表达猪流行性腹泻病毒S1基因植物乳酸杆菌工程菌的免疫原性分析

Immunogenicity of engineered Lactobacillus plantarum expressing porcine epidemic diarrhea virus S1 gene

为探究表达猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)S1基因的植物乳杆菌工程菌是否对动物产生保护力,利用构建的含重组质粒pVE5523-S1乳杆菌口服免疫豚鼠3次,每次间隔14 d,每次2×10^(8) CFU/只。并以植物乳杆菌及含表达质粒pVE5523植物乳杆菌为阴性对照,进行相同处理。用猪流行性腹泻、猪传染性胃肠炎二联活疫苗(HB08株+ZJ08株)肌肉注射接种豚鼠,免疫3次,每次间隔14 d,每次0.2 mL/只,作为阳性对照。分别在免疫后的0 d、7 d、14 d、24 d、31 d、41 d及48 d对4组试验豚鼠心脏采血并分离血清,进行酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)检测抗体和中和试验分析,同时无菌采豚鼠脾脏,进行脾细胞增殖实验分析。结果显示,工程菌能刺激机体产生分泌性抗体sIgA、特异性中和抗体,还可刺激机体IL-4和IFN-γ的增长以及脾细胞的增殖。说明该PEDVS1基因植物乳杆菌工程菌使豚鼠对PEDV产生了特异性免疫力,为后续口服疫苗的研究奠定了基础。

To investigate whether the engineered Lactobacillus plantarum expressing the porcine epidemic diarrhea virus(PEDV)S1 gene can protect animals against PEDV,guinea pigs were fed with recombinant L.plantarum containing plasmid PVE5523-S1,with a dose of 2×10^(8) CFU/piece,three times a day,at 14 days intervals.Guinea pigs fed with wild type L.plantarum and the engineered L.plantarum containing empty plasmid pVE5523 were used as negative controls.For positive control,another group of guinea pigs were injected with live vaccine for porcine epidemic diarrhea and porcine infectious gastroenteritis(HB08+ZJ08)by intramuscular injection,with a dose of 0.2 mL/piece,three times a day,at 14 days intervals.Blood samples were collected from the hearts of the four groups of guinea pigs at 0 d,7 d,14 d,24 d,31 d,41 d and 48 d,respectively,and serum samples were isolated for antibody detection and neutralization test analysis by enzyme-linked immunosorbent assay(ELISA).The spleens of guinea pigs were also aseptically collected to perform spleen cells proliferation assay.The results showed that the engineered bacteria could stimulate the production of secretory antibody sIgA and specific neutralizing antibody,and stimulate the increase of IL-4 and IFN-γ,as well as the proliferation of spleen cells.These results indicated that the engineered L.plantarum containing PEDV S1 induced specific immunity toward PEDV in guinea pigs,which laid a foundation for subsequent oral vaccine development.