可溶性转氢酶促进链球菌异柠檬酸脱氢酶基因表达的研究

Improvement of Gene Expression of Isocitrate Dehydrogenase from Streptococcus Mutans by Soluble Transhydrogenase

目的构建携带E．coli可溶性吡啶核苷酸转氢酶（UdhA）基因的双启动子双基因表达载体，鉴定此载体系统对表达效率不高的链球菌异柠檬酸脱氢酶（SmIDH）的促进表达。方法PCR法扩增SmIDH基因，插入pBluescript ⅡSK（＋）得表达载体pSX。从E．coli基因组扩增UdhA基因，插入pBluescriptⅡSK（＋），构建表达载体pUX。再用以pUX为模板PCR扩增lac启动子和udM基因片段，插入pSX中，获得双启动子双基因表达载体pUS。IPTG诱导表达，SDS—PAGE鉴定SmIDH的表达。结果酶切证实UdhA和SmIDH双基因正确克隆到pBluescriptⅡSK（＋）中，序列测定显示双基因与GenBank报道一致及两个lac启动子方向相同，并且初步的表达实验证明UdhA促进了SmIDH的表达。结论UdhA的表达促进了SmIDH基因的表达，为该表达策略的通用性研究奠定了基础。

Objective To construct expression vector with double-promoters containing the gene of E. coli soluble pyridine nucleotide transhydrogenase （ UdhA）, with the aim of promoting expression of isocitrate dehydrogenase from Streptococcus mutans （SmIDH） which is difficult to reach good expression. Methods First, SmlDH gene was amplified by PCR and inserted into the plasmild pBluescript 77 SK （ ＋ ） to create pSX. And UdhA gene was obtained by PCR and ligated to pBluescript ？？ SK （ ＋ ）, resulting in pUX. Next, the fragments of lac promoter and UdhA gene in pUX were amplified by PCR and inserted into pSX, creating the expression vector pUS containing double lac promoters and double genes. The expression of SmIDH was determined by SDS-PAGE with IPTG induction. Results Restriction digestion and gene sequencing confirmed the insertion and correct orientation of the double-promoters and double-genes in pUS. The preliminary experiment showed that the expression of SmIDH was promoted by the expression of UdhA. Conclusion Gene expression of isocitrate dehydrogenase from Streptococcus mutans was improved by the expression of E. coli solube transhydrogenase, providing a common strategy of UdhA expression.