鼠傷寒沙門氏菌菌毛FimA與PefA蛋白的原核表達及純化
發(fā)布時間：2018-08-19 13:39
【摘要】：沙門氏菌病又名副傷寒。有些沙門氏菌血清型可以引起牛沙門氏菌病。目前由于抗生素的長期濫用導(dǎo)致沙門氏菌耐藥性普遍存在,不僅影響該病的防治,更對公共衛(wèi)生造成威脅。隨著規(guī)模化、集約化舍飼養(yǎng)牛方式的不斷發(fā)展,牛沙門氏菌病成為阻礙其健康發(fā)展和影響食品安全的一個因素。沙門氏菌Ⅰ型菌毛無論是在菌毛裝配上還是在基因、氨基酸序列方面,都較為保守。fimA是編碼Ⅰ型菌毛主要的菌毛亞單位。菌毛Pef具有良好的特異性。pefA為其主要的菌毛亞單位。本試驗通過傳統(tǒng)檢測方法對寧夏地區(qū)牛源沙門氏菌進行分離鑒定并確定了沙門氏菌的優(yōu)勢血清型;然后探索四對特異性引物用于PCR快速檢測的可行性;最后利用原核表達的方法得到純化的鼠傷寒沙門氏菌上述兩種菌毛主要亞單位蛋白,以期望在后續(xù)的抗原性分析、診斷以及預(yù)防鼠傷寒沙門氏菌病提供參考依據(jù)�；谏鲜鲅芯績�(nèi)容作了以下三個方面的工作:1寧夏地區(qū)牛源沙門氏菌的分離鑒定為了分析寧夏地區(qū)引起奶牛臨床乳房炎、隱性乳房炎、犢牛腹瀉、成母牛流產(chǎn)的病原體。本試驗通過采集病料,利用一系列微生物診斷方法和分子生物學(xué)方法對樣品中沙門氏菌進行分離鑒定。結(jié)果表明四種特異性引物PCR擴增結(jié)果與預(yù)期一致,可以用于后續(xù)沙門氏菌PCR快速檢測的建立。627份病牛樣品中有33份鑒定為沙門氏菌,分離率為5.26%。乳樣中檢出7種沙門氏菌血清型,組織樣中檢出2種沙門氏菌血清型,均為我國常見血清型。其中鼠傷寒沙門氏菌為優(yōu)勢血清型,其次為紐波特沙門氏菌。2鼠傷寒沙門氏菌菌毛FimA、PefA蛋白的原核表達為了獲得鼠傷寒沙門氏菌菌毛FimA、PefA的重組蛋白。本階段試驗首先對兩種菌毛亞單位進行B細(xì)胞表位分析,設(shè)計并合成引物,通過PCR技術(shù)擴增目的基因。利用T4連接酶構(gòu)建重組質(zhì)粒。將構(gòu)建好的重組質(zhì)粒導(dǎo)入到大腸桿菌中,并對誘導(dǎo)條件(IPTG濃度、誘導(dǎo)時間)進行優(yōu)化。最后利用SDS-PAGE分析重組蛋白的表達形式。結(jié)果表明兩種重組載體全部構(gòu)建成功,重組蛋白全部表達。兩種重組蛋白FimA、PefA相對分子質(zhì)量分別約為36kDa、33kDa且與預(yù)期一致,說明表達的蛋白是目的蛋白。重組蛋白FimA和PefA在大腸桿菌內(nèi)部積累,FimA蛋白主要以包涵體形式存在,而PefA蛋白主要以可溶形式存在。3菌毛亞單位蛋白FimA、PefA的純化為了進行后續(xù)的抗原性分析需要純化的重組蛋白FimA、PefA。利用Ni-AgaroseResin分離純化FimA、PefA重組蛋白。利用Western blot對純化的FimA、PefA重組蛋白進行分析。蛋白電泳和Western blot都顯示為一條清晰的蛋白條帶。結(jié)果顯示它們的大小分別約為36 kDa、33 kDa。本試驗獲得純度較高的重組蛋白FimA、PefA可以為進一步抗原性分析以及研發(fā)鼠傷寒沙門氏菌的疫苗和檢測提供原材料。
[Abstract]:Salmonellosis is also known as paratyphoid fever. Some salmonella serotypes can cause bovine salmonellosis. At present, drug resistance of Salmonella is widespread due to the long-term abuse of antibiotics, which not only affects the prevention and treatment of the disease, but also poses a threat to public health. With the development of scale and intensive feeding of cattle, bovine salmonellosis has become a factor that hinders its healthy development and affects food safety. Salmonella type I fima is the main fima subunit encoding type I pili, both in its assembly and in gene and amino acid sequence. Pef has a good specificity. PefA is the main subunit of its pili. In this experiment, the traditional detection method was used to isolate and identify Salmonella from cattle in Ningxia area, and the predominant serotype of Salmonella was determined, and the feasibility of using four pairs of specific primers for rapid detection of Salmonella was explored. Finally, the purified protein of the two main subunits of Salmonella typhimurium mentioned above were obtained by prokaryotic expression, in order to provide reference for the subsequent antigenicity analysis, diagnosis and prevention of Salmonella typhimurium. In order to analyze the pathogens of clinical mastitis, recessive mastitis, diarrhea of calves and abortion of adult cows in Ningxia, the following three aspects of work were done: 1) isolation and identification of Bovine Salmonella from Ningxia area in order to analyze the pathogens of clinical mastitis, recessive mastitis, diarrhea of calves and abortion of adult cows. A series of methods of microbiological diagnosis and molecular biology were used to isolate and identify Salmonella in the samples. The results showed that the results of PCR amplification with four kinds of specific primers were consistent with expectations, and could be used for the rapid detection of salmonella by PCR. 33 out of 627 samples of infected cattle were identified as Salmonella, and the isolation rate was 5.26. Seven Salmonella serotypes were detected in milk samples and two Salmonella serotypes were detected in tissue samples, all of which were common serotypes in China. Among them, Salmonella typhimurium was the dominant serotype, followed by Newport Salmonella. 2. The prokaryotic expression of FimA PefA protein of Salmonella typhimurium in order to obtain the recombinant protein of FimA PefA from Salmonella typhimurium pili. In this stage, B cell epitopes of two subunits were analyzed, primers were designed and synthesized, and the target genes were amplified by PCR. The recombinant plasmid was constructed by using T4 ligase. The constructed recombinant plasmid was introduced into E. coli and the induction conditions (IPTG concentration, induction time) were optimized. Finally, the expression of recombinant protein was analyzed by SDS-PAGE. The results showed that the two recombinant vectors were constructed successfully and the recombinant proteins were all expressed. The relative molecular weight of the two recombinant proteins FimA PefA was about 36 kDa and 33kDa respectively, which indicated that the expressed protein was the target protein. The recombinant protein FimA and PefA accumulated in E. coli mainly in the form of inclusion body, while the PefA protein mainly existed in the form of soluble form. The recombinant protein was purified by Ni-AgaroseResin. Western blot was used to analyze the purified FimAminopefA recombinant protein. Both protein electrophoresis and Western blot showed a clear protein band. The results showed that their sizes were about 36 kDa and 33 kDa. respectively. In this study, the recombinant protein FimA PefA with high purity could be used as a raw material for further antigenicity analysis and the development of vaccine and detection of Salmonella typhimurium.
【學(xué)位授予單位】：寧夏大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.61
本文編號：2191818
[Abstract]:Salmonellosis is also known as paratyphoid fever. Some salmonella serotypes can cause bovine salmonellosis. At present, drug resistance of Salmonella is widespread due to the long-term abuse of antibiotics, which not only affects the prevention and treatment of the disease, but also poses a threat to public health. With the development of scale and intensive feeding of cattle, bovine salmonellosis has become a factor that hinders its healthy development and affects food safety. Salmonella type I fima is the main fima subunit encoding type I pili, both in its assembly and in gene and amino acid sequence. Pef has a good specificity. PefA is the main subunit of its pili. In this experiment, the traditional detection method was used to isolate and identify Salmonella from cattle in Ningxia area, and the predominant serotype of Salmonella was determined, and the feasibility of using four pairs of specific primers for rapid detection of Salmonella was explored. Finally, the purified protein of the two main subunits of Salmonella typhimurium mentioned above were obtained by prokaryotic expression, in order to provide reference for the subsequent antigenicity analysis, diagnosis and prevention of Salmonella typhimurium. In order to analyze the pathogens of clinical mastitis, recessive mastitis, diarrhea of calves and abortion of adult cows in Ningxia, the following three aspects of work were done: 1) isolation and identification of Bovine Salmonella from Ningxia area in order to analyze the pathogens of clinical mastitis, recessive mastitis, diarrhea of calves and abortion of adult cows. A series of methods of microbiological diagnosis and molecular biology were used to isolate and identify Salmonella in the samples. The results showed that the results of PCR amplification with four kinds of specific primers were consistent with expectations, and could be used for the rapid detection of salmonella by PCR. 33 out of 627 samples of infected cattle were identified as Salmonella, and the isolation rate was 5.26. Seven Salmonella serotypes were detected in milk samples and two Salmonella serotypes were detected in tissue samples, all of which were common serotypes in China. Among them, Salmonella typhimurium was the dominant serotype, followed by Newport Salmonella. 2. The prokaryotic expression of FimA PefA protein of Salmonella typhimurium in order to obtain the recombinant protein of FimA PefA from Salmonella typhimurium pili. In this stage, B cell epitopes of two subunits were analyzed, primers were designed and synthesized, and the target genes were amplified by PCR. The recombinant plasmid was constructed by using T4 ligase. The constructed recombinant plasmid was introduced into E. coli and the induction conditions (IPTG concentration, induction time) were optimized. Finally, the expression of recombinant protein was analyzed by SDS-PAGE. The results showed that the two recombinant vectors were constructed successfully and the recombinant proteins were all expressed. The relative molecular weight of the two recombinant proteins FimA PefA was about 36 kDa and 33kDa respectively, which indicated that the expressed protein was the target protein. The recombinant protein FimA and PefA accumulated in E. coli mainly in the form of inclusion body, while the PefA protein mainly existed in the form of soluble form. The recombinant protein was purified by Ni-AgaroseResin. Western blot was used to analyze the purified FimAminopefA recombinant protein. Both protein electrophoresis and Western blot showed a clear protein band. The results showed that their sizes were about 36 kDa and 33 kDa. respectively. In this study, the recombinant protein FimA PefA with high purity could be used as a raw material for further antigenicity analysis and the development of vaccine and detection of Salmonella typhimurium.
【學(xué)位授予單位】：寧夏大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.61
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