重组抗原LTB-UreB-HpaA对幽门螺杆菌感染小鼠的免疫保护性及内置佐剂LTB的免疫增强作用(英文)

Immunoprotection of recombinant antigen LTB-UreB-HpaA in Helicobacter pylori infected mice and immunoenhancement of the inner adjuvant LTB

目的构建大肠杆菌不耐热肠毒素B亚单位(LTB)和幽门螺杆菌(Hp)尿素酶B亚单位(UreB)、幽门螺杆菌粘附素A(HpaA)的融合基因ltBureBhpaA及其原核表达系统,鉴定重组表达产物rLTBUreBHpaA的免疫原性、佐剂活性及对Hp感染小鼠的保护作用。方法采用连接引物PCR构建ltBureBhpaA融合基因,TA克隆后测序。采用pET42a质粒及其宿主菌E.coliBL21DE3亚克隆构建原核表达系统pET42altBureBhpaAE.coliBL21DE3,并用不同浓度IPTG诱导表达。SDSPAGE用于检测rLTBUreBHpaA的表达及其产量,免疫双扩散试验及Western印迹法检测rLTBUreBHpaA抗原性和免疫反应性。建立牛GM1的ELISA(GM1ELISA)检测重组蛋白中rLTB的佐剂活性。采用HpSS1株BaLb/C小鼠感染模型,检测rLTBUreBHpaA的免疫保护作用。结果ltBureBhpaA核苷酸序列与各原始基因序列完全相同。不同浓度的IPTG均可诱导pET42altBureBhpaAE.coliBL21DE3表达rLTBUreBHpaA,其产量约为细菌总蛋白的15%。rLTBUreBHpaA家兔抗血清的双扩效价为1∶8。商品化兔抗Hp全菌抗体、兔抗UreB或HpaA均能识别rLTBUreBHpaA并与之结合。GM1ELISA结果证实rLTBUreBHpaA仍具有结合牛GM1的活性。rLTBUreBHpaA(200μg/每只小鼠)免疫后,可使小鼠100%免于HpSS1株的感染。10μgrLTB与rUreB或rHpa共同免疫小鼠,可使保护率分别从66.7%提高至81.8%和83.3%。结论本研究成功地构建了ltBureBhpaA融合基因及其原核表达系统。目的表达产物rLTBUreBHpaA有良好的免疫原性、佐剂活性及免疫保护效果,具有作为Hp基因工程疫苗的应用前景。

In order to increase antigenicity of H. py/ori-specific antigens and decrease the cost of further industrial production, we used a special PCR with linking primers to construct a fusion gene containing H. pylori ureB and hpaA genes and E. coli ltB gene, and to costract its prokaryotic expression system pET42a-ltB-ureB-hpaA-E, coliBL21DE3. The sequencing result indicated the 100 % nucleotide sequence homology of the constructed ltB-ureB-hpaA fusion gene compared to those of the original separated genes.Output of the target recombinant protein rLTB-UreB-HpaA was approximate 15 % of the total bacterial proteins measured by SDSPAGE. The rLTB-UreB-HpaA could induce the immunized rabbits to produce specific antibodies with immunodiffusion titer of 1：8,and could combine to the commercial rabbit antibody against the whole cell of H. pylori as well as rabbit anti-UreB and anti-HpaA sera by using Western bolt assays. Using GM1-ELISA, the ability of rLTB-UreB-HpaA binding to bovine GM1 was confirmed. And rLTB-UreB-HpaA （200μg per mouse） could prevent 100% of the immunized BaLb/C mice from H. pylori strain SS1 infection. The co-administration with 10 μg rLTB, the rUreB or rHpa could increase its protective rates in the immunized mice from 66.7% to 81.8% and 83.3 %, respectively. All these data leads a conclusion that rLTB-UreB-HpaA is a great potential as a practical genetic engineering vaccine to prevent H. pylori infection.