Fusion Expression Of The Extracellular Carbohydrate Recognition Domain Of Mouse DECTIN-1 And Its Recognition Of Î'-GLUCANS In The Cell Wall Of Fungi

With the use of large quantities of immunosuppressive drugs, patients with invasive fungal infections are increasingly high, for whom the rapid detection of the invasive pathogens is a prerequisite of successful treatment. At present, various methods for the rapid diagnosis of invasive fungal infection have been reported based on the detection of the fungal antigens or metabolites from the body fluids, among which the detection of the fungal cell wall componentβ-glucans is widely employed. However, due to the interference from bacterial endotoxins, high false positivity is one of the main defects of the present methods. Thus, there is an urgent need to develop novel detection strategies with high specificity for the diagnosis of invasive fungal infections.β-Glucans are conserved components of fungal cell walls which consist ofβ-1,3-linkedβ-D-glucopyranosyl units that form backbone containing randomly dispersedβ-1,6-linked side chains.β-Glucans can be recognized by the cell surface pattern recognition receptors (PRRs) as pathogen associated molecular patterns (PAMPs), triggering immune responses and anti-infection effects.β-glucans are also biological markers of fungal infections. Dectin-1 (Dentritic cell-associated C-type lectin-1), a type-II transmembrane protein containing a single extracellular C-type lectin-like carbohydrate recognition domain (CRD) and a cytoplasmic domain with an immunoreceptor tyrosine-based activation-like motif (ITAM), has been identified as the majorβ-glucan receptor on leukocytes, which can specifically recognize a variety ofβ-1,3-linked andβ-1,6-linked glucans, as well as yeast particles. The present study is initiated to (1) construct the prokaryotic expression vector for the extracellular carbohydrate recognition domain (CRD) of Dectin-1 from the mouse peritoneal macrophages, (2) to generated the Dectin-1 CRD fusion protein through in vitro induction and affinity purification, and (3) to investigate its ability to recognizeβ-glucans in fungal cell walls. Finally, the specificity of Dectin-1 CRD in the diagnosis of fungal infection was further evaluated.The present study was divided into the following three parts:(1) Construction and identification of the prokaryotic expression vector for the mouse Dectin-1fusion proteinThe dectin-1 CRD gene was amplified by RT-PCR from RNA of mouse peritoneal macrophages and cloned into prokaryotic expression vector pET28a (+), the constructed pET28-Dectin-1 recombinant plasmid was identified by colony PCR, restriction enzyme digestion and DNA sequencing. The sequence analysis showed that the amplified dectin-1 CRD gene was completely correct (genbank accession No. AF262985), and its reading frames were coincident with those of the expression vector, thus suggesting that the recombinant expression plasmid pET28-Dectin-1 was successfully constructed.(2) Expression, purification, renaturation and identification of the mouse Dectin-1 fusion proteinThe constructed recombinant plasmid was than transformed into Escherichia coli BL21(DE3)strain and induced by IPTG at different temperatures for a variety of time durations. The optimal induction conditions for the fusion protein were determined by SDS-PAGE and Western blot. The results showed that the fusion protein showed a specific positive signal at a relative molecular weight of 22 KD, and the positive signals of the inclusion bodies were stronger than those of the supenatants, suggesting that the constructed recombinant plasmid pET28-Dectin-1 could express mouse Dectin-1 fusion protein in Escherichia coli BL21(DE3), and the fusion protein mainly existed in the form of inclusion bodies. The proteins from the inclusion bodies were collected and soluble fusion proteins were approached after affinity purification and renaturation.(3) Initial exploration of the possible role of mouse Dectin-1fusion protein to recognizeβ-glucans from the common fungal cell walls in vitroThe ability of the Dectin-1 fusion protein to recognizeβ-glucans from 8 common fungi such as candia albicans, smooth Candida mycoderma bacteria and aspergillus fumigatus was evaluated by both immunofluorescent microscopy and flow cytometry. Results showed that the soluble fusion protein can specifically bind toβ-glucans from fungal cell walls in vitro.Here we have successfully generated the prokaryotic expression vector for the extracellular carbohydrate recognition domain (CRD) of Dectin-1, the receptor for fungalβ-glucans, and the protein was efficiently induced to express in E.Coli. Its ability to recognize and bind toβ-glucans was further investigated, thus laying a good foundation for fungal detection.