Construction Of Recombinant Plasmid Of Extracellular Domain Structure Of Human CD200R

Background and Objective:The CD200 molecule and its receptor CD200 R protein are members of a highly conserved immune superfamily,a type I membrane glycoprotein with two immunoglobulin-like domains in the extracellular domain.CD200 is expressed in both humans and rodents and is widely distributed in neurons of the central nervous system,activated T cells,B cells,follicular dendritic cells,eosinophils,and mast cells.CD200 R expression is relatively limited,mainly distributed in myeloid-derived cells（such as dendritic cells,macrophages,neutrophils,mast cells,etc.）and lymphocytes.Studies have shown that CD200/CD200 R plays an important role in many diseases such as autoimmune diseases,inflammatory reactions,tumorigenesis,development,and metastasis.The interaction between CD200 and CD200 R activates an intracellular inhibitory signaling pathway that recruits Ras GAP and ultimately produces a cytostatic effect.The activation of CD200 R promotes the differentiation of T cells into Treg direction,differentiates Th1 cells into Th2 cells,and promotes the production of anti-inflammatory cytokines IL-10 and TGF-β,which is necessary for the organism to maintain immune homeostasis.In this study,the extracellular domain structure of CD200 R protein was expressed and purified,and the expressed and purified protein was verified by Western blotting,which provided tools for the further study of the biological function of CD200 R.Methods:Part1: Construction and expression of eukaryotic expression plasmid p Sectag2A-CD200 R.The extracellular amino acid sequence of human CD200 R was searched in the NCBI database.After obtaining the accounting sequence by codon optimization,the complete gene sequence was synthesized to obtain the CD200 R extracellular domain c DNA fragment,which was inserted into the eukaryotic expression vector p Sectag2 A.The recombinant plasmid p Sectag2A-CD200 R was sequenced correctly and then transferred into CHO cells to obtain a highly expressing cell line.Part 2: Purification and Verification of Recombinant CD200 R ProteinThe recombinant plasmid was transferred into CHO cells,and the supernatant was collected and concentrated to obtain purified recombinant CD200 R protein after affinity chromatography.Research results:1.The c DNA of extracellular domain of CD200 R was synthesized in the whole gene and the recombinant plasmid p Sectag2A-CD200 R was successfully constructed.The sequencing results were consistent with those published in the NCBI database（EAW79659.1,NP₆20161.1）.2.The constructed recombinant plasmid p Sectag2A-CD200 R was transfected into CHO cells with MAX reagent,and the cell culture supernatant was purified using a nickel column.A band was observed at 45 k Da by SDS-PAGE.The specificity of the same size was confirmed by Western-Blot analysis.The band,which can specifically bind to the histidine（HIS）tag,indicates that the cell line CHO/p Sectag2A-CD200 R was successfully established and the human CD200 R protein was obtained.Conclusions:1.Genes of extracellular domain of CD200 R were obtained by whole gene synthesis and inserted into p Sectag2 A secretory expression plasmid by molecular cloning technique.A recombinant eukaryotic expression vector p Sectag2A-CD200 R was successfully constructed.2.Recombinant plasmid was transfected into CHO cells with MAX reagent to obtain CHO/p Sectag2A-CD200 R cell line stably expressing CD200 R ectodomain protein.Using protein affinity means,a sufficient amount of active recombinant CD200 R was prepared.3.The purified protein was validated by the Western Blot method,which provided a tool for further study of the biological function of cd200R.