甲型副傷寒桿菌ompA基因分布及重組表達(dá)產(chǎn)物的免疫學(xué)鑒定
發(fā)布時(shí)間：2018-08-15 11:57
【摘要】： 背景和目的近年我國南方部分地區(qū)發(fā)生了甲型副傷寒桿菌(Salmonellaparatyphi A)感染引起的甲副傷寒暴發(fā)流行。由于甲型副傷寒桿菌無莢膜多糖,故目前采用的莢膜多糖Vi疫苗無預(yù)防甲型副傷寒的效果。因此,尋找甲型副傷寒桿菌表面抗原,研制甲型副傷寒疫苗,對于預(yù)防和控制甲型副傷寒的流行具有重要意義。而外膜蛋白(outer membrane proteins,OMP)通常是沙門菌等革蘭陰性菌的主要表面抗原成分。 本研究中,我們構(gòu)建了甲型副傷寒桿菌ompA基因原核表達(dá)系統(tǒng),鑒定了重組表達(dá)產(chǎn)物rOmpA的免疫原性,檢測了我國甲型副傷寒桿菌臨床菌株ompA基因攜帶率,采用小鼠感染模型初步了解了rOmpA的免疫保護(hù)作用,以期為rOmpA作為甲型副傷寒桿菌基因工程疫苗的候選抗原提供依據(jù)。 內(nèi)容和方法采用PCR從甲型副傷寒桿菌臨床株JH01中擴(kuò)增ompA基因,T-A克隆后測序并構(gòu)建ompA基因原核表達(dá)系統(tǒng)。采用SDS-PAGE和BioRad凝膠圖象分析系統(tǒng)檢測rOmpA表達(dá)情況及其產(chǎn)量,采用免疫擴(kuò)散法、微量肥達(dá)試驗(yàn)和Western blot鑒定其抗原性和免疫反應(yīng)性。采用PCR檢測98株甲型副傷寒桿菌臨床菌株ompA基因攜帶率。采用小鼠感染模型,了解rOmpA對甲型副傷寒桿菌50001株感染的免疫保護(hù)作用。 結(jié)果與報(bào)道的相關(guān)序列比較,所克隆的ompA基因核苷酸和氨基酸序列相似性均為100%。rOmpA表達(dá)量約為細(xì)菌總蛋白的65%。rOmpA免疫家兔可產(chǎn)生抗體,并能與其兔抗血清和甲型副傷寒桿菌全菌抗血清產(chǎn)生陽性Western雜交信號。94.9%(93/98)甲型副傷寒桿菌臨床菌株含有ompA基因。100μg和200μgrOmpA對感染小鼠的免疫保護(hù)率分別為41.7%(5/12)和58.3%(7/12)。rOmpA免疫小鼠或保護(hù)試驗(yàn)存活小鼠血清對各副傷寒桿菌H抗原的凝集效價(jià)為1:5～1:40。 結(jié)論ompA基因在甲型副傷寒桿菌臨床菌株中分布廣泛。rOmpA有良好的免疫原性和一定的免疫保護(hù)作用,可作為甲型副傷寒桿菌基因工程疫苗候選抗原。
[Abstract]:Background and objective A paratyphoid fever outbreak caused by paratyphoid A (Salmonellaparatyphi A) infection has occurred in some parts of southern China in recent years. Because paratyphoid A has no capsule polysaccharide, the current vaccine Vi has no preventive effect on paratyphoid A. Therefore, the search for surface antigen of paratyphoid A and the development of paratyphoid A vaccine are of great significance in preventing and controlling the epidemic of paratyphoid A. Outer membrane protein (outer membrane proteinsOMP) is usually the main surface antigen of gram-negative bacteria such as Salmonella. In this study, we constructed the prokaryotic expression system of ompA gene of paratyphoid A, identified the immunogenicity of the recombinant expression product rOmpA, and detected the carrying rate of ompA gene of paratyphoid A clinical strain in China. In order to provide evidence for rOmpA as a candidate antigen for genetic engineering vaccine of paratyphoid A, the immune protective effect of rOmpA was preliminarily understood by using mouse infection model. Content and methods ompA gene was amplified from JH01 of paratyphoid A clinical strain by PCR and sequenced, and the ompA gene prokaryotic expression system was constructed. The expression of rOmpA and its yield were detected by SDS-PAGE and BioRad gel image analysis system. The antigenicity and immunoreactivity of rOmpA were identified by immunodiffusion assay, micro-Wheatty test and Western blot. The ompA gene carrying rate of 98 clinical strains of paratyphoid A was detected by PCR. To investigate the protective effect of rOmpA on 50001 strains of paratyphoid A infection in mice. Results compared with the reported sequences, the nucleotide and amino acid sequence similarity of the cloned ompA gene was that 65%.rOmpA, which expressed about the total bacterial protein, could produce antibodies in rabbits. And it can produce positive Western hybridization signal with rabbit antiserum and paratyphoid A antiserum. 94.9% (93 / 98). The protective rates of ompA gene. 100 渭 g and 200 渭 grOmpA in infected mice were 41.7% (5/ 12) and 58.3% (7 / 12), respectively. ROmpA was small immunized by clinical strains of paratyphoid A (93 / 98). The agglutination titer of serum against H antigen of paratyphoid bacillus in mice or survival mice was 1: 5: 1: 40. Conclusion ompA gene is widely distributed in paratyphoid A clinical strains. ROmpA has good immunogenicity and protective effect. It can be used as candidate antigen of paratyphoid A gene engineering vaccine.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
本文編號：2184129
[Abstract]:Background and objective A paratyphoid fever outbreak caused by paratyphoid A (Salmonellaparatyphi A) infection has occurred in some parts of southern China in recent years. Because paratyphoid A has no capsule polysaccharide, the current vaccine Vi has no preventive effect on paratyphoid A. Therefore, the search for surface antigen of paratyphoid A and the development of paratyphoid A vaccine are of great significance in preventing and controlling the epidemic of paratyphoid A. Outer membrane protein (outer membrane proteinsOMP) is usually the main surface antigen of gram-negative bacteria such as Salmonella. In this study, we constructed the prokaryotic expression system of ompA gene of paratyphoid A, identified the immunogenicity of the recombinant expression product rOmpA, and detected the carrying rate of ompA gene of paratyphoid A clinical strain in China. In order to provide evidence for rOmpA as a candidate antigen for genetic engineering vaccine of paratyphoid A, the immune protective effect of rOmpA was preliminarily understood by using mouse infection model. Content and methods ompA gene was amplified from JH01 of paratyphoid A clinical strain by PCR and sequenced, and the ompA gene prokaryotic expression system was constructed. The expression of rOmpA and its yield were detected by SDS-PAGE and BioRad gel image analysis system. The antigenicity and immunoreactivity of rOmpA were identified by immunodiffusion assay, micro-Wheatty test and Western blot. The ompA gene carrying rate of 98 clinical strains of paratyphoid A was detected by PCR. To investigate the protective effect of rOmpA on 50001 strains of paratyphoid A infection in mice. Results compared with the reported sequences, the nucleotide and amino acid sequence similarity of the cloned ompA gene was that 65%.rOmpA, which expressed about the total bacterial protein, could produce antibodies in rabbits. And it can produce positive Western hybridization signal with rabbit antiserum and paratyphoid A antiserum. 94.9% (93 / 98). The protective rates of ompA gene. 100 渭 g and 200 渭 grOmpA in infected mice were 41.7% (5/ 12) and 58.3% (7 / 12), respectively. ROmpA was small immunized by clinical strains of paratyphoid A (93 / 98). The agglutination titer of serum against H antigen of paratyphoid bacillus in mice or survival mice was 1: 5: 1: 40. Conclusion ompA gene is widely distributed in paratyphoid A clinical strains. ROmpA has good immunogenicity and protective effect. It can be used as candidate antigen of paratyphoid A gene engineering vaccine.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 朱水榮,張政,莫順堂,陳秀英;浙江省傷寒、甲型副傷寒沙門氏菌耐藥性監(jiān)測分析[J];疾病監(jiān)測;2003年04期
，本文編號：2184129
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