乳酸菌融合表达传染性法氏囊病毒VP2蛋白与大肠杆菌LTB及其诱导免疫应答的研究

为研究传染性法氏囊病毒(IBDV)保护性抗原VP2与E.coli不耐热肠毒素B亚单位(LTB)在戊糖乳杆菌(L.pentosus)中的共表达及其免疫原性,本研究以乳酸杆菌表面表达型和分泌表达型质粒pPG-1和pPG-2为载体,以VP2为目的基因,构建VP2基因单独表达及与LTB基因融合表达的4种重组L.pentosus,分别命名为pPG-1-VP2/L.pentosus、pPG-1-VP2-LTB/L.pentosus、pPG-2-VP2/L.pentosus及pPG-2-VP2-LTB/L.pentosus。表达的重组蛋白分子量大小分别约为47 ku、55 ku、45 ku及53ku。将构建的重组L.pentosus分别口服免疫SPF雏鸡,以IDEXX试剂盒测定体液免疫应答水平,结果显示,不同表达方式的重组L.pentosus均可以刺激机体产生特异性循环抗体和分泌抗体(sIgA),其中pPG-2-VP2-LTB/L.pentosus诱导产生的抗体滴度高于其它组。MTT法检测不同表达方式的重组乳酸菌免疫雏鸡外周血淋巴细胞增殖反应,结果显示特异性抗原对免疫雏鸡淋巴细胞的增殖指数显著高于未免疫组,表明重组菌能够刺激机体产生特异性细胞免疫应答。这些结果表明4种重组菌均能够刺激机体产生局部黏膜免疫和全身系统免疫应答;并且带有黏膜免疫佐剂LTB融合表达试验组高于VP2蛋白单独表达组。

Co-expression of IBDV VP2 protein with E.coli LTB protein in Lactobacillus and the immune response induction in chickens

To investigate the immune effect of VP2 protein of infectious bursal disease virus(IBDV) and E.coli heat-labile toxin B subunit(LTB) fusion protein expressed in Lactobacillus pentosus,four recombinant L.pentosus expressing VP2 and LTB gene were constructed with lactobacillus based expressing plasmids of pPG-1 and pPG-2,respectively,which were designated pPG-1-VP2/L.pentosus,pPG-1-VP2-LTB/L.pentosus,pPG-2-VP2/L.pentosus and pPG-2-VP2-LTB/L.pentosus.They were induced by 2% lactose in MRS medium and identified by SDS-PAGE and western blot.The results showed that the four recombinant proteins were approximately 47 ku,55 ku,45 ku and 53 ku respectively.Chickens were inoculated with the recombinant strains by oral administration,respectively.The results showed that all he strains were able to induce specific serum IgG and mucosal IgA and the antibody titer induced by pPG-2-VP2-LTB/L.pentosus was higher than by others strains.Moreover,the lymphocyte proliferation index was higher in the immunized chickens than that in the control group after stimulated with specific antigen.In conclusion,these results indicated that all the recombinant L.pentosus was able to induce both mucosal and systemic immune response,and the immune responses with the LTB fusion proteins were stronger than the VP2 lonely.