目录
摘要Ⅱ
关键词Ⅱ
AbstractⅢ
引言
引言（或绪论）1
文献综述2
1 多杀性巴氏杆菌概述2
多杀性巴氏杆菌特性及培养2
多杀性巴氏杆菌流行病学2
荚膜血清型2
耐药情况2
2 多杀性巴氏杆菌的致病机制及毒力因子3
2.1 多杀性巴氏杆菌致病机制3
2.2 多杀性巴氏杆菌的毒力因子3
2.2.1 荚膜3
2.2.2 脂多糖 4
2.2.3 外膜蛋白4
2.2.4 转铁结合蛋白4
2.2.5 毒素4
3 多杀性巴氏杆菌的诊断和治疗5
3.1 多杀性巴氏杆菌的诊断5
3.1.1 萎缩性鼻炎的诊断方法5
3.1.2 肺炎性巴氏杆菌病5
3.2 多杀性巴氏杆菌的治疗5
4 多杀性巴氏杆菌疫苗研究及应用5
4.1 灭活疫苗5
4.2 弱毒疫苗6
4.3 亚单位疫苗6
5 总结6
第二章 材料与方法6
1材料与方法6
1.1 材料 6
1.1.1 实验菌株 6
1.1.2 实验仪器及来源6
1.1.3 主要实验试剂配制7
1.2 实验方法8
1.2.1菌种的复苏及培养8
1.2.2 多杀性巴氏杆菌的培养特性8
1.2.3 重组质粒的构建8
1.2.4 重组质粒的原核表达9
1.2.5 SDSPAGE电泳9
1.2.6 WesternBlotting检测诱导表达的重组蛋白9
1.2.7 目的蛋白的纯化 10
2 结果与分析 10
2.1 多杀性巴氏杆菌的鉴定 10
2.2多杀性巴氏杆菌的培养特性10
2.3 PCR扩增结果10
2.4 重组质粒的双酶切鉴定 12
2.5 重组蛋白的表 *51今日免费论文网|www.jxszl.com +Q: ￥351916072￥ 
达、纯化及鉴定12
3 讨论 13
3.1 研究结果分析 13
3.2 展望和未来 13
致谢14
参考文献15
表21 引物设计8
图21 油镜下Pm的形态10
图22 Pm的生长曲线及OD600与菌落数的关系11
图23 PCR扩增11
图24 重组质粒双酶切鉴定12
图25 重组蛋白的SDSPAGE及WB鉴定12
猪进行性萎缩性鼻炎灭活疫苗中重组蛋白的构建与表达
摘 要
巴氏杆菌毒素（P. multocida toxin ，PMT）也被称为皮肤坏死毒素、鼻甲萎缩毒素或溶骨毒素，是引发猪进行性萎缩性鼻炎的主要致病因素。鉴于PMT优良的免疫原性，通过福尔马林灭活后可以诱导机体产生抗猪萎缩性鼻炎的特异性抗体。但天然PMT的含量在巴氏杆菌所含蛋白总量中占比不足0.6%，所以很难大规模从产毒素性巴氏杆菌中纯化全长的PMT。为了解决在制备猪萎缩性鼻炎灭活疫苗过程中天然PMT含量不足的问题，本实验将编码PMT的toxA基因以及其N端toxN（11461 bp）和C端toxC（29583858 bp）经PCR扩增后分别将目的基因克隆到原核表达载体pET32a中，通过优化诱导剂的浓度和诱导时间进行蛋白诱导表达，结果表明3个重组蛋白在大肠杆菌中均以可溶性形式表达，其分子量大小分别为176 kDa、88 kDa和67 kDa，重组蛋白纯化效果良好，目标蛋白清晰，杂蛋白含量低。研究证明，rPMT与天然PMT具有相同的生物学功能，该研究的成功开展将为后续利用表达蛋白制备出猪萎缩性鼻炎疫苗提供可靠的生物材料。
EXPRESSION OF RECOMBINANT PROTEINS FOR PREPARATION OF INACTIVATED VACCINE FOR PORCINE PROGRESSIVE ATROPHIC RHINITIS
ABSTRACT
Pasteurella toxin (PMT) is also known as skin necrosis toxin, atrophic toxin of turbinate or osteolytic toxin, which is the main pathogenic factor of progressive atrophic rhinitis in pigs. In view of the excellent immunogenicity, PMT can be inactivated with formalin and used to induce specific antibodies against porcine atrophic rhinitis. Unfortunately, the content of natural PMT is less than 0.6% of the total protein content of Pasteurella, so it is difficult to purify and inactivate the fulllength PMT from Pasteurella on a large scale. In order to solve the problem of the insufficient natural PMT content in the preparation of inactivated vaccine for porcine atrophic rhinitis, the ToxA gene of PMT and its Nterminal (11461bp) and Cterminal (29583858bp) were amplified using PCR and cloned intto pET32a. The protein expression was induced by optimizing IPTG concentration and induction time. The results showed that the three recombinant proteins were expressed in soluble form in E.coli. The molecular mass of three proteins is 176 kDa, 88 kDa, and 67 kDa, respectively. The purification result showed that the obtaining recombinant protein has good purity and the target fragment is clear with less heteroprotein. It has been proved that rPMT has the same biological function as natural PMT. The successful development of this study will provide reliable biomaterials for the preparation of Swine Atrophic Rhinitis Vaccine by using the expressed proteins.

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