含IL-2基因的乙型肝炎病毒DNA疫苗的构建及其免疫效果

目的构建含白细胞介素-2(IL-2)基因的乙型肝炎病毒DNA疫苗,并考察免疫效果。方法将微小病毒内部核糖体进入位点(Internal ribosomal entry site,IRES)基因克隆到质粒pVAX1载体多克隆位点处,再将乙肝病毒表面抗原(HB-sAg)基因和IL-2基因分别连接在IRES基因的两侧,构建出乙肝病毒DNA疫苗pHII。将pHII转染COS-7细胞,进行瞬时表达。大量提取质粒后,按0、2、4周程序免疫BALB/c小鼠,以pVAX1质粒为阴性对照,pVAX/HBsAg质粒为阳性对照。第1针免疫1周后开始眼眶采血,检测血清中HBsAg和HBsAb含量。第1针免疫13周后处死小鼠,用流式细胞仪检测其脾脏T细胞表面CD4+、CD8+分子数量。结果在转染pHII质粒的COS-7细胞上清液中检测到HBsAg和IL-2的表达。实验组和阳性对照组小鼠分别于第1针免疫2、4周后,在血清中检测出HBsAb,但阴性对照组未测出。以上3组均未检测出HBsAg。实验组与阳性对照组相比,产生抗体的时间提前2周,抗体阳转率提高2.5倍,产生抗体的量也提高了近3倍。实验组CD4+分子数量和CD4+/CD8+值均高于阳性对照组。结论乙肝病毒DNA疫苗pHII成功诱导出小鼠的免疫应答,其免疫效果明显优于不含分子佐剂的乙肝DNA疫苗。

Construction of Hepatitis B Virus DNA Vaccine Carrying Interleukin-2 Gene

Objective To construct the hepatitis B virus(HBV) DNA vaccine carrying interleukin-2(IL-2) gene and explore its immune effect.Methods Insert internal ribosomal entry site(IRES) gene into the polyclonal site of plasmid pVAX1,then insert HBsAg and IL-2 genes into the same plasmid,at the two sides of IRES gene respectively,to construct HBV DNA vaccine pHII.Transfect COS-7 cells with pHII for transient expression.Immunize BALB/c mice with the extracted plasmid pHII for 3 times at weeks 0,2 and 4 respectively and determine the HBsAg and HBsAb contents in sera starting from week 1,using plasmid pVAX1 as negative control and plasmid pVAX/HBsAg as positive control.Kill the mice at week 13 and determine for the CD4 and CD8 on surface of T lymphocyte in murine spleens by flow cytometry.Results HBsAg and IL-2 were detected in the supernatant of COS-7 cells transfected with plasmid pHII.HBsAbs were detected in the sera of mice in test and positive control groups at weeks 2 and 4 respectively,but not detected in those of mice in negative control group.However,no HBsAg was detected in the three groups.Compared with those in positive control group,the antibody in test group appeared 2 weeks earlier,the antibody positive rate was 2.5 times higher,and the quantity of antibody increased about 3 folds.Both the number of CD4 molecule and the ratio of CD4 to CD8 of mice in test group were significantly higher than those in positive control group.Conclusion The constructed HBV DNA vaccine pHII induced immune response in mice successfully.Compared with that of HBV DNA vaccine without molecular adjuvant,the immune effect of the constructed HBV DNA vaccine was good.