GST-GP302融合蛋白在大肠杆菌中的表达及其抗血清的制备

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

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 。

AIM: To express fusion protein of GST and vWf binding domain(GP302) of platelet GPⅠba 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 GPIbα. CONCLUSION: The preparation of polyclonal antibody against GP302 peptide provides an usefal reagent for the detection of platelet GPIbα.