布鲁氏菌强弱毒株感染羊白细胞层SSH cDNA文库建立及CD96分子初步研究
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摘要
布鲁氏菌病是由布鲁氏菌引起的危害严重的人畜共患病。近年来，我国动物布病的发病率有所回升。当前控制布病的关键问题是布鲁氏菌致病机制和宿主抵抗布鲁氏菌感染机制不完全清楚。但至今，有关羊抵抗布鲁氏菌感染机制的研究报道较少。强毒株感染导致机体患病，弱毒株感染对机体起到保护作用却不引起患病，说明不同毒力株布鲁氏菌感染可引起机体产生不同的应答反应。因此，比较研究不同毒力株感染机体产生的不同应答分子，能够为预防、治疗、检测布鲁氏菌病提供靶标分子。布鲁氏菌可分为6个种。其中，羊种布鲁氏对我国危害较大。在众多布病疫苗中，猪种布鲁氏菌减毒疫苗S2在我国使用较为广泛。因此，选择羊种布鲁氏菌强毒株和猪种布鲁氏菌弱毒株S2进行比较具有一定的代表性，比较结果更加具有现实意义。
为获得布鲁氏菌强弱毒株感染小尾寒羊差异表达基因，本项研究应用抑制性差减杂交(SSH)技术构建布鲁氏菌强弱毒株感染小尾寒羊SSH cDNA文库。采用鸟枪法对447个文库细菌克隆进行测序。利用荧光定量PCR证实免疫相关备选基因CD96基因在羊种布鲁氏菌(B. melitensis)攻毒小尾寒羊第44天的外周血白细胞层中上调表达。应用RACE技术扩增得到CD96基因全长cDNA序列，并利用生物信息学软件预测CD96-DNAX-1蛋白的抗原表位。通过腹水诱生法获得CD96-D1单克隆抗体。验证了小尾寒羊CD96分子与TNF、IL-10、IFN-γ和IL-4的分泌关系。
1、布鲁氏菌强弱毒株感染小尾寒羊SSH cDNA文库
为获得布鲁氏菌强弱毒株感染小尾寒羊差异表达基因，本研究以雄性小尾寒羊为对象，以羊种布鲁氏菌(B. melitensis)为攻毒菌、以猪种布鲁氏菌减毒疫苗株(Brucellosis Vaccine, Live, S2)为接种菌，利用SSH技术首次成功构建布鲁氏菌强弱毒株感染小尾寒羊白细胞层SSH cDNA文库，该文库容量为1.62×106cfu。
2、克隆获得240个小尾寒羊外周血白细胞层表达基因
采用鸟枪法对447个文库细菌克隆进行测序，经序列分析和拼接，共得到240个小尾寒羊外周血白细胞层表达基因，其中有4个rRNA基因，234个cDNA序列。在234个cDNA序列中，有32个cDNA翻译的氨基酸序列可以搜索到保守结构域，占总数240个功能基因的比例为13.7%。
3、确定CD96基因上调表达并获得CD96基因cDNA序列
为验证CD96基因差异表达的特性，利用荧光定量PCR证实CD96基因在羊种布鲁氏菌(B. melitensis)攻毒小尾寒羊第44天的外周血白细胞层中上调表达，在S2接种后44天的外周血白细胞层中上无显著变化。为获得CD96基因cDNA序列，利用RACE技术扩增得到CD96基因全长cDNA序列。预测氨基酸序列具有PolyA结构和加尾信号序列，所编码蛋白无信号肽，表达于细胞膜表面。
4、预测CD96-DNAX-1蛋白的抗原表位并获得CD96-D1单克隆抗体
利用spdbv4.1.0预测CD96-DNAX-1蛋白的三维结构，发现该结构比较松散，与抗体嵌合成为抗原表位的可能性较大。综合Protean工具和BCPREDS工具的对CD96-DNAX-1蛋白的分析结果，预测其抗原表位为SDVNLTCQAQKKGLLVQMQWSKV并命名为CD96-D1。人工合成抗原表位CD96-D1，采用戊二醛法将小分子半抗原CD96-D1分别与卵清蛋白(OVA)、牛血清蛋白(BSA)偶联制备检测抗原和免疫抗原，免疫BALB/c鼠，融合并筛选杂交瘤细胞。获得2株分泌单克隆抗体的杂交瘤细胞。优选1株，采用小鼠腹水诱生法制备腹水单克隆抗体并纯化，纯化后单克隆抗体浓度为1.96mg/mL。为研究CD96分子与细胞因子的分泌关系奠定基础。
5、验证小尾寒羊CD96分子与TNF、IL-10、IFN-γ和IL-4的分泌关系
为了解CD96分子的生物学功能，利用CD96-D1单克隆抗体刺激健康羊外周血混合淋巴细胞，发现CD96-D1单克隆抗体能刺激TNF、IL-10分泌的增加，对IFN-γ和IL-4分泌未产生影响。
综上所述，本文为深入探讨CD96分子抗布鲁氏菌感染免疫机制奠定基础。
Brucellosis is a worldwide zoonotic infectious disease caused by the genus Brucella.In recent years, brucellosis rebounds worldwide, and it is especially serious in China.But, Brucella pathogenesis and host resistance to Brucella infection mechanism arenot completely known. So far, little has been reported about the mechanism of sheepresisting Brucella infection. Virulent strain infection causes the host sick, and weakstrain inoculation plays a protective effect. It implies that the infection of differentvirulent Brucella strains can cause the body to produce different reactions. Acomparative study of which different virulence strains infect and stimulate the body toproduce molecular responses can provide target molecules for the prevention,treatment and detection of brucellosis. The Brucella genus consists of six classicallyrecognized species, of which B. melitensis causes a highly harmful in our country.Among the brucellosis vaccine, B. suis strain2vaccines is the most widely usedvaccines in our country. Therefore, the choice of B. melitensis virulent strain and B.suis strain2attenuated strain has more representative, and the comparison has morepractical significance.
To obtain differentially expressed genes of Small Tail Han sheep infected byBrucella virulent and attenuated strains, suppression subtractive hybridization (SSH)was used to build SSH cDNA library of buffy coat and a shot-gun DNA sequencestrategy was employed.447library clones from the SSH cDNA library weresequenced. Real-time RT-PCR confirmed the upregulation of CD96in peripheralblood leukocytes when B. melitensis infect Small Tail Han sheep after44days. Thefull length of CD96cDNA was cloned using rapid amplification of cDNA end (RACE)technology. Bioinformatics software predicted the epitopes of CD96-DNAX-1. CD96-D1monoclonal antibody was obtained by means of ascites method. Therelationship between activity of CD96molecule and secretion of TNF, IL-10, IFN-γand IL-4from Small Tail Han sheep was confirmed.
1. SSH cDNA library of buffy coat from Brucella virulent and attenuated strainsinfecting Small Tail Han sheep.
To obtain differentially expressed genes of buffy coat from Brucella virulent andattenuated strains infecting Small Tail Han sheep, in this study Small Tail Han sheepwere used as experimental animals, B. melitensis as attack germs and B. suis strain2as vaccine strain. We use suppression subtractive hybridization (SSH) to build SSHcDNA library of buffy coat from infecting Small Tail Han sheep infected by Brucellavirulent and attenuated strains. The library harvested with1.62×106cfu.
2.240genes of Small Tail Han sheep were cloned in peripheral bloodleukocytes.
A shot-gun DNA sequence strategy was employed, in which447library cloneswere sequenced.240genes of Small Tail Han sheep were cloned in peripheral bloodleukocyte, such as5rRNA genes,235cDNA sequences. In the235cDNA sequences,amino acid sequences translated from32cDNAs can be searched for conserveddomains showing ratio of13.6%to the total number of240functional genes.
3. The CD96gene was determined with the upregulation pattern and thefull-length cDNA sequence was obtained.
Real-time PCR confirmed the upregulation of CD96in peripheral bloodleukocytes at44days post B. melitensis infecting Small Tail Han sheep. Therewere no significant differences in gene expression after B. suis strain2infected. Thefull-length CD96cDNA sequence was cloned by rapid amplification of cDNA end(RACE) technology and the predicted amino acid sequence did not show signalpeptides to express and locate on the cell surface.
4. Analysis of CD96-DNAX-1epitope and preparation of a monoclonal antibodyCD96-D1
For CD96-DNAX-1protein comprehensive analysis, we predicted antigenic epitope of SDVNLTCQAQKKGLLVQMQWSKV, and it was named as CD96-D1.Synthetic epitope CD96-D1was prepared for antigen immunization and detection.BALB/c mice, were used to immunized with CD96-D1, and hybridoma cells werefused and sreened. Monoclonal antibodies induced from the mouse ascites wereprepared and purified. After purification, the monoclonal antibody concentration was1.96mg/mL. It was the foundation of study on the relationship between CD96molecule and secreted cytokines.
5. Relationship between the activity of CD96and secretion of TNF, IL-10, IFN-γand IL-4
To understand the biological function of the molecule CD96, we used CD96-D1monoclonal antibody to stimulate healthy sheep peripheral blood includinglymphocyte. Monoclonal antibody CD96-D1was confirmed to show the activity toincrease TNF, IL-10secretion, and to pose no impact on IFN-γ and IL-4secretion.
In summary, this paper will lay a foundation of CD96anti-brucellosis infection.
为获得布鲁氏菌强弱毒株感染小尾寒羊差异表达基因，本项研究应用抑制性差减杂交(SSH)技术构建布鲁氏菌强弱毒株感染小尾寒羊SSH cDNA文库。采用鸟枪法对447个文库细菌克隆进行测序。利用荧光定量PCR证实免疫相关备选基因CD96基因在羊种布鲁氏菌(B. melitensis)攻毒小尾寒羊第44天的外周血白细胞层中上调表达。应用RACE技术扩增得到CD96基因全长cDNA序列，并利用生物信息学软件预测CD96-DNAX-1蛋白的抗原表位。通过腹水诱生法获得CD96-D1单克隆抗体。验证了小尾寒羊CD96分子与TNF、IL-10、IFN-γ和IL-4的分泌关系。
1、布鲁氏菌强弱毒株感染小尾寒羊SSH cDNA文库
为获得布鲁氏菌强弱毒株感染小尾寒羊差异表达基因，本研究以雄性小尾寒羊为对象，以羊种布鲁氏菌(B. melitensis)为攻毒菌、以猪种布鲁氏菌减毒疫苗株(Brucellosis Vaccine, Live, S2)为接种菌，利用SSH技术首次成功构建布鲁氏菌强弱毒株感染小尾寒羊白细胞层SSH cDNA文库，该文库容量为1.62×106cfu。
2、克隆获得240个小尾寒羊外周血白细胞层表达基因
采用鸟枪法对447个文库细菌克隆进行测序，经序列分析和拼接，共得到240个小尾寒羊外周血白细胞层表达基因，其中有4个rRNA基因，234个cDNA序列。在234个cDNA序列中，有32个cDNA翻译的氨基酸序列可以搜索到保守结构域，占总数240个功能基因的比例为13.7%。
3、确定CD96基因上调表达并获得CD96基因cDNA序列
为验证CD96基因差异表达的特性，利用荧光定量PCR证实CD96基因在羊种布鲁氏菌(B. melitensis)攻毒小尾寒羊第44天的外周血白细胞层中上调表达，在S2接种后44天的外周血白细胞层中上无显著变化。为获得CD96基因cDNA序列，利用RACE技术扩增得到CD96基因全长cDNA序列。预测氨基酸序列具有PolyA结构和加尾信号序列，所编码蛋白无信号肽，表达于细胞膜表面。
4、预测CD96-DNAX-1蛋白的抗原表位并获得CD96-D1单克隆抗体
利用spdbv4.1.0预测CD96-DNAX-1蛋白的三维结构，发现该结构比较松散，与抗体嵌合成为抗原表位的可能性较大。综合Protean工具和BCPREDS工具的对CD96-DNAX-1蛋白的分析结果，预测其抗原表位为SDVNLTCQAQKKGLLVQMQWSKV并命名为CD96-D1。人工合成抗原表位CD96-D1，采用戊二醛法将小分子半抗原CD96-D1分别与卵清蛋白(OVA)、牛血清蛋白(BSA)偶联制备检测抗原和免疫抗原，免疫BALB/c鼠，融合并筛选杂交瘤细胞。获得2株分泌单克隆抗体的杂交瘤细胞。优选1株，采用小鼠腹水诱生法制备腹水单克隆抗体并纯化，纯化后单克隆抗体浓度为1.96mg/mL。为研究CD96分子与细胞因子的分泌关系奠定基础。
5、验证小尾寒羊CD96分子与TNF、IL-10、IFN-γ和IL-4的分泌关系
为了解CD96分子的生物学功能，利用CD96-D1单克隆抗体刺激健康羊外周血混合淋巴细胞，发现CD96-D1单克隆抗体能刺激TNF、IL-10分泌的增加，对IFN-γ和IL-4分泌未产生影响。
综上所述，本文为深入探讨CD96分子抗布鲁氏菌感染免疫机制奠定基础。
Brucellosis is a worldwide zoonotic infectious disease caused by the genus Brucella.In recent years, brucellosis rebounds worldwide, and it is especially serious in China.But, Brucella pathogenesis and host resistance to Brucella infection mechanism arenot completely known. So far, little has been reported about the mechanism of sheepresisting Brucella infection. Virulent strain infection causes the host sick, and weakstrain inoculation plays a protective effect. It implies that the infection of differentvirulent Brucella strains can cause the body to produce different reactions. Acomparative study of which different virulence strains infect and stimulate the body toproduce molecular responses can provide target molecules for the prevention,treatment and detection of brucellosis. The Brucella genus consists of six classicallyrecognized species, of which B. melitensis causes a highly harmful in our country.Among the brucellosis vaccine, B. suis strain2vaccines is the most widely usedvaccines in our country. Therefore, the choice of B. melitensis virulent strain and B.suis strain2attenuated strain has more representative, and the comparison has morepractical significance.
To obtain differentially expressed genes of Small Tail Han sheep infected byBrucella virulent and attenuated strains, suppression subtractive hybridization (SSH)was used to build SSH cDNA library of buffy coat and a shot-gun DNA sequencestrategy was employed.447library clones from the SSH cDNA library weresequenced. Real-time RT-PCR confirmed the upregulation of CD96in peripheralblood leukocytes when B. melitensis infect Small Tail Han sheep after44days. Thefull length of CD96cDNA was cloned using rapid amplification of cDNA end (RACE)technology. Bioinformatics software predicted the epitopes of CD96-DNAX-1. CD96-D1monoclonal antibody was obtained by means of ascites method. Therelationship between activity of CD96molecule and secretion of TNF, IL-10, IFN-γand IL-4from Small Tail Han sheep was confirmed.
1. SSH cDNA library of buffy coat from Brucella virulent and attenuated strainsinfecting Small Tail Han sheep.
To obtain differentially expressed genes of buffy coat from Brucella virulent andattenuated strains infecting Small Tail Han sheep, in this study Small Tail Han sheepwere used as experimental animals, B. melitensis as attack germs and B. suis strain2as vaccine strain. We use suppression subtractive hybridization (SSH) to build SSHcDNA library of buffy coat from infecting Small Tail Han sheep infected by Brucellavirulent and attenuated strains. The library harvested with1.62×106cfu.
2.240genes of Small Tail Han sheep were cloned in peripheral bloodleukocytes.
A shot-gun DNA sequence strategy was employed, in which447library cloneswere sequenced.240genes of Small Tail Han sheep were cloned in peripheral bloodleukocyte, such as5rRNA genes,235cDNA sequences. In the235cDNA sequences,amino acid sequences translated from32cDNAs can be searched for conserveddomains showing ratio of13.6%to the total number of240functional genes.
3. The CD96gene was determined with the upregulation pattern and thefull-length cDNA sequence was obtained.
Real-time PCR confirmed the upregulation of CD96in peripheral bloodleukocytes at44days post B. melitensis infecting Small Tail Han sheep. Therewere no significant differences in gene expression after B. suis strain2infected. Thefull-length CD96cDNA sequence was cloned by rapid amplification of cDNA end(RACE) technology and the predicted amino acid sequence did not show signalpeptides to express and locate on the cell surface.
4. Analysis of CD96-DNAX-1epitope and preparation of a monoclonal antibodyCD96-D1
For CD96-DNAX-1protein comprehensive analysis, we predicted antigenic epitope of SDVNLTCQAQKKGLLVQMQWSKV, and it was named as CD96-D1.Synthetic epitope CD96-D1was prepared for antigen immunization and detection.BALB/c mice, were used to immunized with CD96-D1, and hybridoma cells werefused and sreened. Monoclonal antibodies induced from the mouse ascites wereprepared and purified. After purification, the monoclonal antibody concentration was1.96mg/mL. It was the foundation of study on the relationship between CD96molecule and secreted cytokines.
5. Relationship between the activity of CD96and secretion of TNF, IL-10, IFN-γand IL-4
To understand the biological function of the molecule CD96, we used CD96-D1monoclonal antibody to stimulate healthy sheep peripheral blood includinglymphocyte. Monoclonal antibody CD96-D1was confirmed to show the activity toincrease TNF, IL-10secretion, and to pose no impact on IFN-γ and IL-4secretion.
In summary, this paper will lay a foundation of CD96anti-brucellosis infection.
引文
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