禽流感病毒H5N1血凝素蛋白的细胞膜上提纯和结晶

Purification and crystallization of hemagglutiuln expressed on the cellular membrane originated from avian influenza virus H5N1

目的从细胞膜上提纯禽流感病毒H5N1的血凝素蛋白H51151F和H51151F＋A134V＋E186D，并使蛋白结晶。方法细胞内大量增殖重组牛痘病毒，去垢剂提取细胞膜中的HA蛋白，连续蔗糖密度梯度超速离心，Western印迹检测，菠萝蛋白酶等裂解HA，离子交换层析，SDS—PAGE电泳并染色检测蛋白纯度，坐滴气象扩散法结晶。结果从细胞膜中提取了高纯度的血凝素蛋白H51151F和H51151F＋A134V＋E186D，并得到了H51151F蛋白晶体。结论首次获得了禽流感病毒H5N1的I-IS1151F蛋白晶体，为进一步研究禽流感病毒人传人的可能性打下基础。

Objective To purify and crystallize two kinds of H5N1 virus hemagglutinin proteins, H5 I151F and H5 I151F ＋ A134V ＋ E186D, from the cellular membrane. Methods Recombinant vaccinia viruses were massive propagated, hemagglutinin （HA） proteins were extracted from cellular membrane with detergent. HA proteins were concentrated with continuous sucrose gradient ultracentrifugation and detected by Western Blotting. HA proteins were cleavaged with Bromelain, and purified with Ion-exchange chromatography. The protein purity was detected by SDS-PAGE electrophoresis and staining. HA proteins were crystallized by sitting-drop vapour diffusion. Results Hemagglutinin proteins, H5 I151F and H5 I151F ＋ A134V ＋ E186D, were extensively purified. And the crystal of H5 I151F was obtained. Conclusion For the first time, the highly purified H5 I15lF membrane protein crystal was obtained, which provided the basis for further studying the mechanisms of human to human transmission caused by avian influenza viruses.