大肠杆菌偏嗜性密码子组成的人降钙素(hCT)编码序列的克隆

目的：克隆适于在大肠杆菌(Escherichia coli，E．coli)中表达的人降钙素(hCT)基因，为提高其表达水平奠定基础。方法：根据大肠杆菌密码子频率表，在不改变hCT。氨基酸组成的前提下，设计并合成适于在大肠杆菌中表达的编码hCT的DNA序列，经PCR扩增后，与质粒载体pGEM^R-Teasy进行连接。连接产物转化感受态JM109大肠杆菌，筛选阳性菌落，经扩大培养后，对所含重组质粒进行鉴定。结果：经酶切鉴定及DNA序列分析证实，hCT编码DNA序列成功地克隆人pGEM^R-Teasy载体中，其序列与设计的完全一致。结论：完成了大肠杆菌偏嗜性人降钙素基因的克隆，为下一步hCT表达载体的构建及在大肠杆菌中进行表达的研究奠定了基础。

Cloning of human calcitonin (hCT) coding sequence favorited by E.coli

Objective: To construct the E. coli favorite human calcitonin(hCT) gene. Methods: According to the frenquency of E. coli codons, an E. coli favorite DNA fragment , encoding identical amino acids sequence to that of the wild type hCT gene, was designed and synthesized. Having been amplified by PCR, the sequence was cloned into plasmid pGEM -Teasy and introduced into competent cells of E.coli JM109. The positive colonies were screened out and the recombinant plasmid was identified. Results: Identified by digested with restriction enzymes and DNA sequence analysis, the recombinant plasmid pGEM -Teasy/ hCT from positive E.coli contained the correct E.coli favorite coding sequence of hCT. Conclusion: The E.coli favorite hCT gene was cloned successfully, which forms the basis of constructing the expressive system for the hCT expression in E. coli subsequently .