HBsAg/GM-CSF融合蛋白在毕赤酵母中的表达及其免疫原性研究

为探索一种提高乙肝病毒表面抗原免疫原性的新方法,用PCR和基因重组技术构建HBsAg与GM-CSF的融合基因,并在毕赤酵母中分泌表达HBsAg/GM-CSF(S-GM)融合蛋白。表达产物用SDS-PAGE检测,W estern b lot分析,离子交换柱纯化后免疫昆明鼠,ELISA检测免疫小鼠血清中抗HBsAg的抗体水平。结果显示S-GM融合蛋白在毕赤酵母中获得了表达,离子交换柱一步纯化即可得到纯度达90%以上的S-GM。W estern b lot分析S-GM可分别与抗HBsAg及抗GM-CSF的抗体特异结合。ELISA检测发现第一次免疫后4w出现抗HBsAg的抗体,加强免疫后融合蛋白组几乎全部阳转,且抗体水平较HBsAg组(P=0.009<0.05)及HBsAg和GM-CSF的混合物组(P=0.032<0.05)高。HBsAg/GM-CSF融合蛋白能够在毕赤酵母中表达,且可增强HBsAg的免疫原性,为提高乙肝疫苗的免疫效果提供了新的思路与方法。

Expression of fusion protein HBsAg/GM-CSF in the Yeast Pichia pastoris and its immunogenicity

To search for a new method to enhance immunogenicity of HBsAg,the fusion gene composed of HBsAg and GM-CSF was constructed by PCR and genetic recombinant technique and the fusion product HBsAg/GM-CSF was expressed in the Yeast Pichia pastoris.The expressed protein was detected by SDS-PAGE,Western blot and purified by ion exchange chromatography.Then mice were inoculated with the purified fusion protein and the level of anti-HBsAg in mice serum was tested by ELISA.SDS-PAGE showed that the fusion protein S-GM was expressed in the Yeast Pichia pastoris,Western blot showed the exhibited specific reaction with both antibody of anti-HBsAg and anti-GM-CSF.The fusion protein S-GM with a more than 90% purity was obtained by ion exchange method.ELISA showed that anti-HBsAg appeared at the fourth week after the primary inoculation.Then the positive conversion rate for antibody was almost occupied by the fusion protein after the second inoculation(booster).The level of anti-HBsAg induced by S-GM fusion protein was higher than that by HBsAg alone(P=0.009<0.05),and higher than that by a mixture of HBsAg and GM-CSF(P=0.032<0.05).HBsAg/GM-CSF fusion protein could be expressed in the Yeast Pichia pastoris and the immunogenicity of HBsAg enhanced too.It's not only a new idea but also a practical approach for enhancing the immunity effect of HBV vaccine.