摘要
目的建立高效培养,分离纯化埃可病毒30型(echovirus 30, EC30)的有效方法并筛选优化病毒晶体生长条件,为后续病毒疫苗开发及病毒结构功能研究建立基础。方法先利用人胚胎横纹肌肉瘤细胞系(human embryonic rhabdomyosarcoma cell,RD)培养、生产在2010年广西手足口病疫情中分离到的埃可病毒30型毒株;而后使用蔗糖密度梯度离心法分离纯化病毒颗粒;再用透射电镜观察病毒颗粒的形态;最后使用Hampton晶体试剂盒筛选并优化病毒晶体适宜生长条件。结果经透射电镜验证获得完整形态病毒颗粒并在p H 4.6的醋酸钠溶液中培养得到高质量的病毒颗粒晶体。结论通过RD细胞培养结合蔗糖密度梯度离心法可获得高纯度病毒颗粒;利用试剂盒筛选可获得高质量的病毒蛋白晶体。
Objective To establish an efficient method for isolation and purification of echovirus 30 （EC30） and to optimize the growth conditions of viral crystals, and to establish a basis for subsequent viral vaccine development and viral structural function research. Methods The human embryonic rhabdomyosarcoma cell line （RD） was used to culture and produce the Echovirus type 30 strain isolated from the hand-foot-mouth disease epidemic in Guangxi in 2010. The sucrose density gradient centrifugation method was used to isolate and purify the virus particles; the morphology of the virus particles was observed by transmission electron microscopy; finally, the Hampton crystal kit was used to screen and optimize the suitable growth conditions of the virus crystals. Results The intact morphological virus particles were obtained by transmission electron microscopy and cultured in sodium acetate solution at pH 4.6 to obtain high quality virus particle crystals. Conclusion High purity virus particles can be obtained by RD cell culture combined with sucrose density gradient centrifugation; high quality viral protein crystals can be obtained by kit screening.
作者
何娅玲
HE Ya-ling(School of Life Sciences,Fudan University,Shanghai,200433 China)
出处
《中外医疗》
2018年第27期15-17,共3页
China & Foreign Medical Treatment
关键词
埃可病毒30型
蔗糖密度梯度离心
透射电镜
晶体生长条件筛选
Echovirus type 30
Sucrose density gradient centrifugation
Transmission electron microscopy
Screening of crystal growth conditions
