琥珀酰辅酶A转移酶的表达纯化及抗体制备

目的:琥珀酰辅酶A转移酶(SCOT)是酮体代谢过程中的关键限速酶,此酶缺陷多由SCOT基因突变引起,患者多有酮症酸中毒表现。为了进一步研究SCOT的功能,采用原核表达系统表达并纯化重组SCOT,制备SCOT多克隆抗体。方法:选择蛋鸡、肉鸡模式生物为研究对象,通过生物信息学对其抗原性和属间同源性进行分析,通过RT-PCR从鸡的骨骼肌cDNA中扩增了SCOT基因N端半长片段,克隆到表达载体pET28b中,在大肠杆菌BL21(DE3)中诱导表达,并用镍离子螯合柱(Ni-NTA)纯化重组SCOT;用纯化的重组SCOT免疫小鼠后得到多克隆抗体。结果:Western印迹表明,制备的SCOT抗体具有较高的特异性,可特异性识别鸡的SCOT蛋白,同时可特异性识别小鼠和人的相应SCOT蛋白。结论:SCOT多克隆抗体的制备为后续在鸡、鼠和人中研究SCOT基因提供基础。

Expression, Purification of Recombinant Succinyl CoA: 3-Oxoacid CoA Transferase 1(SCOT) and Preparation of the Specific Antibody Against SCOT

Objective:SCOT(succinyl CoA:3-oxoacid CoA transferase 1)which mediates the rate-determining step of ketolysis in extrahepatic tissues is a key enzyme for ketone body catabolism.Hereditary SCOT deficiency in humans causes episodes of severe ketoacidosis.To further study the function of SCOT gene,the polyantibody against SCOT were prepared.Methods:The antigenic of SCOT was analyzed and the half length of SCOT gene was cloned to the pET-28b plasmid.Then the recombinant plasmid was transformed into Escherichia coli BL21(DE3)and the expressed recombinant protein was purified by His.BindTM column.The purified recombinant protein was injected to generate the polyantibody.Then the antibody was analyzed by Western blot.Results:Based on the experiments,the polyantibody was obtained.The Western blot results showed that the antibody could recognize SCOT from chicken,mouse and human tissues specifically.Conclusion:The polyantibody against SCOT were prepared successful,which supplied a good tool for further studying on SCOT.