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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：1560 下载量：676
| 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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