猪瘟病毒E0蛋白在PK-15中的表达与鉴定
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摘要
[目的]研究猪瘟病毒(CSFV)E0基因在PK-15细胞中的表达特性。[方法]通过PCR方法克隆了E0基因全长681 bp的片段,连接到真核表达载体p EGFP-C1上,构建出重组质粒并进行双酶切鉴定。[结果]成功构建出重组质粒p EGFP-E0。通过双酶切鉴定,PCR鉴定和测序确定了目的基因大小一致,插入位置完全正确后,利用Lipofecta mine 2000转染试剂盒将重组质粒转染到猪肾细胞PK-15中,转染48 h后在荧光显微镜下观察,可以看到大多数细胞呈现出绿色荧光,说明转染成功。通过G418筛选后,在荧光显微镜下仍能观察到部分细胞能够呈现出绿色荧光,这表明重组融合蛋白p EGFP-E0在PK-15细胞中得到了表达。[结论]获得的能够表达重组融合蛋白的细胞克隆,为进一步大量表达与纯化E0糖蛋白、制备其单抗以及研究猪瘟病毒E0糖蛋白的生物学功能奠定了基础。
[Objective]The aim was to study the characteristic how gene E0 of classical swine fever virus( CSFV) expressed in PK-15.[Method] E0 gene whose full length was 681 bp fragment was cloned by PCR,and then a recombinant plasmid was constructed with eukaryotic expression vector of p EGFP-C1,double enzyme digestion was conducted.[Result]A recombinant plasmid p EGFP-E0 was sucessfully constructed,by restriction enzyme digestion,PCR identification and sequencing,the size of the target gene and insertion position was determined and was right,then recombinant plasmid were transformed into pig kidney-derived cells PK-15 by the Lipofecta mine 2000 transfection kit,after transfection 48 h,many cells showed green fluorescence under a fluorescence microscope,which indicated the transfection was successful. After screening by geneticin,there were still green fluorescent cells under the fluorescence microscope,which indicated that the recombinant fusion protein of p EGFP-E0 was expressed in the cell PK-15.[Conclusion]The obtain of expressing recombinant fusion protein of cell clones lay a foundation for further mass production of soluble glycoprotein E0,preparing its monoclonal antibody epitope screening and researching the biological function of protein E0.
[Objective]The aim was to study the characteristic how gene E0 of classical swine fever virus( CSFV) expressed in PK-15.[Method] E0 gene whose full length was 681 bp fragment was cloned by PCR,and then a recombinant plasmid was constructed with eukaryotic expression vector of p EGFP-C1,double enzyme digestion was conducted.[Result]A recombinant plasmid p EGFP-E0 was sucessfully constructed,by restriction enzyme digestion,PCR identification and sequencing,the size of the target gene and insertion position was determined and was right,then recombinant plasmid were transformed into pig kidney-derived cells PK-15 by the Lipofecta mine 2000 transfection kit,after transfection 48 h,many cells showed green fluorescence under a fluorescence microscope,which indicated the transfection was successful. After screening by geneticin,there were still green fluorescent cells under the fluorescence microscope,which indicated that the recombinant fusion protein of p EGFP-E0 was expressed in the cell PK-15.[Conclusion]The obtain of expressing recombinant fusion protein of cell clones lay a foundation for further mass production of soluble glycoprotein E0,preparing its monoclonal antibody epitope screening and researching the biological function of protein E0.
引文
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[9]ARAMOUNI M,KEKARAINEN T,GANGES L,et al.Increased viral load and prevalence of Torque teno sus virus 2(TTSu V2)in pigs experimentally infected with classical swine fever virus(CSFV)[J].Virus research,2013,172:81-84.
[10]OMARI K E,IOURIN O,HARLOS K,et al.Structure of a pestivirus envelope glycoprotein E2 clarifies its role in cell entry[J].Cell reports,2013,3(1):30-35.
[11]CALISHER C H,GOULD E A.Taxonomy of the virus family Flaviviridae[J].Advances in virus research,2003,59(1):1-19.
[12]MOSER C,STETTLER P,TRATSCHIN J D,et al.Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus[J].Journal of virology,1999,73(9):7787-7794.
[13]BEHRENS S E,GRASSMANN C W,THIEL H J,et al.Characterization of an autonomous subgenomic pestivirus RNA replicon[J].Journal of virology,1998,72(3):2364-2372.
[2]FERNANDEZ-SAINZ I,RAMANATHAN P,O’DONNELL V,et al.Treatment with interferon-alpha delays disease in swine infected with a highly virulent CSFV strain[J].Virology,2015,483:284-290.
[3]李传兴,周传军.猪瘟的防控措施[J].养殖技术顾问,2009(2):117.
[4]LEIFER I,RUGGLI N,BLOME S.Approaches to define the viral genetic basis of classical swine fever virus virulence[J].Virology,2013,438:51-55.
[5]GLADUE D P,BAKER-BRANSETTER R,HOLINKA L G,et al.Interaction of CSFV E2 protein with swine host factors as detected by yeast twohybrid system[J].PLOS,2014,9:1-9.
[6]王莹,任向阳,张昶,等.猪瘟病毒分子生物学研究进展[J].上海畜牧兽医通讯,2006(9):8-11.
[7]FRANZONI G,KURKURE N V,EDGAR D S,et al.Assessment of the phenotype and functionality of porcine CD8 T cell responses following vaccination with live attenuated classical swine fever virus(CSFV)and virulent CSFV challenge[J].Clinical and vaccine immunology,2013,20:1604-1616.
[8]SHEN H Y,WANG J Y,DONG X Y,et al.Genome and molecular characterization of a CSFV strain isolated from a CSF outbreak in South China[J].Intervirology,2013,56:122-133.
[9]ARAMOUNI M,KEKARAINEN T,GANGES L,et al.Increased viral load and prevalence of Torque teno sus virus 2(TTSu V2)in pigs experimentally infected with classical swine fever virus(CSFV)[J].Virus research,2013,172:81-84.
[10]OMARI K E,IOURIN O,HARLOS K,et al.Structure of a pestivirus envelope glycoprotein E2 clarifies its role in cell entry[J].Cell reports,2013,3(1):30-35.
[11]CALISHER C H,GOULD E A.Taxonomy of the virus family Flaviviridae[J].Advances in virus research,2003,59(1):1-19.
[12]MOSER C,STETTLER P,TRATSCHIN J D,et al.Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus[J].Journal of virology,1999,73(9):7787-7794.
[13]BEHRENS S E,GRASSMANN C W,THIEL H J,et al.Characterization of an autonomous subgenomic pestivirus RNA replicon[J].Journal of virology,1998,72(3):2364-2372.