Abstract

The human Dectin-1 molecule known as a β-glucan receptor is an immune cell surface receptor implicated in the immunological defense against fungal and other pathogens. Dectin-1 is a type II transmemebrane receptor with a single extracellular carbohydrate recognition domain (CRD), a stalk region which separates the CRD from the membrane and an immunoreceptor tyrosine activation motif in its cytoplasmic tail. The objectives of the present study were isolation of the Dectin-1 genes from the human monocyte complementary deoxyribonucleic acid (cDNA), cloning of the isolated human Dectin-1 isoform transcripts into the mammalian expression vector, make a green fluorescent protein (GFP) fusion, expression and sub-cellular localization of the GFP fusion in the mammalian cell line. Reverse transcription-polymerase chain reaction (RT-PCR) and Vector NTI sequence analysis revealed six transcripts from a human monocyte cDNA. Basic local alignment search tool (BLAST) analysis showed the transcripts are member of the human Dectin-1 gene family. Sequence alignment analysis using contig express and ClustalW revealed a 100% sequence similarity with the human Dectin-1 isoform A, B and F with a size of 741, 603 and 232 bp, respectively and another one transcript (193 bp) which do not homologous with the six isoforms. Cloning of the four isolated human Dectin-1 isoform transcripts into the mammalian expression vector at the 3’end of cytomegalovirus promoter (CMVp) and the GFP was ligated at the 3’end of the cloned Dectin-1 gene. The ligation experiment was proven by restriction enzyme digestion. Transient transfection of the plasmid deoxyribonucleic acid (DNA’s) that contain the chimeric human Dectin-1 isoform-GFP fusion transcripts into a Caco-2 cells were conducted and after 24 h of nucleofection. The tissue culture plate cells were examined by fluorescent microscope and all the tested samples showed green fluorescence signal. Transfection efficiency of 40.2 to 58.0% and 70.7 to 82.8% cell viability were recorded using flow cytometry assay at 48 h of post nucleofection on cells that were cultured on tissue culture plate. Localization of fused GFP was assessed using a laser scanning confocal microscopy after 24 h of transfection on cells that were cultured on a chambered tissue culture coverglass and revealed the GFP is localized on the cell membrane.    

Key words: Cloning, GFP, human DECTIN-1 isoforms, localization, RT-PCR, transfection

Abbreviation

CRD, Carbohydrate recognition domain; cDNA, complementary deoxyribonucleic acid; RT-PCR, reverse transcription-polymerase chain reaction; GFP, green fluorescent protein; BLAST, basic local alignment search tool; CMVp,cytomegalovirus promoter; PRRs, pattern recognition receptors; PAMPs, pathogen associated molecular patterns; CTLs, C-type lectins; DCs, dendritic cells; APC,antigen-presenting cells; ITAM, immunoreceptor tyrosine-based activation motif;CTLD, C-type lectin-like domain; NK, natural killer; NKC, natural killer complex; TNF,tumor necrosis factor; PCR, polymerase chain reaction; IPTG, isopropyl-beta-D-thiogalactopyranoside; LB, luria-bertani; CFP, cyan fluorescence protein; YFP, yellowfluorescent protein; EDTA, ethylenediaminetetraacetic acid; FACS, fluorescence-activated cell sorting; NCBI,  national center for biotechnology information; EtBr,ethidium bromide.