乳酸菌素Gassericin T基因的合成及其在大肠杆菌中的融合表达

目的:在大肠杆菌系统中表达有抗菌活性的乳酸菌素Gassericin T。方法:根据乳酸菌素Gassericin T的基因序列,把Gassericin T的结构基因gatA编码的氨基酸的密码子转换成大肠杆菌偏爱的形式;用人工合成的寡核苷酸片段,通过重叠PCR法扩增得到gatA片段(gat基因);将合成的gat基因插入pGEX-4T-1,构建pGEX-4T-1-gat融合表达载体,转化大肠杆菌DH5α株,IPTG诱导表达,经超声裂解后获得包涵体蛋白,经溶解、变性、复性处理后获得GST-Gassericin T融合蛋白;用琼脂扩散法测定其对金黄色葡萄球菌、大肠杆菌、李斯特菌、枯草杆菌等的抗菌活性。结果与结论:采用pGEX-4T-1融合表达系统在大肠杆菌中表达了有活性的Gassericin T,融合蛋白以包涵体形式存在。复性的融合蛋白对金黄色葡萄球菌和大肠杆菌有明显的抑制作用,对李斯特菌的抑制作用不明显。

Fusion Expression of Synthetic Gassericin T Gene in Escherichia coli

Objective:To express the bacteriocin Gassericin T with antimicrobial activities in E.coli.Methods:DNA fragment encoding the mature peptide of Gassericin T was prepared by three rounds of PCR from a total of six 59 nt oligoes after modification of the original sequence to the optional codon usage of E.coli and then cloned into pGEX-4T-1.Clones carrying pGEX-4T-1-gat was induced by IPTG.The bacteria were broken down by ultrasonic and the inclusion bodies obtained underwent denaturation,renaturation and purification to obtain GST-gassericin T fusion protein.The antimicrobial activity of the fusion protein was assayed by agar diffusion method.Results:Gassericin T was expressed as GST-fusion protein in pGEX-4T-1 prokaryotic expression system.The fusion protein was present in the lysate as inclusion bodies.Gassericin T demonstrated antimicrobial activities against gram-positive S.aureus and against gram-negative E.coli.Conclusion:Recombinant Gassericin T with antimicrobial activities was generated in pGEX-4T-1 fusion expression system.