猪传染性胃肠炎病毒间接免疫荧光检测方法的建立
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摘要
为建立猪传染性胃肠炎病毒(TGEV)直观、特异和快速的检测方法,将TGEV接种PK-15细胞后,以抗TGEV N蛋白的单克隆抗体为一抗,荧光素FITC标记的山羊抗小鼠Ig G为二抗,建立了TGEV的间接免疫荧光(IFA)检测方法。结果显示,TGEV的最佳接种效价为1×102TCID50,培养时间为24 h,细胞最佳固定液为40 m L/L多聚甲醛,最佳封闭条件为10 g/L BSA 37℃封闭30 min,一抗的最佳使用浓度为1 ng/L,二抗的最佳稀释度为1∶100。对常见的猪流行性腹泻病毒、猪轮状病毒和猪圆环病毒2型等猪病病原检测均为阴性。结果表明,建立的检测方法对TGEV具有良好的特异性。本研究建立的方法为TGEV的实验室诊断及TGEV在感染细胞中的定位和动态分布提供了有效的检测手段。
In order to develop specific method for detection of transmissible gastroenteritis virus of swine(TGEV) in PK-15 cells,a monoclonal antibody against TGEV N protein was used as the primary antibody,and a FITC labeled goat anti-mouse Ig G used as the second antibody.Then,the optimization of the reaction conditions was performed to establish an indirect immunofluorescence assay(IFA) for detection of TGEV.The results showed that the titer of TGEV was 1×102TCID50,the incubation time of TGEV was24 h,the stationary liquid was 40 m L/L paraformaldehyde,the blocking condition was at 37 ℃ for 30 min with 10 g/L BSA,the concentration of the primary antibody was 1 ng/ L and the dilution of the second antibody was 1 ∶ 100.The IFA constructed in this study was specific for detecting TGEV,and had no cross-reactions with other related porcine pathogens,such as porcine epidemic diarrhea virus,porcine rotavirus and porcine circovirus type 2.The results indicated that the constructed IFA was specificand stable for detecting TGEV in PK-15 cells.
In order to develop specific method for detection of transmissible gastroenteritis virus of swine(TGEV) in PK-15 cells,a monoclonal antibody against TGEV N protein was used as the primary antibody,and a FITC labeled goat anti-mouse Ig G used as the second antibody.Then,the optimization of the reaction conditions was performed to establish an indirect immunofluorescence assay(IFA) for detection of TGEV.The results showed that the titer of TGEV was 1×102TCID50,the incubation time of TGEV was24 h,the stationary liquid was 40 m L/L paraformaldehyde,the blocking condition was at 37 ℃ for 30 min with 10 g/L BSA,the concentration of the primary antibody was 1 ng/ L and the dilution of the second antibody was 1 ∶ 100.The IFA constructed in this study was specific for detecting TGEV,and had no cross-reactions with other related porcine pathogens,such as porcine epidemic diarrhea virus,porcine rotavirus and porcine circovirus type 2.The results indicated that the constructed IFA was specificand stable for detecting TGEV in PK-15 cells.
引文
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[15]许保疆,游一,郭成留,等.单克隆抗体间接免疫荧光检测猪瘟病毒方法的建立[J].中国兽医学报,2010,30(12):1566-1570.XU Bao-jiang,YOU Yi,GUO Cheng-liu,et al.Establishment of indirect immunofluorescence assay for detecting classical swine fever virus[J].Chinese Journal of Veterinary Science,2010,30(12):1566-1570.(in Chinese)
[16]孙东波,冯力,刘杰,等.猪传染性胃肠炎病毒N基因的原核表达及重组蛋白的免疫活性分析[J].病毒学报,2006,22(2):147-148.SUN Dong-bo,FENG Li,LIU Jie,et al.Prokaryotic expression of N gene of transmissible gastroenteritis virus and activity analysis of the recombinant protein[J].Chinese Journal of Virology,2006,22(2):147-148.(in Chinese)
[2]DOYLE L P,HUTCHINGS L M.A transmissible gastroenteritis in pigs[J].J Am Vet Med Assoc,1946,108(4):257-259.
[3]PATON D,IBATA G,SANDS J,et al.Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from PRCV[J].J Virol Methods,1997,66(2):303-309.
[4]ZHANG X S,HASOKSUZ M,SPIRO D,et al.Complete genomic sequences,a key residue in the spike protein and deletions in nonstructural protein 3b of US strains of the virulent and attenuated coronaviruses,transmissible gastroenteritis virus and porcine respiratory coronavirus[J].Virology,2007,358(2):424-435.
[5]SCHWEGMANN-WESSELS C,HERRLER G.Transmissible gastroenteritis virus infection:a vanishing specter[J].Dtsch Tierarztl Wochenschr,2006,113(4):157-159.
[6]LAIMMC,CAVANAGHD.The molecular biology of coronaviruses[J].Adv Virus Res,1997,48(1):35-112.
[7]ALONSO J M M,BALBIN M,GARWES D J,et al.Antigenic structure of transmissible gastroenteritis virus nucleoprotein[J].Virology,1992,188(1):168-174.
[8]陈仕龙,黄梅清,林天龙,等.高致病性猪生殖与呼吸综合征病毒间接免疫荧光鉴别方法的建立[J].中国兽医科学,2009,39(1):41-44.CHEN Shi-long,HUANG Mei-qing,LIN Tian-long,et al.Development of an indirect immunofluorescence assay for differentiation of highly pathogenic PRRSV from classical PRRSV[J].Chinese Veterinary Science,2009,39(1):41-44.(in Chinese)
[9]刘钊,禹海杰,卓洵辉,等.抗弓形虫MIC3单抗的制备及其在速殖子免疫荧光观察中的应用[J].中国兽医科学,2011,41(10):1030-1033.LIU Zhao,YU Hai-jie,ZHUO Xun-hui,et al.Preparation of monoclone antibodies to Toxoplasma gondii microneme protein 3 and the utilization in tachyzoite immunofluorescent observation[J].Chinese Veterinary Science,2011,41(10):1030-1033.(in Chinese)
[10]赵鑫,张鑫,时洪艳,等.抗猪传染性胃肠炎病毒N蛋白单克隆抗体的制备及抗原表位的鉴定[J].中国预防兽医学报,2014,36(4):314-317.ZHAO Xin,ZHANG Xin,SHI Hong-yan,et al.Preparation of monoclonal antibodies against transmissible gastroenteritis virus nucleoprotein(N)and the N B-cell epitope identification[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(4):314-317.(in Chinese)
[11]STRAW B E,D′A LLAIRE S,MENG ELING W L.Diseases of Swine[M].Iowa:Iowa State University Press,1999.
[12]郭万柱,刘亚刚,娄高明.兽医病毒学[M].成都:四川科学技术出版社,2003.GUO Wan-zhu,LIU Ya-gang,LOU Gao-ming.Veterinary Virology[M].Chengdu:Sichuan Science and Technology Press,2003.(in Chinese)
[13]王文秀,沈志强,王善辉,等.猪传染性胃肠炎病毒检测方法的研究进展[C]//中国畜牧兽医学会兽医公共卫生学分会第三次学术研讨会论文集,2012:74-76.WANG Wen-xiu,SHEN Zhi-qiang,WANG Shan-hui,et al.Progress on detection assay of porcine transmissible gastroenteritis virus[C]//Proceedings of the 3rd Symposium on Chinese Veterinary Public Health Association,Chinese Association of Animal Science and Veterinary Medicine,2012:74-76.(in Chinese)
[14]杨丽梅,马力,徐倩倩,等.我国猪病毒性腹泻的诊断与流行病学调查研究概况[J].动物医学进展,2014,35(2):115-119.YANG Li-mei,MA Li,XU Qian-qian,et al.Diagnosis and epidemiological investigation of porcine virus diarrhea overview[J].Progress in Veterinary Medicine,2014,35(2):115-119.(in Chinese)
[15]许保疆,游一,郭成留,等.单克隆抗体间接免疫荧光检测猪瘟病毒方法的建立[J].中国兽医学报,2010,30(12):1566-1570.XU Bao-jiang,YOU Yi,GUO Cheng-liu,et al.Establishment of indirect immunofluorescence assay for detecting classical swine fever virus[J].Chinese Journal of Veterinary Science,2010,30(12):1566-1570.(in Chinese)
[16]孙东波,冯力,刘杰,等.猪传染性胃肠炎病毒N基因的原核表达及重组蛋白的免疫活性分析[J].病毒学报,2006,22(2):147-148.SUN Dong-bo,FENG Li,LIU Jie,et al.Prokaryotic expression of N gene of transmissible gastroenteritis virus and activity analysis of the recombinant protein[J].Chinese Journal of Virology,2006,22(2):147-148.(in Chinese)