Construction Of Suppression Subtractive CDNA Libraries For Buffy Coat Of Sheep Inoculated With Virulent And Avirulent Brucella And Preliminary Function Reseach On CD96

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-D1For 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-4To 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.