【作者】 幺乃全；

【导师】 王全凯；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2013， 博士

【摘要】 鸭疫晨氏杆菌感染是由鸭疫里氏杆菌(Riemerella anatipestifer, RA)引起家鸭、鹅、火鸡及其他家禽和野禽的一种接触性传染病,可导致2-3周龄雏鸭大批发病和死亡,生长发育严重受阻,并已成为影响养鸭业最为严重的细菌性传染病之一。目前该病在世界各养鸭地区均有流行,给养鸭业造成了严重的经济损失。当前控制RA感染的有效方法是从流行地区的发病鸭体内分离致病菌,制备灭活油佐剂苗并进行免疫注射。但由于鸭疫里氏杆菌血清型多达21种,不同血清型间缺乏交叉保护,使灭活油佐剂苗的应用受到限制。当前对RA疫苗的研究多是集中于对具有交叉保护作用的抗原成分的筛选,已有资料报道的各种抗原成分如OmpA、pN45、Camp和Vap等均被用于RA亚单位疫苗的研究,但保护效果均不够理想,因此筛选出一种或几种具有交叉保护性抗原成分用于RA基因工程疫苗的开发仍是当前研究的重点。微孔蛋白(Porins)是革兰阴性菌普遍含有的重要外膜蛋白成分,诸多研究表明微孔蛋白基因具有高度保守性,可用于菌株的鉴定,且是菌体重要的抗原成分,能够为各型菌株提供交叉保护。本文以RA外膜微孔蛋白(Porin)为研究对象,设计特异性引物对RA外膜porin基因进行扩增,并于血清1、2、4、6、10、11、13、14和17型RA菌株基因组中均成功扩增出1222bp的目的基因,序列比对分析显示这8种血清型RA与RA-JL-1、RA-JL-2及GenBank中登录的RA-CH-2(CP004020.1)、RA-GD (CP002562.1)、DSM15868(CP002346.1)、ATCC11845(CP003388.1) Porin基因的同源性均为100%,仅与RA-CH-1(CP003787.1)的porin基因存在部分碱基序列的差异,同源性为93.8%；Blast分析该基因与黄杆菌科其它菌属细菌porin的同源性均低于70%,表明该基因具有高度的保守性。相比于16S rDNA和外膜蛋白A (OmpA)更适合用于RA菌株的鉴定。根据porin基因保守性高的357bp序列设计特异性引物,建立了检测RA外膜porin的PCR方法,与细菌分离鉴定符合率达到了100%,具有良好的特异性和敏感性。同时利用生物学分析软件对RA外膜Porin蛋白的二级结构、信号肽和跨膜区等进行预测和三级结构同源建模。结果显示RA外膜Porin是以p折叠为主要成分的桶状结构蛋白,以p折叠形式与细胞膜进行整合而形成跨膜区,仅存的1个α螺旋为蛋白的信号肽。DNASTAR软件分析蛋白抗原性,结果显示RA的外膜Porin蛋白中存在有14个潜在的B细胞抗原优势表位和15个潜在的T细胞抗原识别位点。本文构建了pET28a-porin重组原核表达质粒,并诱导目的基因的表达,结果显示诱导表达的RA外膜Porin蛋白分子量大小约为48.5ku,可溶性分析显示该蛋白是以不溶性的包涵体形式进行表达。Western blot分析显示纯化的表达蛋白能够与1、2、10、11、17型RA菌株全菌体阳性血清均发生特异性的免疫反应,证实具有良好的反应原性。在此基础上构建了pVAX1-porin重组真核表达质粒,以脂质体法转染至Marc-145细胞系中,通过RT-PCR和间接免疫荧光法分别从转录水平和蛋白水平鉴定出该基因在真核细胞中获得了表达。以构建的pVAX1-porin重组真核表达质粒免疫BALB/c小鼠,结果显示该质粒能够诱导小鼠产生特异性抗体,且显著高于pVAXl空载体和生理盐水对照组(P<0.05),表明RA外膜Porin能够诱导小鼠产生特异性的体液免疫反应。MTS法检测淋巴细胞增殖结果显示,pVAX1-porin真核表达质粒免疫组小鼠脾淋巴细胞在RA菌体裂解抗原刺激下发生明显增殖。同时检测小鼠血清中IL-2和IL-4含量均显著升高,与pVAX1空质粒和盐水组相比均差异显著(P<0.05),表明RA外膜Porin蛋白能够诱导小鼠产生细胞免疫应答。对RA外膜porin的初步研究证实该基因为各血清型RA菌株共有,且具有高度的同源性,依此建立的PCR方法能够用于RA菌株的鉴定。构建的porin真核表达质粒能够诱导小鼠产生体液免疫和细胞免疫应答,具有良好的免疫原性。研究结果为后续RA基因工程疫苗的开发奠定了一定的理论基础。更多还原

【Abstract】 The infection of Riemerella anatipestifer was a contagious disease of domestic ducks, geese, turkeys, other poultry and wild birds caused by Riemerella anatipestifer and can lead to high incidence, death rate and slow growth of2-3weeks old ducklings. It has become one of the most serious bacterial infectious diseases for duck industry and caused serious economic losses to the duck industry for its distribution in most countries. The most effective control method of RA infection was injection of inactivated vaccine with strains isolated from endemic area. But as many as21kinds of serotypes and lack of cross-protection between serotypes, application of inactivated vaccine was limited. This problem can be solved by finding a useful subunit component of cross protection for each serotype RA strains. According to study data show subunit ingredients have been reported such as OmpA, pN45, Camp and Vap etc with development value and no ideal protective effect. So screening subunit composition with protective effect for each serotype is still the focus of study. Porins were generally major outer membrane protein composition of Gram-negative bacteria and existing research shows it highly conserved among strains can be used for detection and bacterial antigenic components can provide cross-protection for each type strain.Study for outer membrane porin of RA was carried out for the first time. Porin gene of1222bp was successfully amplified by specific primers designed in serotype1,2,4,6,10,11,13,14and17of RA strains and sequence alignment analysis showed homology between8serotypes RA and RA-JL-1、RA-JL-2and RA-CH-2(CP004020.1)、RA-GD (CP002562.1)、DSM15868(CP002346.1)×ATCC11845(CP003388.1) reported in Genbank were100%. Only some base sequence of RA-CH-1(CP003787.1) exist difference and homology was93.8%. The porin homology of RA with other bacteria of Flavobacterium was lower than70%by blast analysis. Result indicated that the gene has a very high conservatisim and homology can be used for the identification of the RA strains better than16S rDNA and OmpA. Specific primers according to a highly conserved357bp sequence of porin gene were designed and PCR method for detection was established. The PCR method was proven to have good specificity, sensitivity and100%coincidence with bacteriological identification. The PCR method can be used for rapid detection RA infection. Secondary structure, signal peptide, transmembrane region and tertiary structure model of Porin were predicted by biological analysis software. Result showed Porin was a barrel protein mainly constituted by β-pleated sheet and the transmembrane region was formed by integration of (3-pleated sheet with cellular membrane. Only one a-helix was the signal peptide of Porin. The antigenic analysis by DNASTAR software showed14potential B cell antigen epitops and15potential T cell antigen recognition sites in Porin.Prokaryotic expression plasmids pET28a-porin were constructed and induced for expression. Result showed that molecular size of expressed Porin was approximately48.5ku and soluble analysis showed the protein was expressed with form of insoluble inclusion bodies. Expressed products were purified and analysed by Western blot. The analysis showed specific immune response occued between expressed protein and positive serum of serotype1,2,10,11and17RA strains whole cell. This indicated Porin has a good immunological cross reaction. Eukaryotic pVAX1-porin recombinant expression plasmid was constructed on this basis and then transfected into Marc-145cells with liposomes method. Porin expression in eukaryotic cells were identified by RT-PCR and indirect immunofluorescence method from gene transcription and protein level.BALB/c mice were immunized with eukaryotic expression plasmid pVAXl-porin and the results showed pVAX1-porin eukaryotic expression plasmid can induce specific antibody of mice and was significantly higher than pVAXl empty vector and saline control group (P<0.05). This indicated that Porin of RA was able to induce specific humoral immune response in mice. The MTS assay results showed significantly proliferation of mice spleen lymphocytes immunized with pVAX1-porin eukaryotic expression plasmid stimulated by RA antigen and simultaneous detection of IL-2and IL-4levels in serum were significantly elevated, compared with pVAXl empty vector and saline group(P<0.05). Result indicated that Porin of RA can induce cellular immune responses of mice.Preliminary study on porin of RA confirmed that the gene existed in the RA strains of each serotype with a high degree of homology and conservation and can be used for the identification of RA strains. Porin eukaryotic expression plasmid induced humoral and cellular immune responses of mice and indicated the Porin has a good immunogenicity. The findings laid theoretical foundation for the development of subsequent RA genetic engineering vaccine.更多还原