白毛乌骨鸡ChIFN-β基因克隆与生物信息学分析

Cloning and Bioinformatics Analysis of ChIFN-β Gene in Black-bone Chicken with White Feather

为研究白毛乌骨鸡β干扰素的生物学特性,利用RT-PCR技术扩增了白毛乌骨鸡肝脏β干扰素(ChIFN-β)基因,将其连接到pGEM-T Easy载体上进行转化,重组质粒PCR鉴定后进行序列测定,同时利用BLAST、SignalP 4.1 Server、TMHMM Server v.2.0和DNAStar等软件对ChIFN-β进行生物信息学分析。结果表明:ChIFN-β基因编码区长度为612 bp,编码203个氨基酸,分子量为23.686 ku,等电点为10.3;ChIFN-β与已报道的其它鸡的IFN-β氨基酸序列具有较高的同源性;白毛乌骨鸡ChIFN-β氨基酸序列和绿头鸭首先聚类,与其它动物的亲缘关系较远;ChIFN-β存在跨膜结构,为分泌蛋白,前26个氨基酸为信号肽序列;ChIFN-β蛋白具有干扰素αβ的匹配区域,同时含有干扰素αβ受体复合物两个跨膜亚基IFNAR-1和IFNAR-2的结合位点,预测ChIFN-β蛋白属于IFαβ超家族。研究结果为白毛乌骨鸡ChIFN-β基因的结构和功能研究提供了参考。

ChIFN-βgene from the liver of black-bone chicken with white feather was amplified by RT-PCR to study the biological characteristics of Interferon beta.The sequence of ChIFN-βwas verified after being connected with pGEM-T Easy vector,transformated into competent cell,and identificated by bacterial fluid PCR.Additionally,bioinformatics analysis of ChIFN-βwere performed by BLAST,SignalP 4.1 Server,TMHMM Server v.2.0,DNAStar and other softwares.The results showed that ChIFN-βgene was 612 bp in length and encoded 203 amino acids,the molecular weight and isoelectric point were predicted to be 23.686 ku and 10.3 respectively.IFN-βhad high similarity between black-bone chicken with white feather and other reported breeds of chicken.ChIFN-βwas clustered together with mallard duck firstly,while had a distant relationship with IFN-βfrom other animals.ChIFN-βwas a secreting protein with a transmembrane structure,and the first 26 amino acids of which were signal peptide sequences.ChIFN-βprotein was predicted to be belonged to IFαβsuperfamily because of conservative region of interferonαβ,IFNAR-1 and IFNAR-2 receptor binding sites.These results could provide reference for further study on the structure and function of ChIFN-βgene in black-bone chicken with white feather.