摘要
从鸡骨髓细胞中分离提取总RNA,经过RT-PCR扩增出鸡β-防御素-6(AvBD6)基因。经克隆测序表明扩增出的cDNA碱基数为204bp,编码68个氨基酸残基。根据已发现的禽β-防御素和鼠β-防御素-6的氨基酸序列构建系统进化树。发现鸡AvBD6氨基酸序列与鸡AvBD7氨基酸序列同源性最高,为62.7%。将该基因克隆到大肠杆菌原核表达载体pGEX-6p-1上,进行原核表达。SDS-PAGE电泳表明,表达的重组鸡AvBD6融合蛋白分子量约为32ku。对该重组蛋白进行纯化与体外抗菌活性的测定。结果表明,重组鸡AvBD6融合蛋白对多杀性巴氏杆菌、金黄色葡萄球菌、枯草芽胞杆菌有较高抗菌活性,对大肠杆菌的抗菌活性较弱,对猪霍乱沙门氏菌无抗菌活性。重组鸡AvBD6蛋白对上述细菌的最小抑菌浓度范围为31.25μg/mL～250μg/mL。此外,重组鸡AvBD6蛋白对温度和酸碱度有较高的稳定性。
The chicken β-defensin 6 （AvBD6） cDNA fragment was amplified by reverse transcription polymerase chain reaction（RT-PCR） from the bone marrow tissue of chicken. The complete nucleotide sequence of the gene contained a 204 bp open reading frame （ORF） encoding 68 amino acids. Phylogenetic tree analysis of AvBD genes and mouse beta defensin-6 showed that the gene shared the highest percentage of amino acid homology （62.7 %） with chicken AvBD7. The chicken AvBD6 gene was cloned into pGEX-6p-1 vector and a 32 ku protein recombinant protein was expressed in E. coli. The antimicrobial activity of the recombinant fusion protein was evaluated in vitro, which exhibited high antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, and Pastcurella multocida, but low antimicrobial activity against Escherichia coil, and no antimicrobial activity against Salmonell choleraesuis. The minimal inhibitory concentration （MIC） of the recombinant protein against above bacteria ranged from 31.25μg/mL to 250 μg/mL.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2009年第6期476-480,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(30600435)
NOVUS国际科学研究基金
黑龙江省教育厅科研重点项目(No.1153lz05)
黑龙江省博士后启动基金
