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猪唾液酸粘附素的分段表达及功能鉴定

发表时间:2012-06-15  浏览量:1089  下载量:517
全部作者: 肖一红,邱洪凯,刘岳,崔然,周恩民
作者单位: 山东农业大学动物科技学院;西北农林科技大学动物医学院,兽医免疫研究所
摘 要: 为验证猪唾液酸粘附素(porcine sialoadhesin,pSn)和猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)囊膜糖蛋白GP5的关系,提取猪肺泡巨噬细胞(porcine alveolar macrophage,PAM)总RNA,用分段RT- PCR方法得到pSn全长基因。将5个分段基因分别克隆入pET- 28a载体,转化大肠杆菌进行诱导表达和纯化,用十二烷基硫酸钠- 聚丙烯酰胺凝胶电泳(SDS- PAGE)和Western blotting方法对表达的蛋白进行鉴定,用ELISA方法检测pSn的 5个分段蛋白与分段表达的谷胱甘肽转移酶(GST)融合GP5分段蛋白的相互关系。结果表明:扩增出了pSn的5个分段基因,pSn与GenBank中登录的序列同源性为99%. 5个分段基因分别克隆入pET- 28a,经异丙基硫代半乳糖苷(IPTG)诱导后用SDS- PAGE和Western blotting验证均得到了表达。ELISA结果表明:pSn- 3能与GP5- 1(约27~200氨基酸)蛋白结合,为了解PRRSV的感染细胞机制奠定了基础。
关 键 词: 兽医学;猪繁殖与呼吸综合征病毒;唾液酸粘附素;GP5;表达;功能
Title: Expression and function identification of PRRSV sialoadhesin fragments
Author: XIAO Yihong, QIU Hongkai, LIU Yue, CUI Ran, ZHOU Enmin
Organization: College of Veterinary Medicine, Shandong Agricultural University; Veterinary Immunobiology Research Institute, College of Veterinary Medicine, Northwest A & F University
Abstract: To identify the relationship of sialoadhesin and envelope glycoprotein GP5, 5 fragments of porcine sialoadhesin (pSn) were cloned from porcine alveolar macrophage (PAM) by RT- PCR. After all the fragments were cloned into pET- 28a vector and expressed. The purified proteins were identified by SDS- PAGE and Western blotting. The relationship of fragments proteins and fragments of GST- GP5 were analyzed by ELISA. The results showed that all the fragments were successfully cloned and expressed identified by SDS- PAGE and Western blotting. The binding of pSn with GP5 was confirmed by identification of ELISA. The results of ELISA showed that fragment of pSn- 3 could bind with GP5- 1 which could help to learn more about the infection mechanism of porcine reproductive and respiratory syndrome virus (PRRSV).
Key words: veterinary medicine; porcine reproductive and respiratory syndrome virus; sialoadhesin; GP5; expression; function
发表期数: 2012年6月第11期
引用格式: 肖一红,邱洪凯,刘岳,等. 猪唾液酸粘附素的分段表达及功能鉴定[J]. 中国科技论文在线精品论文,2012,5(11):1075-1080.
 
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