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1.
应用PCR方法从牛分枝杆菌Vallee株基因组中扩增获得mpb70、mpb83和esat-6三个目的基因片段。采用重叠延伸剪接技术（splicing by overlap extension，SOE）获得融合基因mpb70-mpb83后，将mpb70-mpb83和esat-6串连于同一表达载体pET32a（＋）中得到重组质粒pET70-83-E6。转化BL21（DE3）大肠杆菌感受态后，经IPTG诱导以可溶的形式表达融合蛋白。用Ni^2＋亲合层析的方法纯化该融合蛋白。Western blot分析显示：该融合蛋白能与抗牛分枝杆菌阳性血清发生特异性反应，而与牛副结核病阳性血清不反应。用该纯化蛋白初步建立了间接ELISA方法，并检测了117份临床血清样本（其中67份为PPD阳性牛血清），阳性率为39．32％（46／117）份，与PPD皮试诊断的符合率为82．05％（96／117）。 相似文献
2.
利用DNAStar对牛结核分枝杆菌的3个主要抗原mpb70、mpb83、esat-6进行预测分析,将预测的抗原指数高的抗原表位经全基因合成后,克隆到表达载体pET28a(+)中,构建重组质粒pET28a-mpb-70-83-esat-6,转化入大肠杆菌BL21(DE3)中诱导表达,表达产物经SDS-PAGE鉴定,结果检测到相对分子质量约为21 000的重组蛋白。经NI-NTA纯化重组蛋白后进行Western-blot鉴定,结果显示该重组蛋白可被牛结核分枝杆菌的多克隆抗体识别,表明该蛋白具有良好的反应原性。 相似文献
3.
牛结核杆菌MPB70-ESAT-6融合蛋白的原核表达及抗原反应性分析 总被引：1，自引：0，他引：1
为增强单个蛋白的抗原性,利用(Gly4Ser)3柔性连接肽将牛结核杆菌MPB70和ESAT-6融合。采用重叠延伸PCR技术将牛结核杆菌mpb70与esat-6基因连接,获得融合基因mpb70-esat-6,连接至T-Vector pMD19中,获得克隆质粒pMD-70-esat-6。经Bam HⅠ、EcoRⅠ酶切、纯化,并与p ET28a(+)载体连接,构建了pET-70-esat-6重组表达质粒。SDS-PAGE发现,在27.2 ku处表达了融合蛋白MPB70-ESAT-6,Western blotting证实,MPB70-ESAT-6与牛结核阳性血清反应性良好。MPB70-ESAT-6融合蛋白的研究为牛结核病诊断抗原及相关疫苗研究奠定了基础。 相似文献
4.
以牛分枝杆菌Vallee株基因组DNA为模板,应用PCR扩增获得mpb64和Ag85B两个目的基因片段,采用重叠延伸剪接技术（SOE）剪接mpb64和Ag85B,得到融合基因mpb64-Ag85B;将融合基因片段先克隆于pMD 18-T载体,再亚克隆到表达载体pET32a（＋）中,得到重组质粒pET64-85.该重组质粒经核苷酸序列测定,显示其中的外源片段与期望的序列一致;其BL21（DE3）转化菌经IPTG诱导表达带有6个组氨酸标签的融合蛋白;用Ni2＋螯合层析方法纯化融合蛋白,Western-blotting分析结果显示,该融合蛋白能与抗牛分枝杆菌阳性血清发生反应. 相似文献
5.
牛分枝杆菌mpb64-ag85b-esat6融合基因的分子克隆及表达 总被引：2，自引：0，他引：2
以牛分枝杆菌valleeⅢ株基因组DNA为模板,PCR扩增mpb64、ag85b和esat6 3个目的基因片段,采用重叠延伸剪接技术（SOE）和基因重组技术获得融合基因mpb64-ag85b,将mpb64-ag85b和esat6串联到同一原核表达载体pET32a（＋）中获得重组质粒pET64-85b-e6。将其转化到大肠杆菌感受态细胞BL21（DE3）中,以IPTG（终浓度1 mmol/L）进行诱导,SDS-PAGE电泳分析表达情况,以Ni^2＋亲和层析柱纯化表达的融合蛋白和Western blot分析融合蛋白的免疫活性。结果表明：表达的融合蛋白大约为76 Ku,与预测大小相符,以Ni^2＋亲和层析柱纯化的融合蛋白能与牛结核病阳性血清发生反应。 相似文献
6.
以牛分枝杆菌DNA为模板,克隆了牛分枝杆菌MPB70基因,构建了克隆载体pGEM-MPB70和表达载体pET30a-MPB70,经IPTG诱导在大肠杆菌BL-21中表达,用SDS-PAGE和免疫印迹分析表达产物并进行蛋白纯化。试验结果表明,牛分枝杆菌MPB70基因体外扩增产物与预期值相符,约582 bp;所构建表达质粒pET30a-MPB70经测序,结果与预期一致;SDS-PAGE分析表明,该融合蛋白以包涵体的形式表达,其分子质量约为29 kD,蛋白表达量约占菌体总蛋白的20%;该蛋白经电洗脱纯化后,纯度达85%以上;免疫印迹分析表明,原核表达的融合蛋白可与兔抗牛分枝杆菌多克隆抗体结合,并且具特异的免疫反应性。 相似文献
7.
为提高牛分枝杆菌(M.bovis)单一抗原的抗原性,本研究以重叠延伸拼接PCR技术将M.bovis mpb51与mpb63基因连接,并克隆至pMD18-T中,构建重组质粒pMD-51-63。以BamHⅠ和EcoRⅠ双酶切pMD-51-63和pET28a(+),将纯化的mpb51-63融合基因亚克隆至pET28a(+)中,获得了重组表达质粒pET-51-63。该重组质粒在大肠杆菌BL21(DE3)中经IPTG诱导和SDS-PAGE分析,表达约46 ku的外源融合蛋白,免疫印迹实验证实,该融合蛋白具有M.bovis的抗原反应性。为进一步研究mpb51-63融合基因及其表达产物作为牛结核病亚单位疫苗、核酸疫苗以及特异的诊断试剂奠定了基础。 相似文献
8.
用PCR技术和重叠延伸剪接技术获得牛分枝杆菌esat-6基因和mpb70-mpb83融合基因，连接真核表达载体pcDNA3．1（＋），构建了重组质粒pCE6和pC70-83-E6。分4组免疫小鼠：pCE6组、pC70—83-E6组、pcDNA3．1（＋）和PBS对照组，采用间接EI。ISA法检测免疫小鼠血清特异性抗体水平，MTT法检测免疫小鼠脾淋巴细胞增殖情况和IFN—y分泌情况。结果表明，2重组质粒免疫组小鼠的血清抗体水平持续上升，而2对照组始终维持在较低水平，且pC70-83-E6组小鼠的抗体水平高于pCE6组。经PPD刺激后，pCE6组和pC70—83-E6组小鼠的SI值与2对照组均差异显著（P〈0．05），2重组质粒免疫组间差异不显著（P〉0．05）；2重组质粒免疫小鼠脾细胞产生的IFN—y均显著高于2对照组（P〈0．05），且pC70-83-E6组明显高于其他3组（P〈0．05）。证实本试验构建的2种牛分枝杆菌DNA疫苗可有效诱导实验动物产生体液免疫和细胞免疫应答。 相似文献
9.
本研究以牛分枝杆菌Vallee111染色体DNA为模板,以MPB70成熟蛋白基因特异性引物进行PCR扩增,获得约500bp的DNA片段.通过T-A克隆技术,将PCR产物克隆至pGEM-T Vector中,成功地构建出克隆载体pGEM-T-70.以BamH Ⅰ和EcoR Ⅰ双酶切pGEM-T-70和pET28a( ),并将纯化的MPB70基因亚克隆至pET28a( )中,构建出原核表达载体pET28a-70.将pET28a-70转化至感受态E.coli BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约25Ku外源蛋白带.Western blot分析发现,该蛋白具有牛分枝杆菌抗原性,从而为进一步研究MPB70的亚单位疫苗及DNA疫苗奠定基础. 相似文献
10.
为了研究牛结核病新型诊断抗原,试验根据GenBank中Mycobacterium bovis基因序列设计1对引物,将牛结核分枝杆菌MPB70基因构建到pET22b(+)原核表达载体上,将重组载体转到E.coliBL21(DE3)中,经IPTG诱导获得高效表达,并进行SDS-PAGE和Westen-blot分析。结果表明:MPB70蛋白以可溶形式在细胞周质中表达,有部分蛋白以包涵体形式在细胞质中表达,其分子质量约为30ku,蛋白表达量占菌体总蛋白的20%;重组MPB70蛋白可与牛分枝杆菌阳性血清发生特异性反应。说明重组MPB70蛋白能够作为诊断抗原。 相似文献
11.
Tuberculosis in domesticated red deer: Comparison of purified protein derivative and the specific protein MPB70 for in vitro diagnosis 总被引：7，自引：0，他引：7
The use of a Mycobacterium bovis-specific protein, mycobacterial protein bovis 70 (MPB70), was compared with complex, M bovis-derived purified protein derivative (bovine PPD), for its ability to improve the diagnostic precision of in vitro assays for tuberculosis in farmed deer. A combination of lymphocyte transformation and enzyme-linked immunosorbent assay (ELISA) was used to differentiate between specific M bovis reactivity and crossreactivity due to sensitisation with saprophytic mycobacteria such as Mycobacterium avium. In the lymphocyte transformation assay the response of mononuclear cells, from red deer, to MPB70 was found to be more specific, but less sensitive, as an indicator of infection by M bovis when compared with the complex antigen bovine PPD. When used in conjunction with bovine PPD alone, MPB70 was found to increase the specificity of the ELISA in diagnosing animals with disease. 相似文献
12.
为了建立针对牛分枝杆菌分泌抗原MPB64的检测手段,制备该抗原的单克隆抗体,构建真核表达载体pcDNA-mpb64,以原核表达并纯化的融合蛋白H-MPB64包被ELISA板,建立单克隆抗体检测方法。利用基因免疫方法免疫BALB/c小鼠,通过杂交瘤技术筛选出针对牛分枝杆菌分泌蛋白MPB64的单克隆抗体一株,并制备了腹水,经鉴定腹水效价达到1∶10240,采用硫酸铵沉淀法纯化腹水,纯化抗体效价为1∶8000,为今后研制分枝杆菌鉴定技术奠定基础。 相似文献
13.
Carla D. Marassi Jim McNair John Pollock Paula Ristow Leila S. Fonseca Walter M.R. Oelemann Walter Lilenbaum 《Comparative immunology, microbiology and infectious diseases》2010,33(6):485-489
In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p < 0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases. 相似文献
14.
低分子质量的蛋白抗原CFP-10是一种重要的牛分支杆菌早期分泌蛋白.为了检测该蛋白和其他几种抗原在牛结核病诊断中的临床应用,对CFP-10的基因进行克隆,鉴定,并在原核系统中表达.PCR法扩增cfp-10基因片段,连接到pET-22b( )原核表达载体中,再转入表达宿主E.coli BL21(DE3)PlysS菌株内,用IPTG诱导,进行蛋白表达、纯化.分别以CFP-10、ESAT-6、MPT83、MPT70、牛PPD、CFP-10与ESAT-6混合蛋白,MPT83与MPT70混合蛋白为抗原,用间接ELISA法诊断牛结核病.结果表明,以CFP-10与ESAT-6混合蛋白作为抗原检测牛结核病的特异性和敏感性分别达到了100%和63.6%,均超过了其他单抗原或抗原组合,为以筛选合适抗原为基础的血清学诊断技术提供了有力的支持并创造了良好的应用前景. 相似文献
15.
牛传染性鼻气管炎病毒gE基因的截短克隆与表达 总被引：6，自引：0，他引：6
以牛传染性鼻气管炎病毒Baaha Nu/67株的DNA作为模板，用PCR扩增gE基N并克隆至pGEM-T Easy裁体，再以此质粒作为模板将gE基因分成6个片段，分别插入原核表达载体pET32a并在大肠杆菌中进行了表达。蛋白电泳结果表明6个片段中有2个片段以可溶形式表达，1个片段以包涵体形式表达，另外3个片段没有表达。采用固定化金属离子亲和层析法在非变性条件下对两个可溶性片段进行了纯化。经免疫印迹试验，间接ELISA和交叉试验证明，两个纯化的重组蛋白均与牛传染性鼻气管炎阳性血清样品发生反应，而与牛传染性鼻气管炎阴性血清无任何反应，显示其具有良好的抗原性和特异性，可用于牛传染性鼻气管炎gE-ELISA诊断方法的建立。 相似文献
16.
Amadori M Tagliabue S Lauzi S Finazzi G Lombardi G Teló P Pacciarini L Bonizzi L 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2002,49(2):89-96
Because of the frequent exposure of cattle to mycobacteria of the avium/intracellulare group, an investigation was carried out into the possible repercussions thereof on the diagnosis of bovine tuberculosis. Three calves from a bovine tuberculosis-free herd, scored avian reactors in the gamma-interferon assay for bovine tuberculosis, were sedated and inoculated endotracheally with a virulent Mycobacterium bovis strain. Then, three other avian reactors were housed with the above donor calves. Mycobacterium bovis was isolated from the nasal swabs of the three endotracheally infected, donor calves. On these samples, TB complex-specific polymerase chain reaction (PCR) tests for IS6110 were also positive, albeit with a different time kinetics. The three contact-infected calves showed clear immunological signs of infection; however, their nasal swabs were always PCR-negative and only Mycobacterium avium was isolated. In the endotracheally infected donor calves there was a rise of the gamma-interferon responses to avian and bovine purified protein derivative (PPD) tuberculins, which reached the same stable plateau levels over the whole experiment. The above effect was also observed in the contact-infected calves, even though the response to avian PPD tuberculin always remained at a higher level. By using conventional bovine and avian PPD tuberculins, the comparative intradermal test was generally positive in endotracheally infected, as opposed to contact-infected calves; a positive intradermal test for M. bovis was obtained in two contact-infected calves by different bovine PPD tuberculins based on M. bovis bacillus Calmette-Guerin (BCG) secreted or somatic antigens. It was concluded that M. bovis infection may be concealed for some time in cattle sensitized by mycobacteria of the avium/intracellulare group and that different diagnostic procedures should be adopted for such animals. 相似文献
17.
牛结核病抗体胶体金快速检测技术的建立和应用 总被引：10，自引：0，他引：10
为了建立一种快速检测牛分支杆菌抗体的新方法用于诊断牛结核病,利用胶体金免疫层析技术原理,用原核诱导表达的牛分支杆菌抗原蛋白MPB83和MPB70分别作为胶体金标记抗原和检测线上的捕获抗原,制备牛结核抗体检测试纸条.结果表明,粒径为40 nm的胶体金制备的试纸条敏感性最高,胶体金最佳标记pH为6.0,MPB83抗原最适标记量为每毫升胶体金6.5 μg,MPB70抗原的最适包被浓度为3.0 mg/mL,抗MPB83蛋白IgG的最佳包被浓度为2.5 mg/mL,交叉试验证明试纸条不与牛的其他非相关疾病的阳性血清反应,具有较高的特异性.比较试验证明其敏感性显著高于韩国进口试纸条.在上述试验条件下生产了一批胶体金试纸条进行临床样品检测,并与细菌分离培养、结核菌素皮内变态反应(TST)和韩国试纸条比较.本试纸条与牛分支杆菌分离培养的符合率为85%,与TST的符合率为79.73%,与韩国试纸条的符合率为98.75%.快速检测牛结核抗体的免疫层析试纸条具有敏感、特异、简便、快速的特点,适用于对牛结核病进行普查和检疫,也可作为TST的辅助诊断方法,在牛结核病根除计划中具有良好的应用前景. 相似文献
18.
Casal C Bezos J Díez-Guerrier A Álvarez J Romero B de Juan L Rodriguez-Campos S Vordermeier M Whelan A Hewinson RG Mateos A Domínguez L Aranaz A 《Preventive veterinary medicine》2012,105(1-2):149-154
The intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay are the principal tests used worldwide for the ante-mortem diagnosis of bovine tuberculosis. The conventional reagent currently in use in these tests is purified protein derivative (PPD) tuberculin obtained from Mycobacterium bovis culture. The components of PPD are poorly characterized and difficult to standardize. To overcome this issue, antigens specific to the Mycobacterium tuberculosis complex are being studied. Here we have assessed the biological potency of ESAT-6, CFP-10 and Rv-3615c presented as peptide or recombinant protein cocktails in comparison with the standard bovine PPD used routinely in Spanish eradication campaigns. The study was performed in cattle (n=23) from a herd with natural M. bovis infection. Animals were simultaneously injected with PPD and the peptide and protein cocktails. The percentages of cattle reacting positively to single intradermal test were 60.9% (bovine PPD), 47.8% (peptide cocktail) and 60.9% (protein cocktail), with no significant difference between the actual skin fold thickness increases (p>0.05). The IFN-γ assay detected 60.9% of animals when stimulation was performed with bovine PPD, but decreased to 52.2% when stimulation was performed with the peptide cocktail and to 47.8% when stimulation was performed with the protein cocktail. However, no significant differences were found between IFN-γ responder frequencies (p>0.05). These results show a potential use of these defined reagents for in vivo tuberculosis diagnosis. 相似文献