人R_O蛋白(60kD)编码序列的PCR产物在E.coli中的克隆与表达

通过聚合酶链反应 ( PCR)自人脾 c DNA扩增 RO 蛋白 ( 60 k D)编码 DNA片段 .将该片段定向插入麦芽糖结合蛋白 ( MBP)融合系统的 p MALTM- c载体中并转化 E.coli ( DH5α) ,通过酶联免疫印迹 ( IBT)筛选出具有 RO 抗原性的阳性克隆 .绝大部分阳性克隆表达完整的 RO 融合蛋白 ( 1 0 0k D) .但在传代过程中表达水平很快降低 ;少数阳性克隆虽然表达非完整 RO 融合蛋白 (分子量 <80k D) ,但表达水平却高而且稳定 .同时保留了很强的 RO 抗原性 .产生非完整融合蛋白的一个原因是 ,PCR碱基错配引起 RO 编码 DNA的序列缺失 ;另一原因是融合蛋白在表达过程中被降解

Cloning and Expression in E.coli of PCR Products of DNA Sequence Encoding Human R_O Protein (60kD)

The DNA fragment encoding R O protein (60 kD) was amplified by Polymerase Chain Reaction (PCR) and inserted into a fusion protein vector,pMAL TM c,transformed competent DH5α.Positive clones expressing R O fusion proteins were selected using Immuno blot technique (IBT).The majority of the clones could express integral R O fusion protein (100 kD),but the level of expression dropped down rapidly after generation;Few clones expressed fusion proteins with low molecular weight (<80 kD) but the levels of expression were high.One reason for producing low molecular weight fusion protein was that there was DNA sequence deficiency of the insert in the clone. Another reason was that the degradation occurred during expression.The fusion protein of low molecular weight exhibited high specificity and sensitivity for the detection of anti R O antibodies in IBT and was easy to purify by affinity chromatography.