猪A组轮状病毒主要蛋白的编码基因DNA疫苗联合免疫的研究

摘要

To evaluate the efficacy of combined immunization of recombinant plasmid DNA vaccines expressing hemagglutinin (VP4),group antigen (VP6) and neutralizing antigen (VP7) proteins of porcine rotavirus (PRV),The eukaryotic recombinant expression plasmids of pVAX1-VP4,pVAX1-VP6 and pVAX1-VP7 were constructed and transient expression of target proteins were verified by indirect immunofluorescence assay after transfecting into MA-104 cells,respectively.BALB/c mice were immunized with 100 μg of pVAX1-VP4+pVAX1-VP7 (group B) and 100 μg of pVAX1-VP4+pVAX1-VP6+pVAX1-VP7 (group A) via intramuscular route,two boosters were given by the same way at 2 weeks interval for each group.Blood serum samples were collected at different days post immunization and tested for ELISA antibody against PRV-A,the results showed that the antibodies against rotavirus of mice turned positive in group B on 14 and 42 days (P/N ≥ 2),while antibody in group A turned positive on 14,28 and 42 days (P/N ≥ 2),respectively.Results of spleen immune related cells tests showed that lymphocyte ratio of CD4+/CD8+ and the number of CD19+ B cells in group A were higher than that in group B,and there were significant differences (p＜0.05) between the group A,group B and control group.The results of lymphocyte transformation test showed that the average value of SI in group A were higher than that in group B generally,and there were significant differences (p＜0.05) between group A and group B on 42 days.All the results showed that the combined immunization with 3 recombinant DNA vaccine were able to induce strong humoral and cell-mediated immune responses in mice,these results lay foundations for developing PRV-A vaccine.%为研究猪A组轮状病毒(PRV-A)血凝蛋白(VP4)、群抗原蛋白(VP6)和中和抗原蛋白(VP7)编码基因疫苗联合免疫的效力,本研究将表达PRV-A VP4、VP6、VP7蛋白基因的真核表达质粒pVAX1-VP4、pVAX1-VP6、pVAX 1-VP7以pVAX 1-VP4+pVAX 1-VP7 (B组)和pVAX1-VP4+pVAX1-VP6+pVAX 1-VP7 (A组)设计试验组,分别以100 μg/只经肌肉注射途径免疫BLAL/c小鼠,间隔2周以相同剂量、途径加强免疫,共免疫3次,免疫后不同时间采血,采用ELISA检测血清抗体.结果显示,B组在免疫后14 d和42 d检测到RV抗体阳性(P/N≥2),A组在免疫后14d、28 d、42 d均检测到RV抗体阳性(P/N≥2);脾脏免疫相关细胞测定结果表明,A组的CD4+/CD8+T淋巴细胞比值和CD19+B细胞数量均高于B组,两组与对照组相比差异显著(p＜0.05);淋巴细胞转化试验显示A组的SI值均高于B组,并且42 d时A、B两组差异显著(p＜0.05).上述结果表明,3组分联合免疫的DNA疫苗可以诱导小鼠产生较强的体液和细胞免疫应答,为PRV-A的核酸疫苗研究奠定了基础.