动态模板PCR合成分泌性降钙素基因及其克隆  

Synthesis and cloning of human calcitonin and insulin secretory signal peptide gene

作　　者：王秀利[1] 王义生[1] 

机构地区：[1]郑州大学一附院骨科,450052

出　　处：《中国骨肿瘤骨病》2005年第3期154-157,共4页Chinse Journal Of Bone Tumor And Bone Disease

基　　金：国家自然科学基金资助项目(30271316)

摘　　要：目的用动态模板PCR基因合成法合成与克隆含人胰岛素分泌信号的人降钙素(humancalcitoninhCT)基因并经序列分析予以确证。方法根据Genebank的人胰岛素分泌信号和人的hCT基因的序列,将其分成6个片断合成单链寡核苷酸作为PCR的引物和cDNA合成的模板。其中,在互相邻近的每两条引物之间均有19～20个碱基互补,6条合成的单链寡核苷酸在PCR反应中相邻的两条单链寡核苷酸互为引物和模板,经过3个PCR循环,直接扩增出人胰岛素和降钙素的编码序列,经过25～30循环,获得足量的PCR产物,即含人胰岛素分泌信号的hCT基因。采用琼脂糖凝胶回收法,回收目的片段胰岛素分泌信号和hCT基因全长DNA。纯化后,将此段DNA插入到克隆载体PMD18-T载体上。转化用CaCl2法制备的感受态细菌JM109,恒温震荡培养箱培养1h后,涂在含氨苄青霉素和X-gal的LB固体培养基上培养。利用氨苄抗性筛选和蓝白斑筛选实验,筛选出阳性克隆,用碱裂解法提取质粒,应用双酶切和DNA测序鉴定重组质粒。结果经动态模板PCR基因合成法合成获得212bp产物,经酶切鉴定和DNA测序确定所合成片断为胰岛素分泌信号和hCT基因全长,证明克隆到的含人胰岛素分泌信号和降钙素的基因为编码人胰岛素分泌信号和人降钙素的全长DNA序列。结论本研究为基因合成提供了一种新的可靠的方法,成功地克隆了含人胰岛素分泌信号的人降钙素基因,为人降钙素基因与后续研究提供了新的研究方法。Objective A gene coding for human calcitonin （hCT）and insulin secretory signal peptide （ISSP） was divided into six fragments synthesis by changing template PCR technique and cloned into PMD18 -T vector. The 211bp hCT and ISP gene synthesis does not rely on the DNA ligase, but relies on fidelity DNA polymerase LA Taq^TM. Methods The hCT and ISSP gene was assembled through three round of PCR from a total of only six40 -60bp oligos. The synthesized gene was inserted into PMD18 -T vector and then transferred into Ecoli JM 109. Finally the nucleotide sequence of synthesized hCT and ISSP gene was determined with the dedeoxychaing- termination method combing with PCR technique. Results The result indicates that the nucleotide sequence of the hCT and ISSP gene is the same as the sequence designed Human calcitonin and ISSP gene was cloned and show to encode the 32 amino acids of hCT. And for the further study a stop codon （TAA） was introduced to the expected 3 terminus before the endonuclease cut site of the inserted gene. The result demonstrates that a reliable and forthright method of synghesis of oligo - peptide gene （ or cDNA） and cloning is provided . Conclusions This paper is a basic work for expression and secretion of hCT or hCT or further study on hCT - associated disease.

关 键 词：降钙素 聚合酶链反应 基因 克隆 降钙素基因 PCR基因 克隆载体 合成法 模板 分泌性 

分 类 号：R346[医药卫生—基础医学]