The Cross-Reactivity Of IL-21 Between Tree Shrew And Human

Objectives:Currently, in vitro studies have indicated the expression of interleukin-21 (IL-21) are associated with the pathogenesis and prognosis of several human liver diseases. As alternative animal models, tree shrews have been widely used in the fields of many human diseases, especially including liver diseases. In order to explore an experimental feasibility for further IL-21-related immunological study in vivo in tree shrews, the homology and cross-reactivity of IL-21 between tree shrew and human should be firstly understood.Methods:1. The homology of tree shrew and human in IL-21 was analysed. Phylogenetic trees were constructed via MEGA5.0; The alignments of a variety of experimental animals and human were compared in IL-21 gene by DNAMAN6.0, then the difference of IL-21 CDS and amino acid sequence between tree shrew and human were further analysed; Tree shrew IL-21 protein three-dimensional structure model was construsted by Discovery Studio, then the difference between tree shrew and human IL-21 molecules in hydrophobicity, secondary structure and surface charge distribution were analysed and predicted.2. A real-time quantitative PCR method to detect cytokines of splenic lymphocytes mRNA expression in tree shrew was established. Specific primers of tree shrew cytokines were designed by refering GenBank; Tree shrews’splenic lymphocytes IL-2, IFN-y, TNF-a, IL-4, IL-10, IL-17, IL-21 and GAPDH were detected by the established method; The influence of ConA on tree shrew’s spleen lymphocytes was detected by the new method.3. A pEGFP-N3/tsIL-21 eukaryotic expression vector of tree shrew IL-21 was constructed and identified. TsIL-21 cDNA was amplified from ConA-activated tree shrew lymphocytes by RT-PCR, then the eukaryotic expression plasmid pEGFP-N3/tsIL-21 was constructed through introducing the IL-21 gene into the HindⅢ/KpnⅠ site of pEGFP-N3, connection target gene fragment and pEGFP-N3, transformation recombinant plasmids into E.coli DH5a competent cells, screening pEGFP-N3/tsIL-21 plasmids and identification them; The pEGFP-N3/tsIL-21 plasmids were transfected into Huh7 cells via Lipofectamine 3000, and the expression of GFP label protein were observed by fluorescent microscope; Total RNA was isolated from pEGFP-N3/tsIL-21 transfected Huh7 cells to detect the expession of tsIL-21 gene by established method.4. The cross-reactivity of IL-21 between tree shrew IL-21 and anti-human IL-21 antibody was analysed by flow cytometry, enzyme-linked immunosorbent assay and western blot after transfection for 48 hours; The influence of recombinant human IL-21 on cytokine profiles of tree shrew splenic lymphocyte preliminarily in vitro was studied by established method.Results:1. The results of phylogenetic trees showed that tree shrew IL-21 clustered with primate IL-21, however, rodents, birds, amphibians and fish were more distance from primate; The nucleotide and amino acid sequence analysis showed that the alignment of tree shrew, rat and mouse with human IL-21 were 69.93%,64.33%, 53.59%at the amino acid level respectively, and 83.33%,68.61%,64.90%at the nucleotide level respectively; Secondary structure, hydrophobicity and surface charge in tree shrew IL-21 molecule were similar to human IL-21 molecule.2. The melting curve of established method of real-time quantitative PCR showed specific single peak with no primer dimer neither nonspecific amplification products; The correlation coefficient Rsq of the amplification curve were all above 0.990, and the amplification efficiency of Eff was about 1.0; The variation coefficients of intra-and inter-group were less than 3.5%.3. It showed that the eukaryotic plasmid pEGFP-N3/tsIL-21 we constructed was transfected into Huh7 cells successfully, and green fluorescent marker protein could be expressed normally; Huh7 cells grew well in the EG group, CG group and BG group, and dense monolayer could be formed in 48H, which indicated that there was no significant difference in the morphology and proliferation rate between the three groups; Real-time quantitative PCR analysis revealed that the expression of the pEGFP-N3/tsIL-21 in Huh7 cells was excellent and significant compared the control group.4. The results of FCM and ELISA and WB detection to tree shrew IL-21 showed that with the increase of the proportion of recombinants pEGFP-N3/tsIL-21, the expression of tree shrew IL-21 increased, which suggested that a good immunological cross reactivity between anti human IL-21 antibody and tree shrew IL-21; The results of human IL-21 ELISA kit detection to the supernatant of IL-21 levels showed that IL-21 concentrations were from 18.35 pg/ml to 47.11 pg/ml after ConA stimulation for 24 hours, while tree shrew IL-21 levels were not detected in control group; The results of tree shrew IL-21 mRNA expression detected by established qPCR method showed that tree shrew IL-21 mRNA expression was significantly higher in ConA-induced group than that in the control group (P<0.05); The results of the influence of recombinant human IL-21 on cytokine profiles of tree shrew splenic lymphocytes showed that the expression of seven cytokines in ConA group were significantly increased (P<0.05) compared to control group, while only IFN-γ and IL-10 were significantly increased in rhIL-21 group (P<0.05); Recombinant human IL-21 could induced comparably high expression of IL-10 with ConA-induced tree shrew spleen lymphocytes in vitro (P<0.05), rather than the expression of IL-2, IL-4, IL-17, TNF-α and IL-21 (P>0.05), while there was no significant difference between the two groups in the expression of IFN-γ (P<0.05).Conclusions:This study indicates well homology, structural similarity and immunological cross reactivity of IL-21 between human and tree shrew, and both these findings and new established test method suggest that a well experimental feasibility for further IL-21-related immunological in vivo study in tree shrew.