RA CH3株ompA基因缺失株的構(gòu)建及其部分生物學(xué)特性的研究
發(fā)布時(shí)間：2018-08-12 12:40
【摘要】：鴨疫里默氏桿菌(Riemrella anatipestifer,RA)病是一種接觸性細(xì)菌性傳染病。目前可以確定的RA血清型有21種,但血清型之間的交叉免疫保護(hù)性極差。由于RA中的ompA基因序列的保守性非常高,許多研究學(xué)者對(duì)ompA基因產(chǎn)生了興趣,尤其是對(duì)其免疫學(xué)功能的研究,希望通過對(duì)RA不同的血清型中ompA基因研究來解決RA不同血清型的缺乏交叉免疫保護(hù)這一難題。另外已經(jīng)有5株RA的全基因組序列已提交到GenBank中,分析結(jié)果表明編碼的蛋白預(yù)測(cè)約有2000個(gè),但對(duì)于RA中的蛋白生物學(xué)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血清型中有特別高的同源性。研究ompA基因的生物學(xué)特性為OmpA蛋白制成新型亞單位疫苗及為RA病預(yù)防治療奠定基礎(chǔ)。此外,OmpA蛋白是外膜蛋白主要成份,在細(xì)菌物質(zhì)的運(yùn)輸、細(xì)菌信號(hào)的傳遞、細(xì)菌結(jié)構(gòu)的保持等方面均起重要作用。為了了解鴨疫里默氏桿菌ompA基因缺失對(duì)RA生物學(xué)特征的影響,本研究通過同源重組的方法用自殺質(zhì)粒構(gòu)建了ompA基因缺失株CH3ΔompA株。用pDS132自殺質(zhì)粒連接用PCR方法擴(kuò)增的ompA基因左右兩側(cè)的同源臂和壯觀霉素抗性基因表達(dá)盒,獲得pDS132-ompA-LSR的重組自殺質(zhì)粒。然后將此重組質(zhì)粒通過接合轉(zhuǎn)導(dǎo)的方法轉(zhuǎn)入到CH3親本株中,用含Kan和Spc篩選TSA培養(yǎng)基,篩選可能的基因缺失株,并用PCR和Western blot進(jìn)行鑒定,獲得基因缺失株CH3ΔompA。然后將完整的ompA基因克隆到pRES0穿梭載體上,用結(jié)合傳導(dǎo)的方法回復(fù)到CH3ΔompA中,從而得得到缺失株的互補(bǔ)株CH3ΔompA(pRES0-ompA)。本研究采用紙片法對(duì)CH3、CH3ΔompA和CH3ΔompA(p RES0-ompA)菌株進(jìn)行藥敏試驗(yàn),分析CH3、CH3ΔompA和CH3ΔompA(pRES0-ompA)的耐藥性差別。結(jié)果表明,野生株CH3與CH3ΔompA相比,野生株CH3對(duì)氯霉素、紅霉素和四環(huán)素等抗生素的耐藥性比缺失株CH3ΔompA要強(qiáng)�；パa(bǔ)株CH3ΔompA(pRES0-ompA)對(duì)四環(huán)素、鹽酸多西環(huán)素、紅霉素和氯霉素的耐藥性介于缺失株CH3ΔompA與CH3野生株之間,其對(duì)青霉素類抗生素的耐藥性增強(qiáng)可能是由于在大腸桿菌-RA中穿梭表達(dá)質(zhì)粒pRES0上攜帶了氨芐抗性bla基因所引起。野生株CH3、缺失株CH3ΔompA及其互補(bǔ)株CH3ΔompA(pRES0-ompA)對(duì)氨基苷類抗生素和甲氧芐啶均為耐受。試驗(yàn)結(jié)果表明,CH3ΔompA基因缺失株與野生株CH3相比繁殖速度減慢,對(duì)NaCl(鹽離子濃度)的耐受能力降低,對(duì)部分抗生素的敏感性增加,而對(duì)去污劑SDS、EDTA和酸堿(pH)耐受性沒有明顯的影響。CH3ΔompA基因缺失株比CH3野生株形成生物被膜的能力顯著增強(qiáng)。
[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.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.61
本文編號(hào)：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.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.61
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