幽门螺杆菌hpaA基因在乳酸菌中的表达及免疫原性分析

摘要 目的 在乳酸菌中表达幽门螺杆菌(HP)hpaA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基因hpaA,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hpaA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-hpaA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hpaA基因并构建了重组原核表达质粒pNICE:sec-hpaA,转化乳酸菌NZ9000后经nisin诱导可表达Mr分子量约29KD的重组蛋白,表达量约占菌体总蛋白量的9.3%,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-hpaA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水平明显高于其它组（P<0.05）。结论 表达hpaA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。

Expression of Helicobacterpylori hpaA Gene in Lactococcus lactis and Its Immunogenicity Analysis

Abstract AIM To develop an oral vaccine against Helicobacter pylori infection, the H. pylori hpaA gene, was expressed in a live delivery vehicle lactococcus lactis. METHODS The immunogenicity of the recombinant hpaA was evaluated after the lactococcus lactiswas administrated orally into mice. First, the hpaA gene of Helicobacter pylori was amplified by PCR and cloned into the prokaryotic expressive vector pNICE:sec. Second, the recombinantvector pNICE:sec-hpaA was transformated into Lactococcus lactis strain NZ9000 to express hpaA protein. Then the recombinant hpaA was induced to express and was identified by SDS-PAGE and Western blot. Finally, ICR mice were randomly divided into 4 groups and administrated orally with PBS, L. lactis pNICE:sec, L. lactis pNICE:sec-hpaA, and the inactivated H. pylori respectively. The specific IgG and IgA were identified after 7 times immunization. RESULTS The results were described as follows. The hpaA was amplified and cloned into the vector pNICE: sec successfully. The fusion protein (29kDa) was expressed in L. lactis by the induction of the nisin. The quantity of expression accounted for 9.3 % of the total bacterial protein. Western bolt analysis confirmed that fusion protein could be recognized specifically by the serum of anti-H. pylori. Anti-hpaA IgG titers in the serum of L. lactis pNICE: sec-hpaA group was higher than the L. lactis pNICE:sec group and no obviously difference with the inactivated H. pylori. The anti-hpaA IgA titer in the intestinal mucosa of L lactis pNICE:sec-hpaA group was higher than other groups. CONCLUSION The results suggest that the recombinant L. lactis which expresses hpaA, may be applicable as an oral vaccine to induce protective immunity against H. pylori.