Attacin抗菌肽基因体内抗菌活性研究

In vivo Antibacterial Activity of Antibacterial Peptide Attacin Gene

目的建立Attacin基因体内抗菌活性检测系统,并初步研究其抗菌活性。方法PCR扩增Attacin编码区目的序列,并构建原核表达重组质粒,转化大肠埃希菌,在体内检测Attacin的抗菌活性,同时SDS-PAGE分析融合蛋白表达情况。结果与未诱导对照相比,包含重组质粒的宿主菌生长受到抑制,含pET30a(+)/Attacin宿主菌诱导表达后,提纯不到His-Attacin融合蛋白,而含pGEX-4T-1/Attacin宿主菌可获得GST-Attacin融合蛋白。结论建立灵敏、简便的Attacin体内抗菌活性检测方法,为下一步研究Attacin的生物学功能及其应用开发奠定基础。

OBJECTIVE To clone the cDNA sequence of Attacin from Musca domestica larvae, construct prokaryotic fusion expression plasmids, establish in vivo monitoring system for antibacterial activity analysis, and then preliminarily study its antibacterial activity. METHODS The cDNA fragment encoding Attacin was amplified from pLTCm-T/Attacin by PCR with specific primers and then cloned into pET30a（a＋） and pGEX-4T-1 vector respectively. These recombinant plasmids were then transformed into E. coli to express the relative fusion proteins which were analyzed by SDS-PAGE respectively. According to the growth curve of transformants carrying recombinant plasmids, the antibacterial activity of Attacin in Escherichia coli was monitored by an in vivo system. RESULTS Transformation assay of E. coli with the recombinant plasmids revealed that, after IPTG induction and compared to non-induced control, the growth of host cells containing pET30a （＋）/Attacin was significantly suppressed, with no His-Attacin band on SDA-PAGE as expected. Although the growth of host cells containing pGEX-4T-1/Attacin was also inhibited, the GST-Attacin fusion protein was obtained. CONCLUSIONS An in vivo antibacterial activity monitoring system of Attacin is established, and provided a reliable tool for further study of biological functions and application of Attaicn.