Abstract
Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame (ORF) of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers (CD40, CD54, CD80, and CD86), and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses.
Highlights
The cotton rat (Sigmodon hispidus) was first used in polio research in the 1930s [1], and throughout the last century, it has proven to be an excellent model for biomedical research [2, 3, 4]
A sequence corresponding to nucleotides 535 through to the poly-A tail was obtained using the 3’ RACE kit and mRNA as starting material, which was isolated from cotton rat splenocytes and a rodent consensus sequence as a primer
The 5’ end of the protein was obtained in the step by PCR amplification of the cDNA obtained in the first step with the 3’ RACE kit and the reverse complement of the consensus sequence primer and a second consensus sequence primer designed to bind to the beginning of the CD40 ligand (CD40L) mRNA
Summary
The cotton rat (Sigmodon hispidus) was first used in polio research in the 1930s [1], and throughout the last century, it has proven to be an excellent model for biomedical research [2, 3, 4]. Cotton rat CD40 ligand and ophthalmic) [6], influenza (orthomyxovirus) [7, 8], HIV-1 [9], RSV (respiratory syncytial virus) [10], adenovirus [11, 12], human parainfluenza [13], and human metapneumovirus [14] This model has been valuable for adenovirus-based gene replacement therapy research [15, 16], and was proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies Depending on the post-translational modification, the murine CD40L is a 32–39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4+ T cells [21, 22]. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production
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