DNA疫苗预敏蛋白疫苗增强策略对乙型肝炎病毒表面抗原蛋白免疫应答的影响

Immune Responses to HBV Surface Protein Using DNA Prime and Protein Boost Strategy in BALB/c Mice

目的:观察核酸疫苗预敏,乙型肝炎病毒HBsAg蛋白疫苗增强的免疫对Balb/c小鼠免疫应答的影响。方法:以Transfection TM脂质体将乙型肝炎病毒表面抗原中蛋白(MHBs)核酸疫苗pSW3891/MHBs/ad(r简称为adr),及空载体pSW3891(简称vector)体外转染293T细胞。免疫印迹法(Westernblot)检测adr,vector的体外表达;动物体内研究选用Balb/c小鼠共18只,每组6只,编号后随机分为3组,即空载体质粒组(vector+vector组)、adr核酸疫苗+HBsAg蛋白疫苗(adr+protein组)、HBsAg蛋白疫苗+HBsAg蛋白疫苗(Protein+Protein组);于第0周肌肉注射法分别以vector、adr及HBsAg蛋白疫苗免疫小鼠,于第4周肌内注射法分别以vector、HBsAg蛋白疫苗、及HBsAg蛋白疫苗免疫小鼠。采用酶联免疫吸附试验(ELISA)检测小鼠血清HBsAg特异性抗体、酶联免疫斑点(ELISPOT)法检测小鼠脾细胞HBsAg多肽特异性IFN-γ分泌细胞。结果:adr体外转染293T细胞后,能够表达乙型肝炎病毒表面抗原中蛋白(MHBs);体内研究结果显示:除vector+vector组外,adr+protein组、Protein+Protein组小鼠均能检出血清抗-HBs,Protein+Protein组抗-HBs终点滴度比adr+protein组高,但无统计学意义;三组中vector+vector组没有检测到特异性INF-γ分泌的脾细胞,而adr+protein组、Protein+Protein组小鼠均能检出,且adr+protein组特异性细胞数量显著高于protein+protein组(P<0.001),具有统计学意义。结论:乙型肝炎病毒表面抗原中蛋白(MHBs)核酸疫苗预敏,能明显增强Balb/c小鼠对乙型肝炎HBsAg蛋白疫苗细胞免疫应答水平。

Objective： To observe the effect on the immune response to HBV surface protein vaccine after DNA prime and HBV protein boost strategy in BALB/c mice. Methods： PSW3891 （vector） and HBV DNA vaccine pSW3891/MHBs/adr （adr） which express the HBV surface middle protein were transfected into 293T cells with Transfection TM Liposome. The expression of adr in vitro was tested by western blot analysis. 18 Balb/c mice were selected and divided into 3 groups： Vector ＋Vector group,adr ＋ HBV protein vaccine group, and HBV protein vaccine＋HBV protein vaccine group. The mice were given an intramuscular injection with Vector, adr, Protein at week 0, and Vector, Protein, Protein at week 4, separately. Titres of anti-HBs were tested by ELISA. The murine spleen cells of the HbsAg peptide targeting IFN-y was tested by ELISPOT. Results： Expression of adr and vector in vitro can be tested by Western blot analysis. The study in vivo showed that the anti-HBs can be examined in all except the vector ＋ vector group. The top titre of anti-Hbs was achieved in two weeks after BALB/c mice being second immuned. The highest titre of anti-HBs was the Protein ＋Protein group, and the DNA ＋Protein group was the second. And the DNA ＋Protein group was the highest in all of the cell immunity, while the Protein ＋Protein group was the second. Conclusions： HBsAg DNA prime and protein boost could significantly improve the cell immunity in BALB/c mice.