人降钙素基因的克隆及其序列分析

CLONING AND SEQUENCING OF HUMAN CALCITONIN GENE

目的：克隆人降钙素基因（ｈＣＴ）并经序列分析予以确证。方法：首先自新鲜全血中用含ＴｒｉｔｏｎＸ－１００的缓冲液提取基因组ＤＮＡ，经酒精沉淀和洗涤后，作为模板，加入上下游引物，应用聚合酶链反应直接扩增出ｈＣＴ的编码序列。并将此段ＤＮＡ插入克隆载体ｐＵＣ１８的ＥｃｏＲＩ和ＳｍａＩ位点之间。最后用ＰＣＲ－双脱氧末端终止法进行序列分析。结果：成功地克隆了ｈＣＴ，证实所得ｈＣＴ基因克隆含编码人降钙素３２个氨基酸的全长ＤＮＡ序列９６ｂｐ。结论：已获得人降钙素基因克隆。

Objective:Calcitonin(CT)is one of three mainly polypeptide hormones which regulate calcium and phosphate metabolism,together with 1,25(OH)2 Vitamin D3 and parathyroid hormone (PTH), being called “calcitrophic hormones”Not only calcitonin gene probe but also certain amount of the purified hormone are requiredThe first step of gene engineering is to clone human calcitonin geneMethods:Owing to the location of the encoding DNA fragment of hCT132 in the same extron,it was separated directly from peripheral blood cell genomic DNA by means of PCR method,and identified by 2% agrose gel electrophoresis (Figure 1)The PCR product was inserted between the Sma I and EcoRI sites of pUC18 vectorThe positive clones containing an inserted DNA fragment were screened after being digested with the endonuclease PvuⅡThe nucleotide sequence of the DNA fragment was determined with the dedeoxychaintermination method combining with PCR techniqueResults:Human calcitonin gene was cloned and shown to encode the 32 amino acids of hCTAnd,for further study,a stop codon(TAA)was introduced to the expected 3' terminus of the inserted gene (Figure 2,3)Conclusion:Human calcitonin gene was cloned,which was an preliminary work of producing hCT polypeptide,and laid a forundation for the further study about the hormone action on calcium and phosphate metabolism and its action mechanism