鸭疫里氏杆菌野生型质粒的分离、序列分析和DPP Ⅳ缺失株的构建
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摘要
鸭疫里氏杆菌病是由鸭疫里氏杆菌(Riemerella anatipestifer, RA)引起的家鸭、鹅、火鸡和多种禽类的一种以败血性渗出为主要特征的传染病。临床剖检症状主要表现为纤维素性心包炎、肝周炎或气囊炎,部分病例出纤维素性脑膜炎、输卵管炎或关节炎。该病给世界各国养鸭业造成了巨大经济损失。目前已报道的鸭疫里氏杆菌血清型有21种,各血清型之间交叉保护率低,使得疫苗免疫防治比较难以进行。在大多数鸭场,鸭疫里氏杆菌感染一旦发生很难彻底清除,呈现持续性感染的状态。因此加强RA分子生物学、致病和免疫机制的研究,对从根本上防治该病的发生具有重要的现实意义。
本研究利用碱裂解法抽提RA临床分离株野生型质粒,通过酶切分析,引物延伸的方法获得质粒的全序列,并对其进行生物信息学分析,为质粒改造奠定基础。另外利用自杀性载体首次成功构建了RA-YM菌株的DPPⅣ缺失株,为进一步研究DPPⅣ在RA致病机制中的作用奠定基础。本研究取得的结果如下：
1.鸭疫里氏杆菌野生型质粒的分离、序列分析
应用碱裂解方法,对鸭疫里氏杆菌临床分离株RA-JX进行质粒抽提,获得了纯度较高的质粒。采用XbaⅠ限制性酶切,胶回收酶切片段,再与同样酶切的载体pUC18连接,得到的阳性克隆送去测序,再用引物延伸的方法,测定整个质粒的序列。分析结果显示,RA-JX菌株中含有的质粒大小为8048 bp。
对质粒pRA-JX进行生物信息学分析,包括质粒全序列的ORF的搜索,ORF的同源性比较,复制子的分析等。质粒pRA-JX上存在着分别编码Rep.3, Transposase_11,COG2378和Cas2_Ⅰ_Ⅱ_Ⅲ的主要ORF。对质粒pRA-JX ORF之外的序列BlastN比对、分析,推测质粒pRA-JX的复制子,包括质粒的复制起始蛋白以及其上游的一段序列,大小为2449 bp。本试验为进一步从分子生物学水平研究鸭疫里氏杆菌提供了技术支持。
2.鸭疫里氏杆菌血清1型云梦分离株DPPⅣ缺失株的构建
参照鸭疫里氏杆菌血清1型云梦分离株DPPⅣ序列,从RA-YM基因组中扩增出DPPⅣ同源臂片段,在其同源臂之间插入壮观霉素抗性基因,连接到自杀性质粒pRE112上,构建重组自杀性质粒pRE-LSR。重组自杀性质粒转化大肠杆菌X7213,以大肠杆菌X7213阳性克隆为供体菌,与受体菌RA-YM进行接合转移,涂布壮观霉素(Spc)抗性平板筛选出Spc抗性的接合子,并用PCR鉴定重组自杀性质粒已经整合到染色体中。本试验为进一步深入研究RA的分子致病机理奠定基础以及鸭疫里氏杆菌基因工程疫苗的设计与研发提供理论依据。
The Riemerlla anatipestifer infection is a communicable disease of domestic ducks, gose, turkeys, and various other birds, which is characterized by septicemia anserum exsudativa. Infected ducks have fibrinous pericarditis, perihepatitis and capsulitis, some have fibrinous meningitis, salpingitis and arthritis. It accounts for significant economic losses to the duck industry throughout the world. Presently,21 serotypes of R.anatipestifer have been identified and no significant cross-protection was reported. It lead to the difficulty of using vaccines to immunize duck against infection. R.anatipestifer infection has been a continued problem in many duck farms. Therefore, emphasis on the study of molecular biology, pathopoiesis and immunologic mechanism, which play a very important role in control and prevent the disease.
In this study, we isolated the plasmid from R.anatipestifer wild type strain. Using the experiment of digestion single restriction endonuclease and the primer walking method, we obtained the complete sequence of the wild type plasmid. The bioinformatics analysis of the complete sequence laid a foundation for the reconstruction of the plasmid. Otherwise, using suicide vector, we obtained DPP IV deleted mutant from R.anatipestifer strain RA-YM. The mutant wil be helpful for the further study of DPPⅣas a virulence factor. The research contents are summarized as follows:
1. Isolation and analysis of the complete sequence of plasmid from R.anatipestifer wide type strain
Using alkaline lysis, we isolated the high purity plasmid from R.anatipestifer wild type strainRA-JX. The plasmid was digested with single restriction endonuclease Xba I, and ligated to the cloning vector pUC18 digested with the same enzyme. The recombinatant plasmid was isolated and sequenced. Squencing of the complete sequence of the plasmid used the primer walking method. The result showed that the plasmid was 8048 base pairs in length.
The bioinfromatics analysis showed that there are some primary open reading frames were identified. These putative gene products showed significant homology to Rep-3, Transposase-11, COG2378 and Cas2.Ⅰ.Ⅱ-Ⅲ. BlastN analysis showed the minimal replication was located in the fragment containing Rep and its upstream sequence. It was 2449 base pairs in length. This result makes it possible to continue research R.anatipestifer in molecular lever.
2. Construction of DPP IV deleted mutant from R.anatipestifer strain RA-YM
Accoding to the sequence of DPP IV, the homologous arm gene were respectively amplified from RA-YM genome, added to the spectinomycin resistance gene, subcloned into suicide plasmid pRE112. The recombination suicide plasmids were designated as pRE-LSR. The DPP IV deleted mutant was constrcted first. The E.coli donor strain X7213 transformed with the suicide plasmid pRE-LSR was conjugated with the recipient strain, the R.anatipestifer strain RA-YM. After transconjugation, spectinomycin-resistant transconjugants in which the whole plasmid had been incorporated into the recipient chromosome were analyzed by PCR. This laid a foundation for the molecular pathopoiesis mechanism and provide a theoretical basis for the design and research genetically engineering vaccine of R.anatipestifer.
本研究利用碱裂解法抽提RA临床分离株野生型质粒,通过酶切分析,引物延伸的方法获得质粒的全序列,并对其进行生物信息学分析,为质粒改造奠定基础。另外利用自杀性载体首次成功构建了RA-YM菌株的DPPⅣ缺失株,为进一步研究DPPⅣ在RA致病机制中的作用奠定基础。本研究取得的结果如下：
1.鸭疫里氏杆菌野生型质粒的分离、序列分析
应用碱裂解方法,对鸭疫里氏杆菌临床分离株RA-JX进行质粒抽提,获得了纯度较高的质粒。采用XbaⅠ限制性酶切,胶回收酶切片段,再与同样酶切的载体pUC18连接,得到的阳性克隆送去测序,再用引物延伸的方法,测定整个质粒的序列。分析结果显示,RA-JX菌株中含有的质粒大小为8048 bp。
对质粒pRA-JX进行生物信息学分析,包括质粒全序列的ORF的搜索,ORF的同源性比较,复制子的分析等。质粒pRA-JX上存在着分别编码Rep.3, Transposase_11,COG2378和Cas2_Ⅰ_Ⅱ_Ⅲ的主要ORF。对质粒pRA-JX ORF之外的序列BlastN比对、分析,推测质粒pRA-JX的复制子,包括质粒的复制起始蛋白以及其上游的一段序列,大小为2449 bp。本试验为进一步从分子生物学水平研究鸭疫里氏杆菌提供了技术支持。
2.鸭疫里氏杆菌血清1型云梦分离株DPPⅣ缺失株的构建
参照鸭疫里氏杆菌血清1型云梦分离株DPPⅣ序列,从RA-YM基因组中扩增出DPPⅣ同源臂片段,在其同源臂之间插入壮观霉素抗性基因,连接到自杀性质粒pRE112上,构建重组自杀性质粒pRE-LSR。重组自杀性质粒转化大肠杆菌X7213,以大肠杆菌X7213阳性克隆为供体菌,与受体菌RA-YM进行接合转移,涂布壮观霉素(Spc)抗性平板筛选出Spc抗性的接合子,并用PCR鉴定重组自杀性质粒已经整合到染色体中。本试验为进一步深入研究RA的分子致病机理奠定基础以及鸭疫里氏杆菌基因工程疫苗的设计与研发提供理论依据。
The Riemerlla anatipestifer infection is a communicable disease of domestic ducks, gose, turkeys, and various other birds, which is characterized by septicemia anserum exsudativa. Infected ducks have fibrinous pericarditis, perihepatitis and capsulitis, some have fibrinous meningitis, salpingitis and arthritis. It accounts for significant economic losses to the duck industry throughout the world. Presently,21 serotypes of R.anatipestifer have been identified and no significant cross-protection was reported. It lead to the difficulty of using vaccines to immunize duck against infection. R.anatipestifer infection has been a continued problem in many duck farms. Therefore, emphasis on the study of molecular biology, pathopoiesis and immunologic mechanism, which play a very important role in control and prevent the disease.
In this study, we isolated the plasmid from R.anatipestifer wild type strain. Using the experiment of digestion single restriction endonuclease and the primer walking method, we obtained the complete sequence of the wild type plasmid. The bioinformatics analysis of the complete sequence laid a foundation for the reconstruction of the plasmid. Otherwise, using suicide vector, we obtained DPP IV deleted mutant from R.anatipestifer strain RA-YM. The mutant wil be helpful for the further study of DPPⅣas a virulence factor. The research contents are summarized as follows:
1. Isolation and analysis of the complete sequence of plasmid from R.anatipestifer wide type strain
Using alkaline lysis, we isolated the high purity plasmid from R.anatipestifer wild type strainRA-JX. The plasmid was digested with single restriction endonuclease Xba I, and ligated to the cloning vector pUC18 digested with the same enzyme. The recombinatant plasmid was isolated and sequenced. Squencing of the complete sequence of the plasmid used the primer walking method. The result showed that the plasmid was 8048 base pairs in length.
The bioinfromatics analysis showed that there are some primary open reading frames were identified. These putative gene products showed significant homology to Rep-3, Transposase-11, COG2378 and Cas2.Ⅰ.Ⅱ-Ⅲ. BlastN analysis showed the minimal replication was located in the fragment containing Rep and its upstream sequence. It was 2449 base pairs in length. This result makes it possible to continue research R.anatipestifer in molecular lever.
2. Construction of DPP IV deleted mutant from R.anatipestifer strain RA-YM
Accoding to the sequence of DPP IV, the homologous arm gene were respectively amplified from RA-YM genome, added to the spectinomycin resistance gene, subcloned into suicide plasmid pRE112. The recombination suicide plasmids were designated as pRE-LSR. The DPP IV deleted mutant was constrcted first. The E.coli donor strain X7213 transformed with the suicide plasmid pRE-LSR was conjugated with the recipient strain, the R.anatipestifer strain RA-YM. After transconjugation, spectinomycin-resistant transconjugants in which the whole plasmid had been incorporated into the recipient chromosome were analyzed by PCR. This laid a foundation for the molecular pathopoiesis mechanism and provide a theoretical basis for the design and research genetically engineering vaccine of R.anatipestifer.
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