副结核分支杆菌MAP0862蛋白的表达及临床应用
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摘要
本研究以腹泻牛粪便基因组DNA为模板,扩增MAP0862基因,构建重组表达载体pET-30a(+)-MAP0862和pEGX-6P-1-MAP0862,通过原核表达系统表达His-MAP0862和GST-MAP0862蛋白,经纯化后制备兔源高免血清,建立ELISA特异性检测方法,最后将纯化蛋白和ELISA方法用于牛副结核病临床样品检测,并对检测数据进行分析。结果表明,以MAP0862蛋白为检测原建立的ELISA检测方法在临床样品检测中具有较高的特异性和敏感性,说明MAP0862蛋白适用于副结核病抗体检测。本研究建立的ELISA检测方法可有效检测临床样品中副结核病的感染。
The MAP0862 gene was cloned from fecal genome of diarrhea cattles. The pET-320(+)-MAP0862 and pEGX-6 P-1-MAP0862 expression vector were constructed to express the His-MAP0862 and GST-MAP0862 proteins. The recombinant proteins were used to obtain immune serum after purification, and the ELISA method was established. The MAP0862 protein and ELISA method were used to detect paratuberculosis. The results showed that there was highly specificity and sensitivity in ELISA test using MAP0862 protein as stimulator, so it was confirmed that MAP0862 protein was optimal for antibody detection of paratuberlosis. The ELISA method established based on MAP0862 protein could detect paratuberculosis effectively in clinical samples.
The MAP0862 gene was cloned from fecal genome of diarrhea cattles. The pET-320(+)-MAP0862 and pEGX-6 P-1-MAP0862 expression vector were constructed to express the His-MAP0862 and GST-MAP0862 proteins. The recombinant proteins were used to obtain immune serum after purification, and the ELISA method was established. The MAP0862 protein and ELISA method were used to detect paratuberculosis. The results showed that there was highly specificity and sensitivity in ELISA test using MAP0862 protein as stimulator, so it was confirmed that MAP0862 protein was optimal for antibody detection of paratuberlosis. The ELISA method established based on MAP0862 protein could detect paratuberculosis effectively in clinical samples.
引文
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[15] Bannantine J P, Barletta R G, Stabel J R, et al. Application of the genome sequence to address concerns that Mycobacterium avium subspecies paratuberculosis might be a foodborne pathogen[J]. Foodborne Pathog. Dis., 2004, 1:3-15.
[16] Wang J, Pritchard J R, Kreitmann L, et al. Disruption of Mycobacterium avium subsp. paratuberculosis-specific genes impairs in vivo fitness[J]. BMC Genomics, 2014, 15:415.
[17] Bannantine J P, Paustain M L, Waters W R, et al. Profiling bovine antibody responses to Mycobacterium avium subsp. paratuberculosis infection by using protein arrays[J]. Infect. Immun., 2008, 76(2):739-749.
[18] Paustain M L, Amonsin A, Kapur V, et al. Characterzation 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.
[2] Whittington R J, Sergeant E S. Progress towards understanding the spread, detection and control of Mycobactrium avium subsp. paratuberculosis in animal population[J]. Aust. Vet. J., 2001, 79:267-278.
[3] Motiwala A S, Amonsin A, Strother M, et al. Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species[J]. J. Clin. Microbiol., 2004, 42:1703-1712.
[4] Chacon O, Bermudez L E, Barletta R G. Johne’s disease, inflammatory bowel disease, and Mycobacterium paratuberculosis[J]. Annu. Rev. Microbiol., 2004, 58:329-363.
[5] Davis W C, Madsen-Bouterse S A. Crohn’s disease and Mycobacterium avium subsp. paratuberculosis: the need for a study is long overdue[J]. Vet. Immunol. Immunopathol., 2012, 145:1-6.
[6] Paccagnini D, Sieswerda L, Rosu V, et al. Linking chronic infection and autoimmune disease: Mycobacterium avium subspecies paratuberculosis, SLC11A1 polymorphisms and type-1 diabetes mellitus[J]. PLoS One, 2009, 4:e7109.
[7] Sisto M, Cucci L, D’Amore M, et al. Proposing a relationship between Mycobacterium avium subspecies paratuberculosis infection and Hashimoto’s thyroiditis[J]. Scand. J. Infect. Dis., 2010, 42:787-790.
[8] Arru G, Caggiu E, Paulus K, et al. Is there a role for Mycobacterium avium subspecies paratuberculosis in Parkinson’s disease?[J]. J. Neuroimmunol., 2016, 293:86-90.
[9] Niegowska M, Delitala A, Pes G M, et al. Increased seroreactivity to proinsulin and homologous mycobacterial peptides in latent autommune diabetes in adults[J]. PLoS One, 2017, 12:e0176584.
[10] Coussens P M. Model for immune responses to Mycobacterium avium subspecies paratuberculosis in cattle[J]. Infect Immun., 2004, 72:3089-3096.
[11] Stabel J P. Transition in immune response to Mycobacterium paratuberculosis[J]. Vet. Microbiol., 2000, 77: 465-473.
[12] Koo H C, Park Y H, Hamilton M J. Analysis of the immune response to Mycobacterium avium subsp. paratuberculosis in experimentally infected calves[J]. Infect Immun., 2004, 72: 6870-6883.
[13] Arsenault R J, Maattanen P, Daigle J, et al. From mouth to macrophage: mechanism of innate immune subversion by Mycobacterium avium subsp. paratuberculosis[J]. Vet. Res., 2014, 45(54):1-15.
[14] Kuehnel M P, Goethe R, Habermann A, et al. Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal PH and fusogenicity in J774 macrophages compared with other mycobacteria[J]. Cell Microbiol., 2001, 3(8):551-556.
[15] Bannantine J P, Barletta R G, Stabel J R, et al. Application of the genome sequence to address concerns that Mycobacterium avium subspecies paratuberculosis might be a foodborne pathogen[J]. Foodborne Pathog. Dis., 2004, 1:3-15.
[16] Wang J, Pritchard J R, Kreitmann L, et al. Disruption of Mycobacterium avium subsp. paratuberculosis-specific genes impairs in vivo fitness[J]. BMC Genomics, 2014, 15:415.
[17] Bannantine J P, Paustain M L, Waters W R, et al. Profiling bovine antibody responses to Mycobacterium avium subsp. paratuberculosis infection by using protein arrays[J]. Infect. Immun., 2008, 76(2):739-749.
[18] Paustain M L, Amonsin A, Kapur V, et al. Characterzation 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.