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1.
猪肺炎支原体P216基因片段的表达及黏附活性研究 总被引：1，自引：0，他引：1
本研究旨在研究猪肺炎支原体P216蛋白的黏附活性并初步建立猪肺炎支原体蛋白黏附模型。根据软件分析和文献报道从P216全基因中选取亲水性、抗原性、黏附性较好的目的片段,利用PCR从Mhp NJ株扩增P216基因片段,克隆到表达载体pET-32a(+)中,获得重组质粒pET-32a(+)/P216,经IPTG诱导获得重组蛋白,通过Western blot和间接免疫荧光试验检测重组蛋白的免疫原性及黏附活性。结果表明,PCR扩增的目的基因大小为l 636bp;SDS-PAGE检测重组蛋白相对分子质量为80.1ku;Western blot检测表明重组蛋白能与Mhp阳性血清发生特异性反应;间接免疫荧光试验表明该蛋白对猪肺炎支原体黏附SJPL细胞产生占位抑制作用。结果提示,P216蛋白具有良好的黏附活性,能黏附SJPL细胞,从而为猪肺炎支原体其他黏附因子的研究奠定基础。 相似文献
2.
p97蛋白是猪肺炎支原体表面的黏附因子,是主要的致病物质,它可以刺激机体首先产生抗p97的IgA和IgM,且这些抗体比其他相关抗体早产生30d~60d。研究表明,p97具有良好的免疫原性,采用p97作为猪肺炎支原体的早期诊断和疫苗研制靶标具有重要意义。论文对p97的分子结构特征和免疫原性等进行了系统阐述,以期为猪支原体肺炎诊断试剂盒和疫苗的研制提供参考。 相似文献
3.
根据GenBank中猪肺炎支原体P97基因序列设计1对特异性引物,通过PCR扩增出猪肺炎支原体168弱毒株特异性黏附因子P97抗原决定簇基因部分序列,经测序确认后,克隆入原核表达载体pET-32a,转化宿主菌BL21,将筛选出的阳性克隆用IPTG诱导,通过SDS-PAGE进行鉴定,Western blot证明所表达的重组蛋白具有猪肺炎支原体抗原性,这为猪肺炎支原体免疫检测与诊断提供了重要条件。 相似文献
4.
采用气管结扎、链霉蛋白酶灌注冷消化法分离猪气管上皮细胞,并以对数生长期的猪肺炎支原体(Mhp)感染猪气管上皮细胞,通过间接免疫荧光技术定性定量研究不同滴度的猪肺炎支原体对猪气管上皮细胞的黏附及相同滴度的猪肺炎支原体随时间变化对猪气管上皮细胞的黏附。结果表明,分离的猪气管上皮细胞存活率为95%以上,广谱角蛋白染色阳性;Mhp野毒株XLW-1与猪气管上皮细胞37℃作用30min就能看到少量的支原体黏附,作用4h以上时黏附更明显,并且,二者作用1、2、4、6、10h,XLW-1对猪气管上皮细胞的黏附与阴性对照相比差异极显著(P0.01);1×107、2.5×106 CCU/mL的XLW-1与猪气管上皮细胞37℃作用6h,能看到支原体黏附于细胞表面,并且与阴性对照相比差异极显著(P0.01),而其他低滴度的XLW-1则看不到黏附。 相似文献
5.
为了解猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)P159蛋白的黏附活性,本实验利用PCR从Mhp NJ株扩增P159基因片段(3’端),克隆于pET-28a(+)进行表达,并用表达的截短蛋白进行黏附试验。结果表明,PCR扩增的目的基因片段约为450 bp;SDS-PAGE检测重组蛋白分子量为46 ku;western blot检测表明该重组蛋白能够与Mhp阳性血清发生特异性反应,表明该蛋白具有良好的免疫反应原性;表达的截短蛋白与Mhp的黏附试验结果表明,该重组蛋白可以粘附于猪肺上皮细胞(SJPL)表面,并部分抑制Mhp对SJPL的粘附作用。本研究结果为Mhp致病机制的进一步研究奠定了基础。 相似文献
6.
牛支原体致病机制研究进展 总被引：1，自引：0，他引：1
《动物医学进展》2015,(7)
牛支原体是引起牛呼吸系统疾病的主要病原之一,给养牛业造成了严重的经济损失。论文对近期国内外关于其致病机制的研究进展进行梳理,以期为牛支原体肺炎的防控提供参考。对牛支原体致病机制的研究可为研制有效的疫苗和新型绿色药物及其他防控途径提供理论依据,进而在临床上有效控制牛支原体引发的疾病。论文涉及三方面内容:一是牛支原体黏附蛋白的黏附作用;二是牛支原体在黏附基础上其代谢产物对宿主细胞的损伤和相关蛋白介导的宿主细胞凋亡;三是免疫学致病机制,包括黏附蛋白的免疫原性及免疫逃避机制、宿主的免疫应答及炎症损伤和宿主的抗炎反应及免疫抑制机制。 相似文献
7.
为进一步探究绵羊肺炎支原体P128蛋白的相关功能,在进行生物信息学分析的基础上,对编码p128的部分基因片段进行了PCR扩增、克隆和原核表达,并采用Western-blot方法对其免疫原性进行了分析。结果显示,p128基因与猪肺炎支原体黏附素基因p146高度同源;克隆基因在大肠杆菌Rosetta(DE3)中成功获得表达,重组蛋白以包涵体的形式存在;经纯化的重组蛋白与抗绵羊肺炎支原体全菌血清发生结合反应,表明P128蛋白是良好的免疫原。研究结果为进一步分析其功能及绵羊肺炎支原体血清学诊断技术的建立提供了有用的信息。 相似文献
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9.
猪肺炎支原体是引起猪地方性肺炎的主要病原,流行于世界各地,给养猪业造成重大经济损失。本文综述了猪肺炎支原体的主要传播途径以及引起的临床症状和肺部病变,总结了其主要致病机理,分析了综合控制该疫病的方法。猪肺炎支原体的主要传播途径是与感染猪密切接触,也可通过空气远距离传播,但无垂直传播相关报道;猪肺炎支原体感染的主要临床特征是间歇性、强度可变的干咳,但在与其他病原继发或混合感染后,临床症状和肺部病变可能会加重;猪肺炎支原体主要通过释放黏附素引起纤毛发生病变、死亡,导致上皮细胞受损或脱落从而影响纤毛功能,也能通过产生剧毒的H2O2等代谢产物引发毒性效应;防治该病首先要通过生物安全措施控制猪肺炎支原体的传入,其次是采用抗菌药物进行治疗,最后是通过接种疫苗降低其传播率。本文为猪养殖场做好猪肺炎支原体病防控提供了参考。 相似文献
10.
牛支原体是严重危害养牛业的重要病原体,阐明其毒力因子和致病机制有助于防控技术的研发。内肽酶O(PepO)参与肺炎链球菌在猪体的致病过程,本研究旨在探讨PepO在牛支原体中是否具有毒力相关功能。利用巢式PCR将牛支原体PepO中的色氨酸密码子TGA突变为大肠杆菌的色氨酸密码子TGG,进一步克隆至大肠杆菌原核表达载体,成功表达重组蛋白PepO。利用重组蛋白免疫BALB/c小鼠制备针对PepO的高免血清,经Western-blot分析显示出多抗血清结合,可激活PLG的结构域,从而发挥降解特异性底物S-2251的纤溶酶功能。比较分析牛支原体PepO突变菌株T5.37与野毒株HB0801的生长及对牛肺上皮细胞EBL的黏附特性,其结果显示PepO缺失显著降低牛支原体对EBL的黏附。本研究证实了牛支原体PepO具有活性,参与宿主细胞黏附过程,是一个潜在的毒力相关因子,为进一步研究阐明牛支原体致病机制奠定了基础。 相似文献
11.
Marchioro SB Simionatto S Galli V Conceição FR Brum CB Fisch A Gomes CK Dellagostin OA 《Veterinary microbiology》2012,155(1):44-52
Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), a chronic respiratory disease which causes significant economic losses to the swine industry worldwide. More efficient strategies for controlling this disease are necessary. In this study, we cloned17 genes coding for transmembrane proteins from M. hyopneumoniae, among which six were successfully expressed in Escherichia coli and had their immunogenic and antigenic properties evaluated. All proteins were immunogenic in mice and sera from naturally infected pigs reacted with the recombinant proteins, suggesting that they are expressed during infection. These antigens may contribute for the development of new recombinant vaccines and diagnostic tests against EP. 相似文献
12.
为了解猪肺炎支原体对抗菌药物的敏感性,采用宏量肉汤稀释法对4株猪肺炎支原体的最小抑菌浓度（MIC）值进行了测定。结果表明,猪肺炎支原体对泰妙菌素和环丙沙星最为敏感,MIC≤0．03μg／mL,其次分别为四环素类药物（包括四环素和多西环素）、林可霉素和泰乐菌素,而对氟苯尼考的敏感性则较差。本研究可为猪支原体肺炎的防控以及抗菌药物的合理使用提供参考。 相似文献
13.
Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia in swine. Various reports indicate that different strains are circulating in the swine population. We investigated the variety of M. hyopneumoniae strains by a newly developed genetic typing method based on the polyserine repeat motif of the LppS homolog P146. PCR amplification using M. hyopneumoniae specific, conserved primers flanking the region encoding the repeat motif, followed by sequencing and cluster analysis was carried out. The study included strains isolated from different geographic regions as well as lysates from lung swabs from a series of pig farms in Switzerland. High diversity of M. hyopneumoniae was observed but farms being in close geographic or operative contact generally seemed to be affected by the same strains. Moreover, analysis of multiple samples from single pig farms indicated that these harbored the same, farm-specific strain. The results indicate that multiple strains of M. hyopneumoniae are found in the swine population but that specific strains or clones are responsible for local outbreaks. The method presented is a highly reproducible epidemiologic tool allowing direct typing of M. hyopneumoniae from clinical material without prior isolation and cultivation of strains. 相似文献
14.
C H Armstrong A B Scheidt H L Thacker L J Runnels M J Freeman 《Canadian journal of veterinary research》1984,48(3):278-281
Ten swine from each of five herds believed to be affected with mycoplasmal pneumonia of swine and ten swine from each of five herds believed to be mycoplasmal pneumonia-free were selected for postmortem study. Lungs from the 100 swine were examined; grossly and microscopically for lesions typical of mycoplasmal pneumonia of swine and culturally and by an indirect immunofluorescent procedure for the presence of Mycoplasma hyopneumoniae. Nineteen of the lungs had both gross and microscopic lesions typical of mycoplasmal pneumonia of swine and 13 (68%) of these were infected, i.e. were culturally and/or indirect immunofluorescent positive. Absence of gross lesions did not prove freedom from mycoplasmal pneumonia, 14 of 73 (19%) grossly normal lungs were found to be infected with M. hyopneumoniae. Comparison of the indirect immunofluorescent and cultural examination, as methods of diagnosing mycoplasma pneumonia, revealed that neither procedure alone was reliable in the case of negative results. Ten lungs were indirect immunofluorescent negative and culturally positive and seven were culturally negative and indirect immunofluorescent positive (11 lungs were positive by both procedures). It was concluded that a definitive diagnosis of mycoplasmal pneumonia of swine requires that M. hyopneumoniae be visualized in indirect immunofluorescent stained lung sections or that it be recovered culturally. 相似文献
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16.
猪肺炎支原体P46基因的原核表达与间接ELISA方法的建立 总被引：3，自引：1，他引：2
猪肺炎支原体是猪喘气病的病原体,本研究选择猪肺炎支原体P46膜蛋白基因亲水区序列进行克隆,并将其内3个编码Trp的TGA突变成TGG,然后再克隆到pET28a(+)载体中,在大肠杆菌BL21 (DE3)细胞内实现了高效表达,表达产物相对分子质量约为31 ku,约占菌体总蛋白35％,表达形式为包涵体,通过Western blotting 证明表达产物与猪肺炎支原体高免血清具有很好的反应原性和特异性.将大肠杆菌表达的猪肺炎支原体P46重组蛋白经过洗涤、过柱纯化后,作为间接ELISA包被抗原用于检测猪血清中猪肺炎支原体抗体,通过对各参数和试剂的优化建立了rP46-ELISA方法,获得了较好的效果,通过与现有ELISA检测方法的比较,结果表明二者间具有较高的符合率. 相似文献
17.
Diagnosis of Mycoplasma hyopneumoniae 总被引：1，自引：0，他引：1
Thacker EL 《Animal health research reviews / Conference of Research Workers in Animal Diseases》2004,5(2):317-320
Mycoplasma hyopneumoniae, the cause of enzootic pneumonia, remains an important pathogen in the swine industry. This small, complex organism colonizes the ciliated cells of the respiratory tract, resulting in little exposure to the immune system. Confirming the presence of M. hyopneumoniae, as well as identifying its role in respiratory disease and pneumonia, remains challenging to the veterinary profession. While culture of the organism remains the gold standard for identification, the use of serology, the polymerase chain reaction and various assays to detect the presence of M. hyopneumoniae in tissue is common in diagnostic laboratories. Because of the role M. hyopneumoniae plays in increasing the severity of pneumonia associated with concurrent bacterial and viral infections, understanding the pathogenesis and diagnostic assays available is critical for developing effective intervention strategies to control respiratory disease on a herd basis. 相似文献
18.
J D McKean J J Andrews D O Farrington 《Journal of the American Veterinary Medical Association》1979,174(2):177-180
The complement-fixation test (CFT), a latex-agglutination test (LAT), and Mycoplasma hyopneumoniae isolation procedures were compared with gross and microscopic lung evaluations for the routine diagnosis of mycoplasmal pneumonia (MP) of swine. The difficulty and variability of M hyopneumoniae isolation rendered this technique unacceptable. In market-weight swine, the finding of typical gross lesions was correlated with CFT results (P less than 0.005). The MP status of a herd could not be determined by use of these techniques in 4- to 9-week-old pigs. No technique could be used alone to establish a diagnosis. Gross and microscopic examination of all slaughter specimens, CFT, and clinical evaluation of the herd, used in combination, were the most useful means of determining the MP status of the herd. 相似文献
19.
A tissue culture system to study respiratory ciliary epithelial adherence of selected swine mycoplasmas 总被引：2，自引：0，他引：2
An in vitro culture system for swine tracheal epithelial cells was developed to study the adherence of swine mycoplasmas. Swine tracheal epithelial cells were isolated by enzymatic digestion and cultured on microporous membranes. Growth medium was placed under the membrane support to create air-liquid interface feeding resulting in the cells growing cilia and microvilli on the apical surface. Two strains of Mycoplasma hyopneumoniae (pathogenic strain 91-3 and non-pathogenic type strain J) and two strains of Mycoplasma flocculare (type strain Ms42 and field isolate 7160T) were used in this study. The morphology of the cultured tracheal cells was evaluated by transmission electron microscopy. Adherence of M. hyopneumoniae and M. flocculare and damage to the cilia were demonstrated using scanning electron microscopy. The pathogenic M. hyopneumoniae strain 91-3 adhered to cilia inducing obvious damage. The non-pathogenic M. hyopneumoniae strain J did not adhere to mature cilia. Both M. flocculare strains Ms42 and 7160T adhered to mature and budding cilia. No obvious ciliary damage was observed with strain Ms42. Minimal damage consisting of a slight tangling of the cilia occurred after adherence by strain 7160T. This model will enable us to further study the role of adherence of mycoplasmas on the pathogenesis of swine pneumonia. 相似文献
20.
Lungs from 191 slaughter pigs with gross lesions indicative of enzootic pneumonia of pigs (EPP) and 80 grossly normal lungs, all originating from 9 different herds, were subjected to microbiological and pathological examinations. The microbiological studies included both bacterial and mycoplasmal culture and also testing for Mycoplasma hyopneumoniae antigen in tissue by indirect immunofluorescent technique. M. hyopneumoniae, Pasteurella multocida and Mycoplasma hyorhinis were detected in 83%, 43% and 37% of the pneumonic lungs, respectively. Mycoplasma flocculare was the most frequently isolated organism in the non-pneumonic lungs. The greatest amounts of macroscopic pneumonia (25.2%) were recorded in lungs with all the three agents M. hyopneumoniae, P. multocida and M. hyorhinis present. The amounts of pneumonia in lungs with M. hyopneumoniae alone and in concurrence with P. multocida, were 9.3% and 15.6%, respectively. M. hyorhinis was also, in this study, associated with higher frequency of diffuse pleuritis. These findings indicate that M. hyorhinis might be involved in the pathogenesis of pneumonia in slaughter pigs. Ninety-six per cent of the isolates of P. multocida from pneumonic lungs could be characterized as type A. In the herds which had the most severe pneumonia problems, toxin production was detected in 83% of the P. multocida strains while only 28% were toxigenic in herds with subclinical to moderate pneumonia problems. 相似文献