H3N2亚型禽流感病毒核蛋白基因的克隆及其在大肠杆菌中的表达  

Cloning and Expression of Nucleoprotein Gene of H3N2 Subtype Avian influenza virus in Escherichia coli

作　　者：柳斌[1] 杨志新[1] 李文平[2] 屈孝初[2] 周晓巍[1] 黄培堂[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]湖南农业大学动物科技学院,湖南长沙410128

出　　处：《生物技术通讯》2007年第3期419-422,共4页Letters in Biotechnology

摘　　要：目的:获得H3N2亚型禽流感病毒核蛋白(NP)全长基因,并在大肠杆菌中表达,以用于对NP功能的研究。方法:从感染了H3N2亚型禽流感病毒的MDCK细胞培养液中收获病毒,提取病毒RNA,用RT-PCR扩增出NP基因的编码区序列,将其定向克隆到pTIG-TRX原核表达载体并测序,在大肠杆菌BL21(DE3)plysS中表达,用SDS-PAGE和Western印迹检测表达产物。结果:所克隆的核苷酸片段包含了NP基因编码区完整阅读框架,编码498个氨基酸;构建的重组表达载体在大肠杆菌BL21(DE3)plysS中表达出相对分子质量约66000的重组蛋白。结论:克隆和表达了禽流感病毒核蛋白编码区基因,为获得大量NP以制备抗体,以及对其进行功能性研究奠定了基础。Objective： To clone the complete gene of nucleoprotein（NP） of H3N2 subtype avian influenza virus, and to express recombinant NP in E.coli for the function research. Methods： The complete NP gene was amplified from supernatant of MDCK cells infected with H3N2 subtype avian influenza virus by RT-PCR. The complete encoding gene was inserted into prokaryotic expression vecotr pTIG-TRX, and then was transformed and expressed in E.coli BL21 （DE3）plysS. The amplified NP gene was identified by sequencing. The expressed protein was detected by Western blotting. Results： The cloned eDNA fragments covered the whole open reading frame of NP gene and encoded 498 amino acids. The relative molecular weight of expressed recombinant protein was about 66 000. Conclusion： The NP encoding gene of avian influenza virus is cloned and expressed successfully. The study lays a foundation of preparating antibody and earring on analysising function of NP protein for avian influenza virus.

关 键 词：禽流感病毒 核蛋白 原核表达 大肠杆菌 

分 类 号：Q78[生物学—分子生物学]