降钙素基因相关肽表达载体的构建

Construction of an expression vector of calcitonin gene-related peptide gene

背景:为研究降钙素基因相关肽基因治疗在骨质疏松中的应用,首先要建立高效的降钙素基因相关肽基因载体。目的:构建降钙素基因相关肽真核表达载体pBaBb-puro-CGRP。方法:设计引物并通过PCR扩增出降钙素基因相关肽,酶切后用T4DNA连接酶将其与pBaBb-puro进行连接,经过转化、阳性克隆筛选后,进行酶切和测序鉴定。结果与结论:经PCR扩增获得了含430bp的降钙素基因相关肽基因编码序列,经过酶切、连接、转化后,挑选10个菌落进行PCR检测,筛查到8个菌落含有重组质粒,进一步行酶切和测序鉴定,结果与理论预期完全相符。提示实验成功构建了pBaBb-puro-CGRP真核表达载体。

BACKGROUND： Efficient calcitonin gene-related peptide （CGRP） gene vector should be established first to study the application of CGRP gene therapy in osteoporosis. OBJECTIVE： To construct an expression vector of pBaBb-puro-CGRP METHODS： Primers were designed. Then, CGRP was obtained by PCR amplification and it was connected to pBaBb-puro after enzyme digestion. After transformation and positive clone screening, enzyme digestion and sequence identification were performed. RESULTS AND CONLUSlON： 430 bp coding sequence of CGRP gene was obtained by PCR amplification. After enzyme digestion, connection and transformation, the clones were obtained. Next, 10 clones were picked up and examined by PCR, and eight clones were found the recombinant plasmid. The result was in accord with expectation through further enzyme digestion and sequencing. These findings suggest that expression vector of pBaBb-puro-CGRP was successfully constructed.