利用重组表位蛋白map0862-2154c检测牛副结核血清抗体胶体金免疫层析方法的建立（英文）
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摘要
筛选牛副结核分枝杆菌的2个主要抗原map0862、map2154c中抗原指数高的抗原表位基因,将多表位基因序列合成,与原核表达载体pET28a(+)连接,构建重组质粒pET28a-map0862-2154c。重组质粒转化至大肠杆菌感受态细胞BL21(DE3)中诱导表达,以表达的重组表位蛋白为抗原建立检测牛副结核分枝杆菌抗体的胶体金免疫层析方法(GICA,map0862-2154c-GICA),同时对河北省部分牛场采集的242份副结核病奶牛血样进行血清抗体检测。结果表明,牛副结核分枝杆菌表位蛋白map0862-2154c成功表达,表达产物能与牛副结核分枝杆菌阳性血清反应。临床血清样本检测结果表明,其敏感性、特异性和符合率分别为91.86%(79/86),94.23%(147/156)和93.38%(226/242),该方法可用于牛副结核病的血清抗体检测。
The experiment was conducted to develop a colloidal gold immunochromatographic method for the serodiagnosis of the bovine paratuberculosis with the recombinant epitope antigen map0862-2154 c.The recombinant epitopes gene of the map0862-2154 cof the Mycobacterium paratuberculosis(M.paratuberculosis)was synthesized and cloned into the pET28 a(+)expression vector to construct the recombinant plasmid called pET28 a-map0862-2154 cand expressed in E.coli BL21(DE3)strain.A colloidal gold immunochromatographic assay(GICA,map0862-2154 c-GICA)for the detection of antibody against M.paratuberculosis was established by the purified recombinant epitope protein map0862-2154 cas the coating antigen.242 sera samples from cows in the field were tested for the antibody against M.paratuberculosis by the map0862-2154 c-GICA strip and the IDEXX HerdChek ELISA kit,simultaneously to compare the specificity,sensitivity and accuracy.The results showed that the recombinant plasmid map0862-2154 c-GICA could be expressed successfully in E.coli and the expressed protein could react to serum from M.paratuberculosis.The map0862-2154 c-GICA showed 94.23%(147/156),91.86%(79/86)and 93.38%(226/242)in terms of specificity,sensitivity and accuracy compared to the IDEXX ELISA test kit.The map0862-2154 c-GICA has high sensitivity and specificity and can be used for the detection of the antibody against the M.paratuberculosis.
The experiment was conducted to develop a colloidal gold immunochromatographic method for the serodiagnosis of the bovine paratuberculosis with the recombinant epitope antigen map0862-2154 c.The recombinant epitopes gene of the map0862-2154 cof the Mycobacterium paratuberculosis(M.paratuberculosis)was synthesized and cloned into the pET28 a(+)expression vector to construct the recombinant plasmid called pET28 a-map0862-2154 cand expressed in E.coli BL21(DE3)strain.A colloidal gold immunochromatographic assay(GICA,map0862-2154 c-GICA)for the detection of antibody against M.paratuberculosis was established by the purified recombinant epitope protein map0862-2154 cas the coating antigen.242 sera samples from cows in the field were tested for the antibody against M.paratuberculosis by the map0862-2154 c-GICA strip and the IDEXX HerdChek ELISA kit,simultaneously to compare the specificity,sensitivity and accuracy.The results showed that the recombinant plasmid map0862-2154 c-GICA could be expressed successfully in E.coli and the expressed protein could react to serum from M.paratuberculosis.The map0862-2154 c-GICA showed 94.23%(147/156),91.86%(79/86)and 93.38%(226/242)in terms of specificity,sensitivity and accuracy compared to the IDEXX ELISA test kit.The map0862-2154 c-GICA has high sensitivity and specificity and can be used for the detection of the antibody against the M.paratuberculosis.
引文
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[13]GAOA,MUTHARIA L,RAYMOND M,et al.Improved template DNA preparation procedure for detection of Mycobacterium avium subsp.paratuberculosis in milk by PCR[J].J Microbiol Methods,2007,69(2):417-420.
[14]STEWART LD,FODDAI A,ELLIOTT CT,et al.Development of a novel phage-mediated immunoassay for the rapid detection of viable Mycobacterium avium subsp.paratuberculosis[J].J Appl Microbiol,2013,115(3):808-817.
[15]TRIPATHI B N,PERIASAMY S,PALIWAL O P,et al.Comparison of IS900tissue PCR,bacterial culture,johnin and serological tests for diagnosis of naturally occurring paratuberculosis in goats[J].Vet Microbiol,2006,116(1):129-137.
[16]IMIRZALIOGLU C,DAHMEN H,HAIN T,et al.Highly specific and quick detection of Mycobacterium avium subsp.paratuberculosis in feces and gut tissue of cattle and humans by multiple real-time PCR assays[J].J Clin Microbiol,2011,49(5):1843-1852.
[17]BANNANTINE J P,BAYLES D O,WATERS W R,et al.Early antibody response against Mycobacterium avium subspecies paratuberculosis antigens in subclinical cattle[J].Proteome Sci,2008,6(1):5-6.
[18]LYASHCHENKO K P,SINGH M,COLANGELI R,et al.A multi-antigen print immunoassay for the development of serological diagnosis of infectious diseases[J].J Immunol Methods,2000,242(1/2):91-100.
[19]PAUSTIANML,AMONSIN A,KAPUR V,et al.Characterization of novel coding sequences specific to Mycobacterium avium subsp.paratuberculosis:implications for diagnosis of johne's disease[J].J Clin Microbiol,2004,42(6):2675-2681.
[20]GASTEINER J,WENZL H,FUCHS K,et al.Serological cross-sectional study of paratuberculosis in cattle in Austria[J].Zentralbl Veterinarmed B,1999,46(7):457-466.
[21]STROMMENGER B,STEVENSON K,GERLACH G F.Isolation and diagnostic potential of ISMav2,a novel insertion sequence-like element from Mycobacterium avium subspecies paratuberculosis[J].FEMS Microbiol Lett,2001,196(1):31-37.
[22]GLANEMANN B,HOELZLE LE,WITTENBRINK M M.Bacteriological investigations about paratuberculosis in dairy herds in Switzerland[J].Deutsche tierrztliche Wochenschrift,2002,109(12):528-529.
[2]COLLINS M T,SOCKETT D C.Accuracy and economics of the USDA-licensed enzme-linked immunosorbent assay for bovine paratuberculosis[J].Am Vet Med Assoc,1993;203(10):1456-1463.
[3]JOSEPH L,GYORKOS T W,COUPAL L.Bayesian estimation of disease prevalence and the parameters of diagnostic tests in the absence of a gold standard[J].Am J Epidemiol,1995;141(3):263-272.
[4]LOMBARDJ E.Epidemiology and economics of paratuberculosis[J].Vet Clin North Am Food Anim Pract,2011;27(3):525-535.
[5]GRANTI R,O’RIORDAN L M,BALL H J,et al.Incidence of Mycobacterium paratuberculosis in raw sheep and goats’milk in England,Wales and Northern Ireland[J].Vet Microbiol,2001,79(2):123-131.
[6]AYELEWY,SVASTOVA P,ROUBAL P,et al.Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow's milk in the czech republic[J].Appl Environ Microbiol,2005,71(3):1210-1214.
[7]ELLINGSONJ L,ANDERSON J L,KOZICZKOWSKI J J,et al.Detection of viable Mycbacterium avium subsp.paratuberculosis in retail pasteurized whole milk by two culture methods and PCR[J].J Food Prot,2005,68(5):966-972.
[8]GARRY F.Control of paratuberculosis in dairy herds[J].Vet Clin North Am Food Anim Pract,2011,27(3):599-607.
[9]WHITTINGTON R J and SERGEANT E S.Progress towards understanding the spread,detection and control of Mycobacterium avium subsp paratuberculosis in animal populations[J].Aust Vet J,2001,79(79):267-278.
[10]WHITTINGTON R J,HOPE A F,MARSHALL D J,et al.Molecular epidemiology of Mycobacterium avium subsp.paratuberculosis:I S900 restriction fragment length polymorphism and IS1311polymorphism analyses of isolates from animals and a human in Australia[J].J Clin Microbiol,2000,38(9):3240-3248.
[11]WHITLOCK R H,BUERGELT C.Preclinical and clinical manifestations of paratuberculosis(including pathology)[J].Vet Clin North Am Food Anim Pract,1996,12(2):345-356.
[12]HARRISN B and BARLETTA R G.Mycobacterium avium subsp.paratuberculosis in veterinary medicine[J].Clin Microbiol Rev,2001,14(3):489-512.
[13]GAOA,MUTHARIA L,RAYMOND M,et al.Improved template DNA preparation procedure for detection of Mycobacterium avium subsp.paratuberculosis in milk by PCR[J].J Microbiol Methods,2007,69(2):417-420.
[14]STEWART LD,FODDAI A,ELLIOTT CT,et al.Development of a novel phage-mediated immunoassay for the rapid detection of viable Mycobacterium avium subsp.paratuberculosis[J].J Appl Microbiol,2013,115(3):808-817.
[15]TRIPATHI B N,PERIASAMY S,PALIWAL O P,et al.Comparison of IS900tissue PCR,bacterial culture,johnin and serological tests for diagnosis of naturally occurring paratuberculosis in goats[J].Vet Microbiol,2006,116(1):129-137.
[16]IMIRZALIOGLU C,DAHMEN H,HAIN T,et al.Highly specific and quick detection of Mycobacterium avium subsp.paratuberculosis in feces and gut tissue of cattle and humans by multiple real-time PCR assays[J].J Clin Microbiol,2011,49(5):1843-1852.
[17]BANNANTINE J P,BAYLES D O,WATERS W R,et al.Early antibody response against Mycobacterium avium subspecies paratuberculosis antigens in subclinical cattle[J].Proteome Sci,2008,6(1):5-6.
[18]LYASHCHENKO K P,SINGH M,COLANGELI R,et al.A multi-antigen print immunoassay for the development of serological diagnosis of infectious diseases[J].J Immunol Methods,2000,242(1/2):91-100.
[19]PAUSTIANML,AMONSIN A,KAPUR V,et al.Characterization of novel coding sequences specific to Mycobacterium avium subsp.paratuberculosis:implications for diagnosis of johne's disease[J].J Clin Microbiol,2004,42(6):2675-2681.
[20]GASTEINER J,WENZL H,FUCHS K,et al.Serological cross-sectional study of paratuberculosis in cattle in Austria[J].Zentralbl Veterinarmed B,1999,46(7):457-466.
[21]STROMMENGER B,STEVENSON K,GERLACH G F.Isolation and diagnostic potential of ISMav2,a novel insertion sequence-like element from Mycobacterium avium subspecies paratuberculosis[J].FEMS Microbiol Lett,2001,196(1):31-37.
[22]GLANEMANN B,HOELZLE LE,WITTENBRINK M M.Bacteriological investigations about paratuberculosis in dairy herds in Switzerland[J].Deutsche tierrztliche Wochenschrift,2002,109(12):528-529.