携带GFP基因的重组狂犬病病毒的拯救及其生物学特性研究

doi:10.16431/j.cnki.1671-7236.2018.07.030

摘要/Abstract

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。

关键词: 狂犬病病毒; 反向遗传; GFP

Abstract:

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 GFP gene 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 level did 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.

Key words: rabies virus; reverse genetic; GFP

中图分类号:

S852.65

刘翠, 殷相平. 携带GFP基因的重组狂犬病病毒的拯救及其生物学特性研究[J]. 《中国畜牧兽医》, 2018, 45(7): 1965-1971.

LIU Cui, YIN Xiangping. Rescue and Biological Characteristics Analysis of Recombinant Rabies Virus Containing GFP Gene[J]. , 2018, 45(7): 1965-1971.

参考文献

[1] POULIOTT C,DUPUIS M,APPLER K,et al.Susceptibility of neuroblastoma cells to rabies virus may be affected by passage number[J].Journal of Virological Methods,2017,247:28-31.
[2] MARTINEZ L.Global infectious disease surveillance[J].International Journal of Infectious Diseases,2000,4(4):222-228.
[3] TORDO N,POCH O,ERMINE A,et al.Walking along the rabies genome:Is the large GL intergenicregion a remnant gene[J].Proceedings of the National Academy of Sciences,1986,83(11):3914-3918.
[4] CONZALMANN K K,COX J H,SCHNEIDER L G,et al.Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B19[J].Virology,1990,175(2):485-499.
[5] TORDO N,POCH O,ERMINE A,et al.Primary structure of leader RNA and nucleoprotein genes ofthe rabies genome:Segmented homology with VSV[J].Nucleic Acids Research,1986,14(6):2671-2683.
[6] TORDO N,POCH O,ERMINE A,et al.Completion of the rabies virus genome sequence determination:Highly conserved domains among the L (polymerase) proteins of unsegmented negative-strand RNA viruses[J].Virology,1988,165(2):565-576.
[7] 汪孟航,朱洪伟,何民辉,等.狂犬病病毒糖蛋白生物学功能研究进展[J].中国畜牧兽医,2016,43(12):3349-3355. WANG M H,ZHU H W,HE M H,et al.Advances in biological function of rabies virus glycoprotein[J].China Animal Husbandry & Veterinary Medicine,2016,43(12):3349-3355.(in Chinese)
[8] 王朝,周明,傅振芳,等.狂犬病病毒逃逸宿主天然免疫反应的研究进展[J].生命科学,2017,29(3):237-244. WANG C,ZHOU M,FU Z F,et al.The research progress on the mechanism of evasion from the host innate immune response by rabies virus[J].Chinese Bulletin of Life Sciences,2017,29(3):237-244.(in Chinese)
[9] SEO W,PREHAUD C,KHAN Z,et al.Investigation of rabies virus glycoprotein carboxyl terminus as an in vitro predictive tool of neurovirulence.A 3R approach[J].Microbes and Infection,2017,19(9-10):476-484.
[10] BEN AZOUN S,BEN ZAKOUR M,SGHAIER S,et al.Expression of rabies virus glycoprotein in the methylotrophic yeast Pichia pastoris[J].Biotechnology and Applied Biochemistry,2017,64(1):50-61.
[11] AlBERTINI A A V,SCHOEHN G,WEISSENHORN W,et al.Structural aspects of rabies virus replication[J].Cellular and Molecular Life Sciences,2008,65(2):282-294.
[12] WIRBLICH C,COELEMON C M,KURUP D,et al.One-health:A safe,efficient,dual-use vaccine for humans and animals against Middle East respiratory syndrome coronavirus and rabies virus[J].Journal of Virology,2017,91(2):e02040-16.
[13] LUO J,ZHAO J,TIAN Q,et al.A recombinant rabies virus carrying GFP between N and P affects viral transcription in vitro[J].Virus Genes,2016,52(3):379-387.
[14] ELMORE S A,CHIPMAN R B,SLATE D,et al.Management and modeling approaches for controlling raccoon rabies:The road to elimination[J].PLoS Neglected Tropical Diseases,2017,11(3):e0005249.
[15] AMANN R,ROHDE J,WULLE U,et al.A new rabies vaccine based on a recombinant ORF virus (parapoxvirus) expressing the rabies virus glycoprotein[J].Journal of Virology,2013,87(3):1618-1630.
[16] CONZELMANN K K,SCHNELL M.Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins[J].Journal of Virology,1994,68(2):713-719.
[17] WILLET M,KURUP D,PAPANERI A,et al.Preclinical development of inactivated rabies virus-based polyvalent vaccine against rabies and filoviruses[J].Journal of Infectious Diseases,2015,212(Sppl_2):S414-S424.
[18] SHUAI L,WANG X,WEN Z,et al.Genetically modified rabies virus-vectored Ebola virus disease vaccines are safe and induce efficacious immune responses in mice and dogs[J].Antiviral Research,2017,146:36-44.
[19] APPOLINARIO C M,ALLENDORF S D,PERES M G,et al.Evaluation of short-interfering RNAs treatment in experimental rabies due to wild-type virus[J].Brazilian Journal of Infectious Diseases,2015,19(5):453-458.
[20] XUE X,ZHENG X,LIANG H,et al.Generation of recombinant rabies virus CVS-11 expressing eGFP applied to the rapid virus neutralization test[J].Viruses,2014,6(4):1578-1589.
[21] KHAWPLOD P,INOUE K,SHOJI Y,et al.A novel rapid fluorescent focus inhibition test for rabies virus using a recombinant rabies virus visualizing a green fluorescent protein[J].Journal of Virological Methods,2005,125(1):35-40.
[22] 冯鹏,杨增岐,吴晓平.犬用狂犬病疫苗免疫效果的评价[J].中国兽医科学,2017,47(10):1337-1340. FENG P,YANG Z Q,WU X P.Evaluation of immune efficacy of rabies vaccines in dogs[J].Chinese Veterinary Science,2017,47(10):1337-1340.(in Chinese)
[23] APPOLINÁRIO C,ALLENDORF S D,VICENTE A F,et al.Fluorescent antibody test,quantitative polymerase chain reaction pattern and clinical aspects of rabies virus strains isolated from main reservoirs in Brazil[J].Brazilian Journal of Infectious Diseases,2015,19(5):479-485.