结核分枝杆菌Rv1400c多抗制备及反应原性分析
|
摘要
将pET28b-Rv1400c重组表达质粒转化到大肠杆菌BL21(DE3)中,筛选出阳性菌株并增菌培养后IPTG诱导表达,离心后沉淀用SKL变性溶解,用亲和层吸树脂纯化Rv1400c蛋白,通过SDS-PAGE和Western-blot分析目的蛋白表达与纯化情况;利用Rv1400c蛋白免疫新西兰兔,制备多克隆抗体;Western-blot分析鹿结核阳性血清的反应情况。SDS-PAGE和Western-blot结果显示:Rv1400c以包涵体形式表达,蛋白分子质量为39 ku;目的蛋白免疫新西兰兔后,ELISA检测血清效价达到1∶51 200。纯化的Rv1400c重组抗原蛋白能与鹿的阳性血清发生体外结合反应。
The recombinant plasmid pET-28b-Rv1400 c was transformed into E.coliBL21(DE3)for expression induced with IPTG(1mmol/ L). The expressed product was purified by Ni-NTA af-finity chromatography. The recombinant protein Rv1400 c was analyzed by SDS-PAGE and Western-blot. The recombinant protein Rv1400 c was immunized the New Zealand rabbits to prepare polycio-nal antibody. The positive deer serum was analyzed by Western-blot. The result showed that theRv1400 c was successfully expressed in the form of inclusion body. The molecular weight of proteinwas 39 ku. The New Zealand rabbits immunized the purified recombinant protein could produce highspecific antibodies with the titer to 1∶51 200 and the antibodies expressed good specificity, and therecombinant protein could be combined by the positive deer serum in vitro.
The recombinant plasmid pET-28b-Rv1400 c was transformed into E.coliBL21(DE3)for expression induced with IPTG(1mmol/ L). The expressed product was purified by Ni-NTA af-finity chromatography. The recombinant protein Rv1400 c was analyzed by SDS-PAGE and Western-blot. The recombinant protein Rv1400 c was immunized the New Zealand rabbits to prepare polycio-nal antibody. The positive deer serum was analyzed by Western-blot. The result showed that theRv1400 c was successfully expressed in the form of inclusion body. The molecular weight of proteinwas 39 ku. The New Zealand rabbits immunized the purified recombinant protein could produce highspecific antibodies with the titer to 1∶51 200 and the antibodies expressed good specificity, and therecombinant protein could be combined by the positive deer serum in vitro.
引文
[1]陶荣珊,李金伟,刘思国,等.结核分枝杆菌酯酶Rv1400c原核表达及表达产物的酶活性分析[J].吉林农业大学学报,2014-07-23.
[2]曾范得,王文玉,宋纪伟,等.鹿结核分枝杆菌流行株Rv3874-Rv3875融合基因的克隆及原核表达[J].经济动物学报,2014-09-11.DOI:10??13326/j.jea.2014??1030.
[3]Koch R.Die Aetiologie der Tuberculose[J].Berliner Kli-nische Wochenschrift,1882,19:221-230.
[4]Tollefsen S,Vordeermeier M,Ulsen I,et al.DNA injection incombination with electroporation:A novel method for vaccina-tion of farmed ruminants[J].Scand J Immunol,2003,57(3):229-238.
[5]Ducati R,Ruffino-Netto A,Basso L,et al.The resumption ofconsumption-a review on tuberculosis[J].Mem Inst OswaldoCruz,2006,101(7):697-714.
[6]Lu M X.The tuberculosis epidemic situation and preventionmeasures[J].Joumal of China Traditional Chinese MedicineInformation,2012,44(4):73-74.
[7]Zumla A,Nahid P,Cole S T.et al.Advances in the development of new tuberculosis drugs and treatment regimens[J].Nat Rew Drug Discow,2013,12(5):388-404.
[8]Dillon D C,Alderson M R,Day C H,et al.Molecular and immunological characterization ofMycobacterium tuberculosisCEP-10,an immunodiagnostic antigen misssing inMycobacterium[J].Clim Microbiol,2003,38(5):3285-3290.
[9]Liebana E,Aranaz A,Francis B,et al.Assessment of genetic markers for species differentiation within theMycobacterium tuberculosiscomplex[J].Clin Microbiol,1996,34(4):933-938.
[10]沈国妙.结核病复发及结核分枝杆菌耐药机制的研究[J].复旦大学,2006,37(7):676-680.
[11]Due A V,Kuper J,Geerlof A.et al.Wilmanns M:Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosisby active site metamorphosis[J].Proc Natl Acad Sci USA,2001,108(9):3554-3559.
[12]Carrey E A,Dietz C,Glubb D M,et al.Detettion and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa reproduction[J].Tubercle,2002,123(6):757-768.
[13]Brock I,Weldingh K,Leythn S,et al.Specific T-cell epitopes for immunoassay-based diagnosis ofMycobacterium tuberculosis infection[J].J Clin Microbiol,2004,42(6):2378-2387.
[14]Feng X,Xiu B,Chen K,et al.Enhanced serodiagnostic utility of novelMycobacterium tuberculosispolyproteins[J].The Journal of Infection,2013,66(4):366-370.
[15]Ireton G C,Greenwald R,Liang H,et al.Identification ofMycobacterium tuberculosisantigens of high serodiagnotic value[J].Clin Vaccine Immunol,2010,17(10):1539-1547.
[2]曾范得,王文玉,宋纪伟,等.鹿结核分枝杆菌流行株Rv3874-Rv3875融合基因的克隆及原核表达[J].经济动物学报,2014-09-11.DOI:10??13326/j.jea.2014??1030.
[3]Koch R.Die Aetiologie der Tuberculose[J].Berliner Kli-nische Wochenschrift,1882,19:221-230.
[4]Tollefsen S,Vordeermeier M,Ulsen I,et al.DNA injection incombination with electroporation:A novel method for vaccina-tion of farmed ruminants[J].Scand J Immunol,2003,57(3):229-238.
[5]Ducati R,Ruffino-Netto A,Basso L,et al.The resumption ofconsumption-a review on tuberculosis[J].Mem Inst OswaldoCruz,2006,101(7):697-714.
[6]Lu M X.The tuberculosis epidemic situation and preventionmeasures[J].Joumal of China Traditional Chinese MedicineInformation,2012,44(4):73-74.
[7]Zumla A,Nahid P,Cole S T.et al.Advances in the development of new tuberculosis drugs and treatment regimens[J].Nat Rew Drug Discow,2013,12(5):388-404.
[8]Dillon D C,Alderson M R,Day C H,et al.Molecular and immunological characterization ofMycobacterium tuberculosisCEP-10,an immunodiagnostic antigen misssing inMycobacterium[J].Clim Microbiol,2003,38(5):3285-3290.
[9]Liebana E,Aranaz A,Francis B,et al.Assessment of genetic markers for species differentiation within theMycobacterium tuberculosiscomplex[J].Clin Microbiol,1996,34(4):933-938.
[10]沈国妙.结核病复发及结核分枝杆菌耐药机制的研究[J].复旦大学,2006,37(7):676-680.
[11]Due A V,Kuper J,Geerlof A.et al.Wilmanns M:Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosisby active site metamorphosis[J].Proc Natl Acad Sci USA,2001,108(9):3554-3559.
[12]Carrey E A,Dietz C,Glubb D M,et al.Detettion and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa reproduction[J].Tubercle,2002,123(6):757-768.
[13]Brock I,Weldingh K,Leythn S,et al.Specific T-cell epitopes for immunoassay-based diagnosis ofMycobacterium tuberculosis infection[J].J Clin Microbiol,2004,42(6):2378-2387.
[14]Feng X,Xiu B,Chen K,et al.Enhanced serodiagnostic utility of novelMycobacterium tuberculosispolyproteins[J].The Journal of Infection,2013,66(4):366-370.
[15]Ireton G C,Greenwald R,Liang H,et al.Identification ofMycobacterium tuberculosisantigens of high serodiagnotic value[J].Clin Vaccine Immunol,2010,17(10):1539-1547.