用指示基因法定量分析副粘病毒包膜糖蛋白所致的细胞融合  

Quantitative analysis of cell fusion caused by glycoprotein of paramyxoviruses with reporter gene method

作　　者：王志玉[1] 于修平[1] 

机构地区：[1]山东大学(西校区)病毒学研究室,济南250012

出　　处：《中华实验和临床病毒学杂志》2001年第2期179-181,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金资助 (395 70 0 32

摘　　要：目的　建立一种定量分析副粘病毒包膜糖蛋白融合细胞功能的方法。方法　将vTF 7重组痘苗病毒感染BHK2 1细胞后 ,分别转染待测基因DNA和pG1NT7β galDNA ,16h后将两个细胞群混合 ,37℃ 15h后用NP 40裂解细胞 ,取上清进行显色反应 ,在SOFTMAX软件支持下用ELISA自动测定仪在 5 70nm测吸光度A值。结果　指示基因法与细胞计数法有密切相关关系 ,r =0 9890 (P <0 0 1) ;与面积判断法的符合率达 10 0 %。最佳主要反应条件包括 :16mmol L底物浓度 ;显色反应 2 0～2 5min ;1μgDNA转染量 ;每种细胞群接种 1× 10 5 个。结论　指示基因法是一种非常敏感、特异、可重复性好的细胞融合定量分析方法 ,可用于副粘病毒细胞融合作用功能的研究 ,并可作为其他病毒细胞融合作用研究的参考 。Objective To establish a kind of quatitative assay method for analyzing the reporter gene assay for fusion was established.Methods Two populations of BHK21 cells were infected with vTF\|7 recombinant and wild\|type vaccinia viruses respectively and transfected with paramyxovirus F and HN cDNA and plasmid pG1NT7 β\|gal respectively.After 16 h,the two cell populations were removed from the wells by trypsinization.Equal numbers of the two populations were mixed in wells of a 96\|well flat\|bottom plate after washing and pelleting.After 15 hours' incubation at 37 ℃,the cells were lysed with Nonidet P\|40 and A values were read at 570 nm with an ELISA reader supported by SOFTMAX software.Results Reporter gene method had a close relationship with cell count method, r=0 9890(P <0 01).Positive and negative conincidence rates were 100%.The best main reaction conditions are as follows:substrate concentration,16 mmol/L;color reaction time,20-25 min;amount of DNA for transfection, 1 μg;cell numbers for each population,1×10 5.Conclusion Reporter gene method is a kind of very sensitive,specific and repeatable quantiative analysis for cell fusion and can be used in the studies on membrane fusion caused by any virues.

关 键 词：副粘病毒 细胞融合 定量分析 

分 类 号：R373[医药卫生—病原生物学]