人骨髓单核细胞表面分化抗原CD14基因的扩增、克隆和鉴定

Amplification Cloning and Identification of Gene Encoding the Human Myelomonocytic Surface Differentiation Antigen CD14

根据人骨髓单核细胞表面分化抗原ＣＤ１４（ｈＣＤ１４）基因的核苷酸序列，设计ｈＣＤ１４基因５＇端和３＇端的两个引物．以人血中提取的基因组总ＤＮＡ为模板，利用聚合酶链式反应（ＰＣＲ）技术特异性扩增该基因１２４５加的编码序列．扩增产物经Ｘｂａｌ、ＫｐｎⅠ双酶切后，克隆到ｐＵＣ１８质粒ＸｂａＩ－ＫｐｎＩ位点间，随后转化大肠杆菌ＪＭ１０９．用ＰＣＲ方法筛选出重组菌落．经酶切和序列分析方法检测后，证明获得了含ｈＣＤ１４基因的重组克隆ｐＨＣＤ１４．巳测出的插入片段５＇端部分中包含着人ＣＤ１４基因４８８ｂｐ的序列，与巳报道的相应ＤＮＡ序列相比具有９９％的同源性．

On the basis of the nucleotide sequence encoding the human myelomonocytic surface differentiation antigen CD14,two primers were designed and synthesized. Using the human genomic DNAfrom whole blood as template, 1245hp fragment,containing the encoding region of the CD14 gene,was amplified by Polymerase Chain Reaction (PCR). After digested by XbaI and KpnI,the fragment was inserted between the XbaI and KpnI site of pUC18 to construct pHCD14 recombinantplasmid,which was transfered into Escherichia coli bacterial strain JM109, Recombinant cloneswere screened by PCR. The results of restriction analysis and DNA sequencing showed that therecombinant plasmids containing hCD14 gene were obtained. The 488hp nucleotide sequence represented 5' regions of hCD14 gene shows approximately 99% homology with that of corresponding sequence of hCD14 gene reported,only 5 nucleotides viers found different.