【作者】 唐先春；

【导师】 吴斌； 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】 多杀性巴氏杆菌(Pasteurella multocida，Pm)是一种重要的病原菌，按产毒素与否，可将其分为非产毒素多杀性巴氏杆菌(Non-toxingenic Pasteurella multocida，TPm)和产毒素多杀性巴氏杆菌(Toxingenic Pasteurella multocida，T~+Pm)。T~-Pm往往导致猪肺疫、禽霍乱、牛羊出血性败血症等畜禽传染病；T~+Pm是猪传染性萎缩性鼻炎(Swine infectious atrophic rhinitis，AR)的主要病原菌。 用特异性荚膜抗原可将Pm分为A，B，D，E和F 5个血清群(型)；利用菌体抗原可将Pm分为1到16个菌体型。Pm的菌体最主要的外膜蛋白是OmpH，各血清型的OmpH蛋白同源性较高，而且能诱导高水平的保护性抗体。 T~+Pm产生的多杀性巴氏杆菌毒素(Pasteurella multocida toxin，PMT)是一种约145kDa的皮肤坏死毒素，该毒素由toxA基因编码。T~-Pm缺少toxA基因，不能导致严重的萎缩性鼻炎症状，而只注射提纯的PMT就可以导致SPF猪出现萎缩性鼻炎症状。可见PMT在AR的产生过程中起着重要的作用。研究还表明：PMT与细胞结合并转入细胞的活性位点位于N端，而发挥毒性催化作用的位点在C端；PMT全毒素是有致病性的，而适当截短的PMT能失去致病性、保留免疫原性，可用于疫苗且比天然毒素易于获得。 本研究针对Pm及PMT主要开展了以下工作： 1、建立了猪多杀性巴氏杆菌的PCR检测方法，应用该检测方法并结合细菌的生化试验，从临床样品中分离鉴定了66株Pm。其中24株分离于肺脏，42株分离于鼻拭子样品。同时对每一分离菌株进行了药敏实验。 2、参照文献设计了5对PCR引物，它们是分别针对多杀性巴氏杆菌A、B、D、E、F 5个荚膜血清型的特异基因。从而建立了猪多杀性巴氏杆菌的PCR分型方法，用该方法对每一分离菌株进行了荚膜分型。结果表明，在分离的66株Pm中有46株为D型，18株为A型，1株为B型，1株无法定型，没有E、F型。在46株D型Pm中有28株分离于鼻拭子样品，18株分离于肺脏；18株A型Pm中有14株分离于鼻拭子样品，4株分离于肺脏：1株B型Pm分离于肺脏：1株未定型的Pm分离于肺脏。 3、设计了一对特异扩增T~+Pm的toxA基因的引物，建立了产毒素多杀性巴氏杆菌的PCR检测方法。用该方法对分离菌株进行了毒素检测，结果表明，所分离的66株Pm有8株为产毒素菌株。这8株T~+Pm都分离于有AR症状猪的鼻拭子样品，均为D型。 4、参照GenBank已发表的多杀性巴氏杆菌ompH基因序列，设计了一对引物，华中农业大学硕士毕业论文通过PCR的方法，克隆了Pm的。mPH基因，以pMD18一T为载体，构建了克隆载体pMD一口仰H并进行了序列测定。比较分析表明，所克隆的口mpH基因与NcBI上所报道的7个。呷H基因序列:us琴208、u52200、u522一3、AJ4597s5、u522ox、工二丝匹、旦业塑互的大小完全一致，为1114bP。核昔酸同源性达到%%一99%。然后构建了原核表达载体PET-o即H，转化大肠杆菌B玩，，实现了口朋卯H基因在大肠杆菌BL2.中的高效表达。表达蛋白约35kDa，W七Stem一blot检测表明，表达的蛋白具有生物学活性。 5、参照GenBank己发表的toxA基因序列，设计了一对带有酶切位点的引物，通过pCR的方法，克隆了完整的toxA基因(扩增片段为4024bp，o盯为3858bp)。构建了原核表达载体pET28b一toxA，并对其序列进行测定，比较分析表明，该才。工A基因与NCBI上所报道的5个toxA基因序列:225388、X57775、x52478、^J566llo、迷返工丝鱼的大小完全一致，核昔酸同源性都在99%以上。将表达载体pET28b一toxA转化大肠杆菌BLZ，，但是没有得到表达。然后，在质粒pET28b一toxA基础上，用酶切方法构建了4个亚克隆表达载体:①pET28b一N1513;②pET28a一1514C;③pET28b一N3 173;④pET28b一N3389，用于表达截短的toxA基因。分别转化大肠杆菌BLZ，，诱导表达。结果只有质粒①pET28b一1 513和②pET28a一1514C得到了表达，分别表达了toxA基因5’端的1 513个碱基和3’端的2345个碱基，二者相加正好是全毒素。表达蛋白分别约57kDa和87kDa，W七stem一blot检测表明，表达的蛋白具有生物学活性。 本研究分离鉴定了66株Pm，其中8株是产毒素菌株，为进一步研究提供了材料:所建立的Pm鉴定、Pm分型、T+Pm鉴定方法有助于临床诊断及细菌分离;对口mPH基因的克隆表达为研制猪多杀性巴氏杆菌病的ELISA诊断方法和新型基因工程疫苗莫定了基础;才口义A全毒素基因的克隆以及剪切后毒素基因片段的表达为研制AR的EUSA诊断方法和亚单位疫苗奠定了基础。更多还原

【Abstract】 Pasteurella multocida is an important pathogenic bacterium and can be classified into toxigenic P. multocida(T+Pm) and non-toxigenic P. multocida(TPm). Usually, TPm can cause pneumonic pasteurellosis in pig, fowl cholera in poultry, hemorrhagic septicemia in cattle and sheep. T^Pm is a major pathogen of swine infectious atrophic rhinitis.P. multocida strains are separated into serogroup A, B, D, E, F basing on capsular antigen and serotype 1 to 16 basing on somatic antigen. OmpH is a major outer membrane protein of P. multocida. The OmpHs of different serotype are highly homologous, and can stimulate high titers of protective antibodies.T+Pm generates P. multocida toxin(PMT) which is a dermonecrotic toxin about 145kDa and is encoded by toxA gene. TPm does not have this toxA gene and can not cause severe AR. But injection of purified PMT alone often results in typical nasal turbinate atrophy in gnotobiotic piglets. It is abvious that PMT plays a key role in the course of AR. Extensive research indicats that the N terminal of PMT possesses cell-binding activity and the C terminal has catalytic activity; the holotoxin of PMT is pathogenic while the properly truncated toxin may lose pathogenic activity and remain immunogenic activity. The truncated toxin can be easily obtained to use in vaccines.The focus of this research is Pm and PMT. The projects are as following:1. A PCR method is established to detect P. multocida from swine. With this method and biochemical reaction, 66 strains of P. multocida were isolated from clinic samples. Every isolate was then studied on sentivity to antibiotics.2. Based on literature, 5 sets of PCR primers were designed which are respectively specific to the capsular gene of serogroup A, B, D, E, F of P. multocida, then PCR typing method for P. multocida was established. PCR typing assay indicated that, among 66 isolates, forty-six were serogroup D(28 from nasal swabs and 18 from lung), eighteen were serogroup A(14 from nasal swabs and 4 from lung), one was serogroup B(from lung) and one was untypable(from lung).3. A PCR method is established to detect Tl’m. The PCR results indicated that, among 66 strains of P. multocida, 8 isolates are toxingenic. The 8 isolates are all serogroup D and from the nasal swabs of swine suffering atrophic rhinitis.4. Basing on the nucleotide sequences of ompH gene published in GenBank, a pairof primers was designed to amplify the ompti gene of P. multocida. This ompti gene was cloned into pMD18-T to get pMD-ompH and was then sequenced. The homology of nucleotide sequence with other seven published ompH sequences on GenBank was 96%-99%. The expression vector named pET-ompH was constructed and was then transformed into E.coli ~BLi\. SDS-PAGE and Western-blot indicated that the ompH gene is highly expressed in BL2i as a 35kDa protein which has biological activity.5. Basing on the nucleotide sequences of tax A published in GenBank, a pair of primers with restriction sites was designed to amplify toxA gene of T+Pm. The PCR product was then cloned into pET-28b to obtain pET28b-to;cA. Sequencing result revealed that its size was identical to the five published toxA sequences in GenBank and the homology of nucleotide sequence is higher than 99%. The expression vector of pET28b-/oxA was transformed into E.coli BL21. But SDS-PAGE showed no expression of the holotoxin. Based on the plasmid of pET28b-roxA, four subcloned expressing vectors were then constructed with restriction enzymes: (DpET28b-N1513; (2) pET28a-1514C; (3)pET28b-N3173; ﹑ET28b-N3389. All these vectors were used to express truncated PMT. They were transformed into E.coli BLai. But only (D pET28b-N1513 and (2)pET28a-1514C resulted expression in E.coli BL2i, whichseparately expressed 1513 base pairs of 5’ terminal and 2345 base pairs of 3’ terminal of toxA. The expressed proteins are 57kDa and 87kDa respectively. Western-blot indicated that both recombinant proteins have biological activity.In summary, in this study, we isolated 66 strains of P. multocida in which 8 isolates are tox更多还原