大肠杆菌表达的乙型肝炎表面抗原124aa分子的纯化和免疫学鉴定

Purification and characterization of the hepatitis B surface antigen fragment with 124 amino acids expressed in E.coli

目的 探索大肠杆菌表达的乙型肝炎表面抗原 (HBsAg) 12 4aa分子作为疫苗成分应用的可能性。方法 以超声破碎法获取包涵体 ,用HisTrapTM试剂盒亲和层析纯化目的蛋白 ,加入聚乙二醇 (PEG40 0 0 )以提高变性蛋白的复性效率 ;以抗HBsAga抗原决定簇单克隆抗体 (McAb)和抗HBsAg多克隆抗体 (PcAb)作为包被抗体 ,采用夹心ELISA法分析其抗原性 ;以纯化产物免疫BALB/c小鼠 ,RIA法测定小鼠血清中抗HBs抗体。结果 通过HisTrapTM 试剂盒亲和层析纯化的HBsAg12 4aa分子经反相高效液相色谱 (RP HPLC)分析 ,纯度达 95 %以上 ;经复性后的蛋白具有抗原性和免疫原性。结论 HBsAg12 4aa分子在大肠杆菌中的成功表达 。

Objective To study the potential of HBsAg 124aa fragments expressed in E.coli as a component of hepatitis B vaccine.Methods The inclusion bodies of HBsAg 124aa fragments were collected from the engineered bacteria after sonofication,and then dissolved in buffers contatining 8mol/L of urea in buffers containing 8mol/L of urea and purified by immobilized metal Ni2+ chelation affinity chromatography in one step.The purified proteins were treated for molecular refold in the presence of polyethylene glycol (PEG) and further analysed by reverse phase-high performance liquid chromatography(RP-HPLC).Antigenicity of the protein was tested by ELISA and immunogenicity was demonstrated in BALB/c mice.Results RP-HPLC analysis revealed that the purity of the proteins was over 95%,and that the renaturalized protein can be recognized by polyclonal antibody and monoclonal antibody against HBsAg a determinant and could elicite antibody response against HBsAg in BALB/c mice.Conclusion The hepatitis B surface antigen fragment with 124 amino acids expressed in E.coli could be a promising candidate as a new immunogen of hepatitis B vaccines.