重组乙肝表面抗原真核表达质粒pVAX-S_2S的构建及DNA免疫

Construction of recombinant eukaryotic expression vector pVAX-S_2S containing the gene of hepatitis B virus surface antigen and DNA-mediated immunization

目的 :为探索更安全的乙型肝炎病毒 (HBV)DNA疫苗 ,构建编码乙肝表面抗原中蛋白的卡那霉素抗性真核表达质粒 ,并观察其诱导BALB c小鼠产生体液免疫应答情况。方法 :采用限制性内切酶从重组的真核表达质粒pcDNA S2 S中分离出乙肝表面抗原中蛋白 (preS2 +S)基因片段 ,将其亚克隆于pVAX1真核表达载体 ,酶切鉴定 ,按不同剂量一次性肌内注射免疫小鼠 ,ELISA法检测小鼠血清抗 HBs。结果 :酶切鉴定重组质粒pVAX S2 S为正向插入的阳性克隆。HBVDNA疫苗 (pVAX S2 S)高 (10 0 μg 只 )、中 (5 0 μg 只 )、低 (10 μg 只 )三组剂量一次性免疫健康BALB c小鼠 ,均能在 2w诱导抗 HBs产生 ,抗体效价随时间延长而增长。血清抗体水平比较 ,高剂量组 (97 83± 38 78)mU ml较中剂量组 (45 13± 2 1 12 )mU ml、低剂量组 (19 74±11 92 )mU ml差异均具显著性 (P <0 0 5 ) ,以后的 4,8w高、中剂量组间差别缩小 ,但两者较低剂量组差异均具显著性 (P <0 0 5 )和非常显著性 (P <0 0 1)。结论 :卡那霉素抗性的重组质粒pVAX S2 S能有效诱导正常小鼠产生体液免疫应答。

Objective:In order to obtain safer HBV DNA vaccine,recombinant kanamycin resistance eukaryotic expression plasmid containing the gene of HBV surface antigen was constructed and used to study humoral immune response in BALB/c mice.Methods:HBV antigen middle protein(preS2+S) encoding gene fragment was isolated from the recombinant eukaryotic expression plasmid pcDNA-S 2S;subcloned into eukaryotic expression vector pVAX1.After confirmed the presence of the gene by restriction endonuclease analysis,it was used to immunize the healthy BALB/c mice at different doses,and the levels of anti-HBs in serum was determined by ELISA.Results:Restriction endonuclease analysis confirmed recombinant plasmid pVAX-S 2S was what was expected.Serum anti-HBs was detected at the 2nd week after DNA vaccination at all three doses(100 μg,50 μg,10 μg per mice)and their titers were increased with time.Quantitative comparison of the serum anti-HBs levels revealed significant(P<0.05)difference betwee the high dose group(97.83±38.78)mU/ml and the median dose group(45.13±21.12)mU/ml or the lower dose(19.74±11.92)mU/ml.On the 4th and 8th week following the induction the high and the media dose group displayed less difference whereas they displayed significant(P<0.05) and highly significant(P<0.01) differences with the lowest dose group respectively.Conclusion:Recombinant kanamycin resistance eukaryotic expression plasmid pVAX-S 2S can effectively induce humoral immune response in healthy mice.