血型A抗原的模拟多肽的表达及功能研究

Expression and characteristic of the peptide mimics of blood group A antigen

目的融合表达血型A抗原的模拟多肽,初步鉴定融合蛋白结合抗A抗体的能力。方法PCR克隆扩增血型A抗原模拟多肽(P5)和谷胱苷肽S-转移酶(GST)编码基因,并连接成P5-GST。双酶切后克隆入原核表达质粒pET28b,经E.coli DH5a扩增后纯化并酶切、测序鉴定。以BL21(DE3)为表达菌,在IPTG诱导下表达P5-GST融合蛋白,Ni-NTA柱纯化蛋白。红细胞凝集抑制实验分析融合蛋白模拟血型A抗原的能力。ABO-ELISA初步分析P5-GST融合蛋白检测血型抗体的效能。结果成功构建P5基因和GST基因融合的原核表达质粒pET28b-P5-GST,目的基因可以高效表达,纯化的融合蛋白具有良好的模拟血型A抗原的能力。基于P5-GST融合蛋白的ABO-ELISA具有一定的检测血浆抗A抗体的能力。结论血型A抗原的模拟多肽序列与GST的融合表达蛋白具有特异性结合抗A抗体的特性,本融合蛋白具有替代血型A多糖抗原潜能,为发展新型的抗A抗体检测方法提供依据。

Objective To express the blood group A mimitope and GST fusion protein and determine the mimic ability of the fusion protein. Methods The P5 and GST genes were amplified by PCR and ligated as P5-GSF gone. The P5-GST gene was cloned into pET28b plasmid and named pET28b-P5-GST.Restrict digest and sequencing were used to identify the correct pET28b-P5-GST plasmid.Then the pET28b-P5-GST was transformed into E.coli BI21（DE3）,P5-GST fusion protein was induced by WIG and purified by Ni-NTA resin.The inhibit agglutination assay was used to determine the group A-mimic ability of the fusion protein. ABO-ELISA based on P5-GST fusion protein was compared with classic haemagglutination. Results The pET28b-P5-GST vector was constrncted suecessfully and could express the fusion protein efficiently. The purified fusion protein maintained blood group A antigenic activity. Compared with haemagglutination,ABO-ELISAs based on P5-GST fusion protein have the ability to detect anti-A antibedy.Conclusion The P5-GST fusion protein can mimic the blood group A antigen and has the potential to instead of natural blood group A antigen.