酿酒酵母磷酸胆碱胞苷转移酶基因(cct)的克隆表达及活性测定

Cloning,Expression of Cholinephosphate Cytidylyltransferase of Saccharomyces Cerevisiae in Escherichia Coli and Evaluation of the Expressed Product

以酿酒酵母(Saccharomyces cervisiae)基因组DNA为模板,克隆并表达了编码磷酸胆碱胞苷转移酶的基因cct。根据NCBI提供的序列信息,设计扩增cct基因的引物,经PCR扩增、酶切后,将cct插入到表达载体pET-29a(+)中,构建了重组质粒pET-29a-cct,经过酶切验证和测序后转化到大肠杆菌BL21(DE3)中,在30℃条件下经IPTG诱导表达后,SDS-PAGE结果显示构建的工程菌可高效表达相对分子质量约45 k的蛋白,与理论值相符。对粗酶液进行活性测定,表达产物可检测到CCT酶活性,即可转化CTP和磷酸胆碱为CDP-胆碱,此结果可为CDP-胆碱的生物合成提供依据。

To study the cholinephosphate cytidylyltransferase gene from Saccharomyces cerevisiae,the conservative region named cct was amplified by PCR. The primers were designed according to the sequence of cct reported on the NCBI. Then,the target gene was transferred to prokaryotic expression vector pET-29a （ ＋ ） and the recombinant plasmid named pET-29-cct was constructed. The vector pET-29a-cct was verified by sequencing and restriction analysis. Recombinant plasmid pET-29a-cct was transformed to E. coli BL21 （DE3）, and induced with IPTG at 30 ~C. As a result, the acquired cct gene expressed at a high level in E. coli BI21 （DE3） and showed a 45 k band which was equivalent to the calculated molecular mass by SDS-PAGE band scanning analysis. More impor- tantly, the expressed protein proved to have cholinephosphate cytidylyltransferase activity of transforming CTP and phosphorylcholine into cytidine-5＇-diphosphate choline （CDP-choline）. The cloning and expression of cct gene provide an important basis for the synthetic biology research and low-cost production of CDP-choline.