羊布鲁氏菌Omp25、VjbR与小鼠巨噬细胞互作蛋白的筛选鉴定
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摘要
布鲁氏菌病（Brucellosis，简称布病）是由布鲁氏菌引起的一种世界性人兽共患传染病。目前，人畜布病主要集中在发展中国家，2000年以后，我国人畜布病有逐年上升的趋势，给畜牧业和人的健康造成了巨大危害。布病难以防控的原因是多方面的，其致病机制、胞内寄生机制不清楚是主要原因。布鲁氏菌能够感染专职巨噬细胞和非专职巨噬细胞，并能在其中生存复制，其中巨噬细胞是布鲁氏菌的主要感染对象。布鲁氏菌之所以能在巨噬细胞内寄生，推测是细菌蛋白与宿主细胞蛋白相互作用的结果。当前，布鲁氏菌蛋白和巨噬细胞蛋白相互作用的机制还不清楚。
Omp25属于布鲁氏菌第3组外膜蛋白Omp25/Omp31家族中的一员，研究发现缺失Omp25基因的羊布鲁氏菌突变株不会引起宿主流产，且能够在宿主体内增殖，与其亲本株相比毒力减弱且激发体液免疫的能力也减弱，说明Omp25既是毒力因子又是免疫原性蛋白。VjbR是密度感应系统（Qurum sensing，QS）LuxR蛋白家族的一个转录激活因子，能够影响IV型分泌系统(T4SS)和鞭毛基因表达，并且它在布鲁氏菌毒力和胞内寄生中都发挥了重要作用。鉴于此，本研究构建了羊布鲁氏菌16M株感染小鼠巨噬细胞cDNA文库，并以Omp25、VjbR为诱饵蛋白从该文库中筛选鉴定互作蛋白，为进一步研究布鲁氏菌的分子致病机制和胞内寄生机制奠定基础。
首先，利用分子克隆技术对羊布鲁氏菌16M株Omp25、VjbR进行克隆、测序，将其连接到pSos载体上，构建诱饵载体，并检测诱饵载体毒性、自激活性以及膜定位。
其次，建立羊布鲁氏菌16M株感染小鼠巨噬细胞模型，提取总RNA，分离、提纯mRNA，构建羊布鲁氏菌16M株感染后的小鼠巨噬细胞cDNA文库，将文库连接到pMyr猎物载体上。
然后，将诱饵质粒和pMyr-cDNA文库质粒共转化cdc25H酵母感受态细胞进行互作蛋白筛选，并应用免疫共沉淀技术进行验证。
通过以上实验研究，得到如下结果：
1、经过测序表明羊布鲁氏菌16M株Omp25、VjbR基因克隆正确，并且成功连接到pSos载体上，构建的诱饵载体对酵母细胞无毒副作用，无自激活性，膜上定位正确，能够用于酵母双杂交实验。
2、利用间接免疫荧光实验和透射电镜观察，确立细菌和细胞按照500︰1的比例进行感染，在感染4小时后可以成功建立羊布鲁氏菌16M株感染小鼠巨噬细胞模型。
3、成功构建了羊布鲁氏菌16M株感染后小鼠巨噬细胞cDNA文库，并将文库与猎物载体pMyr相连，构建了pMyr-cDNA质粒文库。质粒库容为1.6×109cfu/mL，重组率较高。
4、将诱饵质粒和pMyr-cDNA文库质粒共转化cdc25H酵母感受态细胞进行酵母双杂交，结果筛选到2个阳性克隆，经测序两个基因分别为NDAH还原酶装备因子2（Mus musculus NADH dehydrogenase (ubiquinone)1alpha subcomplex,assembly factor2（NM_001127346.1），Ndufaf2)和核糖体蛋白S5（Mus musculusribosomal protein S5（NM_009095.2），RPS5）。经免疫共沉淀验证Ndufaf2能够与Omp25发生相互作用，RPSs5能够与VjbR发生相互作用。通过上述试验结果，我们提出如下假设：（1）Omp25和Ndufaf2结合后，可以促进细胞中NADH浓度增加，提高NADH活性，降低巨噬细胞对布鲁氏菌的氧化杀伤，从而创造了有利于布鲁氏菌胞内生存的条件。（2）VjbR与RPS5结合后，抑制了RPS5活性，从而抑制了小鼠巨噬细胞的自然凋亡，创造了有利于布鲁氏菌长期感染的环境。这为布鲁氏菌致病机理、胞内寄生机制研究提供了新的思路。
本研究为绘制布鲁氏菌与小鼠巨噬细胞相互作用蛋白的网络提供了实验依据，为进一步研究布鲁氏菌的分子致病机制和胞内寄生机制奠定了基础。
Brucellosis is a worldwide zoonosis caused by brucella. At present, brucellosis isalways outbreak in developing countries. The disease is increasing from2000, whichcauses tremendous harm to animal and human. The obstacles for prevention andcontrolling of brucellosis are mainly unclear on the mechanism of pathogenesis andintracellular parasitic. Brucella could infect allied and non-allied macrophage, andthen survive and replicate. Macrophage is the first object of infection. The interactionof bacterial protein and host cell proteins might provide the condition for brucellaintracellular survival, but the mechanism is not uncovered.
Omp25is one of Omp25/Omp31family proteins, which belong to the thirdgroup outer membrane protein of brucella. Brucella melitensis Omp25mutant couldnot cause portion, and could survive and replicate in vivo. The virulence and humoralimmunity of Omp25mutant compared parent strain was weakened. All thesesuggested that Omp25was not only virulent factors, but also immuno-protein. VjbR isa transcription factor of Qurum sensing system (QS) belonging to LuxR family, whichaffects IV secretion system (T4SS) and flagellum expression and play important rolesin virulence and intracellular survival.
In this paper, we constructed cDNA library of Brucella melitensis16M infectingmurine macrophage, and screened and identified interaction proteins using Omp25and VjbR as bait protein. All these could lay the foundation for researching thepathogenesis and intracellular survival mechanism.
First, we cloned Omp25and VjbR, and ligased to pSos vector and constructedbait vecter after detecting bait vector toxicity, activation and membrane localization.
Second, we constructed the model of Brucella melitensis infecting murinemacrophage, and extracted total RNA and prepared mRNA for cDNA libraryconstruction, and then cDNA library was convered to pMyr vector.
Finally, Co-transformed bait vectors and pMyr-cDNA into cdc25HSaccharomyces cerevisiae competents for screening interaction proteins, andverificated by co-immunoprecipitation.
Results:
1. Omp25and VjbR gene were cloned and connected with pSos vector, and thebait vectors were non-toxicity, non-self reactivation, and located at right membraneposition suitable for yeast hybrid test.
2. By indirect immunofluorescence test and transmission electron microscopetechnology, the model of Brucella melitensis16M infecting murine macrophage wasestablished at the bacteria and cells ratio was500:1.
3. cDNA library was constructed by Stratangen mRNA kits, and convered cDNAlibrary to pMyr bait vectors, and constructed pMyr-cDNA library. The plasmidcapacity was1.6×109cfu/mL, and recombination ratio was very high.
The bait plasmid and pMyr-cDNA library were co-transformed into cdc25Hyeast competent for yeast hybrid. Two positive clones were screened, and thesequence showed that they were Mus musculus NADH dehydrogenase (ubiquinone)1alpha subcomplex, assembly factor2（NM_001127346.1），Ndufaf2, and （Musmusculus ribosomal protein S5（NM_009095.2），RPSs5. Ndufaf2could interact withOmp25, and RPSs5could interact with VjbR by co-immunoprecipitation. Datashowed:(1) Omp25interacted with Ndufaf2could enhance NADH concentration, andincreased NAHD activity, reducing the oxidative derivatives for brucella intracellularsurvival.(2) VjbR connected RPS5could inhibite RPS5activity, and inhibit apoptosisof murine macrophage providing brucella infection environment.
In this study, the foundation was established for paint the network of proteinsinteraction of brucella and murine macrophage, which provide foundations for futurestudy on pathogenesis and intracellular parasitic mechanisms.
Omp25属于布鲁氏菌第3组外膜蛋白Omp25/Omp31家族中的一员，研究发现缺失Omp25基因的羊布鲁氏菌突变株不会引起宿主流产，且能够在宿主体内增殖，与其亲本株相比毒力减弱且激发体液免疫的能力也减弱，说明Omp25既是毒力因子又是免疫原性蛋白。VjbR是密度感应系统（Qurum sensing，QS）LuxR蛋白家族的一个转录激活因子，能够影响IV型分泌系统(T4SS)和鞭毛基因表达，并且它在布鲁氏菌毒力和胞内寄生中都发挥了重要作用。鉴于此，本研究构建了羊布鲁氏菌16M株感染小鼠巨噬细胞cDNA文库，并以Omp25、VjbR为诱饵蛋白从该文库中筛选鉴定互作蛋白，为进一步研究布鲁氏菌的分子致病机制和胞内寄生机制奠定基础。
首先，利用分子克隆技术对羊布鲁氏菌16M株Omp25、VjbR进行克隆、测序，将其连接到pSos载体上，构建诱饵载体，并检测诱饵载体毒性、自激活性以及膜定位。
其次，建立羊布鲁氏菌16M株感染小鼠巨噬细胞模型，提取总RNA，分离、提纯mRNA，构建羊布鲁氏菌16M株感染后的小鼠巨噬细胞cDNA文库，将文库连接到pMyr猎物载体上。
然后，将诱饵质粒和pMyr-cDNA文库质粒共转化cdc25H酵母感受态细胞进行互作蛋白筛选，并应用免疫共沉淀技术进行验证。
通过以上实验研究，得到如下结果：
1、经过测序表明羊布鲁氏菌16M株Omp25、VjbR基因克隆正确，并且成功连接到pSos载体上，构建的诱饵载体对酵母细胞无毒副作用，无自激活性，膜上定位正确，能够用于酵母双杂交实验。
2、利用间接免疫荧光实验和透射电镜观察，确立细菌和细胞按照500︰1的比例进行感染，在感染4小时后可以成功建立羊布鲁氏菌16M株感染小鼠巨噬细胞模型。
3、成功构建了羊布鲁氏菌16M株感染后小鼠巨噬细胞cDNA文库，并将文库与猎物载体pMyr相连，构建了pMyr-cDNA质粒文库。质粒库容为1.6×109cfu/mL，重组率较高。
4、将诱饵质粒和pMyr-cDNA文库质粒共转化cdc25H酵母感受态细胞进行酵母双杂交，结果筛选到2个阳性克隆，经测序两个基因分别为NDAH还原酶装备因子2（Mus musculus NADH dehydrogenase (ubiquinone)1alpha subcomplex,assembly factor2（NM_001127346.1），Ndufaf2)和核糖体蛋白S5（Mus musculusribosomal protein S5（NM_009095.2），RPS5）。经免疫共沉淀验证Ndufaf2能够与Omp25发生相互作用，RPSs5能够与VjbR发生相互作用。通过上述试验结果，我们提出如下假设：（1）Omp25和Ndufaf2结合后，可以促进细胞中NADH浓度增加，提高NADH活性，降低巨噬细胞对布鲁氏菌的氧化杀伤，从而创造了有利于布鲁氏菌胞内生存的条件。（2）VjbR与RPS5结合后，抑制了RPS5活性，从而抑制了小鼠巨噬细胞的自然凋亡，创造了有利于布鲁氏菌长期感染的环境。这为布鲁氏菌致病机理、胞内寄生机制研究提供了新的思路。
本研究为绘制布鲁氏菌与小鼠巨噬细胞相互作用蛋白的网络提供了实验依据，为进一步研究布鲁氏菌的分子致病机制和胞内寄生机制奠定了基础。
Brucellosis is a worldwide zoonosis caused by brucella. At present, brucellosis isalways outbreak in developing countries. The disease is increasing from2000, whichcauses tremendous harm to animal and human. The obstacles for prevention andcontrolling of brucellosis are mainly unclear on the mechanism of pathogenesis andintracellular parasitic. Brucella could infect allied and non-allied macrophage, andthen survive and replicate. Macrophage is the first object of infection. The interactionof bacterial protein and host cell proteins might provide the condition for brucellaintracellular survival, but the mechanism is not uncovered.
Omp25is one of Omp25/Omp31family proteins, which belong to the thirdgroup outer membrane protein of brucella. Brucella melitensis Omp25mutant couldnot cause portion, and could survive and replicate in vivo. The virulence and humoralimmunity of Omp25mutant compared parent strain was weakened. All thesesuggested that Omp25was not only virulent factors, but also immuno-protein. VjbR isa transcription factor of Qurum sensing system (QS) belonging to LuxR family, whichaffects IV secretion system (T4SS) and flagellum expression and play important rolesin virulence and intracellular survival.
In this paper, we constructed cDNA library of Brucella melitensis16M infectingmurine macrophage, and screened and identified interaction proteins using Omp25and VjbR as bait protein. All these could lay the foundation for researching thepathogenesis and intracellular survival mechanism.
First, we cloned Omp25and VjbR, and ligased to pSos vector and constructedbait vecter after detecting bait vector toxicity, activation and membrane localization.
Second, we constructed the model of Brucella melitensis infecting murinemacrophage, and extracted total RNA and prepared mRNA for cDNA libraryconstruction, and then cDNA library was convered to pMyr vector.
Finally, Co-transformed bait vectors and pMyr-cDNA into cdc25HSaccharomyces cerevisiae competents for screening interaction proteins, andverificated by co-immunoprecipitation.
Results:
1. Omp25and VjbR gene were cloned and connected with pSos vector, and thebait vectors were non-toxicity, non-self reactivation, and located at right membraneposition suitable for yeast hybrid test.
2. By indirect immunofluorescence test and transmission electron microscopetechnology, the model of Brucella melitensis16M infecting murine macrophage wasestablished at the bacteria and cells ratio was500:1.
3. cDNA library was constructed by Stratangen mRNA kits, and convered cDNAlibrary to pMyr bait vectors, and constructed pMyr-cDNA library. The plasmidcapacity was1.6×109cfu/mL, and recombination ratio was very high.
The bait plasmid and pMyr-cDNA library were co-transformed into cdc25Hyeast competent for yeast hybrid. Two positive clones were screened, and thesequence showed that they were Mus musculus NADH dehydrogenase (ubiquinone)1alpha subcomplex, assembly factor2（NM_001127346.1），Ndufaf2, and （Musmusculus ribosomal protein S5（NM_009095.2），RPSs5. Ndufaf2could interact withOmp25, and RPSs5could interact with VjbR by co-immunoprecipitation. Datashowed:(1) Omp25interacted with Ndufaf2could enhance NADH concentration, andincreased NAHD activity, reducing the oxidative derivatives for brucella intracellularsurvival.(2) VjbR connected RPS5could inhibite RPS5activity, and inhibit apoptosisof murine macrophage providing brucella infection environment.
In this study, the foundation was established for paint the network of proteinsinteraction of brucella and murine macrophage, which provide foundations for futurestudy on pathogenesis and intracellular parasitic mechanisms.
引文
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