摘要
本研究旨在构建一株携带绿色荧光蛋白(GFP)的重组病毒,以便更快速、准确地检测疫苗效价。利用基因重组技术构建含有GFP基因的重组质粒,并利用反向遗传学操作进行重组病毒的拯救,在Vero细胞进行病毒感染并收集蛋白样品进行Western blotting试验,检测外源蛋白GFP的表达情况,测定一步法生长曲线,比较与原始毒株的生物学特性差异。结果显示,本试验成功拯救出携带GFP基因的重组狂犬病病毒毒株SAD-GFP;Western blotting试验结果显示,重组病毒可表达GFP,且随着病毒感染时间的延长,蛋白表达增多;病毒结构蛋白G蛋白表达并未受外源蛋白表达的影响,重组毒株与原始毒株的生长并无明显差异;传代后GFP仍可在病毒中稳定表达。在连续传代过程中,本试验成功拯救出SAD-GFP重组毒株,GFP基因并未丢失,且蛋白表达量并未降低,证明该位点表达的外源蛋白在病毒传代过程中可持续稳定表达,且对病毒生长特性并未产生影响。
The aim of this study was to construct a recombinant virus carrying green fluorescent protein（GFP）protein,and detect vaccine titer more rapidly and accurately.Recombinant plasmids containing GFPgene were constructed by gene recombination technology and rescued by reverse genetics.The viral infection in Vero cells and protein samples were collected for Western blotting assay to detect the expression of foreign protein GFP.One-step growth curve was compared with the biological characteristics of the original strain.The results showed that we successfully rescued the recombinant GFP-expressing recombinant rabies virus SAD-GFP using the constructed recombinant plasmids.Western blotting result showed that the recombinant virus could express GFP,and the expression of GFP increased with the time of virus infection.The expression of structural protein G was not affected by the expression of foreign proteins.There was no significant difference between the recombinant strains and the original strains.After passage,GFP could stably express in the virus.In this experiment,the recombinant SAD-GFP strain was successfully rescued.In the continuous passage,the GFP gene was not lost,and the protein expression leveldid not decrease,which proved that the foreign protein expressed at this site could sustainedly and stablly express in the course of virus passage,and did not affect the growth characteristics of the virus.
作者
刘翠
殷相平
LIU Cui;YIN Xiangping(State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinaese Academy of Agricultural Sciences,Lanzhou 730046,China)
出处
《中国畜牧兽医》
CAS
北大核心
2018年第7期1965-1971,共7页
China Animal Husbandry & Veterinary Medicine
基金
国家重点研发计划(2016YFE0204100)
