Molecular Cloning, Expression And Biological Function Of Programmed Cell Death10from Sheep (Ovis Aries)

Brucellosis, caused by Brucella spp., remains the most common bacterialzoonosis in the word. The disease poses a severe health threat and public healthsecurity. At present, the clinical diagnosis of brucellosis mainly relies on serologicaltesting that is hard to discern infected animals from vaccinated animals. Thus, it isnecessary to establish effective diagnosis method to discern infected animals fromvaccinated animals. To solve the problem, a subtracted cDNA library wasconstructed by suppression subtractive hybridization （SSH） in our previous studies.A pool of cDNA from the buffy coat of sheep challenged with Brucella melitensiswas used as the driver, and the ones from the buffy coat of sheep vaccinated withBrucella suis S2as the tester. PDCD10was one of the differential genes from thesubtracted cDNA library. Therefore, in this study, we cloned PDCD10cDNA fromthe buffy coat of Small Tail Han sheep （Ovis aries）, named OaPDCD10. Then,differential expression levels of OaPDCD10mRNA between infected sheep（challenged with B. melitensis） and vaccinated sheep （vaccinated with B. suis S2）were analyzed. Finally, the biological function of OaPDCD10was conducted byRNAi. Our studies will lay a stable basis on further studies to shed light on therelation between OaPDCD10and the pathogenic mechanisms of Brucella spp, and todiscover potential diagnostic biomarkers of brucellosis to discern infected sheepfrom vaccinated sheep.一、Full-length cDNAcloning of OaPDCD10and sequence analysisBased on the partial sequence of OaPDCD10from the SSH cDNA library todesign the primers,5’-and3’-rapid amplification of cDNA ends （RACE） wereperformed to obtain the full-length cDNA of OaPDCD10, which was analyzed using various bioinformatics softwares. The results showed that the full-length cDNA ofOaPDCD10（GenBank accession number KC425616） was1343bp in lengthcontaining a5’-untranslated region （UTR） of273bp, an ORF of639bp, and a3’-UTR of431bp with a poly A tail. The ORF encoded a putative protein of212amino acid residues with a theoretical isoelectric point of8.20and a deducedmolecular weight of24.71kDa. Multiple alignments revealed that OaPDCD10wasso highly conserved among various species, the highest percentage of identity was100%with Mus musculus （GenBank accession number NP₀62719）. Additionally,OaPDCD10was predicted to be an adaptor protein composed of seven-helixes,have five Ser phosphorylated sites, and without catalytic structural domain,transmembrane domains as well as signal peptide.二、Verification and analysis of differential expression of OaPDCD10mRNAReverse transcription quantitative real-time PCR （RT-qPCR） was carried out toinvestigate tissues distribution and the differential expression of OaPDCD10mRNAbetween infected sheep and vaccinated sheep. Based on the full-length cDNA ofOaPDCD10, the primers for RT-qPCR were designed. The results of tissuesdistribution revealed that OaPDCD10mRNAs were ubiquitously expressed in alltested tissues. The highest expression was observed in the heart, followed by rumen,kidney, liver, spleen and skeletal muscle. The lowest expression was in the lung,buffy coat and small intestine with no significant differences among them. Theresults of differential expression showed that, compared to the control, OaPDCD10mRNAs from infected sheep or vaccinated sheep were both significantlyup-regulated （p<0.05or p<0.01） in a time-dependent manner. Moreover,OaPDCD10mRNAs from vaccinated sheep were higher than ones from infectedsheep. OaPDCD10mRNAs between infected and vaccinated sheep were differentsignificantly （*p<0.05） or most significantly （**p<0.01） after30dayspost-inoculation. These result suggested that OaPDCD10possibly play an importantrole on pathogenic process of Brucella spp.. 三、Expression, purification and characterization of OaPDCD10The recombinant plasmid of OaPDCD10（pET30a-OaPDCD10） wasconstructed, and then expression and purification of the recombinant OaPDCD10（rOaPDCD10） were performed. The results showed that the highest product ofrOaPDCD10were obtained at28°C,0.4mM IPTG for4h. The band of the samesize at²8kDa were detected by SDS-PAGE and Western blot, longer than thepredicted24.71kDa. The purified rOaPDCD10was single band, the concentrationof it was0.4mg/ml. To determine biological activity of rOaPDCD10,293T cellswere treated with the purified rOaPDCD10（50μg/ml） to analyze the effect ofrOaPDCD10on cell proliferation and apoptosis. The results showed that thepercentage of apoptotic293T cells treated with the rOaPDCD10proteins （50μg/ml）was respectively89.07%,86.85%,84.22%at12h,24h,36h post-treatment, largerthan the control （77.89%）. Compared to the control, the viability of293T cellstreated with rOaPDCD10（50μg/ml） increased significantly （p<0.05or p<0.01）.四、The preparation of McAb against OaPDCD10The purified rOaPDCD10proteins were used as immunogen to inoculate themouse, cell fusion was conducted by the classic lymphoid cell fusion technology andhybridoma cell lines were selected by indirect ELISA. Finally, two hybridoma celllines could secrete McAb against OaPDCD10stably were successfully prepared.Moreover, the McAb against OaPDCD10was highly specific and could recognizeboth the recombinant OaPDCD10and natural OaPDCD10.五、Effect of OaPDCD10overexpression on cell proliferation and apoptosisTo elucidate the function of OaPDCD10, we constructed the expression plasmidpLV5-OaPDCD10-GFP to transfect293T cells to analyze the effects of OaPDCD10overexpression on cell proliferation and apoptosis. Western blot were carried out toanalyze the expression levels of OaPDCD10protein. The viability of293T cells wasassessed by the MTT assay. Apoptosis was assessed by two assays: thehoechst33342/propidium iodide （PI） staining assay and Annexin V-PE/7-AAD assay.The results showed that the expression levels of OaPDCD10in293T cellstransfected with overexpressed vector of OaPDCD10increased significantly at48h post-transfection, were1.55folds （p<0.05） than the control. Compared to the control,293T cells transfected with overexpressed vector of OaPDCD10demonstratedincreased cell vitality （p<0.05）. The percentage of apoptotic293T cells transfectedwith overexpressed vector of OaPDCD10was81.3%, less than the negative control（GFP）（85.37%）, but more than the control （77.89%）, suggesting that OaPDCD10overexpression was able to promote cell proliferation and inhibit cell apoptosis.六、Effect of PDCD10knockdown on cell proliferation and apoptosisTo further identify the function of PDCD10, we constructed the shRNAinterference plasmid silencing PDCD10to transfect293T cells to analyze the effectsof PDCD10knockdown on cell proliferation and apoptosis. Western blot werecarried out to analyze the expression levels of PDCD10protein. The viability of293T cells was assessed by the MTT assay. Apoptosis was assessed by two assays:the hoechst33342/propidium iodide （PI） staining assay and the AnnexinV-PE/7-AAD assay. The results showed that, compared to the control, at48hpost-transfection, the expression levels of PDCD10in293T cells transfected withsh1or sh2vector were reduced up to76.1%（p<0.05） and25.7%（p<0.01）respectively. Thus, sh2was selected to further identify the function of PDCD10.Compared to the control,293T cells transfected with shPDCD10vectordemonstrated decreased cell vitality （p<0.01）. The percentage of apoptotic293Tcells transfected with shPDCD10vector was90.73%, more than the negative control（shNC）（84.96%）, and much more than the control （77.89%）, suggesting thatPDCD10knockdown was able to inhibit cell proliferation and promote cellapoptosis.