Cloning Of Human CD200 Gene And Construction Of Eukaryotic Expression Vector

Background: The CD200 gene is a newly found member of the immunoglobulin superfamily( IgSF ), which was called MOX2,MRC OX2 antigen. The CD200 gene has regulation function on immune system and can be widely expressed on a variety of cells, which includes human thymocytes and peripheral B lymphocytes, activated T cells, and follicular dendritic cells. The expressing sites of CD200 indicate that the distribution of CD200 gene is related to immune tolerance induced by the gene. The interaction between CD200 and it's receptor (CD200R) expressed on myeloid cells can depress he myeloid cell activity, regulate cytokine network, interfere with the development and differentiation of help T cells by altering secretion of cytokine, and then promote the immune tolerance. CD200-CD200R interaction can alleviate the symptom of autoimmune disease, enhance graft survival chance, and prevent from abortion caused by pre-inflammatory factors and break tumor immune tolerance. In the present, the therapeutic studies have been carrying out in Canada. These study data strongly indicate that the CD200-CD200R interaction is involved in the negative or restrictive control of myeloid cellular function in both healthy and disease states. Objective: the present study was undertaken to clone the human CD200 gene, construct the CD200 eukaryotic expression vector to transfect the U937 cell, and to observe the transfection efficiency and CD200 expression measured by flow cytometry, therefore to lay a theoretical foundation for assessment of biological activities of the recombinant CD200 plasmid as well as the it's effects on immune tolerance and clinical application. Methods: Total RNA was extracted from human peripheral blood white cell stimulated by Con A in present experiment, and amplified human CD200 gene fragment by twice steps RT-PCR technique as following : Target gene was cloned into pMD18T vector and after sequence analysis CD200 gene was inserted orientally into downstream of human cytomegalovirus (hCMV) promoter in pcDNA3 and construct recombinant eukaryotic expression plasmid pcDNA3-CD200 which was screened and validated by restriction analysis and PCR. By MiniBEST Plasmid Purification Kit Ver.2.0 method with sterility, the recombination vector pcDNA3-CD200 was transfected by electroporation to the alternated strain U937 cell. After 24 hours the cells were cultured with selective IMDM medium containing G418 (800μg/ml) to selecting positive cell clones. The cells were cultured to 30 generations until they were able to express human CD200 in stability and efficiency in vitro. Results: Human CD200 gene fragment was cloned successfully (810 bp) in this study .pMD18T sequencing analysis of this gene is identical with the gene in GenBank (NM₀05944). The construction of pcDNA3 –CD 200 eukaryotic express vector was accomplished. We detected the results by flow cytometry after transfected the vector to U937 cell by electropo -ration. These results were analyzed by statistical methods. Compared with pcDNA3 group, there is no significant difference in the percentage of positive cell of expressing CD200 gene in control group. Human CD200 gene expression in pcDNA3-CD200 group (72.76%±3.34%) is significantly higher than those in the pcDNA3 group and control group which respectively is 6.36%±2.12% and2.98%±1.45% respective.(both p﹤0.01).It indicates that CD200 was transfected successfully and had stable expressing...