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目的 :构建乙型肝炎病毒 (HBV)变异s基因真核表达载体 ,检测其诱导小鼠产生特异性体液免疫应答。方法 :利用限制性内切酶定位克隆构建s基因nt5 87G→A的真核表达载体pCMV S2 .S +14 5R(PR)。用其转染人肝癌细胞系HepG2后 ,用EIA、ELISA及免疫细胞化学法 ,观察其抗原性。以重组变异型s基因真核表达载体 (PR)和载体pcDNA3.0分别免疫C5 7BL/6小鼠各 5只。每只小鼠各肌肉注射纯化质粒 10 0 μg。用ELISA法检测血清抗 HBs及抗 HBs2抗体的效价。结果 :体外实验证实 ,变异型HBsAg可与抗 HBs结合 ;PR免疫小鼠可诱导其产生抗 HBs抗体及抗 HBs2抗体 ,但抗 HBs2抗体的出现早于抗 HBs抗体约 1～ 2wk。结论 :HBV变异s基因(nt5 87G→A)的真核表达载体的表达产物具有良好的抗原性 ,能够诱导C5 7BL/6小鼠产生体液免疫应答
Objective: To construct eukaryotic expression vector of hepatitis B virus (HBV) variant s gene and detect the specific humoral immune response induced by it in mice. Methods: The eukaryotic expression vector pCMV S2 .S +14 5R (PR) of nt5 87G → A was constructed by restriction endonuclease mapping. After being transfected into HepG2 cell line, the antigenicity was observed by EIA, ELISA and immunocytochemistry. Five of 5 7BL / 6 mice were immunized with recombinant s gene eukaryotic expression vector (PR) and vector pcDNA3.0 respectively. Each mouse was injected intramuscularly with 10 μg of purified plasmid. Serum anti-HBs and anti-HBs2 antibody titers were measured by ELISA. Results: In vitro experiments confirmed that variant HBsAg could bind to anti-HBs. PR vaccinated mice could induce anti-HBs antibodies and anti-HBs2 antibodies, but anti-HBs2 antibodies appeared earlier than anti-HBs antibodies for about 1 ~ 2 weeks. Conclusion: The eukaryotic expression vector of HBV variant s gene (nt5 87G → A) has good antigenicity and can induce humoral immune response in C5 7BL / 6 mice