Construction Of Murine Mannose Recptor CTLD4-7 Domain Expression Vector And Gene Expression

Mannose receptor (CD206) is one member of the mannose receptor family and belongs to C-type lectin receptor. It was initially founded in rat liver Kupffer cell. Mannose receptor was also founded distributing extensively on many other kinds of tissue cell in vivo. Its major biological functions display in mediating the internalization and clearance of endogenous or exogenous polysaccharide, polypeptide, protein and nucleic acid, eliminating pathogen, conveying and presenting antibody and initiating immune response. It is one of the conservative pattern recognition receptors (PPR).Mannose receptor is a 175 kDa I type trans-membrane receptor consisting of five domains: a cysteine-rich amino terminus; a fibronectin type II repeat region; 8 C-type lectin-like carbohydrate recognition domains (CTLD1-8); a trans-membrane domain and a cytoplasmic domain. Mannose receptor can bind endogenous or exogenous ligands through different domains.It is reported that mannose receptor expressed on human lymphatic endothelium is a noval ligand of L-selectin. The interaction between the two molecules mediates lymph cell homing. The early study work of our laboratory indicates that the lymphatic metastasis capability of mouse high and low metastasis hepatoma carcinoma cell HCa-F and HCa-P is related to their L-selectin expression level . In lymphoma P388D1, this phenomenon also exist. So, we deduce that the interaction between mannose receptor and L-selectin is involved in tumor lymphatic metastasis.Objective: In this thesis we are to construct a chimeric gene of the mannose receptor C-type lectin-like domain 4-7 fused to rabbit secreting signal peptide gene and the Fc segment gene of human IgG. The chimeric gene named Sig-CTLD4-7-Fc is to be inserted in vector pcDNA3.1, then the secreted chimeric protein is to be transient expressed in 293T cells . It may provide a basis for purifying this chimeric protein by using Protein-A bind , identifying if MR can interact with L-selectin through adhesion experiment . The observation of the two molecules'interaction may give us some hints for further research of the mannose receptor's function in the process of tumor lymphatic metastasis.Methods:1.We are to testify pMD-18T/ CTLD1-8 vector of our laboratory by enzyme cutting .Then we amplify CTLD4-7 of MR from the vector of pMD-18T/ CTLD1-8 by PCR to inserte it into pMD-18T clone vector and identify sequence after testifing by enzyme cutting.The right MR CTLD4-7 gene is to be inserted into pX vector(pX vector contains secreting signal sequence in upriver position and Fc gene of human IgG in downriver position).Then testify the aim vector by enzyme cutting. It is to be named pX/ CTLD4-7.The right Sig-CTLD4-7-Fc chimeric gene is to be obtained by single NotI enzyme cutting pX/ CTLD4-7 ,then inserted in pcDNA3.1 expression vector. It constructs the recombinant expression vector pcDNA3.1/ Sig-CTLD4-7-Fc.2.Culture 293T cell. Then we transfect the highly-purified plasmid pcDNA3.1/ Sig-CTLD4-7-Fc into 293T cells with TM2000. The transient expression product of 24h, 48h, 72h is to be detected with negtive and positive control by ELISA. At the same time, cell immunochemistry is to be done for qualitative assay of transient expression.Results:1.MluI single enzyme and MluI, KpnI double enzyme cutting pMD-18T/ CTLD1-8 plasmid generate prospective 3420 bp, 2800 bp and 2800bp, 2120bp,1300bp segments respectively. AatII, SalI double enzyme cutting pMD-18T/ CTLD4-7 generate prospective 2200bp,1695bp, 498bp segments, and the CTLD4-7 gene sequence is identified exactly. AatII , SalI double enzyme and NotI single enzyme cutting pX/CTLD4-7 generate prospective 3621bp, 1695bp and 2865bp, 2451bp segments respectively. And NotI single enzyme cutting pCDNA3.1/Sig-CTLD4-7-Fc generate prospective 5522 bp, 2451 bp segments. The direction of Sig-CTLD4-7-Fc in pCDNA3.1/Sig-CTLD4-7-Fc is identified by PCR and the sequence by sequencing. All indicate we construct the eukaryotic expression vector pCDNA3.1/Sig-CTLD4-7-Fc exactly2.After transfecting 293T cell, ELISA experimental result manifests chimeric protein starts to secrete at the time of 24h, and the amount obviously rises at the time of 48h,72h;cell immunochemistry identifys that it strats to express chimeric protein CTLD4-7-Fc at the time of 24h, and it continues to express at the time of 48h,72h.Conclusions:1.We have successfully constructed the recombinant expression vector pCDNA3.1/ Sig-CTLD4-7-Fc.2.The recombinant expression vector can be successfully expressed in the 293T cells by lipid transfecting.