重组人TFF2乳酸链球菌表达载体pTRTFF2的构建

Construction of Lactococcus lactis expression vector of recombinant human trefoil factor family 2

目的构建带c-myc分子标签的人三叶因子家族2(hTFF2)融合基因c-myc-hTFF2的乳酸链球菌表达载体,以便进一步构建能表达活性c-myc-hTFF2融合蛋白的乳酸链球菌工程菌。方法根据hTFF2的氨基酸表达序列和乳酸链球菌的氨基酸密码子,以c-myc作为分子标签,设计c-myc-hTFF2的cDNA序列,并在其5'端和3'端添加SalI和BamHI酶切位点;将目的基因片段设计成14个相互互补的寡核苷酸,经聚合酶链式反应,得到目的基因c-myc-hTFF2;将c-myc-hTFF2连入pBluescriptIIsk(+)载体,构建其克隆载体pBS-TFF2并进行酶切鉴定及DNA测序;分别用SalI/BamHI和XhoI/BamHI对pBS-TFF2和pNBC1000进行双酶切反应,再通过连接反应将c-myc-hTFF2融合基因插入pNBC1000,构建c-myc-hTFF2大肠杆菌表达载体(pNTFF2);用XbaI对pNTFF2和穿梭载体pTRKH2单酶切反应,并通过连接反应将c-my-hTFF2的表达载体连入穿梭载体pTRKH2,完成乳酸链球菌表达载体pTRTFF2的构建,并进行酶切鉴定。结果成功构建了c-myc-hTFF2的乳酸链球菌表达载体pTRTFF2。结论体外基因拼装是构建目的基因有效的方法;pTRTFF2的构建为构建乳酸链球菌工程菌奠定了基础。

Objective To construct a Lactococcus lactis expression vector of c-myc-tagged human trefoil factor family 2 （hTFF2） fusion gene to prepare for genetic modification ofLactococcus lactis that can secrete bioactive c-myc-hTFF2 protein. Methods Based on the amino sequence of hTFF2 and optimal Lactococcus lactis codon usage, the cDNA of hTFF2 was designed and extended at their 5＇ ends with a sequence encoding c-myc as the molecular tag. According to the restriction sites of pBluescript II sk （＋）, the Sa/I and BamHI sites were arranged at the 5＇ and 3＇ ends of the fusion gene respectively. The sequence of the fusion gene c-myc-hTFF2 was designed as 14 oligonucleotides that overlapped with each other, and by means of PCR, all the oligonucleotides were spliced to complete the construction of c-myc-hTFF2 fusion gene. The target gene of c-myc-hTFF2 was inserted into pBluescript Ⅱ sk （＋） to construct the cloning vector pBS-hTFF2 of c-myc-hTFF2 followed by verification by enzyme digestion and DNA sequencing. By digestion of pBS-TFF2 with Bam HI/Sa/I and of pNBC 1000 with BamHI/XhoI, we connected c-myc-hTFF2 with pNBC1000 to construct the expression vector c-myc-hTFF2 in E. coli named as pNTFF2. After digestion of pNTFF2 and pTRKH2 with XbaI, the target gene was subcloned into pTRKH2 and the construction of the expression vector pTRTFF2 in Lactococcus lactis was completed. The constructed vector was identified by restriction enzyme digestion. Results and Conclusion The expression vector pTRTFF2 of c-myc-hTFF2 fusion gene has been successfully constructed. Assembly of oligonucleotides in vitro is an effective means to synthesize the target fusion gene and this prepares the ground for constructing engineered bacterium ofLactococcus lactis.