奶牛乳房炎肠球菌ESP基因真核重组质粒的构建与表达
发布时间：2018-12-08 17:14
【摘要】：奶牛乳房炎泛指能引起奶牛乳腺的各种疾病,不仅是奶牛最为常见的疾病之一,也是给养殖业带来巨大损失的一种疾病。奶牛乳房炎分布广泛,病因复杂,其病因、病情严重程度以及结果都与周围环境、病原菌和宿主本身有关,但病原微生物是引发该疾病的主要原因。肠球菌是存在于人和动物肠道中的正常菌群,常在所分离的能引起腹腔及盆腔感染的混合菌中发现,一般认为是对人畜不致病的共栖菌,但近几年的研究已证实肠球菌也拥有致病力。肠球菌能引起尿路感染以及皮肤感染,严重时甚至还能引发危及性命的疾病,例如腹腔感染、心内膜炎、败血病、脑膜炎等。而肠球菌性乳房炎主要是由肠球菌为主的多种病原菌混合而引起的感染,肠球菌只占引起奶牛乳房炎病原菌的一小部分,是其中的一种菌。尽管肠球菌性乳房炎的危害不及由主要病原菌引起的奶牛乳房炎,但是其能引起的牛奶质量下降和对奶牛乳腺造成损伤,使肠球菌性乳房炎不仅给养殖业造成严重的经济损失,同时也带来公共卫生安全和人类健康等诸多问题。因此,本研究以研究与研发新型奶牛乳房炎基因工程疫苗为目标,基于本实验室分离并鉴定的肠球菌为基础,运用分子生物学、基因工程和DNA重组技术等理论和方法,开展抗肠球菌性奶牛乳房炎基因工程疫苗的相关研究。首先,本研究采用美国临床检验标准委员会(NCCLS)推荐的微量肉汤稀释法,以本实验室分离、鉴定并保存的50株肠球菌为研究对象,检测其对临床常用的11种抗生素的敏感性,以期为该病的预防和临床选药提供依据。其次,本研究以本实验室分离并鉴定的肠球菌为基础,甄选出肠球菌表面蛋白(esp),采用PCR方法获得目的基因,利用T/A克隆将esp基因转入到pCR@2.1Vector,用限制性内切酶EcoRI和KpnI双酶切真核载体pcDNA3.1-HisB和含有esp基因的pCR@2.1 Vector,分别得到线型pcDNA3.1-HisB基因和esp基因,并且其都具有EcoRI和KpnI限制性酶酶切末端。用T4连接酶连接两个线型基因,重新构建了真核载体pcDNA3.1 HisB-rib esp,转化入感受态细胞,筛选阳性克隆。最后,通过脂质体将目的基因转化入MCF-7真核细胞内,G418筛选后,得到稳定转染细胞系。用PCR、Western blot等方法检测,在约46kDa处有一新生蛋白条带,与理论蛋白分子量一致,所构建的真核重组表达质粒能在真核细胞中正确表达目的蛋白,结果显示真核重组质粒pcDNA3.1 HisB-rib esp构建成功。本研究将为肠球菌性奶牛乳房炎新型基因工程疫苗的研究提供了新的突破性思路,积累实验数据,建立技术平台,本实验结果也有望为肠球菌新型疫苗的动物保护实验和临床前研究奠定基础,为采取科学、合理的综合防治措施,从根本上控制奶牛乳房炎的发生和流行提供科学依据,开辟新的途径。
[Abstract]:Cow mastitis is a kind of disease which can cause dairy cow mammary gland. It is not only one of the most common diseases in dairy cattle, but also a disease that brings great loss to the breeding industry. Dairy cow mastitis is widely distributed and the etiology is complicated. The etiology, severity and result of mastitis are related to the surrounding environment, pathogenic bacteria and host itself, but pathogenic microorganisms are the main causes of the disease. Enterococcus is a normal group of bacteria found in human and animal intestines, often found in isolated mixed bacteria that cause abdominal and pelvic infections, and is generally considered to be a common bacteria that is not pathogenic to humans and animals. But recent studies have shown that Enterococcus is also virulent. Enterococci can cause urinary tract and skin infections and even life-threatening diseases such as abdominal infection endocarditis septicaemia meningitis and so on. Enterococcal mastitis is mainly caused by a mixture of enterococcal pathogens. Enterococcus is only a small part of the pathogenic bacteria causing cow mastitis and is one of them. Although the harm of enterococcal mastitis is less than that of cow mastitis caused by main pathogenic bacteria, it can cause milk quality decline and damage to cow mammary gland. At the same time, it also brings many problems, such as public health safety and human health. Therefore, the aim of this study was to study and develop a novel genetic engineering vaccine for dairy cow mastitis. Based on the enterococcus isolated and identified in our laboratory, the theories and methods of molecular biology, genetic engineering and DNA recombination were used. Studies on genetic engineering vaccine against enterococcal cow mastitis were carried out. First of all, the microbroth dilution method recommended by (NCCLS) was used to detect the sensitivity of 50 strains of Enterococcus isolated, identified and preserved in our laboratory to 11 kinds of antibiotics commonly used in clinic. In order to provide the basis for the prevention of the disease and clinical drug selection. Secondly, based on the Enterococcus isolated and identified in our laboratory, the surface protein (esp), of Enterococcus was selected to obtain the target gene by PCR method, and the esp gene was cloned into pCR@2.1Vector, by T / A clone. The eukaryotic vector pcDNA3.1-HisB and pCR@2.1 Vector, containing esp gene were digested with restriction endonuclease EcoRI and KpnI, respectively. The linear pcDNA3.1-HisB gene and esp gene were obtained, respectively, and both of them had EcoRI and KpnI restriction endonuclease digests. Two linear genes were ligated by T4 ligase. The eukaryotic vector pcDNA3.1 HisB-rib esp, was transformed into receptive cells to screen positive clones. Finally, the target gene was transformed into MCF-7 eukaryotic cells by liposome. After G418 selection, stable transfection cell lines were obtained. PCR,Western blot and other methods were used to detect a new protein band at about 46kDa, which was consistent with the molecular weight of the theoretical protein. The constructed eukaryotic recombinant expression plasmid could correctly express the target protein in eukaryotic cells. The results showed that the eukaryotic recombinant plasmid pcDNA3.1 HisB-rib esp was successfully constructed. This study will provide a new breakthrough idea for the study of new genetic engineering vaccine of enterococcal cow mastitis, accumulate experimental data and establish technical platform. The results of this study are expected to lay a foundation for animal protection experiments and preclinical studies of new enterococcal vaccines, and provide scientific basis for scientific and rational comprehensive prevention and control of the occurrence and prevalence of dairy cow mastitis. Open up new ways.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23
本文编号：2368699
[Abstract]:Cow mastitis is a kind of disease which can cause dairy cow mammary gland. It is not only one of the most common diseases in dairy cattle, but also a disease that brings great loss to the breeding industry. Dairy cow mastitis is widely distributed and the etiology is complicated. The etiology, severity and result of mastitis are related to the surrounding environment, pathogenic bacteria and host itself, but pathogenic microorganisms are the main causes of the disease. Enterococcus is a normal group of bacteria found in human and animal intestines, often found in isolated mixed bacteria that cause abdominal and pelvic infections, and is generally considered to be a common bacteria that is not pathogenic to humans and animals. But recent studies have shown that Enterococcus is also virulent. Enterococci can cause urinary tract and skin infections and even life-threatening diseases such as abdominal infection endocarditis septicaemia meningitis and so on. Enterococcal mastitis is mainly caused by a mixture of enterococcal pathogens. Enterococcus is only a small part of the pathogenic bacteria causing cow mastitis and is one of them. Although the harm of enterococcal mastitis is less than that of cow mastitis caused by main pathogenic bacteria, it can cause milk quality decline and damage to cow mammary gland. At the same time, it also brings many problems, such as public health safety and human health. Therefore, the aim of this study was to study and develop a novel genetic engineering vaccine for dairy cow mastitis. Based on the enterococcus isolated and identified in our laboratory, the theories and methods of molecular biology, genetic engineering and DNA recombination were used. Studies on genetic engineering vaccine against enterococcal cow mastitis were carried out. First of all, the microbroth dilution method recommended by (NCCLS) was used to detect the sensitivity of 50 strains of Enterococcus isolated, identified and preserved in our laboratory to 11 kinds of antibiotics commonly used in clinic. In order to provide the basis for the prevention of the disease and clinical drug selection. Secondly, based on the Enterococcus isolated and identified in our laboratory, the surface protein (esp), of Enterococcus was selected to obtain the target gene by PCR method, and the esp gene was cloned into pCR@2.1Vector, by T / A clone. The eukaryotic vector pcDNA3.1-HisB and pCR@2.1 Vector, containing esp gene were digested with restriction endonuclease EcoRI and KpnI, respectively. The linear pcDNA3.1-HisB gene and esp gene were obtained, respectively, and both of them had EcoRI and KpnI restriction endonuclease digests. Two linear genes were ligated by T4 ligase. The eukaryotic vector pcDNA3.1 HisB-rib esp, was transformed into receptive cells to screen positive clones. Finally, the target gene was transformed into MCF-7 eukaryotic cells by liposome. After G418 selection, stable transfection cell lines were obtained. PCR,Western blot and other methods were used to detect a new protein band at about 46kDa, which was consistent with the molecular weight of the theoretical protein. The constructed eukaryotic recombinant expression plasmid could correctly express the target protein in eukaryotic cells. The results showed that the eukaryotic recombinant plasmid pcDNA3.1 HisB-rib esp was successfully constructed. This study will provide a new breakthrough idea for the study of new genetic engineering vaccine of enterococcal cow mastitis, accumulate experimental data and establish technical platform. The results of this study are expected to lay a foundation for animal protection experiments and preclinical studies of new enterococcal vaccines, and provide scientific basis for scientific and rational comprehensive prevention and control of the occurrence and prevalence of dairy cow mastitis. Open up new ways.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23
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