摘要
为研究不同动物源性K99菌毛基因的差异,以含K99菌毛基因的鸭源大肠埃希氏菌为研究对象,通过PCR方法扩增出不含信号肽K99菌毛基因片段,分别克隆至pMD19-T和pET-32a载体,进行基因序列测定及免疫印迹分析。结果表明,所获得2个不同的K99基因序列,长度均为498 bp,除去酶切位点编码氨基酸159个,理论等电点为8.96,均带正电荷,脂肪系数均为58.43。编码蛋白均为稳定的亲水性蛋白,具有相同B细胞抗原位点。2个K99菌毛基因序列之间的同源性为99.6%,与GenBank的序列比对,鸭源K99菌毛基因与牛源、猪源的K99菌毛基因的同源性较高,说明不同动物源K99菌毛基因之间变异小,相对保守。所表达的重组蛋白能与兔抗K99单因子血清发生特异性反应。研究结果为检测不同动物来源的产肠毒素大肠埃希氏菌及相关生物制品开发应用奠定基础。
To study the difference of K99 fimbrial gene in different sources of ETEC （enterotoxigenic Escherichia coli）. 2 strains of duck Escherichia coli contained K99 fimbrial gene were chosen as the object in this study, a K99 fimbrial subunit gene without signal peptide sequence were amplified by PCR and cloned into vector pMD19-T and pET32a（＋） respectively, analyzed by sequencing and Western blot. The results showed that two acquired K99 gene sequences＇ open reading frames （ORF）contained 159 amino acids （excepted for the restriction enzyme cutting site）, with the same length of 498 bp. And the isoelectric point of this protein was 8.96, both carrying positive charge. The fat coefficients was 58.43 as well. The results of protein prediction indicated that the fimbrial proteins was hydrophilicity and possess the same predicted B cell antigen sites. Sequence homology analysis showed that homology in two K99 fimbrial genes of duck E.coli strains was 99.6%. Besides, K99 fimbrial gene from duck ETEC had higher homology with ETEC from cattle, pig and other species among the 6 sequences, showing relative conservation among K99 fimbrial genes from different species. The results of Western blot confirmed that the recombinant K99 proteins could be inhibited with K99 mono-factor serum by showing specific antigenicity. The results laid a solid foundation to detect the different types of enterotoxigenic Escherichia coli and develop relevant biological products.
出处
《中国家禽》
北大核心
2016年第21期29-33,共5页
China Poultry
基金
国家农业科技成果转化资金项目(2013GB2F000422)
关键词
鸭源大肠埃希氏菌
K99菌毛基因
克隆表达
生物信息学分析
duck enterotoxigenic Escherichia coli
K99 fimbrial gene
cloning and expression
bio-information analysis
