【作者】 郁川；

【导师】 程相朝；

【作者基本信息】 河南科技大学 ， 基础兽医学， 2010， 硕士

【摘要】 猪霍乱沙门氏菌( Salmonella Choleraesuis )是引起仔猪副伤寒的主要病原之一,可引起断奶仔猪大批发病,常因伴发或继发感染其它疾病或治疗不及时而大批死亡,给养猪业造成重大损失。动物生前感染或食品受到污染,可使人发生食物中毒,在公共卫生上也具有十分重要意义。疫苗接种是防控仔猪副伤寒的最适宜策略。在我国,研制仔猪副伤寒活疫苗比较成功的是有关专家将抗原性良好的猪霍乱沙门氏菌强毒株C78-1接种含有醋酸铊的培养基中传数百代后,选出了免疫原性良好的弱毒株C500。但该菌株是经化学方法传代致弱,对其致弱的遗传背景和引起免疫保护的机理目前仍不清楚。另外,该疫苗菌株具有一定的残余毒力,且存在毒力返强的风险。本研究旨在研制更加安全有效的猪霍乱沙门氏菌疫苗弱毒株,并为进一步将其开发为适于粘膜免疫的口服疫苗活载体奠定一定基础,以crp(cAMP receptor protein,crp)基因作为猪霍乱沙门氏菌C78-1株减毒基因,运用重组自杀性质粒介导细菌的等位交换技术两步法筛选猪霍乱沙门氏菌C78-1株crp基因工程缺失突变株,并对其生物学特性进行了初步研究。主要内容包括:1.crp基因的克隆与序列分析参照鼠伤寒沙门氏菌LT2株(GenBank No:AE008859)crp基因序列,从猪霍乱沙门氏菌C78-1基因组中扩增出crp基因。通过测序和序列对比分析显示,与C500株crp基因全序列完全相同,与Genbank上登录的其它全序列已知的3株沙门氏菌crp基因高度保守。2.重组自杀性质粒pREΔcrp的构建以猪霍乱沙门氏菌C78-1基因组为模板,分别扩增crp上下游片段并将其克隆入自杀性质粒pRE112上,转化大肠杆菌χ7213,构建含缺失320bp的crp基因重组自杀性质粒pREΔcrp。3.接合转移与缺失菌株ΔcrpC78-1的筛选鉴定以转化了重组自杀性质粒pREΔcrp的大肠杆菌χ7213为供体菌,C78-1为受体菌进行接合转移。利用重组自杀性质粒介导细菌基因等位交换技术两步法筛选crp基因缺失突变株,并进行PCR和测序鉴定,以确定缺失菌株ΔcrpC78-1的构建成功。4.缺失菌株ΔcrpC78-1生物学特性研究对缺失菌株ΔcrpC78-1表型、遗传稳定性、生长特性及对小鼠的毒力等生物学特性进行了鉴定,结果发现:缺失菌株?crpC78-1失去了利用麦芽糖、蔗糖、果糖与甘露醇等碳源的能力,不能分解H2S,但保留了利用葡萄糖的能力;血清型与亲本菌株C78-1一致,且能够稳定遗传缺失的crp基因;小鼠致死性试验结果表明其毒力较C78-1降低约750倍。更多还原

【Abstract】 Salmonella choleraesuis is one of the main pathogens of piglet’s paratyphoid, and it affect mainly weaned piglets. A large number of weaned piglets can be dead when secondary infection with other pathgens, or not treated in time. In addition, this pathogen play a significant role in public health for causing human food poisoning through the infected animals or the contaminated products. Vaccination against S. choleraesuis is an appropriate strategy for control and prevention of this disease. In China, the most effective attenuated paratyphoid fever vaccine strain was C500 strain. It was attenuated from highly virulent S. choleraesuis C78-1 strain by maintaining in the thallium acetate’s culture medium for hundreds of generations, and had stronger immunogenicity. However, the genetic background and the mechanism of immune protection of C500 strain attenuated by chemical methods remain still unknown. In addition, the vaccine strain remains some residual virulence and risk of back mutation.In the current study, a safer attuanted S. choleraesuis vaccine was developed through deleting the crp gene, which coding a cAMP receptor protein. The ?crpC78-1 mutant was constructed through the two-step method introduced by the transduction of recombinant suicide plasmid. The biological characteristics of the ?crpC78-1 was further analysed. The main research was described as follows:1. Cloning and sequential analysis of crp geneThe crp gene were cloned from genomic DNA of C78-1 strain according to the corresponding sequences of S. typhimurium LT2 strain (AE008859) from GenBank. The results of sequence alignment analysis showed that the C78-1 crp gene was completely same as C500 strain’s and highly conserved compared with other three salmonella whose whole genome sequences were published.2. Construction of recombination suicide plasmids pREΔcrpThe upstream and downstream fragements of crp gene were amplified from genomic DNA of S. choleraesuis C78-1, then successfully subcloned into suicide plasmid pRE112, transformed into E. coliχ7213. The recombination suicide plasmid were designated as pREΔcrp which contained 320bp-deleted crp fragement.3. Transconjugation and identification of theΔcrpC78-1 mutantThe E. coil donor strainχ7213 transformed with recombination suicide plasmid pREΔcrp was conjugated with the recipient S. choleraesuis C78-1 strain. The ?crp mutant of S. choleraesuis C78-1 was constructed by the allelic exchange introduced by the transduction of the recombination suicide plasmid. Then, theΔcrpC78-1 mutant was determined by PCR and sequencing analysis.4. Analysis for Biological Characteristics ofΔcrpC78-1 mutantThe phenotypes, genetic stability, growth properties and virulence in mice ofΔcrpC78-1 mutant were characterized. The O and H antigens of the mutant was 6,7:C:1,5, identical to the parent C78-1 strain. TheΔcrpC78-1 mutant was able to ferment glucose, not maltose, mannose and xylose, while the parental strain C78-1 did. The mutant was stable with the recombinant ?crp gene in vitro. However, fermentation patterns and growth rate of the mutant were differed from the parent C78-1 strain obviously. The mouse lethal test showed that the virulence of the muntant was lower 750 times than C78-1.更多还原