猪肺炎支原体P46蛋白单克隆抗体制备

P46蛋白是猪肺炎支原体的主要免疫优势蛋白,且与其他支原体无交叉反应,常作为猪肺炎支原体检测的靶蛋白。制备针对该蛋白的特异性单抗能为猪肺炎支原体的检测及机理研究提供有效的资源。本研究取猪肺炎支原体全菌抗原免疫小鼠,用大肠杆菌表达的P46蛋白作为筛选抗原制备了2株特异性单克隆抗体1A4和3C11。结果显示,2株单抗ELISA效价最高可达1∶64 000。Western-blot结果表明,2株单抗均能与原核表达的P46蛋白及猪肺炎支原体全菌蛋白中46 000大小的蛋白发生特异性反应;2株杂交瘤细胞株经3个月冻存或连续传代3个月均能保持较高的滴度。本研究成功制备了2株针对猪肺炎支原体P46蛋白的特异、稳定且高滴度的单克隆抗体。     

Immune Responses to the Attenuated Mycoplasma hyopneumoniae 168 Strain Vaccine by Intrapulmonic Immunization in Piglets

To investigate the immune responses to the attenuated Mycoplasma hyopneumoniae 168 strain vaccine, 8-15 d old piglets were immunized with M. hyopneurnoniae 168 strain vaccine by intrapulmonic route. And the specific IgG antibody in serum, lymphoproliferation, IFNT, and specific secretory IgA （SIgA） antibody in bronchoalveolar lavage fluid were detected on 30 and 60 d post-immunization （DPI）, respectively. On 60 DPI, all the pigs except for those in health control group were challenged with a field M. hyopneumoniae strain JS. Necropsy was performed on 30 d post-challenge （DPC）. The results showed that IFN7 and specific SIgA were stimulated on surface of respiratory tract after immunization. And peripheral blood mononuclear cells could also be proliferated about 1.81 and 2.12 fold on 30 and 60 DPI when stimulated by M. hyopneumoniae protein in vitro. However, no serum IgG antibody against M. hyopneumoniae was detected during the whole immune phage. After challenge, vaccinated pigs were observed with only very slight histological lesion in individual lobes. None of vaccinated pigs showed any clinical signs. While the unvaccinated pigs from challenge control group showed varying degrees of clinical sign and severe macroscopical lesion of mycoplasmal pneumonia of swine （MPS）. The result suggested that the attenuated M. hyopneumoniae 168 strain vaccine inoculated by intrapulmonic route could activate the systemic cellular immunity, the local mucosal immunity and IFNγ secretion in respiratory tract to against M. hyopneumoniae infection in piglets.     

猪肺炎支原体PCR检测方法的建立及初步临床应用

根据GenBank中猪肺炎支原体（Mhp）J株P36蛋白基因（登录号X67286）的核苷酸序列设计1对引物,建立了快速检测Mhp的PCR方法。该方法能扩增出948bp的Mhp特异性条带,其敏感性达到可检测出0．735ng的MhpDNA,但对鸡毒支原体、猪伪狂犬病毒、猪圆环病毒、猪流感病毒、猪呼吸与繁殖综合征病毒均未检测出相应目的条带;将克隆获得的目的片段与GenBank已发表的MhpJ株的P36基因进行比较,同源性达到99．9％。采用所建立的PCR方法对42份疑似猪支原体肺炎（MPS）病料进行临床诊断,发现有13份呈阳性（31．0％）。可见,该PCR方法适合于MPS的临床诊断,可为其防控提供可靠依据。     

霉形体在原代细胞内增殖的观察

对接种霉形体的原代细胞进行了电镜观察,结果如下:(1)进入细胞内的霉形体,不仅能以包涵体形式存在,也可以散在形式分布,同时还能进入粗面内质网等细胞器内.吸附在细胞膜上的霉形体、包涵体内的霉形体和散在于细胞质中的霉形体均可增殖,而进入细胞核内的霉形体未观察到增殖相.(2)在原代细胞内,正在进行出芽增殖的较大型霉形体内有一个线粒体.(3)进入细胞质中的霉形体,除具有出芽增殖和裂殖增殖形式外,在较大型霉形体内还观察到数个小球形霉形体.霉形体同时可以两种或3种方式增殖.(4)感染霉形体的细胞发生严重的空泡化,细胞体积增大.     

猪肺炎支原体量子点荧光原位杂交研究方法的建立与应用

利用生物素化的猪肺炎支原体核酸探针和量子点荧光显色系统,对人工感染组、自然感染组和健康对照组的试验猪肺样品,以及临床样品进行量子点荧光原位杂交检测。结果表明：按照探针质量浓度为1mg／L、80～90℃变性10min、37℃杂交10h和QDs—SA复合物浓度为10nmol／L的条件进行猪肺炎支原体的量子点荧光原位杂交检测结果比较理想;而人工感染组、自然感染组和健康对照组样品的QD-FISH检测结果与分离培养结果和PCR检测结果相比,符合率均达到100％刘占床样品的QD-FISH检测结果与PCR检测结果相比,符合率为83．3％。从而证明猪肺炎支原体量子点荧光原住杂交法是一种直观和敏感的检测方法,可以用于猪肺炎支原体的检测、定位和致病机理研究,也为其他病原的研究提供了参考方法。     

动物支原体相关蛋白的免疫原性研究进展

动物支原体可引起肺炎、关节炎、乳腺炎等一系列重要疾病,临床上常表现为慢性、持续性感染,给养殖业造成严重的经济损失.亚单位疫苗具有安全高效、成本低廉等优点,是支原体疫苗研究的重要发展方向.本文主要介绍了重要动物支原体相关蛋白的免疫原性研究进展,以期为今后动物支原体病亚单位疫苗研究提供参考.     

蛋白质免疫印迹快速测定猪肺炎支原体抗原含量

为建立快速测定猪肺炎支原体抗原含量的方法,试验应用P46单抗建立不同CCU含量的猪肺炎支原体抗原蛋白免疫印迹图谱,测定不同批次的猪肺炎支原体抗原CCU含量,并与Western blot(WB)测定的CCU进行比较.结果 显示:曝光5s,107 CCU/mL含量以上的猪肺炎支原体抗原在46 kDa处有条带,且条带粗细、明...     

犬肾（DK）传代细胞中霉形体的分离与鉴定

取经电镜观察证实有霉形体污染的ＤＫ传代细胞，分别接种于霉形体检验用液体、半流体和固体培养基，证明为霉形体混合感染，即该培养物在液体培养基和半流体培养基上，同时显示出水解精氨酸和发酵葡萄糖２种特性，前者使培养基ｐＨ上升，后者使ｐＨ下降。通过反复挑选单个菌落进行克隆传代和有目的地加入单一种属的霉形体抗血清的方法，将混合的２株霉形体纯化分开。经鉴定，一株为精氨酸霉形体，一株为莱氏无（需）胆甾醇霉形体。     

Mycoplasma Hyorhinis in the Etiology of Serositis among Piglets

In a study on the involvement of Mycoplasma hyorhinis in serositis of piglets, 26 routine diagnostic animals, 3-7 weeks old, with distinct serofibrinous lesions in the pericardial, pleural and peritoneal cavities were examined. M. hyorhinis was isolated in 9 cases, non-haemolytic Escherichia coli in another 9 cases and in 4 cases both species were found. Neither of the microorganisms were found in the remaining 4 cases.The presence of M. hyorhinis in the serous cavities in the absence of non-haemolytic E. coli was always accompanied by a diagnosis of other disease conditions, mainly of the respiratory tract. In the cases infected with non-haemolytic E. coli complicating problems were absent.The pathogenicity of M. hyorhinis was further studied by inoculation of 2 young pigs in which the typical serofibrinous lesions of the serous cavities were produced. It therefore appears that M. hyorhinis can be regarded as a cause of polyserositis in piglets; under field conditions, however, the synergistic presence of other debilitating syndromes appears necessary for its haematogenous spread from the respiratory tract to the serous cavities.     

肺炎支原体感染大鼠实验模型的建立

本试验旨在建立肺炎支原体感染的Wistar大鼠模型 ,为研究肺炎支原体的发病机制及药物治疗理论提供基础。采用滴鼻法对实验大鼠进行肺炎支原体感染 ,利用PCR法进行咽拭子检测 ,并以透射电镜和光学显微镜进行肺部病理组织学检查。结果发现 ,实验大鼠在感染肺炎支原体 1 0d时 ,咽拭子PCR检测结果为阳性 ;透射电镜观察到肺脏细胞膜破裂 ,线粒体变性 ,嵴断裂 ;光镜下可见到支气管及肺血管周围有明显的淋巴细胞浸润 ,形成斑片状间质性支气管肺炎。结果表明了Wistar大鼠对肺炎支原体较易感 ,并产生以间质性肺炎为主要特征的肺部和呼吸道感染 ,也说明本次造模试验成功