人颗粒酶B的表达及抗原性初步分析

目的在原核系统表达人颗粒酶B(GrB)酶原型及活性型基因,并分析表达产物的抗原性。方法从人外周血淋巴细胞中抽提RNA,用RT-PCR方法扩增人颗粒酶B序列,将其克隆至pGEX-5x-1表达质粒,与GST进行融合表达,用SDS-PAGE及Western blot对表达产物进行分析。结果所克隆的GrB序列与已报道的从人体分离出的GrB完全一致。酶原型和活性型GrB与GST的融合蛋白以包涵体的形式表达,其大小与预期结果一致,并能与GrB特异性抗体发生结合反应。结论已成功构建了GrB原核表达系统,酶原型及活性型GrB均具有抗原性。

Expression and Antigenicity of Human Granzyme B

Objective To express the genes encoding human granzyme B(GrB)precursor and active GrB in prokaryotic cells and analyze the antigenicity of expressed product.Methods Extract RNA from human peripheral blood lymphocytes for the amplification of human GrB gene by RT-PCR.Clone the amplified gene fragment into expression vector pGEX-5x-1 for fusion expression with GST.Identify the expressed product by SDS-PAGE and Western blot.Results The sequence of cloned GrB gene was completely consistent with that reported.Both GrB precursor and active GrB were expressed in forms of inclusion bodies,with identical relative molecular mass to those expected,and reacted with GrB specific antibody.Conclusion The prokaryotic expression system of GrB was successfully constructed,and both the expressed GrB precursor and active GrB showed good antigenicity.