Li Bao Cun, Chen Yu Qing, Liu Ping, Zhang Shuang Quan
The Life Science School of Nanjing Normal University, Nanjing 210097.
Fen Zi Xi Bao Sheng Wu Xue Bao. 2007 Apr;40(2):98-102.
According to the amino acid sequence of CM4 and the bias for preferred condons of E. coli, the CM4-like gene was obtained by a recursive PCR (rPCR) strategy using two lapping oligonucleotides. The synthesized gene was coloned into the expression vector pET32(a) and transformed into E. coli BL21 (DE3). Recombinant CM4-like gene expression was driven by the T7 promoter on the vector upon addition of IPTG and high level of expression was achieved. The solube protein was purified by Ni-chelating agarose and treated with formic acid. After cleavege, the recombinant peptide was purified by another Ni(2+)-NTA-Agarose affinity chromatography and cation-exchange chromatography. Results of agarose diffuse assay and liquid turbidity analysis indicated that the recombinant peptide exhibited the antibacterial activity.
根据CM4的氨基酸序列以及大肠杆菌对偏好密码子的偏向性，使用两个重叠的寡核苷酸通过递归PCR（rPCR）策略获得了类CM4基因。合成的基因被克隆到表达载体pET32(a)中，并转化到大肠杆菌BL21(DE3)中。加入IPTG后，载体上的T7启动子驱动重组类CM4基因表达，并实现了高水平表达。可溶性蛋白通过镍螯合琼脂糖进行纯化，并用甲酸处理。切割后，重组肽通过另一种Ni(2+)-NTA-琼脂糖亲和色谱和阳离子交换色谱进行纯化。琼脂糖扩散试验和液体浊度分析结果表明，重组肽具有抗菌活性。
