中国家蚕抗菌肽ABP-CM4在E.coli中的融合表达及活性分析  

EXPRESSION OF THE ANTIBACTERIAL PEPTIDE CM4-LIKE GENE OF CHINESE SILKWORM BOMBYX MORI IN ESCHERICHIA COLI AND ITS ANTIBACTERIAL ACTIVITY ANALYSIS

作　　者：李保存[1] 陈玉清[1] 刘平[1] 张双全[1] 

机构地区：[1]南京师范大学生命科学学院江苏省分子医学生物技术重点实验室,南京210097

出　　处：《分子细胞生物学报》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)。

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.

关 键 词：抗菌肽CM4 融合表达 E.COLI 

分 类 号：Q78[生物学—分子生物学]