奶牛乳房炎腸球菌ESP基因真核重組質(zhì)粒的構(gòu)建與表達(dá)
發(fā)布時間：2018-12-08 17:14
【摘要】：奶牛乳房炎泛指能引起奶牛乳腺的各種疾病,不僅是奶牛最為常見的疾病之一,也是給養(yǎng)殖業(yè)帶來巨大損失的一種疾病。奶牛乳房炎分布廣泛,病因復(fù)雜,其病因、病情嚴(yán)重程度以及結(jié)果都與周圍環(huán)境、病原菌和宿主本身有關(guān),但病原微生物是引發(fā)該疾病的主要原因。腸球菌是存在于人和動物腸道中的正常菌群,常在所分離的能引起腹腔及盆腔感染的混合菌中發(fā)現(xiàn),一般認(rèn)為是對人畜不致病的共棲菌,但近幾年的研究已證實腸球菌也擁有致病力。腸球菌能引起尿路感染以及皮膚感染,嚴(yán)重時甚至還能引發(fā)危及性命的疾病,例如腹腔感染、心內(nèi)膜炎、敗血病、腦膜炎等。而腸球菌性乳房炎主要是由腸球菌為主的多種病原菌混合而引起的感染,腸球菌只占引起奶牛乳房炎病原菌的一小部分,是其中的一種菌。盡管腸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質(zhì)量下降和對奶牛乳腺造成損傷,使腸球菌性乳房炎不僅給養(yǎng)殖業(yè)造成嚴(yán)重的經(jīng)濟損失,同時也帶來公共衛(wèi)生安全和人類健康等諸多問題。因此,本研究以研究與研發(fā)新型奶牛乳房炎基因工程疫苗為目標(biāo),基于本實驗室分離并鑒定的腸球菌為基礎(chǔ),運用分子生物學(xué)、基因工程和DNA重組技術(shù)等理論和方法,開展抗腸球菌性奶牛乳房炎基因工程疫苗的相關(guān)研究。首先,本研究采用美國臨床檢驗標(biāo)準(zhǔn)委員會(NCCLS)推薦的微量肉湯稀釋法,以本實驗室分離、鑒定并保存的50株腸球菌為研究對象,檢測其對臨床常用的11種抗生素的敏感性,以期為該病的預(yù)防和臨床選藥提供依據(jù)。其次,本研究以本實驗室分離并鑒定的腸球菌為基礎(chǔ),甄選出腸球菌表面蛋白(esp),采用PCR方法獲得目的基因,利用T/A克隆將esp基因轉(zhuǎn)入到pCR@2.1Vector,用限制性內(nèi)切酶EcoRI和KpnI雙酶切真核載體pcDNA3.1-HisB和含有esp基因的pCR@2.1 Vector,分別得到線型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連接酶連接兩個線型基因,重新構(gòu)建了真核載體pcDNA3.1 HisB-rib esp,轉(zhuǎn)化入感受態(tài)細(xì)胞,篩選陽性克隆。最后,通過脂質(zhì)體將目的基因轉(zhuǎn)化入MCF-7真核細(xì)胞內(nèi),G418篩選后,得到穩(wěn)定轉(zhuǎn)染細(xì)胞系。用PCR、Western blot等方法檢測,在約46kDa處有一新生蛋白條帶,與理論蛋白分子量一致,所構(gòu)建的真核重組表達(dá)質(zhì)粒能在真核細(xì)胞中正確表達(dá)目的蛋白,結(jié)果顯示真核重組質(zhì)粒pcDNA3.1 HisB-rib esp構(gòu)建成功。本研究將為腸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,積累實驗數(shù)據(jù),建立技術(shù)平臺,本實驗結(jié)果也有望為腸球菌新型疫苗的動物保護實驗和臨床前研究奠定基礎(chǔ),為采取科學(xué)、合理的綜合防治措施,從根本上控制奶牛乳房炎的發(fā)生和流行提供科學(xué)依據(jù),開辟新的途徑。
[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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.23
本文編號：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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.23
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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