Preparation And Identification Of SERCA1 Polyclonal Antibody Against Axolotl

Background:Axolotl is an amphibian with strong regenerative ability,which can regenerate various complex tissues and structures after limb injury.After amputation,the wound epidermis rapidly covered the stump,and within a few days,the wound epithelium thickened to form an Apical epidermal cap（AEC）.AEC-induced residual tissue dedifferentiates and attracts cells,which gather under AEC to form the blastema,which grow and differentiate into missing limb structures.Studies have found that skeletal muscle cells are an important cell source for bud formation,and play an important role in the early regeneration of the limb of axolotl,while calcium signaling plays an important role in the development,regeneration,differentiation and maintenance of equilibrium of skeletal muscle cells.Sarcoplasmic Endoplasmic Reticulum Calcium Atpase1（SERCA1）is a key molecule involved in the regulation of ca²⁺signal and participates in the reconstruction of cytoskeleton and cell membrane.This subject studied the expression of SERCA1 in the process of axolotl limb regeneration by preparing polyclonal antibody against SERCA1,which is of great significance to understand the signal mechanism of axolotl limb regeneration,and also provides an effective tool for studying the biological role of calcium ion signal in regeneration.Methods:In this paper,the limb regeneration model of Axolotl was established,and the morphological and histological dynamics of axolotl limb regeneration were photographed and observed.Then,according to the gene sequence of Axolotl SERCA1,SERCA1 primers were designed and synthesized,and the SERCA1 gene was successfully cloned by PCR.The prokaryotic expression vector pGEX-6p-1/SERCA1was constructed to induce the expression of SERCA1 recombinant protein with a size of 50KD.The recombinant protein was purified with GST-resin column and gel cutting purification method,and then immunized rabbit with purified protein as antigen to prepare polyclonal antibody.The specificity of polyclonal antibody was detected by Western Blot.Results:Through morphological and histological staining observation,it was found that after axolotl amputation,the wound epidermis rapidly covered the stump,and within a few days,the wound epithelium thickened to form an apical epidermal cap（AEC）.The stump tissue induced by AEC dedifferentiated and attracted cells,and these cells gathered under the AEC to form the blastema.That is,about 7 days after amputation,the blastema began to appear,14 days after amputation,the blastema became significantly larger.30 days after amputation,the blastema differentiated into limb tissue.Until 42 days after amputation,the limb after amputation had completely differentiated into four toes.Finally,the polyclonal antibody against SERCA1 of axolotl was prepared,and the expression changes of SERCA1 at the mRNA level and protein level in different stages of limb regeneration of axolotl were studied by QRT PCR,Western blot and immunofluorescence staining.The results showed that SERCA1was significantly up-regulated in the early stage of limb regeneration of axolotl,and the transcription and protein levels reached the peak at 7 days after amputation,and then gradually decreased to the pre-amputation level.Conclusion:In this paper,the SERCA1 antibody of axolotl was successfully prepared,and the expression and deposition of SERCA1 at the mRNA level and protein level during different stages of limb regeneration of axolotl were studied.The results showed that the mRNA and protein levels of SERCA1 increased in the early stage of limb regeneration,especially during the blastema formation stage.This study is of great significance for further understanding the factors that promote regeneration and the transformation to the field of regenerative medicine.