IL-2 preS DNA疫苗免疫正常小鼠和HBV转基因鼠的免疫应答研究

The study of immune response of IL-2 preS DNA vaccine on BALB/c mice and HBV transgenic mice

目的 探讨IL 2preSDNA疫苗作为预防和治疗性疫苗的可行性及作用机理。方法应用基因重组技术 ,构建人白细胞介素 2 (hIL 2 )和前表面抗原 (preS)的真核表达载体 ,将此重组载体用基因枪分别注射正常的BALB c小鼠和HBV转基因小鼠 ,通过ELISA方法检测BALB c小鼠和HBV转基因鼠的抗 preS2、HBsAg及抗 HBs抗体水平 ;荧光定量PCR方法检测HBV转基因鼠血清中HBVDNA拷贝数 ,并用免疫病理HE染色观察小鼠肝组织炎症活动度 ,同时检测肝功能指标。结果 ①基因枪注射真核表达质粒免疫正常小鼠后 ,10 0 %小鼠能在第 4、6周检测到抗preS1抗体 ,持续时间长达10周。②用IL 2preS真核表达质粒基因枪肌肉注射方式优于正常肌肉注射和皮下注射的方式 ,且所需质粒量 (10 μg 只 )仅为后者 (10 0 μg 只 )的 1 10。③在第 4周高峰期检测IgG亚类 ,是诱导以TH1(IgG2a)细胞免疫为主的反应。④基因枪注射真核表达质粒 (1μg 只 )免疫转基因小鼠后 ,80 %的小鼠产生了抗体 ,HBVDNA量下降 ,其中 2 0 %的小鼠HBsAg转阴。⑤HE肝组织染色显示 :肝组织有明显的炎细胞浸润、肝细胞肿大、颗粒样变性、转氨酶升高。结论 IL 2preSDNA疫苗能刺激小鼠机体产生体液和细胞免疫 ,可部分打破小鼠机体的免疫耐受 。

Objective To investigate the feasibility and functional mechanism of hIL-2 preS DNA vaccine as prevention and therapeutic vaccine for hepatitis B. Methods hIL-2 and preS eukaryon expression vector was constructed by using DNA recombinant technology. Normal BALB/c mice and HBV transgenic mice were injected with gene gun. The antibody of anti-preS2 and anti-HBs was detected by ELISA. The HBV DNA copies in HBV transgenic mice serum and liver tissue were detected by real-time PCR. The inflammatory activity, liver function and transaminase were detected at the same time. Results (1) preS1 antibody can be detected at the 4^(th) and 6^(th) week, which can maintain up to 10 wk after the injection of immune-normal mice with eukaryotic expressive plasmid. (2) The injection of eukaryotic plasmid into muscle using gene gun is better than normal muscle injection and subcutaneous. The quality of plasmid (10μg per mouse) is only one tenth of the later (100μg per mouse). (3) When detecting IgG subset at the peak stage of the 4^(th) week, it is a reaction that mainly induces cellular immunity of T_H1 IgG2a cells. (4) After the transgenic mouse was immuned by injection of eukaryotic expression plasmid (1μg per mouse) using gene gun, 80% mice got the antibody, and the copied of HBV DNA decreased. (5) HE stain of liver tissue showed there were obviously inflammatory cells infiltration and hepatocyte liver cell swelling and particulate degeneration. Conclusion Humoral and cellular immunity can be produced and immunological tolerance can be broken partly after the injection of IL-2 preS DNA vaccine. All these provide experimental foundation for the further research of therapeutic HBV DNA vaccine. [