Zebrafish CD36Binds To Bacteria And Pamp And Its Expression Can Be Induced By Fatty Acids

The scavenger receptor (SR) is one subfamily of the pattern recognition receptor(PRR) family, which is involved in the host defense against invading pathogens. SRhomologues also exist in teleost fish including Ictalurus punctatus，Oncorhynchusmykiss and Gadus morhua. However, the characterization and functionalidentification of these molecular in teleost fish is still limited. In particular, a differentmechanism may be involved in their immune response between mammals and fish.Therefore, it is necessary to further study the function and evolution of fish SRs inpathogen recognition and clearance. In an effort to investigate the SR molecular infish, we identified a class B scavenger receptor CD36from zebrafish, an importantmodel fish which is widely used for developmental genetics, biotechnology andphysiology. Then we are interested in exploring whether zebrafish CD36(zCD36) hassimilar structural characteristics and biological functions as that from mammalian.First we builted the pcDNA-CD36eukaryotic expression vector with zebrafishCD36gene ORF and pcDNA3.1/V5-His A and then transfected it into RAW264.7cell line. Stimulating the cell lines with LPS and LTA, the result of quantitativereal-time PCR showed that LPS and LTA can both elevated the expression of CD36gene, consistent with the study in vivo, demonstrating the important role of CD36gene in zebrafish’s natural immune response. Then we used pET-28a/CD36recombinant plasmid which was constructed in the eraly work to express therecombinant protein rzCD36. We expressed and purified a lot of recombinant proteinwhich was identified and analyzed by Western Blot and Mass Spectrometry to makesure the veracity. To explore the role of CD36in zebrafish innate immunity, westudied the binding of recombinant protein rzCD36to S. aureus and E. coli, which arethe models of Gram-positive bacterial and Gram-negative bacteria respectively withtwo ways: FITC Fluorescence Labeling and Western Blot. The experimental resultsshowed that rzCD36can bind to these two kinds of bacteria bothly. To further study the role of CD36in the recognition of exogenous pathogen molecules in zebrafish, weused ELISA technique to explore the capacity of combination between recombinantprotein rzCD36and LTA and LPS, which are the the cytoderm components ofGram-positive bacterial and Gram-negative bacteria respectively. The results showedthat His-CD36had a strong binding ability to these two components, which revealedthe key features of CD36in natural immunity of zebrafish. This result shows thediscrepancy with mammalian. The results showed that the class B SRs can bind LPSand LTA, deepening our understanding of the evolution and function of SRs in theinnate immune response of fish in the process of biological evolution, suggesting thatin the evolutionary process SRs may have played a role in the identification andremoval of pathogensroles starting from the fish.Furthermore, we demonstrated zebrafish CD36mRNA can be upregulated by LAin vitro cell lines. It would be interesting to further check the role of CD36in directlylinking fatty acid metabolism to immunological regulation.