牛病毒性腹泻病毒E0基因重组表达质粒pMG36e-E0的构建与免疫原性分析
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摘要
利用SacⅠ、HindⅢ限制性内切酶分别对pMG36e与pMD19-T-E0进行双酶切,将目的基因E0整合入穿梭表达载体pMG36e,构建牛病毒性腹泻病毒E0基因重组表达质粒pMG36e-E0。提取重组质粒pMG36e-E0,进行双酶切鉴定及PCR鉴定。将阳性重组质粒pMG36e-E0转化到大肠杆菌DH5α中进行表达,对表达产物进行SDSPAGE和Western-blotting鉴定。用表达的E0蛋白二次免疫家兔,间接ELISA法检测其血清抗体水平。结果,重组质粒经双酶切得到大小分别约为3 600、691bp的2个片段,PCR扩增出691bp的片段,双酶切与PCR鉴定结果表明目的基因E0正确插入到载体pMG36e中。SDS-PAGE电泳结果表明E0基因在大肠杆菌获得了表达,表达产物相对分子质量大小约为27 000。Western-blotting分析结果证明表达产物能被BVDV阳性血清所识别。ELISA检测结果证明表达的E0融合蛋白能刺激动物产生抗体,具有天然蛋白的免疫原性。
The pMG36 evector and pMD19-T-E0 were digested by restriction endonuclease SacI and HindⅢ,respectively.The E0 gene was subcloned into the expression vector pMG36 eto constructed recombinant plasmid pMG36e-E0,and then transformed into E.coli DH5αstrain.The constructed recombinant plasmids pMG36e-E0 were extracted and identificated by SacⅠ/HindⅢ double enzyme digestion and PCR.The expression of E0 fusion protein was confirmed by SDSPAGE and Western-blotting.The healthy male rabbits were immunized with recombinant E0 protein,and its antibody levels against BVDV in serum were detected by an indirect ELISA.The SacⅠ/HindⅢ enzyme digestion of the recombinant plasmids obtained a target band of 690 bp and a band of 3 600 bp in size corresponding pMG36 evector genome.PCR amplification of E0 genes using recombinant plasmids as a template resulted in single band of 690 bp in size.The results showed that the constructed recombinant plasmid pMG36e-E0 was positive plasmid by enzyme cleavage and PCR.The E0 fusion protein band of about 27 000 was identificated by SDS-PAGE and Western blotting.The recombinant plasmid pMG36e-E0 was successfully constructed and able to express in E.coli DH5α.Western blotting analysis indicated that the recombinant protein could specifically react to antibodies against BVDV.The results of studies on immunogenicity by indirect ELISA indicated that the expressed E0 protein could induce animals to produce specific antibodies against BVDV
The pMG36 evector and pMD19-T-E0 were digested by restriction endonuclease SacI and HindⅢ,respectively.The E0 gene was subcloned into the expression vector pMG36 eto constructed recombinant plasmid pMG36e-E0,and then transformed into E.coli DH5αstrain.The constructed recombinant plasmids pMG36e-E0 were extracted and identificated by SacⅠ/HindⅢ double enzyme digestion and PCR.The expression of E0 fusion protein was confirmed by SDSPAGE and Western-blotting.The healthy male rabbits were immunized with recombinant E0 protein,and its antibody levels against BVDV in serum were detected by an indirect ELISA.The SacⅠ/HindⅢ enzyme digestion of the recombinant plasmids obtained a target band of 690 bp and a band of 3 600 bp in size corresponding pMG36 evector genome.PCR amplification of E0 genes using recombinant plasmids as a template resulted in single band of 690 bp in size.The results showed that the constructed recombinant plasmid pMG36e-E0 was positive plasmid by enzyme cleavage and PCR.The E0 fusion protein band of about 27 000 was identificated by SDS-PAGE and Western blotting.The recombinant plasmid pMG36e-E0 was successfully constructed and able to express in E.coli DH5α.Western blotting analysis indicated that the recombinant protein could specifically react to antibodies against BVDV.The results of studies on immunogenicity by indirect ELISA indicated that the expressed E0 protein could induce animals to produce specific antibodies against BVDV
引文
[1]Tajima M,Frey H R,Yamato O,et al.Prevalence of genotypes 1and 2of bovine viral diarrhea virus in Lower Saxony,Germany[J].Virus Res,2001,76(1):31-42.
[2]Wengler G,Bradley D W,Collett M S,et al.Classification and Nomenclature of Viruses.Sixth Report on the International Committee on Taxonomy of Viruses[J].Springer Wien New York Arch Virol,1995,10:415-427.
[3]Ridpath J.Bovine viral diarrhea virus:global status[J].Vet Clin North Am Food Anim Pract,2010,26(1):105-121.
[4]Oem J K,Chung J Y,Roh I S,et al.Characterization and phylogenetic analysis of bovine viral diarrhea virus in brain tissues from nonambulatory(downer)cattle in Korea[J].J Vet Diagn Invest,2010,22(4):518-23.
[5]Porter B F,Ridpath J F,Calise D V,et al.Hypomyelination associated with bovine viral diarrhea virus type2infection in a longhorn calf[J].Vet Pathol,2010,47(4):658-663.
[6]Wensvoort G,Teerpstra C.Bovine viral diarrhoea virus infection in piglets born to sows vaccinated against swine fever with contaminated virus[J].Res Vet Sci,1988,45(9):143-148.
[7]Moennig V,Frey H R,Liebler E,et al.Reproduction of mucosal disease with cytopathogenic bovine viral diarrhoea virus selected in vitro[J].Vet Rec,1990,127(8):200-203.
[8]王新平,任文涉,朱维正,等.双抗体夹心ELISA检测牛病毒性腹泻病毒的研究[J].中国兽医学报,1995,15(9):246-249.
[9]Schefers J M,Collins J E,Goyal S M,et al.Detection,characterization,and control of bovine viral diarrhea virus infection in a large commercial dairy herd[J].Can Vet J,2009,50(10):1075-1079.
[10]Intisar K S,Ali Y H,Khalafalla A I,et al.The first report on the prevalence of pestivirus infection in camels in Sudan[J].Trop Anim Health Prod,2010,42(6):1203-1207.
[11]虞蕴如,许炳坤,秦祥勇,等.南京市初生犊牛病毒性腹泻-黏膜病血清学调查[J].中国兽医科技,2003,33(3):46-48.
[12]蒋禄峰,赵月兰,李苏楠,等.牛病毒性腹泻病毒HBDCZ株E0基因的克隆与抗原表位预测[J].中国兽医学报,2013,33(7):979-984.
[13]Bhudevi B,Weinstock D.Fluorogenic RT-PCR assay(TaqMan)for detection and classification of bovine viral diarrhea virus[J].Vet Microbiol,2001,83(1):1-10.
[14]Fulton R W,Burge L J.Bovine viral diarrhea virus types 1and 2antibody response in calves receiving modified live virus or inactivated vaccines[J].Vaccine,2000,19(2/3):264-274.
[15]Thiel H J,Stark R,Weiland E,et al.Hog cholera virus:Molecular composition of virons from a pestivirus[J].J Virol,1991,65(9):4705-4712.
[16]Vilcek S,Paton D J,Durkovic B,et al.Bovine viral diarrhoea virus genotype I can be separated into at least eleven genetic groups[J].Arch Virol,2001,146(1):99-115.
[17]Deng R,Brock K V.Molecular cloning and nucleotide sequences of a pestivirus genome noncytopathic bovine viral diarrhea virus strain SD-1[J].Virology,1992,52(19):867-879.
[18]de Smit A J,Bouma A,van Gennip H G P,et al.Chimeric(marker)C-strain viruses induce clinical protection against virulent classical swine fever virus(CSFV)and reduce transmission of CSFV between vaccinated pigs[J].Vaccine,2001,19(11/12):1467-1476.
[19]Fulton R W,Burge L J.Bovine viral diarrhea virus types 1and 2antibody response in calves receiving modified live virus or inactivated vaccines[J].Vaccine,2000,19(2/3):264-274.
[2]Wengler G,Bradley D W,Collett M S,et al.Classification and Nomenclature of Viruses.Sixth Report on the International Committee on Taxonomy of Viruses[J].Springer Wien New York Arch Virol,1995,10:415-427.
[3]Ridpath J.Bovine viral diarrhea virus:global status[J].Vet Clin North Am Food Anim Pract,2010,26(1):105-121.
[4]Oem J K,Chung J Y,Roh I S,et al.Characterization and phylogenetic analysis of bovine viral diarrhea virus in brain tissues from nonambulatory(downer)cattle in Korea[J].J Vet Diagn Invest,2010,22(4):518-23.
[5]Porter B F,Ridpath J F,Calise D V,et al.Hypomyelination associated with bovine viral diarrhea virus type2infection in a longhorn calf[J].Vet Pathol,2010,47(4):658-663.
[6]Wensvoort G,Teerpstra C.Bovine viral diarrhoea virus infection in piglets born to sows vaccinated against swine fever with contaminated virus[J].Res Vet Sci,1988,45(9):143-148.
[7]Moennig V,Frey H R,Liebler E,et al.Reproduction of mucosal disease with cytopathogenic bovine viral diarrhoea virus selected in vitro[J].Vet Rec,1990,127(8):200-203.
[8]王新平,任文涉,朱维正,等.双抗体夹心ELISA检测牛病毒性腹泻病毒的研究[J].中国兽医学报,1995,15(9):246-249.
[9]Schefers J M,Collins J E,Goyal S M,et al.Detection,characterization,and control of bovine viral diarrhea virus infection in a large commercial dairy herd[J].Can Vet J,2009,50(10):1075-1079.
[10]Intisar K S,Ali Y H,Khalafalla A I,et al.The first report on the prevalence of pestivirus infection in camels in Sudan[J].Trop Anim Health Prod,2010,42(6):1203-1207.
[11]虞蕴如,许炳坤,秦祥勇,等.南京市初生犊牛病毒性腹泻-黏膜病血清学调查[J].中国兽医科技,2003,33(3):46-48.
[12]蒋禄峰,赵月兰,李苏楠,等.牛病毒性腹泻病毒HBDCZ株E0基因的克隆与抗原表位预测[J].中国兽医学报,2013,33(7):979-984.
[13]Bhudevi B,Weinstock D.Fluorogenic RT-PCR assay(TaqMan)for detection and classification of bovine viral diarrhea virus[J].Vet Microbiol,2001,83(1):1-10.
[14]Fulton R W,Burge L J.Bovine viral diarrhea virus types 1and 2antibody response in calves receiving modified live virus or inactivated vaccines[J].Vaccine,2000,19(2/3):264-274.
[15]Thiel H J,Stark R,Weiland E,et al.Hog cholera virus:Molecular composition of virons from a pestivirus[J].J Virol,1991,65(9):4705-4712.
[16]Vilcek S,Paton D J,Durkovic B,et al.Bovine viral diarrhoea virus genotype I can be separated into at least eleven genetic groups[J].Arch Virol,2001,146(1):99-115.
[17]Deng R,Brock K V.Molecular cloning and nucleotide sequences of a pestivirus genome noncytopathic bovine viral diarrhea virus strain SD-1[J].Virology,1992,52(19):867-879.
[18]de Smit A J,Bouma A,van Gennip H G P,et al.Chimeric(marker)C-strain viruses induce clinical protection against virulent classical swine fever virus(CSFV)and reduce transmission of CSFV between vaccinated pigs[J].Vaccine,2001,19(11/12):1467-1476.
[19]Fulton R W,Burge L J.Bovine viral diarrhea virus types 1and 2antibody response in calves receiving modified live virus or inactivated vaccines[J].Vaccine,2000,19(2/3):264-274.