携带空质粒的乳酸菌、携带pNICE:sec-alpA的乳酸菌、灭活的Hp灌胃。免疫7次后检测其特异性IgG和IgA的产生。结果:扩增alpA基因并构建了重组原核表达质粒pNICE:sec-alpA,转化乳酸菌NZ9000后经nisin诱导可表达Mr约56 000的重组蛋白,表达量约占菌体总蛋白量的9.6%,Western blot分析其能与幽门螺杆菌抗血清特异性反应,具有良好的免疫原性。携带pNICE:sec-alpA质粒的乳酸菌刺激产生的IgG水平明显高于携带空质粒组,与灭活Hp组没有明显的差异,但其刺激产生的IgA水平明显高于其他组(P0.05)。结论:表达al-pA蛋白的乳酸菌口服免疫小鼠后,能够刺激小鼠产生特异的IgG和IgA,可作为幽门螺杆菌口服疫苗候选抗原。为乳酸菌作为抗原递送载体的研究和Hp口服疫苗的开发提供一定的实验基础。
[Abstract]:Objective: to express the adhesion al-pA gene of Helicobacter pylori (HP) in lactic acid bacteria and to detect its immunogenicity after oral immunization. The recombinant plasmid was transformed into NZ9000 strain of lactic acid bacteria, and positive colonies were screened and identified. The induced expression of alpA protein was identified by SDS-PAGE and Western blot. The female ICR(CV mice were randomly divided into 4 groups with PBS. Lactobacillus carrying empty plasmids and lactic acid bacteria carrying pNICE:sec-alpA. The production of specific IgG and IgA were detected after 7 times of immunization. Results: the alpA gene was amplified and the recombinant prokaryotic expression plasmid pNICE:sec-alpA was constructed. After the transformation of lactic acid bacteria NZ9000, the recombinant protein of Mr 56 000 was induced by nisin, and the expression amount was about 9.6% of the total bacterial protein. Western blot analysis showed that it could react specifically with Helicobacter pylori antiserum. The level of IgG produced by lactic acid bacteria carrying pNICE:sec-alpA plasmid was significantly higher than that of empty plasmid group, and there was no significant difference between the two groups. However, the level of IgA stimulated by Lactic acid bacteria was significantly higher than that of other groups (P 0.05). Conclusion: lactic acid bacteria expressing al-pA protein can stimulate mice to produce specific IgG and IgA after oral immunization. It can be used as the candidate antigen of Helicobacter pylori oral vaccine, and provide some experimental basis for the study of lactic acid bacteria as antigen delivery carrier and the development of HP oral vaccine.
【作者单位】：中国医学科学院/北京协和医学院医学生物学研究所;
【基金】：云南省自然科学基金资助项目(2007C144M)
【分类号】：R392.11
【正文快照】： 幽门螺杆菌(Helicobacter pylori,Hp)是寄生于胃上皮细胞表面的革兰阴性微需氧菌[1],其感染可导致急、慢性活动性胃炎,消化性溃疡,胃腺癌等疾病。现有的根除Hp的方法主要是三联和四联抗菌疗法,但是Hp的抗感染治疗存在治愈率不高且不能有效阻止Hp的再感染和复发等问题。由于越

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