磷酸铝佐剂对乙型肝炎DNA疫苗抗体应答的增强作用

Enhancement of a hepatitis B DNA vaccine potency using aluminum phosphate in mice

目的 探讨磷酸铝佐剂对乙型肝炎DNA疫苗诱导体液免疫应答的增强作用。 方法 应用基因重组技术构建乙型肝炎表面抗原(HBsAg)真核表达质粒pcDNA 3.1-S,经酶切和测序鉴定无误后,作为乙型肝炎DNA疫苗,将不同浓度的磷酸铝悬液与之混合后免疫小鼠;用酶联免疫吸附法检测重组质粒在小鼠局部肌肉中HBsAg的表达和在血清中HBsAg的含量;并于免疫小鼠6周后检测小鼠血清抗-HBs水平。 结果 与单纯使用质粒pcDNA 3.1-S相比,将质粒pcDNA 3.1-S与磷酸铝悬液混合后免疫小鼠,重组质粒在小鼠局部肌肉中HBsAg表达差异无显著性;HBsAg在血清中的浓度均为阴性。将质粒pcDNA3.1-S与磷酸铝悬液混合后免疫小鼠,6周后检测小鼠血清抗-HBs,每1μl质粒中含1、10、50、100 μg磷酸铝组,小鼠血清抗-HBs抗体的P/N值分别为11.00±6.62、20.30±10.20、49.1 8±24.40和48.68±27.78,单纯使用质粒pcDNA3.1-S组P/N值为11.54±5.60。含1μg和10μg磷酸铝组,抗-HBs抗体P/N值与单纯用质粒pcDNA3.1-S组相比,差异无显著性;50μg和100μg磷酸铝组抗-HBs抗体P/N值高于单纯用质粒pcDNA3.1-S组,但50μg和100μg磷酸铝组二者差异无显著性。 结论 磷酸铝与质粒pcDNA3.1-S混合对pcDNA3.1-S在小鼠局部肌肉中HBsAg的表达无明显增强作用。

Objective To study antibody response to a hepatitis B DNA vaccine by formulation with aluminum phosphate in mice. Methods An eukaryotic expression plasmid inserted HBsAg gene (pcDNA3.1-S) was constructed by cloning technique and the accuracy of the construct was confirmed by restriction enzyme digestion and DNA sequencing, then hepatitis B DNA vaccine formulations were prepared by mixing pcDNA3.1-S with various concentration of aluminum phosphate in 0.9% NaCl. HBsAg expressions were assayed by ELISA in vivo five days after intramuscular injection of pcDNA3.1-S with or without aluminum phosphate. And serum samples were obtained from individual immunized or control mice 6 weeks post injection. Then anti-HBs were assayed in mice sera by ELISA. Results Five days after intramuscular immunization, the levels of HBsAg expression of groups with aluminum phosphate showed no difference from those of control group in tibialis arterials muscles. In sera, HBsAg could not be detectable in all groups. Intramuscular immunization of BABL/C mice with pcDNA3.1-S mixed aluminum phosphate (0 μg, 1 μg, 10 μg, 50 μg, 100 μg) 6 weeks later, the P/N values of anti-HBs in sera were 11.54±5.60, 11.00± 6.62, 20.30±10.20, 49.18±24.40 and 48.68± 27.78, respectively. It showed that pcDNA3.1-S mixing with aluminum phosphate could increase anti-HBs titers in mice. Conclusions No increase of HBsAg expression was observed by mixing plasmid pcDNA3.1-S with various concentration of aluminum phosphate in vivo. But Intramuscular immunization of BALB/C mice with pcDNA3.1-S mixing aluminum phosphate adjuvant can increase anti -HBs titers. It seemed that aluminum phosphate would be valuable for further investigation as a potential adjuvant of hepatitis B DNA vaccines.