用PCR组建巨细胞病毒抗原决定簇基因的表达克隆

CONSTRUCTION OF THE EXPRESSION CLONE OF AN EPITOPE GENE OF HUMAN CYTOMEGALOVIRUS POLYMERASE CHAIN REACTION

巨细胞病毒感染的血清学诊断方法是目前临床诊断中应用最广泛的方法之一。但由于缺少高效价、高纯度和高特异性的抗原，使各种血清学方法在其敏感性和特异性方面均不能令人满意。文献报道，通过分子生物学得到的或化学合成的单一病毒蛋白或部分成份可作为血清学诊断的良好抗原。本文报道用聚合酶链反应扩增了巨细胞病毒主要磷蛋白ｐＰＵＬ３２的一个强抗原决定簇基因，并同时在扩增产物的两端引入了相应的限制性内切酶识别位点序列，经与表达载体连接后转入相应的细菌中，得到了能够表达此抗原决定簇的克隆，其表达的融合蛋白在免疫转印检测中与人巨细胞病毒阳性血清有强特异性反应。

erological diagnosis has been the main method till now for human cytomegalovirus used in clinic.However,this method could not give a satisfactory result in sensitivity and specificity,due to lack of highly purified specificantigen with high titer.It has heen shown that antigenic materials composed of single viral protein,or portion ofthem,produced via molecular biology or chemical synthesis are good antigen in serological diagnosis.In this study ,astrong epitope gene of HCMV basic phosphoprotein,pPUL32,was amplified by PCR. At the meantime,the se-quence whose sites could be recognized by appropriate endonuclease were introduced into the two ends of the PCRproduct.After ligation with expression vector,the recombinant plasmid was electro-transformed into suitable bacte-ria.A clone that could express the epitope was obtained. There were strong specific reaction between the fusionprotein from the clone and HCMV positive sera in immunoblotting assay.