Preparation Of Monoclonal Antibody Against CTGF In Chinese Fire-bellied Newt And Preliminary Study On Limb Regeneration

Research background: Salamanders are amphibians that could regenerate body parts such as the tail,limb and heart.Because of its extraordinary regenerative ability,it has gradually become a model animal for the study of regeneration.Connective tissue growth factor(CTGF)can regulate cell proliferation,differentiation and migration.It also has an important impact on the reconstruction of extracellular matrix.These events not only occur during the limb regeneration of the salamander,but also are the key factors that determine the success of the regeneration.However,whether CTGF plays a role in salamander limb regeneration,and how does it affect the regeneration process,these questions have not been resolved.Methods and results: In this experiment,the pET-28a-CTGF expression vector was constructed first.After inducing the expression of CTGF fusion protein by IPTG,the fusion protein was purified and used as antigen to immunize mice.Finally,the CTGF monoclonal antibody of newt was successfully prepared by hybridoma technology.Subsequently,a regeneration model of limb was established,and samples were taken to study at 6 different times during the regeneration process.The Western Blot and the real-time fluorescence quantitative PCR were used to detect the expression of CTGF in limb regeneration at protein and nucleic acid aspects respectively.The research showed that the expression level of CTGF increased gradually and reached a peak at 42dpa(days post amputation).In addition,in situ hybridization of tissue sections revealed that Ctgf was expressed in the wound epidermis at3 dpa.Ctgf also could be detected in the blastema cells after blastema formation.At 42 dpa,Ctgf was expressed in chondrocytes.In order to further study the effect of CTGF during regeneration,the limbs were electroporated with pDsRed-C1-CTGF recombinant plasmid to increase the expression of CTGF.The results showed that CTGF over-expressing would delay limb regeneration.Conclusion: In this study,the monoclonal antibody against CTGF of Chinese fire-bellied newt was successfully prepared.The expression of CTGF increased after amputation.The spatiotemporal expression profile in the process of limb regeneration was revealed.In addition,this study confirmed that overexpression of CTGF would delay the regeneration of salamander limbs.These results provide a basis for the later study of the effect of CTGF on limb regeneration of salamander.