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

Immune responses of the recombinant plasmids expressing the major proteins of porcine rotavirus A in immunized mice

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-AVP4、VP6、VP7蛋白基因的真核表达质粒pVAX1-VP4、pVAX1-VP6、pVAX1-VP7以pVAX1-VP4+pVAX1-VP7(B组)和pVAX1-VP4+p VAX1-VP6+pVAX1-VP7(A组)设计试验组,分别以100μg/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14 d、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。

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.