幽门螺杆菌napA基因在乳酸菌中的表达及免疫原性分析

本实验目的是在乳酸菌中表达幽门螺杆菌（Helicobacter pylori,H.pylori） 中性粒细胞激活蛋白(NAP)，口服免疫小鼠后检测其免疫原性。在实验中利用了分子克隆技术构建携带nap基因的重组原核表达质粒pNICE:sec-nap,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NAP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-nap的乳酸菌、灭活的H.pylori 灌胃。免疫7次后检测其特异性IgG和IgA的产生。成功扩增了nap基因并构建了重组原核表达质粒pNICE:sec-nap，转化乳酸菌NZ9000后经nisin诱导可表达Mr约17kDa的重组蛋白，表达量约占菌体总蛋白量的9.5%，表达的蛋白能与兔抗H.pylori 血清特异性反应,具有良好的免疫原性。携带pNICE:sec-nap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H.pylori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。以上结果说明表达NAP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，对幽门螺杆菌疫苗的研究开发具有理论的意义。为乳酸菌作为抗原递送载体的研究和H.pylori口服疫苗的开发提供一定的实验基础。

Expression of Helicobacter pylori napA gene in Lactococcus lactis and its immunogenicity analysis

To develop an oral vaccine against Helicobacter pylori infection, the H.pylori napA gene, encoding the neutrophil-activating protein (NAP), was expressed in a live delivery vehicle lactococcus lactis. And the immunogenicity of the recombinant NAP was evaluated after the lactococcus lactis was administrated orally into mice. First, the napA gene of Helicobacter pylori was cloned into the prokaryotic expressive vector pNICE:sec. Second, the recombinant vector pNICE:sec-nap was transformated into Lactococcus lactis strain NZ9000 to express NAP protein. Then the recombinant NAP was induced to express and was identified by SDS-PAGE and Western-bloting. Finally, ICR mice were randomly divided into 4 groups and administrated orally with PBS, L.lactis pNICE:sec, L.lactis pNICE:sec-nap, and the inactivated H.pylori respectively. The specific IgG and IgA were identified after 7 times immunization. The results we got in this experiment were described as follows. The napA was amplified and cloned in the vector pNICE:sec successfully. The fusion protein (17kDa) was expressed in L.lactis by the induction of the nisin. The quantity of expression accounted for 9.5% of the total bacterial protein. Western bolt analysis confirmed that fusion protein could be recognized specifically by the serum of anti-H.pylori. Anti-NAP IgG titers in the serum of L.lactis pNICE:sec-nap group was higher than the L.lactis pNICE:sec group and no obviously difference with the inactivated H.pylori. The anti-NAP IgA titer in the intestinal mucosa of L.lactis pNICE:sec-nap group was higher than other groups. These results suggest that the recombinant L.lactis which expresses napA, may be applicable as an oral vaccine to induce protective immunity against H.pylori.