Cloning Of Human Tim-3 Promoter And Construction Of Retroviral Expression Vector Of Human Tim-3

Newly-found Tim-3 is specifically expressed on terminally differentiated Th1 cells but not on Th2 cells and negatively regulates Th1 responses.Numerous murine and human studies have demonstrated that Tim-3 influences the ability to induce T cell tolerance and many immune diseases, including autoimmunity,asthma and cancer.Tim-3 is an attracting therapeutic target in human various immune-mediated diseases.Blockade of the inhibitory Tim-3 pathway may prove useful in the treatment of a wide array of atopic diseases, most notably asthma,that are associated with a biased Th2 response.Tim-3 was the first,and is presently the only,surface molecule that can specifically identify Th1 cells in both mice and humans,but the regulatory mechanisms of its expression remains unknown.To better understand the molecular regulation of Tim-3 expression and function,three parts as follows were performed:firstly,to establish a cell model for the further exploration on the regulation of human Tim-3 promoter;then,to construct the luciferase reporter plasmids which contain human Tim-3 gene promoter;lastly,to construct the retroviral expression vector of human Tim-3.Part 1ObjectiveTo analyse the expression of Tim-3 in healthy human PBMC stimulated by mitogen and set up a cell model for Tim-3 regulation study.MethodsPBMC were isolated from human peripheral blood and the mononuclear cells were eliminated by attachment.Then cells were stimulated by PHA,LPS for 24h,respectively.The total RNA were prepared for RT-PCR analysis of Tim-3 mRNA.Untreated PBMC were as control.ResultPBMC stimulated by PHA could express Tim-3,but PBMC stimulated by LPS could not, suggesting that activated but not polarized human T cells approach to express Tim-3.ConclusionThis part has established a cell model for the further study on the mechanisms of human Tim-3 transcriptional control.Part 2ObjectiveTo clone and construct luciferase reporter plasmids containing human Tim-3 promoter.Methods1 Prediction of human Tim-3 promoter regionThe 5'-flanking region spanning -2500 to +202 of human Tim-3 gene was submitted and analyzed by online Matlnspector software.2 PCR amplification of Tim-3P1 and Tim-3P2Segment from -2083 to +242 was selected finally and fractionated into two parts to clone, according to the only Sacâ… site in this region.These two fragments are named Tim-3P1(spanning -2083 to -838 calculated from TSS +1)and Tim-3P2(spanning -948 to +242 containing TSS and ATG),respectively.Specific primers for Tim-3P1 and Yim-3P2 were designed by Primer Premier 5.0 software.Tim-3P1 and Yim-3P2 were amplified by PCR using human genomic DNA as template.3 Construction and confirmation of the different luciferase reporter plasmidsThe PCR product of Tim-3P1 was linked into pGEM-T easy vector and then subcloned into the multiple cloning site of a promoterless pGL3-Basic plasmid to construct pGL3-Tim-3P1 with the correct sequence.The amplified PCR product of Tim-3P2 was directionally fused to the luciferase reporter gene to create pGL3-Tim-3P2 after double digestion with Sacâ… and Xhoâ… .The construct was also confirmed by DNA sequencing.In order to enhance the transcriptional activity of the plasmid constructed,SV40 enhancer sequence was introduced into pGL3-Tim-3P2 to generate Enhancer-Tim-3P2.4 Expression of endogenous Tim-3 in different cell linesTotal RNA was isolated from RAW264.7,B16 and COS-7 up to 5×10⁵ cells and RT-PCR was carried out to assess the expression of Tim-3.5 TransfectionThe constructs as described above and pGL3-Basic(negative control)were transiently co-transfected with pRL-TK into RAW264.7,B16 and COS-7 cells,respectively.Luciferase reporter gene assay were conducted 48h after transfection.6 Dual-Luciferase reporter assaysThe protocol as follows.20μl of cell extract,50μl of LARâ…¡and 50μl Stop & Glo^TMReagent by using luminoskan TL plus(Labsystems,Frankfurt,Germany).The firefly luciferase activity was normalized with the renilla reniformis luciferase activity of pRL-TK to control for variations in transfection efficiency.All the data shown were obtained from at least three independent experiments, and each plasmid was established double-wells.7 Analysis of TFBSs within Tim-3P1 and Tim-3P2Both were analyzed for putative regulatory elements by online MatInspector software.Results1 Prediction of human Tim-3 promoter regionThe results showed that there exist many TATA box,CAAT box and important TFBSs within the region.2 Successful generations of the reporter plasmidsAnalysis of DNA sequencing ensured the fidelity of amplification and the correct orientation of the luciferase reporter plasmids constructed.3 Expression of endogenous Tim-3 in different cell linesUsing reverse transcriptase PCR analysis,endogenous expression of Tim-3 was detected in both RAW264.7 and B16,but not in COS-7.4 Determination of the transcriptional activities of the constructsThe results of dual-luciferase reporter assays showed that pGL3-Tim-3P2 could lowly expressed in RAW264.7 and B16 both of which can express Tim-3 endogenously,but not in COS-7 in which no endogenous Tim-3 could be examined.The luciferase activity of pGL3-Tim-3P1 was a little higher than that of pGL3-basic in B16.Furthermore,SV40 enhancer could significantly argument the promoter activtity of Enhancer-Yim-3P2.5 TFBSs analysis of Tim-3P1 and Tim-3P2Interestingly,sequence analysis has revealed the existence of many putative transcription regulatory elements such as c-Myb,Ik-1,CHOP,RFX1,FOXK2,HNF-3,MZF1,GATA-3,p53, CDE/CHR,NFAT,YY1 and TATA box within them.ConclusionsDNA sequencing confirmed the successful generation of the human Tim-3 luciferase reporter plasmids which could promote the luciferase expression in a cell-specific manner.As a first step towards understanding the transcriptional regulation of human Tim-3 gene,the constructed reporter chimerics provide a fundamental framework for the identification of specific factors regulating its gene expression.Part 3ObjectiveTo develope a retroviral expression vector of human Tim-3.MethodsHuman Tim-3 was excised from plasmid hTim-3-pGEM-T by PCR using the corresponding primers designed from the sequence of human Tim-3 mRNA.PCR product was cloned into MCS of pLNCX2 to generate pLNCX2-Tim-3.The recombinant was transiently transfected into PT67 cells with the correct sequence and the expression of Tim-3 was examined by RT-PCR.Untreated PT67 and PT67 transfected with pLNCX2 empty vector were as control.ResultspLNCX2-Tim-3 was successfully created and can express Tim-3 efficiently in packing cell line PT67 contrary to untreated PT67 cells and the PT67 cells transfected with pLNCX2 empty vector.ConclusionsThe recombinant retroviral expression vector containing human Tim-3 has been successfully constrcucted and can effectively express Tim-3.This work would facilitate our subsequent studies on the elucidation of the regulated expression of Tim-3 gene transcription and explore the clinical feasibility of application in human various immune diseases.