H5N1型禽流感NS1基因在HEK293细胞中的表达及稳定细胞系的构建
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摘要
将H5N1型禽流感病毒NS1(non-structural protein 1)基因插入pEGFP-N1真核表达载体中,获得重组质粒pNS1-EGFP,经酶切鉴定并测序后,去内毒素提取pNS1-EGFP质粒,在脂质体转染试剂作用下将pNS1-EGFP质粒转染HEK293细胞,利用倒置荧光显微镜、FCM和Western blot 3种方法检测NS1-EGFP融合蛋白的表达,利用极限稀释的方法获取单细胞,并利用G418抗性筛选获取稳定细胞系。结果显示,NS1基因正确插入pEGFP-N1真核表达载体,NS1-EGFP融合蛋白读码框架正确;3种检测方法表明,NS1-EGFP融合蛋白在HEK293细胞中能够表达;pEGFP-N1和NS1-EGFP稳定细胞系流式细胞仪检测,阳性率分别为95.74%和96.10%,Western blot检测结果发现,HEK293空白细胞和pEGFP-N1稳定细胞系未见任何条带,NS1-EGFP稳定细胞系在51 000处见特异性条带,综上说明稳定细胞系构建成功。本试验为进一步深入研究禽流感病毒NS1基因的生物学功能作铺垫。
The recombinant vector constructed by gene recombinant technology was analyzed by restriction enzyme digestion and DNA sequencing.The recombinant vector was transfected into HEK293 cells by LipofectamineTM2000.The expression of fusion protein AIV NS1 was identified by fluorescent microscope,flow cytometry and Western blot.Single cell was obtained by limiting dilution method and stable cell line was obtained by G418 resistance screening.The recombinant plasmid was successfully constructed,which was proved by restriction enzyme digestion and DNA sequencing.The expression of fusion protein was shown by fluorescent microscope,flow cytometry and western blot.Flow cytometry was used to detect pEGFP-N1 and NS1-EGFP stable cell lines,the positive rates were 95.74%and 96.10%,respectively.Western blot test results found that there was no band of HEK293 blank cells and pEGFP-N1 stable cell lines,and there was a specific band at 51 000 brand in EGFP stable cell line.This experiment paved a way for further research on the biological function of NS1 gene of avian influenza virus.
The recombinant vector constructed by gene recombinant technology was analyzed by restriction enzyme digestion and DNA sequencing.The recombinant vector was transfected into HEK293 cells by LipofectamineTM2000.The expression of fusion protein AIV NS1 was identified by fluorescent microscope,flow cytometry and Western blot.Single cell was obtained by limiting dilution method and stable cell line was obtained by G418 resistance screening.The recombinant plasmid was successfully constructed,which was proved by restriction enzyme digestion and DNA sequencing.The expression of fusion protein was shown by fluorescent microscope,flow cytometry and western blot.Flow cytometry was used to detect pEGFP-N1 and NS1-EGFP stable cell lines,the positive rates were 95.74%and 96.10%,respectively.Western blot test results found that there was no band of HEK293 blank cells and pEGFP-N1 stable cell lines,and there was a specific band at 51 000 brand in EGFP stable cell line.This experiment paved a way for further research on the biological function of NS1 gene of avian influenza virus.
引文
[1]赵冬雪,穆卫涛,王立砺,等.禽流感病毒与microRNA[J].中国兽医学报,2017,37(8):1613-1616.
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[10] GARCIA SA,EGORUV A,MATASSOV D,et al.Influenza A virus lacking the NSl gene replicates in interfemn deficient systems[J].Virology,1998,252(2):324-330.
[11] TABYNOV K,SANSYZBAY A,SANDYBAYEV N,et al.The pathogenicity of swan derived H5N1virus in birds and mammals and its gene analysis[J].Virol J,2014,29(11):207.
[12] KAWAOKA Y,GONNAN O T,ITO T,et al.Influence of host species on the evolution of the nonstructural(NS)gene of influenza A viruses[J].Virus Res,1998,55(2):143-156.
[13] OZAKI H,SUGIURA T,SUGITA S,et al.Detection of antjbodies to the nonstructuraI protein(NSl)of infIuenza A vlrus allows distinctlon between vaccinated and lnfected horses[J].Vet Microbiol,2001,82(2):111-119.
[14]钟如婷,孙淑鹏,丁洁,等.2015—2016年广州市部分活禽市场H9N2亚型禽流感病毒的流行病学调查[J].中国兽医学报,2018,38(4):631-636.
[15] SALVATORE M,BASLER C F,PARISIEN J P,et al.Effects of influenza a virus NSl protein on protein expression:the NSl protein enhances translation and is not required for shutoff ofhost protein synthesis[J].J Virol,2002,76(3):1206-1212.
[2]丛彦龙,钟颖,孙艺学等.H9N2亚型禽流感病毒流行病学及其疫苗的研究进展[J].中国兽医学报,2017,37(2):386-392.
[3]STEPHAN L,STEPHAN P,THORSTEN W.A fatal relationship-influenza virus interactions with the host cell[J].Viral Immunol,1999,12(3):175-196.
[4]SKEHEL J J.Polypeptide synthesis in influenza virus infected cells[J].Virology,1972,49(7):23-26.
[5]KATZ J M,LU X,FRUEE A M,et al.Pathogenesis of and immunity to avian influenza A H5viruses[J].Biomed Pharmacother,2000,54(4):178-187.
[6]彭特,许丽娜,张思远,等.1株HA蛋白S145变异H9N2亚型禽流感病毒的分离鉴定与HA基因序列分析[J].中国兽医学报,2017,37(4):676-681.
[7]HANWEI J,LI D,YONG C H,et al.Eeffective inhibition of mRNA accumulation and protein expression of H5N1avian influenza virus NS1 gene in vitro by small interfering RNAs[J].Folia Microbiol,2013,58(8):335-342.
[8]WEN X,SUN J,WANG X,et al.Identification of a novel linear epitope on the NS1protein of avian influenza virus[J].BMC Microbiol,2015,20(15):168.
[9]MA Y,SUN J,GU L,et al.Annexin A2(ANXA2)interacts with nonstructural protein 1and promotes the replication of highly pathogenic H5N1avian influenza virus[J].BMC Microbiol,2017,17(1):191.
[10] GARCIA SA,EGORUV A,MATASSOV D,et al.Influenza A virus lacking the NSl gene replicates in interfemn deficient systems[J].Virology,1998,252(2):324-330.
[11] TABYNOV K,SANSYZBAY A,SANDYBAYEV N,et al.The pathogenicity of swan derived H5N1virus in birds and mammals and its gene analysis[J].Virol J,2014,29(11):207.
[12] KAWAOKA Y,GONNAN O T,ITO T,et al.Influence of host species on the evolution of the nonstructural(NS)gene of influenza A viruses[J].Virus Res,1998,55(2):143-156.
[13] OZAKI H,SUGIURA T,SUGITA S,et al.Detection of antjbodies to the nonstructuraI protein(NSl)of infIuenza A vlrus allows distinctlon between vaccinated and lnfected horses[J].Vet Microbiol,2001,82(2):111-119.
[14]钟如婷,孙淑鹏,丁洁,等.2015—2016年广州市部分活禽市场H9N2亚型禽流感病毒的流行病学调查[J].中国兽医学报,2018,38(4):631-636.
[15] SALVATORE M,BASLER C F,PARISIEN J P,et al.Effects of influenza a virus NSl protein on protein expression:the NSl protein enhances translation and is not required for shutoff ofhost protein synthesis[J].J Virol,2002,76(3):1206-1212.