【作者】 梁望旺；

【导师】 何启盖； 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae，APP)是猪传染性胸膜肺炎(Porcine Contagious pleuropneumonia)的致病菌，该病是一种呼吸道疾病，影响着全世界养猪业的发展。猪感染此病后会呈现从急性的突然死亡到慢性感染及亚临床感染等症状。急性感染可表现为肺的纤维素性、出血性坏死，死亡率高；慢性感染的猪无明显的临床症状但生长缓慢。发病后存活下来的猪可成为病原的携带者，从而可能引起此病的再次爆发。所以，猪群APP血清抗体的检测对预防和控制该病的爆发具有十分重要的意义。 迄今为止，已报道的APP血清型共有15种，不同血清型之间没有或仅有弱的交叉反应，这给该病的诊断和预防带来了很大的困难。传统的血清学诊断方法，如CFT、IHA等，针对的是菌体抗原诱导产生的抗体，只能对同血清型的APP做出检测。免疫预防也存在着同样的问题，APP的灭活苗具有一定的保护力，但是，也只能对同血清型APP的感染产生保护。本研究主要是针对以上情况，旨在建立一种特异、敏感、能对所有血清型APP做出检测的血清学检测方法。 由于胸膜肺炎放线杆菌的毒素和转铁结合蛋白具有种特异性，所以有希望用于APP诊断方法的建立。 将含有猪胸膜肺炎放线杆菌毒素Ⅱ基因的质粒pET-ApxⅡ转化到大肠杆菌BL21(DE3)。挑选出表达量最高的克隆子，然后于37℃经IPTG诱导表达。对表达条件进行优化，并对包涵体进行了变性和复性。用复性后的蛋白作抗原，建立了检测猪传染性胸膜肺炎放线杆菌抗体的间接酶联免疫吸附试验(ELISA)。与间接血凝(IHA)检测结果作了比较，证明所建立的ELISA诊断方法具有较高的特异性和敏感性，并具有良好的稳定性和可重复性。利用建立的ApxⅡ-ELISA诊断方法对2503份临床送检的各地猪血清进行检测，测得猪传染性胸膜肺炎的阳性率平均为63％以上。 利用地方分离的APP血清型2型菌株，设计一对引物，用PCR的方法扩增了其转铁结合蛋白A基因(tbpA基因)，扩增片段的大小为2726bp，并克隆到pET-28b中构建了重组表达载体。经酶切鉴定及测序分析后，将此表达载体转入大肠杆菌BL21(DE3)中，SDS-PAGE和Western blotting结果表明该基因在大肠杆菌BL21中获得了表达。表达的蛋白经纯化后建立了Tbp-ELISA检测方法，并与IHA和ApxⅡ-ELISA进行了比较，表明该方法可用于APP血清抗体的检测。 上述研究为猪传染性胸膜肺炎血清抗体的检测提供了种特异性方法，为预防、控制乃至根除此病奠定了基础。更多还原

【Abstract】 Actinobacillus pleuropneumoniae ( APP ) is the etiological agent of porcine contagious pleuropneumonia, a severe respiratory contagious disease effecting swine industry worldwide. The clinical signs vary from acute to chronic, and asymptomatic carriers also occur frequently. Acute pleuropneumonia is characterized by necrotic and hemorrhagic lung lesions with fibrinous pleuritis and high mortality. Chronically infected pigs have less obvious clinical signs but show reduced growth rates. The surviving pigs can remain as carriers in the herd and cause a new outbreak when immunity is weakened.Fifteen serotypes of APP have been recognized so far. There is no or weak cross reaction in different serotypes, which brings many difficulties to diagnosis and prevention of the disease. The conventional serological tests, such as Complement Fixation Test (CFT), Indirect Hemagglntination Test (IHA ), only test the antibodies induced by the antigens in the bacterial surface and diagnose Pleuropneumonia caused by homologous serotypes. The same problem exists in prevention by vaccine inoculation. The killed vaccine of APP can induce certain protective immunity, only to homologous serovar, not to heterologous serovar. The goal of the study is to develop a sensitive, species-specific diagnostic method able to detect all serotypes.The expression plasmid pET-ApxII were tansformed into E.coli BL21(DE3).Clones of higher expression level were selected, and grown in the presence of IPTG at 37 C to induce expression. The conditions of expression were optimized and the protein was purified form inclusion bodies, denatured and renatured. Using the renatured protein as antigen an indirect enzyme-linked immunosorbent assay(ELISA) was developed to detect antibodies to Actinobacillus pleuropneumoniae. The comparison between this method and IHA showed that the ELISA had good speciality, sensitivity and reliability for clinic serum samples. The average positive rate of Porcine Contagious pleuropneumonia is above 63% using the Apx II -ELISA method to detect 2503 clinic serum samples.The 2726 bp gene encoding subunit A of transferring- binding protein ( TbpA ) of Actinobacillus pleuropneumoniae serotype 2 was amplified by PCR and cloned into plamid pET-28b. The positive clones were confirmed by enzyme digestion and sequencing analysis, and transformed to E.coli BL21 cells. Protein expression form the clones was analyzed by SDS-PAGE and Western blot. After purification, the protein was applied as antigen to develop indirect ELISA. The comparison between IHA and theTbp-ELISA was performed by simultaneously detecting 117 clinical samples. The experimant demonstrated the diagnostic potential of the transferring-binding protein (Tbp) for actino bacillus pleuropneumoniae.更多还原