医学分子生物学杂志2007，Vol.4Issue(1)：20-26,7.
家蝇幼虫抗菌肽Attacin基因的克隆表达及抑菌生物学活性
Cloning and Expression of Antibacterial Peptide Attacin Gene Isolated from Musca Domestica Larvae and Its Biological Activities
摘要
Abstract
Objective To clone the cDNA sequence of Attacin gene from Musca domestica larvae, construct prokaryotic fusion expression vectors, express and purify the Attacin protein, and preliminarily study its antibiotic biological function. Methods The cDNA fragment encoding Attacin was amplified from recombinant plasmid pUCm-T/Attacin by PCR with specific primers and then cloned into pET30a (a + ) and pGEX-4T-1 vector separately. These prokaryotic expression recombinant plasmids were transformed into E. coli to express the relative proteins respectively. The antibacterial activity of Attacin in E. coli was monitored with an in vivo system. The expressed recombinant products were purified with affinity-chromatograph column and analyzed with SDS-PAGE. Then the biological activities of Attacin were detected by agarose plate antibiotic assay. Results The 674 bp cDNA of Attacin was obtained by RT-PCR. The sequence has been accepted by GenBank (Accession number DQ062744). The pET30a (a + ) /Attacin and pGEX-4T-1/Attacin recombinant plasmids were confirmed by PCR, restriction endonuclease digestion and DNA sequencing, respectively. 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. The GST-Attacin fusion protein then underwent GST affinity-chromatography, and SDS-PAGE analysis confirmed the final purified product had the expected size. The recombinant protein exhibited antibacterial activities when assayed with agarose plates inoculated with bacteria. Conclusion The Attacin expressed in prokaryotic system possesses antibacterial activities, which lays a reliable foundation for further research on biological functions and application of Attaicn.关键词
家蝇/抗菌肽Attacin/基因克隆/生物信息学分析/原核表达Key words
Musca domestica/antibacterial peptide Attacin/gene cloning/bioinformatic analysis/prokaryotic expression分类
生物科学引用本文复制引用
徐建华,朱家勇,金小宝,许琴英..家蝇幼虫抗菌肽Attacin基因的克隆表达及抑菌生物学活性[J].医学分子生物学杂志,2007,4(1):20-26,7.基金项目
广东省科技计划攻关项目(No.2003B31602) This work was Supported by a grant from the Key Science and Technology Program of Guangdong Province ( No. 2003B31602) （No.2003B31602）
