3种纯化重组禽流感病毒NS1抗原方法的比较  

Comparison of Three Methods to Purify Recombinant NS1 Antigen of Avian Influenza Virus

作　　者：霍惠玲 李永清[2] 赵燕岭 张莉[2] 章振华[2] 

机构地区：[1]河北省兽药监察所,河北石家庄050000 [2]北京市农林科学院畜牧兽医研究所,北京100097

出　　处：《生物技术通讯》2007年第5期774-777,共4页Letters in Biotechnology

基　　金：河北省省校合作基金项目(03820417D)

摘　　要：目的:摸索出最佳分离纯化和复性重组禽流感病毒NS1抗原的方法,得到高纯度的重组蛋白。方法:将重组质粒pET32a-NS1转染大肠杆菌BL21(DE3)后获得表达,分别以尿素变性、复性,Ni-NTA His.Bind Resin亲和,以及脱氧胆酸钠-N-十二烷基肌氨酸钠(DOC-SKL)洗涤溶解等3种纯化方法从表达产物包涵体中分离纯化NS1蛋白,并进行比较研究。结果:原核表达得到相对分子质量约45000的目的蛋白;3种纯化方法均能分离和纯化出NS1重组蛋白,其中尿素纯化的蛋白纯度为50%～60%,Ni-NTAHis.Bind Resin亲和纯化的蛋白纯度为80%～90%,DOC-SKL纯化的蛋白纯度达95%以上;Western blot检测表明,复性后的纯化蛋白具有良好的生物学活性。结论:应用十二烷基肌氨酸钠洗涤纯化是最佳的纯化NS1蛋白的方法,所获得的蛋白可作为包被ELISA的抗原。Objective： In order to obtain recombinant NS1 antigen of avian influenza virus with high-purity, the purified methods of fusion protein from bacterial lysates were optimized. Methods： The recombinant prokaryotic expression plasmids pET32a-NS1 was transformed into competent Escherichia coli strain BL21 （DE3） and expressed. The expression products were purified by three methods including urea denaturalization and reversion, Ni-NTA His Bind Resin affinity chromatograph and deoxycholic acid sodium-N-sodium lauryl sarkosinate（DOC-SKL） washing and reversion. Results： The expression products containing fusion protein were obtained. Three above methods could isolate and purified the NS1 fusion protein from bacterial lysates. By using urea denaturalization and reversion, the purity of NS1 fusion protein reached 50%-60%; by using Ni-NTA His Bind Resin affinity chromatograph, the purity of fusion protein reached 80%-90%; by using Doc- SKL washing and reversion, the purity of fusion protein was above 95%. The results of Western blot showed the purified fusion protein with good biologic characteristic. Conclusion： Comparing the three methods, the DOC-SKL washing and reversion was the optimal purified method of fusion protein from bacterial lysates. The fusion protein of NS1 could be a candidate antigen to develop specific diagnosing ELISA kit.

关 键 词：禽流感病毒NS1抗原 重组抗原 纯化 

分 类 号：Q503[生物学—生物化学] Q78