摘要
采用RT-PCR方法,从鸡舌组织中扩增到Gallinacin-9(Gal-9)基因,测序表明Gal-9为201 bp,其成熟肽由67个氨基酸残基组成。进一步将克隆的Gal-9基因亚克隆到大肠杆菌原核表达载体pGEX-6p-1的EcoRⅠ和SalⅠ双酶切位点上,构建重组表达质粒pGEX-Gal-9,经序列分析鉴定确证目的基因克隆入载体的预期位点。将重组质粒转化大肠杆菌BL21,于37℃诱导培养不同时间,SDS-PAGE电泳表明该基因在大肠杆菌中高水平表达,表达的重组鸡Gal-9融合蛋白相对分子质量约为32 ku。重组蛋白占菌体总蛋白的40%。表达的重组鸡Gal-9融合蛋白以包涵体的形式存在。重组蛋白经纯化后,分别以对数生长中期的大肠杆菌BL21(DE3-)株与致病性链球菌CAB株为检测菌,利用薄层平皿琼脂糖孔穴扩散法测定了重组Gal-9蛋白的抗菌活性,结果表明,重组Gal-9对这2种细菌都具有抗菌活性。
Gallinacin(Gal)-9 was cloned from tongue of chicks by RT-PCR and sequenced.The full length cDNA of Gal-9 consists of 201 bp,encoding 67 amino acids.The cDNA of chicken Gal-9 gene was sub-cloned into EcoR Ⅰ and Sal Ⅰ sites of pGEX-6p-1 vector to construct recombinant plasmid pGEX-Gal-9.The recombinant plasmid was transfected into E.coli BL21 and the transformed bacteria was induced with IPTG.It was demonstrated by SDS-PAGE that a 32 kDa protein which was equal to chicken Gal-9 protein in molecular weight was expressed in E.coli BL21.The recombinant fusion protein was purified.The recombinant Gal-9 was expressed as inclusion bodies.The recombinant Gal-9 exhibit expected anti-E.coli and anti-Pathogenic streptococcus activity,as measured by inhibition zone assaying.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2008年第10期1426-1431,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(30600435)
NOVUS国际科研基金
黑龙江省教育厅重点项目(1153LZ05)
