【作者】 李求春；

【导师】 焦新安；

【作者基本信息】 扬州大学 ， 人兽共患病学， 2010， 博士

【摘要】 鸡白痢沙门菌多侵害20日龄以内的幼雏,引起白色下痢,病死率极高。成年鸡感染后虽然不会出现典型的临床症状,但是长期处于带菌状态。在成年鸡的脾脏和生殖道内,细菌可以存活40周以上。在性成熟时期,细菌能侵入卵巢和输卵管,垂直传播给下一代。由于该菌能水平传播和垂直传播,对养禽业可造成极大的危害。目前,在西方发达国家该病已经被消灭或基本消灭,然而在个别小的禽群中仍有发生,国内禽群鸡白痢的爆发仍较多。寻求鸡白痢沙门菌的致病基因对于了解其致病机理及有效地防治鸡白痢具有重要的意义。本研究运用IVIAT(in vivo induced antigen technology)筛选和鉴定了鸡白痢沙门菌的感染相关因子,为全面认识鸡白痢沙门菌的致病机理提供了基础。沙门菌毒力质粒在细菌致病过程中发挥着重要的作用,本研究从鸡白痢沙门菌分离鉴定了一个新的质粒pSPI12,并分析了该质粒上唯一的毒力相关基因ipaJ的功能,为深入了解细菌早期的感染机制提供了有利的材料。1.鸡白痢沙门菌S06004基因组表达文库的构建作为禽的主要病原菌之一,鸡白痢沙门菌的致病机理仍不是很清楚。本实验采用IVIAT(体内抗原诱导技术)筛选鸡白痢沙门菌的体内感染相关因子,以了解细菌体内的基因表达情况。首先,要构建鸡白痢沙门菌的基因组表达文库。将S06004的基因组用Sau3AI酶切后,回收大小在0.1 kb-4 kb的片段。同时用BamHI酶切原核系列表达载体(pET30a,b,c),去磷酸化后,回收线性载体片段。将基因组酶切的片段按适当的比例与原核表达载体连接后,转化大肠杆菌DH5α,挑取部分转化子,液体培养后提取质粒,用载体特异性引物分析插入片段的大小分布和片段的插入率。然后从平板上提取质粒,并将其转化大肠杆菌BL21(DE3),构建了S06004基因组的原核表达文库。2.利用IVIAT技术筛选鸡白痢沙门菌的感染相关因子获取了十份感染了鸡白痢沙门菌的阳性血清,混合后分步与S06004体外培养状态下表达的抗原结合去除相应的抗体,同时利用间接ELISA检测吸附效果。然后将经过吸附处理的血清与S06004基因组表达文库进行免疫筛选,经初次筛选和二次筛选以后,共筛选出45个阳性克隆。对阳性克隆测序后,分析其中所包含的蛋白序列。筛选获得的蛋白涉及生物大分子合成和代谢、转运蛋白、调控因子、能量代谢相关蛋白及功能未知蛋白等,反映了细菌在体内感染过程中,除毒力基因的表达外,细菌需要表达相关蛋白维持正常的生长代谢和应对体内不断变化的体内环境,为揭示细菌的体内活动提供了依据。3.实时荧光定量PCR检测鸡白痢沙门菌S06004感染相关因子的体内外表达差异根据IVIAT筛选获得的阳性克隆的序列和蛋白序列同源比对结果,设计了11对S06004感染相关因子(traV、gatD、pbpC、stbC、deoR、emrB、dapA、phoQ、trkH、adhE、yhaN)的检测引物,同时以鸡伤寒沙门菌gmk基因作为内参。提取鸡白痢沙门菌S06004体外液体LB培养的总RNA,反转录成cDNA;以SPF鸡为动物模型,采用静脉注射的方式将体外培养的S06004细菌免疫,感染后在不同时间段采血,从血液中收集细菌,提取总RNA,反转录成cDNA,利用荧光定量PCR比较感染因子在体内外培养时mRNA的表达水平差异。结果表明:个别基因表现出很高的表达量,如phoQ;部分基因表达呈持续上升趋势如gatD;traV、dapA、deoR、emrB和trkH呈先升后降趋势;adhE、pbpC和stbC呈下降趋势;yhaN呈先降后升趋势。反映了各感染相关因子在体内感染不同时间段mRNA水平并不一致,其表达水平相对于体外培养时有不同程度的上调。4.鸡白痢沙门菌pSPI12质粒的分离与序列分析利用抑制差减杂交的方法,构建了鸡白痢沙门菌与肠炎沙门菌的差减文库,筛选获得了鸡白痢沙门菌的特异核苷酸序列。部分序列拼接形成ipaJ基因与猪霍乱沙门菌C500减毒株质粒pSFD10中的ipaJ高度同源。为了验证鸡白痢沙门菌ipaJ基因是否存在于类似的质粒上,我们将卡那霉素抗性基因插入鸡白痢沙门菌S06004株的ipaJ基因中,构建了插入突变株SIM12。从突变株中提取质粒,转化大肠杆菌DH5α,卡那霉素抗性筛选阳性克隆,从中提取质粒获得了携带ipaJ基因的质粒pSPI12,将质粒构建到pMD18T载体上测序分析获得了pSPI12的完整序列和图谱。同源性分析显示pSPI12和pSFD10高度同源,两者都含有一个毒力相关基因ipaJ。PCR鉴定结果显示本室保存的105株鸡白痢沙门菌分离株都含有该基因,除了猪霍乱沙门菌C500疫苗株外,其它猪霍乱沙门菌菌株和其它血清型肠道沙门菌亚种中未扩增出目的基因。Southern blot分析表明ipaJ基因只存在于质粒上。pSFD10是ColE型质粒,可以在F质粒的介导下进行接合转移,所以我们推测猪霍乱沙门菌C500中的pSFD10质粒可能由鸡白痢沙门菌中的pSPI12转移后部分碱基突变所致。5.鸡白痢沙门菌IpaJ蛋白功能的初步分析志贺氏菌中的ipa负责编码侵袭素,参与调控细菌侵入细胞的过程。为了探究鸡白痢沙门菌中IpaJ蛋白的功能,构建了携带有ipaJ基因的回复质粒PCR?2.1-ipaJ,电转化入ipaJ基因突变株SIM12株中,获得了回复株SIM12(ipaJ)。以禽肾脏上皮细胞(CKC)为上皮细胞感染模型,比较了S06004株、SIM12株和SIM12(ipaJ)株对细胞的侵袭能力和在细胞内的增殖差异,结果显示突变株的侵袭能力和在胞内的增殖能力明显低于野生株和回复株。由于鸡白痢沙门菌以脾脏巨噬细胞作为其寄生场所,本研究以禽巨噬细胞系HD-11为巨噬细胞感染模型,比较三株细菌对细胞侵袭和在胞内增殖能力的差异,突变株的侵袭能力都明显低于野生株和回复株,但在巨噬细胞内的增殖趋势没有明显差异,都是从感染后开始到5 hr呈增长趋势,5 hr后胞内的细菌数开始下降。体内实验分析比较了野生株、突变株和回复株对10日龄海兰白蛋鸡的致病能力,突变株的致死率与野生株相比降低了约14倍,而回复株和野生株相当。从感染的巨噬细胞和鸡的脾脏细胞中提取总RNA,利用RT-PCR从感染了S06004的细胞总RNA中扩增出ipaJ基因,表明在感染过程中该基因得到了表达。Western-blot实验显示了体外表达的IpaJ蛋白可以与鸡白痢沙门菌阳性血清反应,进一步说明IpaJ蛋白可能参与了鸡白痢沙门菌对机体的感染过程,该基因的突变会导致细菌毒力的降低。综上所述,本研究利用IVIAT技术筛选获得了鸡白痢沙门菌体内感染过程中表达的感染相关因子,45个蛋白功能涉及细菌生物大分子的合成和降解、调控蛋白、运输蛋白、能量代谢相关蛋白、噬菌体功能相关蛋白和未知功能蛋白等。选取11个因子进行实时荧光定量PCR分析,结果显示在体内感染过程中,这些基因的表达量与体外培养状态相比,都有一定程度的上调。本研究从鸡白痢沙门菌中分离出了一个新的质粒pSPI12,大小为4080 bp。序列分析显示该质粒上存在细菌毒力相关基因ipaJ,其编码蛋白可能参与了细菌早期感染侵袭细胞的过程。动物实验显示ipaJ基因的突变会导致细菌毒力的降低,所以我们推测ipaJ基因是鸡白痢沙门菌可能的毒力基因。PCR分析结果证明该基因存在于本实验室分离保存的时间跨度达四十多年的105株鸡白痢沙门菌中,而除了在猪霍乱沙门菌疫苗株C500中含有该基因和相似的质粒外,在其它猪霍乱沙门菌菌株和肠道沙门菌亚种菌株中并未分离到该基因。更多还原

【Abstract】 Salmonella enterica serovar Pullorum is the causative agent of pullorum disease in poultry, an acute systemic disease that results in a high mortality rate in young chicks but rarely causes severe clinical disease in adult birds. The bacteria can persist in both the spleen and the reproductive tract for over 40 weeks following experimental infection in chickens. During the period of sexual maturity, Salmonella pullorum colonized both the ovary and the oviduct of hens and led to the laid eggs infected by the bacteria. Because of its vertical transmission and horizonal transmission, it could cause great economic loss in poulty industry. Although pullorum disease has been largely eliminated from many western countries, it still has been happenning in small flocks. The explosion of pullorum disease is popular in China. Searching for virulence genes is very important to understand the pathogenesis of Salmonella pullorum and find therapeutic measures to the disease. In this study, the in vivo induced technology(IVIAT) was used to screen and identify infection-related factors from Salmonella pullorum.The Salmonella virulence plasmids play important roles in the pathogenesis, in our study, a new plasmid pSPI12 was isolated from Salmonella pullorum. There is only one virulence-related gene ipaJ in the plasmid. The function of IpaJ protein was analyzed, and provide information for our understanding of the pathogen’s early infection process. 1. Construction of S06004 genomic expression libraryAs an important pathogen of poulty, the pathogenesis of Salmonella pullorum was still unclear. In this study, the in vivo induced antigen technology(IVIAT) was used to screen the infection-related factors of Salmonella pullorum. Firstly, the pathogen genomic expression library was constructed. The genome of S06004 strain was digested by Sau3AI, then the fragments ranging from 0.1 kb to 4 kb were purified and ligated to pET30a, b, c expression vectors, which were digested by BamHI and dephosphorylated by CIAP. The ligated products were transformed into E. coli DH5α, and the size of fragments inserted into the vector and the insertion efficiency was analyzed by PCR with pET30 vectors specific primers. Then the plasmids were extracted from recombinant E. coli DH5αin the plate and transformed into BL21(DE3) to creat genomic expression library.2. Screening of infection-related factors from S06004 by IVIATThe convalescent sera from chicken challenged with Salmonella pullorum were collected, adsorbed against in vitro-grown S06004 strain to get rid of antibodies reacted with expressed antigens in vitro. The adsorbed sera were identified by ELISA, and then used to screen S06004 genomic expression library. After rigorous selection, 45 positive clones were screened and sequenced. Sequence analysis showed that the 45 proteins were involved in various aspects of molecule synthesis/degradation, transport system, regulation, energy metabolism and unknown functions. During the infection process, the bacteria had many proteins expressed to keep alive and adapt to the changed environment in vivo, which also provided the foundation for our study of the infection process of the pathogen.3. Differential expression of infection-related factors detected by real-time fluorescent quantitative PCR in in vivo and in vitro condition.According to the sequence and homology analysis of screened positive clones by IVIAT, 11 pairs of primers specific to infection-related factors (traV, gatD, pbpC, stbC, deoR, emrB, dapA, phoQ, trkH, adhE, yhaN) were synthesized to analyze their differential expression in in vivo and in vitro conditions, the gene of Salmonella gallinarum gmk was used as reference. The total RNA was extracted from S06004 strain cultured in LB, and then reverse transcribed to cDNA. In vivo, specific–pathogen free (SPF) chickens were used as experimental model to learn about mRNA expression difference under in vivo condition. SPF chickens were challenged with S06004 intravenous injection, then bacterial cells were recovered from blood at 12hr, 24hr and 48hr. RNA was isolated, converted to cDNA and then used as template for assessment of their differential expression by quantitative real-time PCR. The result showed that phoQ gene showed high expression level in vivo compared to in vitro cultivation. gatD upregulated continuously; traV, dapA, deoR, emrB and trkH upregulated before 24hr and then went down; adhE, pbpC and stbC went down continuously; yhaN downregulated before 24hr and then went up from 24hr to 48hr. All these showed that the genes had different expression levels at different time, but they upregulated at different degrees compared to in vitro cultivation.4. Isolation and sequence analysis of plasmid pSPI12 from Salmonella pullorumUsing suppression subtractive hybridization (SSH) technique, the Salmonella pullorum subtractive library was constructed by comparison of genomic differences between Salmonella pullorum and Salmonella enteritidis. The Salmonella pullorum specific sequences were screened from the subtractive library. Parts of sequences were spliced together into the whole sequences of ipaJ, which showed high homology to ipaJ gene in plasmid pSFD10 of Salmonella choleraesuis. In order to find whether the gene located in pSFD10-like plasmid in Salmonella pullorum, a kanamycine resistance gene KmR was inserted into the ipaJ gene of Salmonella pullorum strain S06004, giving rise to a mutant strain SIM12. Plasmids in SIM12 were extracted out and transformed into E.coli DH5α, selecting for positive clones resistant to kanamycine. The plasmid harbouring ipaJ gene in E.coli DH5αwas propagated and extracted, which was named pSPI12. The pSPI12 was then cloned into pMD18T vector for sequencing. Sequence analysis showed that pSPI12 was high homological to pSFD10, and both of them had only one virulence-related gene ipaJ. PCR analysis showed that the gene exists in 105 Salmonella pullorum strains isolated from 1962 to 2009. But the gene couldn’t be isolated from other Salmonella choleraesuis and Salmonella spp strains, except Salmonella choleraesuis vaccine strain C500. Southern blot analysis showed that the gene only located in plasmid, not chromosome. pSFD10 is a ColE-type plasmid, which can be transferred by F plasmid, so it is speculated that the pSFD10 in Salmonella choleraesuis C500 is from pSPI12 with some bases mutated.5. Function analysis of IpaJ protein in Salmonella pullorumIn Shigella, the ipa genes encode invasins, which help the pathogen invading into eukaryotic cells. In order to find out the role of IpaJ protein during Salmonella pullorum infection, a recombinant plasmid PCR?2.1-ipaJ was constructed and transformed into SIM12 strain, giving rise to SIM12(ipaJ). The chicken kidney cells (CKC) were used as epithelia cell model, the ability to invade into and propagate in cells of the three strains: S06004, SIM12 and SIM12(ipaJ) was analyzed. The result showed that the invasion and propagation ability of SIM12 was lower compared to that of S06004 and SIM12(ipaJ). As Salmonella pullorum mainly survived and propagated in macrophage cells of spleen, the HD-11 cells was used as macrophage model in this study, made comparison of the invasion and propagation ability of the three strains. The result showed that the invasion ability of SIM12 strain was much lower to that of S06004 and SIM12(ipaJ), while they showed no obivious difference of propagation ability in HD-11 cells. From the beginning to 5 hr infection, the bacteria numbers were increasing, and after that point, the bacteria started to decrease. Then the virulence abiltiy of the three strains to 10-day-old chicken was identified in vivo, the result showed the mortality rate of mutated strain SIM12 to chicken decreased 14 times compared to that of the wild strain S06004, while the SIM12(ipaJ) strain was almost equal to S06004. In order to identify the expression of ipaJ, total RNA was isolated from HD-11 cells, spleen cells and liver cells infected by Salmonella pullorum. RT-PCR proved that the gene was expressed during the infection. It is concluded that IpaJ may participate into the infection process of Salmonella pullorum, and mutation of the gene led to the degradation of the pathogen’s virulence.In summary, the IVIAT was used to screen infection-related factors from Salmonella pullorum, and 45 proteins were selected, involved in various aspects of molecule synthesis/degradation, transport system, regulation, energy metabolism, phage related and unknown functions. 11 out of 45 identified infection-related factors were selected to analyze their differential expression between in vivo and in vitro condition. Compared to cultivation in vitro, the expression level of these genes upregulated at different contents. In our study, a new plasmid which is 4080bp was isolated and identified from Salmonella pullorum. Sequence analysis show that only one virulence-related gene ipaJ exsits in the plasmid, and the encoded protein IpaJ may participate in the early infection process of the pathogen. In vivo test showed that mutation of ipaJ gene in Salmonella pullorum led to the virulence attenuation of the bacteria. It is speculated that ipaJ is a virulence-related gene of Salmonella pullorum. PCR analysis showed that the gene can be detected in 105 Salmonella pullorum isolates in our stock. Except for its existence in Salmonella choleraesuis C500 strain, the gene couldn’t be isolated from other Salmonella choleraesuis strains and other Salmonella spp strains.更多还原