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Construction and Characteristics of sEGFP-CD40L Fusion Gene

Author: MaJingYing
Tutor: TanXiaoHua
School: Third Military Medical University
Course: Oncology
Keywords: Signal peptide Green Fluorescent Protein CD40L Dendritic cells Cell phenotype Interleukin- 12
CLC: R346
Type: Master's thesis
Year: 2010
Downloads: 13
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Abstract

【Objective : To construct encoding the enhanced green fluorescent protein (EGFP) and human CD40L ectodomain (hecdCD40L) fusion gene composed of eukaryotic expression vector , and explore the fusion protein expression on human dendritic cells (Dendritic cells, DCs ) phenotype and function . 【Methods : RT-PCR method to clone hecdCD40L gene fragments from human peripheral blood mononuclear cells . Mouse vascular endothelial growth factor receptor 2 signal peptide sequence (SigmKDR) and EGFP and hecdCD40L of gene fragments fusion or non- fusion , inserted into the eukaryotic expression vector pcDNA3.1 ( - ) myc - 6 × His construct pcDNA3.1 SigmKDR- the His and pcDNA3.1 sEGFP of - hecdCD40L -myc - 6 × His the EGFP ( sEGFP ) - myc -6 × . Expression vector was transfected into COS-7 cells were cultured in vitro with liposomes , G418 resistance screening , FACS sorting the the the stable expression sEGFP sEGFP-hecdCD40L monoclonal COS-7 cell line . Western Blot the tube Get the purified concentrated sEGFP-hecdCD40L and sEGFP fusion protein in COS-7 cells and supernatant expression nickel column and ultrafiltration fusion protein were incubated with human DCs , flow cytometry and ELISA analysis DCs . uptake rate of the two fusion proteins , DCS phenotypic changes and the cytokine IL-12 secretion . Results : pcDNA3.1 sEGFP - myc - 6 × His or pcDNA3.1 sEGFP - hecdCD40L - myc - 6 × His transfected COS-7 cells , G418-resistant screening and flow cytometry sorting screened efficient monoclonal cell lines expressing the fusion protein , EGFP -positive cells up to 95 % , Western Blot and supernatants were able to detect intracellular fusion protein . the the sEGFP sEGFP-hecdCD40L and DCs were incubated for 2 hours, flow cytometry shows EGFP -positive rate of the former and the latter 28.6% and 56.9% , 24 hours after the analysis of the phenotype of DCs , sEGFP - hecdCD40L fusion protein enhanced DCs of CD80 expression of CD83 and HLA-DR molecules , as well as to stimulate the secretion of IL-12 , and sEGFP had no significant effect on the phenotype of DCs and IL-12 secretion . Conclusion : with SigmKDR , the fusion gene can the EGFP and hecdCD40L constituting expression in eukaryotic cells , such secreted fusion protein can be secreted into the extracellular targeting DCs, induce DCs maturation and raised its surface costimulatory molecules and stimulate IL-12 secretion and expression of MHC II molecules .

Construction and Characteristics of sEGFP-CD40L Fusion Gene

Abstract

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