Cloning, prokaryotic expression and antigenicity analyzing of NS1 gene of H9N2 swine influenza virus
2008
Wang Fangkun | Yuan Xiufang | Wang Yicheng
صينى. 对H9N2亚型猪流感病毒NS1（nonstructural protein 1）基因进行原核表达，获得纯化表达产物，以期为检测猪流感抗体的 ELISA试剂盒的研制奠定基础。采用RT-PCR扩增了猪流感病毒（H9N2）的NS1基因，将其克隆于表达载体pET-28a（+）上构建成重组质粒pET-NS1，转化受体菌E.coli BL21-DE3感受态细胞，经酶切鉴定及序列分析，筛选出正确重组质粒转化子。经终浓度5 mmol•L-1乳糖诱导，SDS－PAGE电泳结果显示，重组蛋白NS1得到大量表达，分子质量约为26kD。经Western-blotting分析，表达蛋白能与阳性血清发生特异性反应，而与阴性血清不反应；ELISA检测显示，在被检血清稀释1 280倍时，阳性血清的OD650值大约是阴性血清的3倍，差异明显。重组蛋白NS1表达量高，易于纯化并且具有良好的血清学反应的特异性。
اظهر المزيد [+] اقل [-]إنجليزي. The NS1 gene of H9N2 subtype influenza A virus was expressed in prokaryotic expression system to obtain recombinant protein. The NS1 gene of H9N2 subtype influenza A virus was amplified by RT-PCR, and then cloned into prokaryotic expression vector pET-28a (+).The recombinant plasmid pET-NS1 was extracted after being transformed into E.coli BL21-DE3 competent cells. Restriction enzymes digestion and sequences analysis demonstrated that the recombinant plasmid carrying NS1 was inserted into correct open reading frame. The recombinant protein of NS1 about 26 kD was produced after induction with 5 mmol•L-1 lactose.The western-blotting test showed that the recombinant protein reacted with positive sera from pigs, not with negative sera. The OD650 value difference was distinctly that the positive sera 1 280 fold dilution is about 3 times of the negative sera. These results have laid a solid foundation for developing an effective diagnostic method.
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