CAJ | 학술논문

目的从细胞膜上提纯禽流感病毒H5N1的血凝素蛋白H5 I151F和H5 I151F+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H5 I151F和H5 I151F+A134V+E186D,并得到了H5 I151F蛋白晶体.结论首次获得了禽流感病毒H5N1的H5 I151F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.
목적 종세포막상제순금류감병독H5N1적혈응소단백H5 I151F화H5 I151F+A134V+E186D,병사단백결정.방법 세포내대량증식중조우두병독,거구제제취세포막중적HA단백,련속자당밀도제도초속리심,Western인적검측,파라단백매등렬해HA,리자교환층석,SDS-PAGE전영병염색검측단백순도,좌적기상확산법결정.결과 종세포막중제취료고순도적혈응소단백H5 I151F화H5 I151F+A134V+E186D,병득도료H5 I151F단백정체.결론 수차획득료금류감병독H5N1적H5 I151F단백정체,위진일보연구금류감병독인전인적가능성타하기출.
Objective To purify and crystallize two kinds of H5N1 vires 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 puri-ty was detected by SDS-PAGE electrophoresis and staining. HA proteins were crystallized by sitting-drop vapour diffusion. Results Hemag-glutinin proteins, H5 I151F and H5 I151F + A134V + E186D, were extensively purified. And the crystal of H5 I151F was obtained. Con-clusion For the first time, the highly purified H5 I151F membrane protein crystal was obtained, which provided the basis for further stud-ying the mechanisms of human to human transmission caused by avian influenza viruses.