A抗原表位模拟多肽融合表达载体的构建及鉴定

Peptide mimic of blood group A antigen:construction of the expression vector and determination of the mimicking ability

目的:构建血型A抗原模拟多肽融合表达载体,初步鉴定融合表达蛋白结合抗A抗体的能力。方法:PCR扩增血型A抗原模拟多肽(P17)和谷胱苷肽S-转移酶(GST)编码基因,并连接成P17-GST。双酶切后克隆入原核表达质粒pET28b,酶切、测序鉴定。以BL21(DE3)为表达菌,在IPTG诱导下表达P17-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。结果:成功构建P17基因和GST基因融合的原核表达质粒pET28b-P17-GST,目的基因可以高效表达,纯化的融合蛋白以浓度依赖的方式抑制A型红细胞的凝集作用。结论:血型A抗原的模拟多肽序列与GST的融合表达蛋白可特异性结合抗A抗体,具有替代血型A多糖抗原潜能,为发展新型的抗A抗体滤除材料提供了依据。

Objective:To construction the blood group A mimitope and GST fusion protein expression vector and determine the mimic ability of the fusion protein.Method:The P17 and GST genes were amplified by PCR and ligated as P17-GST gene. The P17-GST gene was cloned into pET28b plasmid and named pET28b-P17-GST. Restrict digest and sequencing were used to identify the correct pET28b-P17-GST plasmid. Then the pET28b-P17-GST was transformed into E.coli BL21 (DE3), P17-GST fusion protein was induced by IPTG and purified by Ni-NTA resin. The inhibit agglutination assay was used to determine the group A-mimic ability of the fusion protein.Result:The pET28b-P17-GST vector was constructed successfully and could express the fusion protein efficiently. The purified fusion protein could inhibit the group A red blood cell agglutination induced by anti-A antibody in a dose dependent manner.Conclusion:The P17-GST fusion protein can mimic the blood group A antigen and has the potential to instead of natural blood group A antigen.