巴什拜羊ISG15基因的克隆与序列分析

Cloning and Sequence Analysis of Xinjiang Bashenbai Sheep ISG15 Gene

本研究旨在了解新疆巴氏拜羊类泛素蛋白ISG15基因的序列特征。采集巴什拜羊外周血液,从中分离得到淋巴细胞并提取RNA,设计特异性引物进行RT-PCR,克隆出巴什拜羊ISG15基因序列,回收PCR产物与pMD18-T载体连接后转化DH5α,检测阳性克隆、测序并进行序列分析。结果表明,克隆的巴什拜羊ISG15基因编码区全长为522bp,编码172个氨基酸。BLAST结果表明,巴什拜羊ISG15基因与绵羊、山羊、小尾寒羊、水牛、牛和野猪的ISG15基因序列同源性分别为99%、98%、95%、94%、94%和82%。构建基因进化树分析结果显示,巴什拜羊与绵羊先聚为一类,再与小尾寒羊聚为一类,然后和牛聚为一类。该聚类结果与生物学上的分类一致。

In order to study the molecular construction of the ISG15 gene from Xijiang Bashibai sheep, RNA was extracted from the lymphocyte, which was isolated from the peripheral blood of the Bashibai sheep. The specific primer designed was amplified by the RT-PCR method. Then cloned the gene order of Bashibai sheep, PCR products that were reclaimed were cloned into pMD18-T vector, and then transformed into DH5a, detected masculine clone, proceeded sequence analysis. The conclusion showed that total length of the genetic coding region cloned from Bashibai sheep is 522 bp, and 172 amino acids were encoded. BLAST analysis showed that Bashibai sheep ISG15 gene shared 99%, 98%, 95%, 94%, 94% and 82% homology with those of Ovis aries, goat, Ovis aries breed Small Tail Han, Bubalus bubalis, bos and Sus scrofa respectively. Molecular phylogenetic tree analysis showed that Bashibai sheep assembled to Ovis aries, then assembled to Ovis aries breed small Tail Han and bos. The clustering was identical to the biological classification.