本文選題：豬種布魯氏菌 + 口蹄疫病毒VP1基因��； 參考：《山東農業(yè)大學》2017年碩士論文

【摘要】：布魯氏菌病(Brucellosis,簡稱布病)是由布魯氏菌引起的一種重要的人獸共患病,2012年我國國務院印發(fā)了《國家中長期動物疫病防治規(guī)劃(2011-2020年)》,將布病列為優(yōu)先防治的國內16種動物疫病之一,并作為當前防控工作的重中之重。該病對畜牧業(yè)和人類健康均構成嚴重威脅,使用疫苗免疫是防控布氏菌病的重要措施之一。目前,我國用于控制布病的疫苗主要是S2株(豬型2號苗),其具有毒力較弱、免疫效果良好、安全性高等特點。布魯氏菌能夠引起機體強烈的Th1型免疫應答,這使得布魯氏菌成為理想的異源性抗原載體,為了開發(fā)出能高效表達外源基因的布魯氏菌活載體疫苗,在本課題中我們首先使用綠色熒光蛋白作為報告基因,將布魯氏菌組成型啟動子Psod、Pdnaj和Pdnak分別克隆入YGT-p BBR1mcs2,得到重組表達載體YGTp BBR1mcs2-Pdnaj、YGT-p BBR1mcs2-Psod和YGT-p BBR1mcs2-Pdnak并轉化至布魯氏菌。熒光顯微鏡檢測GFP蛋白的表達情況。結果表明,3種不同強度的啟動子均能在布魯氏菌S2中啟動GFP基因的表達且啟動能力由強到弱依次為:Pdnak、Pdnaj、Psod�？谔阋�(Foot and mouth disease,FMD)是由口蹄疫病毒(Foot and mouth disease virus,FMDV)引起的偶蹄動物的一種急性、熱性、高度接觸性傳染病,該病可引起偶蹄類動物的口、足等部位皮膚出現(xiàn)水泡而造成部份動物死亡,嚴重影響畜牧產(chǎn)業(yè)的發(fā)展。VP1蛋白是FMDV的主要結構蛋白,是病毒感染細胞的關鍵,具有與細胞受體結合的位點�？谔阋遃P1蛋白可作為研制口蹄疫基因工程疫苗的首選抗原。本研究以豬種布魯氏菌S2株為親本株,利用蔗糖自殺質粒載體,將FMDV的VP1基因表達框部分替代了布魯氏菌wbo A基因,成功構建了重組口蹄疫病毒VP1基因的布魯氏菌病活疫苗。使用熒光定量PCR的方法對重組菌的VP1基因的轉錄情況進行檢測,發(fā)現(xiàn)VP1基因在宿主菌內有顯著的轉錄變化,說明口蹄疫病毒VP1基因在m RNA水平上表達。將重組菌與原始菌滅活后進行超聲破碎,行進Western Blot驗證,結果顯示,重組菌比原始菌多出一條分子量大小為30k Da左右的條帶,與VP1蛋白理論大小一致,證明了口蹄疫病毒VP1在布魯氏菌體內的表達。本研究初步篩選了布魯氏菌的啟動子,并對其強弱進行驗證;初步建立了以豬種布魯氏菌為載體表達外源基因的方法,為布魯氏菌基因工程活載體疫苗的進一步研究奠定了基礎。
[Abstract]:Brucellosis (brucellosis) is an important zoonosis caused by brucellosis. In 2012, the State Council of our country issued the National medium and long term Animal epidemic Prevention Program 2011-2020, which listed brucellosis as the priority control in China. One of the animal blight species, And as the top priority of the current prevention and control work. The disease poses a serious threat to animal husbandry and human health. Vaccine immunization is one of the important measures to prevent and control brucellosis. At present, the main vaccine used to control brucellosis in China is S2 strain, which has the characteristics of weak virulence, good immune effect and high safety. Brucella can induce a strong Th1 immune response, which makes Brucella become an ideal xenogenic antigen vector, in order to develop brucellosis live vector vaccine which can express foreign gene efficiently. In this study, we first used green fluorescent protein as reporter gene and cloned Psodnaj and Pdnak into YGT-p BBR1mcs2, respectively, to obtain the recombinant expression vectors YGTp BBR1mcs2-PdnajAYGT-p BBR1mcs2-Psod and YGT-p BBR1mcs2-Pdnak and transformed them into Brucella. The expression of GFP protein was detected by fluorescence microscope. The results showed that all three kinds of promoters with different intensities could initiate the expression of GFP gene in Brucella S2, and the sequence of promoter ability from strong to weak was: 1. Foot and mouth disease (FMD) is an acute, feverish, highly contact infectious disease of cloven-hoofed animals caused by foot and mouth disease virus, which can cause blisters in the mouth and foot of cloven-hoofed animals and cause some animals to die. VP1 protein is the main structural protein of FMDV, which is the key of virus infection, and has the site of cell receptor binding. Foot-and-mouth disease (FMD) VP1 protein can be used as the preferred antigen for the development of FMD genetic engineering vaccine. In this study, using S _ 2 strain of porcine brucella as parent strain, using sucrose suicide plasmid vector, the wbo A gene of brucella was partially replaced by VP1 gene expression frame of FMDV, and the brucellosis vaccine of recombinant VP1 gene of foot-and-mouth disease virus was successfully constructed. Fluorescence quantitative PCR was used to detect the transcription of the VP1 gene of the recombinant strain. It was found that the VP1 gene had significant transcriptional changes in the host bacteria, indicating that the VP1 gene of foot-and-mouth disease virus was expressed at m RNA level. After inactivation of recombinant bacteria and original bacteria, ultrasonic fragmentation was carried out, and Western Blot verification was carried out. The results showed that the recombinant bacteria had more bands with molecular weight of about 30kDa than the original bacteria, which was consistent with the theoretical size of VP1 protein. The expression of foot-and-mouth disease virus (FMDV) VP1 in brucella was confirmed. In this study, the promoter of brucella was screened, and its strength was verified, and a method of expressing foreign gene in porcine brucella was established. It lays a foundation for the further research of brucella genetic engineering live vector vaccine.
【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.6

【相似文獻】

相關期刊論文 前10條

1 楊勇;任玉紅;梁寶懷;朱雅寧;梁智選;;雞傳染性貧血病毒VP1基因的克隆表達及抗原性分析[J];中國獸醫(yī)科學;2007年02期

2 康忠惠;王永強;王琦;李久寬;秦立廷;高玉龍;高宏雷;祁小樂;林歡;王笑梅;許修宏;;雞傳染性法氏囊病超強毒VP1基因的原核表達及兔抗血清的制備[J];中國家禽;2011年02期

3 馮昕;榮俊;匡紅艷;;雞傳染性貧血病毒VP1基因的原核表達[J];長江大學學報(自然科學版);2012年03期

4 傅光華;陳紅梅;黃瑜;施少華;彭春香;江斌;程龍飛;萬春和;傅秋玲;林建生;林芳;;雛番鴨胰腺型鴨1型甲肝病毒分離鑒定及VP1基因分析[J];福建農業(yè)學報;2012年09期

5 董志強;張志;李曉成;陳德坤;洪軍;張燕霞;黃保續(xù);;口蹄疫病毒VP1基因的克隆及結構蛋白VP1的原核表達[J];中國動物檢疫;2007年02期

6 寇錚,陳繩亮,鐘靖,李天憲,喻子牛;鸚鵡幼雛病毒結構蛋白VP1基因的克隆及原核表達[J];華中農業(yè)大學學報;2004年06期

7 孔留五;康艷梅;何逸民;李小康;潘全會;張桂紅;;Ⅰ型鴨肝炎病毒VP1基因克隆及其原核表達[J];上海畜牧獸醫(yī)通訊;2009年02期

8 李日順;于淼;黃昌力;曹宗喜;王文華;張超佚;張桂紅;;Ⅰ型鴨肝炎病毒VP1基因串聯(lián)表達載體的構建[J];黑龍江畜牧獸醫(yī);2011年15期

9 呂敏娜;戚南山;覃宗華;廖申權;余勁術;袁建豐;吳彩艷;孫銘飛;;Ⅰ型鴨肝炎病毒VP1基因的克隆和表達[J];動物醫(yī)學進展;2012年02期

10 張婷婷;侯振中;張云;劉明;;Ⅰ型鴨肝炎病毒VP1基因的克隆表達及純化[J];黑龍江畜牧獸醫(yī);2011年21期

相關會議論文 前3條

1 趙武;李斌;梁家幸;梁保忠;白安斌;姚瑞英;黃安國;何穎;蔣玉雯;;豬細小病毒自然弱毒N株(PPV-N株)VP1基因的擴增與生物信息學分析[A];中國畜牧獸醫(yī)學會家畜傳染病學分會第七屆全國會員代表大會暨第十三次學術研討會論文集（上冊）[C];2009年

2 辛英豪;李莎莎;高繼明;劉標;姜世金;;Ⅰ型鴨肝炎病毒VP1基因的克隆與原核表達[A];中國畜牧獸醫(yī)學會家畜傳染病學分會第七屆全國會員代表大會暨第十三次學術研討會論文集（下冊）[C];2009年

3 陳宗艷;李傳峰;劉光清;;Ⅰ型鴨肝炎病毒VP1基因表達及其抗體檢測ELISA方法的建立[A];中國畜牧獸醫(yī)學會禽病學分會第十五次學術研討會論文集[C];2010年

相關碩士學位論文 前3條

1 蔣卓婧;輕癥或重癥手足口病患兒HEV71分離鑒定及其VP1基因型分析[D];浙江大學;2016年

2 李捷;甘草酸二鉀對雞傳染性法氏囊病病毒VP1基因和蛋白表達的影響[D];山西農業(yè)大學;2013年

3 謝守玉;華南地區(qū)口蹄疫流行毒株VP1基因核苷酸序列分析及FMDV衣殼蛋白前體P1在FCV中的表達[D];廣西大學;2011年

，

本文編號：1778776