不同猪源受体菌表达猪流行性腹泻病毒保护性抗原S1诱导免疫应答的比较研究

Comparative Study on the Immune Response Induced by the Different PorcineReceptor Bacteria with Expressing the Protective Antigen S1 ofPorcine Epidemic Diarrhea Virus

旨在比较猪源副干酪乳酪杆菌(Lacticaseibacillus paracasei)、罗伊氏黏液乳杆菌(Limosilactobacillus reuteri)、约氏乳杆菌(Lactobacillus johnsonii)作为口服疫苗载体,表达外源蛋白刺激仔猪产生免疫能力的强弱,以期选取合适乳酸菌作为受体菌载体。本研究首先体外鉴定表达PEDV S1蛋白的重组猪源副干酪乳酪杆菌(pPG-T7g10-S1/L.paracasei 27-2)、猪源罗伊氏黏液乳杆菌(pPG-T7g10-S1/L.reuteri J31)、猪源约氏乳酸杆菌(pPG-T7g10-S1/L.johnsonii 6332)的耐酸耐胆盐能力,考察3株重组菌的抗逆性。结果表明,3株重组菌均能够耐受酸和胆盐环境,且与其野生型菌株没有显著差异。接下来为比较3株重组菌的免疫效果,口服免疫初生仔猪后,利用间接ELISA和中和试验检测仔猪产生的特异性抗体水平及其中和活性;并测定免疫后仔猪血清和肠黏膜中各细胞因子水平。结果显示,口服免疫后,与对照组相比,3株免疫组仔猪血清IgG抗体和鼻拭子、肛拭子、肠黏液中SIgA抗体水平均显著升高,且可持续至第28天左右,其中pPG-T7g10-S1/L.paracasei 27-2组诱导产生的抗体水平显著高于其他两组免疫组(P<0.05);仔猪产生特异性的IgG和SIgA对PEDV均具有中和活性。仔猪血清中细胞因子IFN-γ、IL-2、IL-4、IL-10水平和对照组相比显著升高(P<0.05),但3株重组菌组间细胞因子水平未见明显差异;仔猪空肠黏膜中细胞因子IFN-γ、IL-2、IL-4、IL-5、IL-6、IL-17、IL-21、TGF-β、APRIL和BALL水平与对照组相比有所升高,且pPG-T7g10-S1/L.paracasei 27-2组IL-4、IL-5、IL-6、TGF-β、IL-17、IL-21和BALL相比其他组显著升高(P<0.05)。综上所述,本研究分别将质粒组成型表达PEDV主要保护性抗原S1的重组猪源副干酪乳酪杆菌、猪源罗伊氏黏液乳杆菌和猪源约氏乳酸杆菌口服免疫仔猪,结果显示能够刺激机体产生针对PEDV的黏膜免疫、体液免疫和细胞免疫,且相较于其他2株重组菌,重组猪源副干酪乳酪杆菌pPG-T7g10-S1/L.paracasei 27-2的口服免疫效果最好。该试验结果为构建更为有效的乳酸菌口服疫苗提供了科学数据。

The aim was to compare the strength of Lacticaseibacillus paracasei of porcine origin,Limosilactobacillus reuteri of porcine origin,and Lactobacillus johnsonii of porcine origin as oral vaccine vectors to express exogenous proteins to stimulate immunity production in piglets with a view to selecting suitable lactic acid bacteria as receptor vectors.In this study,the acid and bile salt tolerance of recombinant porcine Lacticaseibacillus paracasei(pPG-T7g10-S1/L.paracasei 27-2),Limosilactobacillus reuteri(pPG-T7g10-S1/L.reuteri J 31)and Lactobacillus johnsonii(pPG-T7g10-S1/L.johnsonii 6332)were measured in vitro to investigate the resistance of the three strains expressing PEDV S1 protein.The results showed that all the three recombinant strains could tolerate acid and bile salt environment,and there was no significant difference with their wild type strains.Then to compare the immune effects,the newborn piglets were orally immunized with the three recombinant strains,separately.Indirect ELISA and neutralization test were used to detect the specific antibodies and neutralizing activity of the piglets,and the levels of each cytokine in serum and intestinal mucosa of the immunized piglets were measured.The results showed that after oral immunization,compared with the control group,the levels of serum IgG antibody and SIgA antibody in nasal swabs,anal swabs and intestinal mucus of piglets in the three recombinant strains groups were significantly increased,lasted to the 28th day.The antibody level induced by pPG-T7g10-S1/L.paracasei 27-2 group was significantly higher than that of the other two groups(P<0.05).Pigley-produced specific IgG and SIgA both showed neutralizing activity against PEDV.The levels of IFN-γ,IL-2,IL-4 and IL-10 in the serum of piglets were significantly higher than those in the control group(P<0.05),but there was no significant difference in the levels of cytokines among the three recombinant strains.The levels of cytokines IFN-γ,IL-2,IL-4,IL-5,IL-6,IL-17,IL-21,TGF-β,APRIL and BALL in the jejunal mucosa of piglets were higher than those in the control group.The levels of IL-4,IL-5,IL-6,TGF-β,IL-17,IL-21 and BALL in pPG-T7g10-S1/L.paracasei 27-2 group were significantly higher than those in other groups(P<0.05).In conclusion,in this study,oral immunization of piglets with recombinant L.paracase i,L.reuteri and L.johnsonii,constitutively expressing the major protective antigen S1 of PEDV,showed that they could stimulate mucosal immunity,humoral immunity and cellular immunity against PEDV.Compared with the other two recombinant strains,the recombinant porcine pPG-T7g10-S1/L.paracasei 27-2 had the best oral immunization effect.The results of this experiment provide scientific data for the construction of a more effective oral vaccine for lactic acid bacteria.