表达高致病性PRRSV GP3和GP5基因的核酸疫苗构建及小鼠免疫效果评价

Construction of a nucleic acid vaccine expressing the GP3 and GP5 genes of highly pathogenic PRRSV and evaluation of immunity in mice

目的构建针对美洲型蓝耳病病毒的重组核酸疫苗,评价其与5种佐剂联合免疫BALB/C小鼠的效果。方法构建含有美洲型蓝耳病病毒GP3基因和GP5基因以及CpG-佐剂序列的重组核酸疫苗pVAX-N35HP,分别加入A1佐剂、A2佐剂、A3佐剂、A4佐剂和A8佐剂,免疫BALB/C小鼠,通过中和性抗体、T淋巴细胞亚群及细胞因子检测分析免疫效果。结果成功构建重组核酸质粒pVAX-N35-HP。用重组核酸质粒转染BHK-21细胞,Western blot显示重组核酸质粒稳定表达目的蛋白。中和性抗体结果显示,重组核酸疫苗免疫后35d,pVAX-N35HP+A1组和pVAXN35HP+A2组抗体滴度为1∶12;pVAX-N35HP+A3组抗体滴度为1∶16。pVAX-N35HP组细胞因子IL-4含量为阴性对照组的1.39倍,CD4+T淋巴细胞亚群百分率为阴性对照组的2.35倍(P<0.01)。重组核酸质粒分别与5种佐剂联合免疫小鼠,pVAX-N35HP+A1组CD8+T淋巴细胞亚群百分率为pVAX-N35HP组的1.93倍;pVAX-N35HP+A3组免疫细胞因子IL-4为pVAX-N35HP组的1.41倍(P<0.05),IFN-γ为pVAX-N35HP组的3.79倍(P<0.01);pVAX-N35HP+A2、pVAX-N35HP+A4和pVAX-N35HP+A8组IFN-γ水平升高,CD8+T淋巴细胞数量增多。结论成功构建重组核酸疫苗pVAX-N35HP。该疫苗具有良好的免疫原性,其中A1、A3免疫佐剂的加入可增强小鼠细胞免疫和体液免疫水平。

Objectives To construct a DNA vaccine against the highly pathogenic PRRSV and to evaluate the effect of co-immunization with the vaccine and 5 adjuvants in BALB/C mice. Methods The recombinant DNA vaccine pVAXN35 HP containing the GP3 gene and the GP5 gene of high pathogenicity PRRSV and a CpG-adjuvant sequence was constructed.Balb/C mice were co-immunized with the adjuvant A1,A2,A3,A4,or A8.and then the effects of immunization were examined by detecting neutralizing antibodies,T-lymphocyte subsets,and cytokines. Results The recombinant nucleic acid plasmid pVAX-N35 HP was successfully constructed.The recombinant nucleic acid plasmid was transfected into BHK-21 cells.Western blotting indicated that the recombinant nucleic acid plasmid stably expressed the target protein.Neutralizing antibody results indicated that 35 days after immunization with the recombinant nucleic acid vaccine,the antibody titer in mice immunized with pVAX-N35 HP＋A1 or pVAX-N35 HP＋A2 was 1∶12 while that in mice immunized with pVAX-N35 HP＋A3 was 1∶16.In mice immunized with pVAX-N35 HP,the level of the cytokine IL-4 was1.39 times that in the negative control group,and the percentage of CD4＋T lymphocyte subsets was 2.35 times that in the negative control group（P〈0.01）.Mice were co-immunized with the recombinant nucleic acid plasmid and the fiveadjuvants.Results indicated that the percentage of CD8＋T lymphocyte subsets in mice immunized with pVAX-N35 HP＋A1 was 1.93 times that in mice immunized with pVAX-N35 HP.In mice immunized with pVAX-N35 HP＋A3,the level of the cytokine IL-4 was 1.41 times（P〈0.05）that in mice immunized with pVAX-N35 HP,and the level of IFN-γwas3.79 times（P〈0.01）that in mice immunized with pVAX-N35 HP.The level of the cytokine IFN-γand the number of CD8＋T lymphocytes increased in mice immunized with pVAX-N35 HP＋A2,pVAX-N35 HP＋A4,or pVAX-N35 HP＋A8. Conclusion The recombinant nucleic acid vaccine pVAX-N35 HP was successfully constructed.The vaccine has good immunogenicity,and addition of the adjuvants A1 and A3 can enhance cellular and humoral immunity in mice.