摘要
参照天然抗菌肽CM4(ABP-CM4)氨基酸序列和大肠杆菌偏爱密码子,采用rPCR法获得CM4基因后重组到表达载体pET32a上,在E.coli中融合表达。表达产物以可溶性存在,经Ni2+-NTA琼脂糖亲和层析获得融合蛋白,再经甲酸切割、亲和层析和阳离子交换层析,得到纯化的重组抗菌肽。琼脂糖扩散法和液相测定法证明了纯化的抗菌肽具有抗菌活性。
According to the amino acid sequence of CM4 and the bias for preferred condons of E.coli, the CM4-1ike 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 CM4-1ike 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 cationexchange chromatography. Results of agarose diffuse assay and liquid turbidity analysis indicated that the recombinant peptide exhibited the antibacterial activity.
出处
《分子细胞生物学报》
SCIE
CAS
CSCD
北大核心
2007年第2期98-102,共5页
Journal of Molecular Cell Biology
基金
国家自然科学基金(30270193)
江苏省自然科学基金(BK2006221)。