RA CH3株ompA基因缺失株的构建及其部分生物学特性的研究
发布时间：2018-08-12 12:40
【摘要】：鸭疫里默氏杆菌(Riemrella anatipestifer,RA)病是一种接触性细菌性传染病。目前可以确定的RA血清型有21种,但血清型之间的交叉免疫保护性极差。由于RA中的ompA基因序列的保守性非常高,许多研究学者对ompA基因产生了兴趣,尤其是对其免疫学功能的研究,希望通过对RA不同的血清型中ompA基因研究来解决RA不同血清型的缺乏交叉免疫保护这一难题。另外已经有5株RA的全基因组序列已提交到GenBank中,分析结果表明编码的蛋白预测约有2000个,但对于RA中的蛋白生物学功能尚不清楚。OmpA蛋白是RA中的主要外膜蛋白,具有免疫原性,ompA基因具有保守性,在不同RA血清型中有特别高的同源性。研究ompA基因的生物学特性为OmpA蛋白制成新型亚单位疫苗及为RA病预防治疗奠定基础。此外,OmpA蛋白是外膜蛋白主要成份,在细菌物质的运输、细菌信号的传递、细菌结构的保持等方面均起重要作用。为了了解鸭疫里默氏杆菌ompA基因缺失对RA生物学特征的影响,本研究通过同源重组的方法用自杀质粒构建了ompA基因缺失株CH3ΔompA株。用pDS132自杀质粒连接用PCR方法扩增的ompA基因左右两侧的同源臂和壮观霉素抗性基因表达盒,获得pDS132-ompA-LSR的重组自杀质粒。然后将此重组质粒通过接合转导的方法转入到CH3亲本株中,用含Kan和Spc筛选TSA培养基,筛选可能的基因缺失株,并用PCR和Western blot进行鉴定,获得基因缺失株CH3ΔompA。然后将完整的ompA基因克隆到pRES0穿梭载体上,用结合传导的方法回复到CH3ΔompA中,从而得得到缺失株的互补株CH3ΔompA(pRES0-ompA)。本研究采用纸片法对CH3、CH3ΔompA和CH3ΔompA(p RES0-ompA)菌株进行药敏试验,分析CH3、CH3ΔompA和CH3ΔompA(pRES0-ompA)的耐药性差别。结果表明,野生株CH3与CH3ΔompA相比,野生株CH3对氯霉素、红霉素和四环素等抗生素的耐药性比缺失株CH3ΔompA要强。互补株CH3ΔompA(pRES0-ompA)对四环素、盐酸多西环素、红霉素和氯霉素的耐药性介于缺失株CH3ΔompA与CH3野生株之间,其对青霉素类抗生素的耐药性增强可能是由于在大肠杆菌-RA中穿梭表达质粒pRES0上携带了氨苄抗性bla基因所引起。野生株CH3、缺失株CH3ΔompA及其互补株CH3ΔompA(pRES0-ompA)对氨基苷类抗生素和甲氧苄啶均为耐受。试验结果表明,CH3ΔompA基因缺失株与野生株CH3相比繁殖速度减慢,对NaCl(盐离子浓度)的耐受能力降低,对部分抗生素的敏感性增加,而对去污剂SDS、EDTA和酸碱(pH)耐受性没有明显的影响。CH3ΔompA基因缺失株比CH3野生株形成生物被膜的能力显著增强。
[Abstract]:Riemrella anatipestifera (RA) disease is a contact bacterial infection. At present, there are 21 serotypes of RA, but the protection of cross immunity between serotypes is extremely poor. Because of the high conserved nature of ompA gene sequence in RA, many researchers are interested in ompA gene, especially its immunological function. It is hoped that the study of ompA gene in different serotypes of RA can solve the problem of lack of cross immune protection in different serotypes of RA. In addition, the whole genome sequence of 5 RA strains has been submitted to GenBank. The analysis results show that the predicted protein is about 2 000, but the biological function of the protein in RA is not clear. OmpA protein is the main outer membrane protein in RA. The immunogenicity of ompA gene is conserved, and the homology is very high in different serotypes of RA. The study of the biological characteristics of ompA gene lays a foundation for the preparation of novel subunit vaccine by OmpA protein and for the prevention and treatment of RA disease. In addition, OmpA protein is the main component of outer membrane protein, which plays an important role in the transportation of bacterial substances, the transmission of bacterial signal, and the maintenance of bacterial structure. In order to understand the effect of ompA gene deletion on the biological characteristics of RA, the ompA gene deletion strain CH3 螖 ompA was constructed by homologous recombination method. The recombinant suicide plasmid of pDS132-ompA-LSR was obtained by using pDS132 suicide plasmid and the homologous arm of ompA gene amplified by PCR method and the expression box of spectinomycin resistance gene. Then the recombinant plasmid was transferred into the parent strain of CH3 by the method of conjugation transduction. The TSA medium containing Kan and Spc was used to screen the possible gene deletion strain. PCR and Western blot were used to identify the gene deletion strain CH3 螖 ompA. Then the complete ompA gene was cloned into the pRES0 shuttle vector and reverted to the CH3 螖 ompA by the method of binding conduction, and the complementary strain CH3 螖 ompA (pRES0-ompA) of the missing strain was obtained. In this study, the drug susceptibility tests of CH3 CH3 螖 ompA and CH3 螖 ompA (p RES0-ompA strains were carried out by disk method, and the difference of drug resistance between CH3 CH3 螖 ompA and CH3 螖 ompA (pRES0-ompA) was analyzed. The results showed that the resistance of the wild strain CH3 to chloramphenicol, erythromycin and tetracycline was stronger than that of the missing strain CH3 螖 ompA compared with CH3 螖 ompA. The resistance of complementary strain CH3 螖 ompA (pRES0-ompA) to tetracycline, doxycycline hydrochloride, erythromycin and chloramphenicol was between CH3 螖 ompA and CH3 wild strain. The increased resistance to penicillin antibiotics may be due to the presence of ampicillin resistant bla gene on the shuttle expression plasmid pRES0 in Escherichia coli-RA. The wild strain CH3, the missing strain CH3 螖 ompA and the complementary strain CH3 螖 ompA (pRES0-ompA) were resistant to aminoside antibiotics and trimethoprim. The results showed that compared with wild strain CH3, CH3 螖 ompA gene deletion strain had slower reproduction rate, lower tolerance to NaCl (salt ion concentration), and higher sensitivity to some antibiotics. However, the tolerance of SDS-EDTA and acid-base (pH) to SDS-EDTA and acid-base (pH) was not significantly affected. The ability of biofilm formation of CH3 wild strain was significantly increased compared with that of CH3 wild strain.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.61
本文编号：2179083
[Abstract]:Riemrella anatipestifera (RA) disease is a contact bacterial infection. At present, there are 21 serotypes of RA, but the protection of cross immunity between serotypes is extremely poor. Because of the high conserved nature of ompA gene sequence in RA, many researchers are interested in ompA gene, especially its immunological function. It is hoped that the study of ompA gene in different serotypes of RA can solve the problem of lack of cross immune protection in different serotypes of RA. In addition, the whole genome sequence of 5 RA strains has been submitted to GenBank. The analysis results show that the predicted protein is about 2 000, but the biological function of the protein in RA is not clear. OmpA protein is the main outer membrane protein in RA. The immunogenicity of ompA gene is conserved, and the homology is very high in different serotypes of RA. The study of the biological characteristics of ompA gene lays a foundation for the preparation of novel subunit vaccine by OmpA protein and for the prevention and treatment of RA disease. In addition, OmpA protein is the main component of outer membrane protein, which plays an important role in the transportation of bacterial substances, the transmission of bacterial signal, and the maintenance of bacterial structure. In order to understand the effect of ompA gene deletion on the biological characteristics of RA, the ompA gene deletion strain CH3 螖 ompA was constructed by homologous recombination method. The recombinant suicide plasmid of pDS132-ompA-LSR was obtained by using pDS132 suicide plasmid and the homologous arm of ompA gene amplified by PCR method and the expression box of spectinomycin resistance gene. Then the recombinant plasmid was transferred into the parent strain of CH3 by the method of conjugation transduction. The TSA medium containing Kan and Spc was used to screen the possible gene deletion strain. PCR and Western blot were used to identify the gene deletion strain CH3 螖 ompA. Then the complete ompA gene was cloned into the pRES0 shuttle vector and reverted to the CH3 螖 ompA by the method of binding conduction, and the complementary strain CH3 螖 ompA (pRES0-ompA) of the missing strain was obtained. In this study, the drug susceptibility tests of CH3 CH3 螖 ompA and CH3 螖 ompA (p RES0-ompA strains were carried out by disk method, and the difference of drug resistance between CH3 CH3 螖 ompA and CH3 螖 ompA (pRES0-ompA) was analyzed. The results showed that the resistance of the wild strain CH3 to chloramphenicol, erythromycin and tetracycline was stronger than that of the missing strain CH3 螖 ompA compared with CH3 螖 ompA. The resistance of complementary strain CH3 螖 ompA (pRES0-ompA) to tetracycline, doxycycline hydrochloride, erythromycin and chloramphenicol was between CH3 螖 ompA and CH3 wild strain. The increased resistance to penicillin antibiotics may be due to the presence of ampicillin resistant bla gene on the shuttle expression plasmid pRES0 in Escherichia coli-RA. The wild strain CH3, the missing strain CH3 螖 ompA and the complementary strain CH3 螖 ompA (pRES0-ompA) were resistant to aminoside antibiotics and trimethoprim. The results showed that compared with wild strain CH3, CH3 螖 ompA gene deletion strain had slower reproduction rate, lower tolerance to NaCl (salt ion concentration), and higher sensitivity to some antibiotics. However, the tolerance of SDS-EDTA and acid-base (pH) to SDS-EDTA and acid-base (pH) was not significantly affected. The ability of biofilm formation of CH3 wild strain was significantly increased compared with that of CH3 wild strain.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.61
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