GST-GP302融合蛋白在大肠杆菌中的表达及其抗血清的制备

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

The expression of fusion protein GST-GP302 in E.coli and its preparation of rabbit anti-serum

AIM: To express fusion protein of GST and vWf binding domain(GP302) of platelet GPIbalpha in E.coli and its preparation of rabbit anti-serum. METHODS: GP302 gene was inserted into pGEX-4T-1. The recombinant vector was identificated by restriction endonuclease digestion analysis. Fusion protein GST-GP302 was expressed in E.coli via IPTG induction. The rabbit antibody against GST-GP302 was prepared by using renatured GST-GP302 as immuneogen and the specificity of polyclonal antibody was identified by Western blot. RESULTS: The restriction endonuclease digestion analysis of recombinant plasmid demonstrated that the GP302 gene had been exactly inserted into pGEX-4T-1. SDS-PAGE analysis showed that the ralative molecular mass(M(r)) of the fusion protein was about 59 000. ELISA analysis proved that the titer of rabbit serum against GST-GP302 was 10(-5). The polyclonal antibody specifically bound to purified platelet GPIbalpha. CONCLUSION: The preparation of polyclonal antibody against GP302 peptide provides an usefal reagent for the detection of platelet GPIbalpha.