【作者】 邓均华；

【导师】 熊焰；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 本研究对猪肺炎支原体MY-99株的超微结构进行了研究，发现其菌体大小范围为0.33-0.48um，缺乏细胞壁，菌体形态多样。 在对其致病性的研究中发现，通过鼻腔接种猪肺炎支原体MY-99株2周后60％的试验猪出现了喘气等典型的猪地方流行性肺炎的临床症状，其中2头伴有轻度的咳嗽，病理切片可见到支气管周围有多量淋巴样细胞浸润，肺泡壁增厚等典型的间质性肺炎的病变，这种病变程度在接种2周后和4周后较严重，6周后有所减轻，并且从试验猪的肺脏中分离出了猪肺炎支原体。同时发现试验猪的平均日增重比对照猪显著降低，接种后0-14天期间降低了31.05％（P＜0.01），15-28天期间降低了42.01％（P＜0.01），29-42天期间降低了0.1％（P＞0.05），试验猪血清中的抗体水平在2周后最高(1：2^4.2)，4周后降至1：2^3.9，6周后最低(1：2^2.5)，显示猪肺炎支原体MY-99株诱发了急性的猪地方流行性肿炎。 采用0.4％TritonX-100结合KI提取猪肿炎支原体MY-99株不同代次的传代株MY-991、MY-993和复壮株MY-992的膜蛋白，并通过SDS-PAGE技术对其膜蛋白进行比较分析发现，在人工培养条件下猪肺炎支原体MY-99株的膜蛋白发生了一定的变异，其中MY-991株的膜蛋白有七条蛋白条带：94.4KD、57.5KD、46.0KD、36.0KD、32.4KD、20.4KD和16.6KD，MY-993株的膜蛋白有七条蛋白条带：94.4KD、57.5KD、46.0KD、41.0KD、36.0KD、32.4KD和19.5KD，MY-992株的膜蛋白只有两条蛋白条带：46.0KD和32.4KD。用猪肺炎支原体MY-991、MY-992和MY-993的抗血清，以及猪肺炎支原体的标准阳性血清和标准阴性血清经免疫印迹试验对猪肺炎支原体MY-991、MY-993和MY-992株膜蛋白的抗原性进行检测，结果发现各菌株的P46能与上述所有血清（除阴性血清外）发生反应。 将猪肺炎支原体MY-99株的P46电转移至PVDF膜，染色干燥后，并标记蛋白条带，由中国医学科学院基础医学研究所中心实验室检测出P46蛋白的N-端10个氨基酸的序列为：Ala-Glu（Gly）-Lys-Cys-Asn-Gln-Asp-Gly（Tyr）-Gln-Gly。更多还原

【Abstract】 According to the ultrastructure of Mycoplasma hyopneumoniae MY-99 strain, we found it ranges from 0.33um to 0.48um without cellwall and its modality various .34-day-old cross-bred pigs were inocuolated Mycoplasma hyopneumoniae MY-99 strain intranasally, and at postinoculation weeks (PIW) 2, the clinical signs of swine enzootic pneumonia (SEP) such as wheeze occurred to 60% inoculated pigs, a few of them coughed; histologic lesions included accumulation of neutrophils and macrophages around bronchus, and the lesions reached the peak at PIW 2 and 4, then increasingly relieved at PIW 6. We isolated Mycoplasma hyopneumoniae from the lungs of the inoculated pigs. Meanwhile, the average daily gain of inoculated pigs decreased significantly compared to the control pigs, 31.05% decreased during postinoculation days 0-14(P< 0.01). 42.01% decreased during postinoculation days I5-28(P<0.01), and 0.1% decreased during postinoculation days 29-42(P>0.05). the liters of the antibody in serum at PIW 2,4,6 were 1:24.2,1:23.9 and 1:22.5 respectively, which indicate that Mycoplasma hyopneumoniae MY-99 strain induced an acute SEP.We extracted membrane protein of Mycoplasma hyopneumoniae MY-99 strain from its different passages’ strains MY-99l,MY-993 and rejuvenescence strain MY-992, then analyzed them by SDS-PAGE, the results show that the membrane protein of Mycoplasma hyopneumoniae MY-99 strain mutated during artificial culturing . According to the electrophoretic profiles, the membrane proteins of MY-991 include 7 protein bands: 94.4KD,57.5KD,46.0KD,36.0KD,32.4KD,20.4KD and 16.6KD. The membrane proteins of MY-993 also include 7 protein bands: 94.4KD,57.5KD, 46KD, 41KD,36.0KD,32.4KD and 19.5KD,but the membrane proteins of MY-992 only include 2 protein bands:46KD and 32.4KD. The immunoblotting to the antiserum of MY-991,MY-992 and MY-993, positive serum and negative serum respectively, show that P46 of all the strains reacted to all the sera but negative serum.We transferred the membrane proteins of Mycoplasma hyopneumoniae MY-99 strain to PVDF membrane, dyed and dried, then marked the P46 and sequenced the N-terminal 10 amino acids of P46 by the central laboratory of fundamental medical institute, Chinese medical academy of science. The sequence is: Ala-Glu(Gly)-Lys- Cys-Asn-Gln-Asp-Gly(Tyr)-Gln-Gly.更多还原