人Ⅰ型丙氨酸氨基转移酶的可溶性表达及抗血清反应模式

Expression of soluble human alanine transaminase Ⅰ and determination of antiserum reactions to isoenzyme

目的 克隆表达重组人I型丙氨酸氨基转移酶 (ALT1)蛋白及制备抗血清 ,探讨建立ALT1特异性检测方法思路。方法 逆转录聚合酶链反应 (RT PCR)扩增人ALT1cDNA ,插入pGEX 2T ,构建原核表达载体 pGEX 2T ALT1,并转入大肠杆菌进行表达。SDS PAGE ,活力测定和活性染色鉴定表达产物。表达产物免疫小鼠所得抗血清对天然ALT1,重组ALT1及重组ALT2进行Westernblot分析。结果 重组质粒测序和酶切结果显示ALT1基因已经成功克隆到pGEX 2T载体。SDS PAGE和活性染色表明所表达蛋白具有天然活性。抗ALT1血清除识别重组ALT1外 ,还可识别天然蛋白和重组ALT2。结论 重组人ALT1得到高效可溶性表达。用目前方法制备的抗血清特异性不高 ,如建立ALT1的免疫学检测方法需要采用特异性识别ALT1的抗体 ,以避免交叉反应引起的假性升高。

Objective To express human Alanine transaminase I (ALT1) and determine antibody reactions to isoenzyme Method Human ALT1 gene was amplified by RT PCR and cloned into pGEX 2T by restriction and subsequent ligation SDS PAGE and enzyme reaction were conducted to identify the product Western blot was performed to determine the antiserum reactions to ALT isoenzyme Result Sequence and restriction analysis revealed ALT1 gene was cloned into frame of pGEX 2T The results of SDS and native PAGE, as well as enzyme determination, showed the expression of soluble potent ALT1 ALT1 antiserum could cross reacted to recombinant ALT2 Conclusion Recombinant human ALT1 with native activity was expressed in satisfactory levels Antibody specific to ALT1 should be employed to avoid the cross reaction to ALT2 in developing ALT1 immunological tests