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目的 :在大肠杆菌中表达血小板糖蛋白GPIbα之vWF结合区(GP30 2 )与谷胱甘肽S 转移酶GST的融合蛋白并制备其抗血清。方法 :将GP30 2片断插入GST融合表达载体 pGEX 4T 1,重组载体酶切鉴定后 ,在大肠杆菌中经IPTG诱导表达获得GST GP30 2融合蛋白 ,SDS PAGE分析表达产物。包涵体经变性复性后免疫新西兰白兔 ,制备抗血清 ,ELISA、Westernblot检测重组抗原的免疫活性。结果 :重组质粒酶切鉴定表明 ,GP30 2基因已正确插入到 pGEX 4T 1中 ,经IPTG诱导后 ,表达出相对分子质量 (Mr)约为 5 90 0 0的融合蛋白 ,获得了ELISA效价为 1× 10 -5的多克隆抗体。Westernblot证明所制备的多抗可以与血小板糖蛋白特异性结合。结论 :GP30 2片断在大肠杆菌中的成功表达及制备得到的多克隆抗体 ,为检测血小板糖蛋白GPIbα及其在其他体系中的表达提供了一种检测途径
OBJECTIVE: To express the fusion protein of vWF binding domain (GP30 2) of glutathione S transferase GST of GPIbα in Escherichia coli and prepare its antiserum. Methods: The GP30 2 fragment was inserted into GST fusion expression vector pGEX 4T 1 and identified by recombinant vector. The fusion protein was induced by IPTG in E. coli and expressed by SDS PAGE. New Zealand white rabbits were immunized with denatured and refolded inclusion bodies to prepare antiserum. ELISA and Western blot were used to detect the immunological activity of recombinant antigens. Results: The recombinant plasmids were identified by enzyme digestion. The GP30 2 gene was inserted into pGEX 4T 1 correctly. After induced by IPTG, the fusion protein with relative molecular mass (Mr) of about 5 90 0 was expressed. The ELISA titer was 1 × 10 -5 polyclonal antibody. Westernblot showed that the prepared polyclonal antibodies could specifically bind to platelet glycoproteins. Conclusion: The successful expression of GP30 2 fragment in Escherichia coli and the prepared polyclonal antibody provide a detection method for detecting platelet glycoprotein GPIbα and its expression in other systems