Production And Identification Of The Monoclonal Antibodys Against CD14 On Macrophage Surface Of Axolotl

Background:When humans and other mammals lose body parts,their ability to regenerate is limited.In contrast,amphibian salamanders have a strong ability to regenerate,and even in adulthood,they can automatically regenerate lost limbs.Because the limbs of salamanders are anatomically similar to human limbs,understanding how they regenerate will provide important clues to regenerative medicine.Studies have confirmed that inflammation may affect wound healing and the beginning and completion of regeneration in the whole process of limb regeneration.As an important source of pro-inflammatory and anti-inflammatory signals,macrophages play a key role in the early stage of limb regeneration of axolotl.However,the role of axolotl macrophages in regeneration needs further study.In this study,monoclonal antibodies against macrophage surface marker CD14 were prepared and identified.The antibody provides an important means for the isolation and functional study of macrophages from axolotl.Methods and Results:In this study,a model for regeneration of amputated limbs of axolotl was established,the CD14 gene was cloned,and the CD14 recombinant protein was expressed.,and successfully prepared the monoclonal antibody against the axolotl CD14.The specificity of the antibody was verified by the macrophage of the axolotl.1.The establishment of the axolotl regeneration model and CD14 transcription expression.After the amputation of the axolotl amputation,7 time points were selected to observe the regeneration situation,and samples were taken at each time point.Total RNA was extracted at the corresponding time point of axolotl regeneration,and then reversed to corresponding c DNA.Using CD14 primers of axolotl,real-time fluorescent quantitative PCR(q RT-PCR)was used to observe that the transcription level of CD14 was the highest at 3dpa of limb regeneration in axolotl.The tissue samples were embedded in paraffin and sectioned.After HE staining and Masson staining,the bud base was observed at 7dpa,elbow joint and four toes began to differentiate at 30dpa,and the differentiation of toe and elbow joint was complete at 42dpa(day post amputation).2.Cloning and identification of CD14 gene.In this study,CD14 primers with restriction sites were designed.Using PCR technology,The 296bp sequence of CD14 with restriction sites was amplified.After double enzyme restriction,CD14 was connected to p ET28a-sumo which is the prokaryotic expression vector,and transformed into the clone E.coli DH5?.The sequencing results showed that pet28a-sumo-CD14 was successfully constructed.3.CD14 protein was expressed and purified.p ET28a-sumo-CD14 was transformed into E.coli BL21(DE3).The recombinant protein CD14 with SUMO was successfully induced by IPTG.Its size was about 22 KD and verified by Western blot.After purification by a nickel column,the ultrafiltration tube was blocked to remove impurities,and the protein purity was verified by SDS-PAGE to be high.4.Preparation and identification of monoclonal antibody against CD14The mice were immunized with adjuvant emulsified CD14 antigen at 0,4 and 7 weeks.The tail vein blood of the mice was extracted,and then the serum was detected by indirect ELISA.The titer level was higher than 1:10~6.Three days before fusion,the mice were immunized by intraperitoneal injection of antigen.The mice with the highest titer were selected and killed to prepare immune spleen cells.The spleen cells were fused with SP2/0 cells by PEG4500 fusion method.After 24 hours of culture in RPMI-1640 medium containing feeder cells,hybridoma cell lines secreting CD14 antibody stably and continuously were obtained through HAT culture medium and three subclones screening.Western blot showed that the specificity of antibody binding to antigen was good.5.Acquisition and identification of macrophages from axolotlThe RPMI-1640 medium was injected into the abdominal cavity of the axolotl,and the ascites was gently drawn.After washing with RPMI-1640 medium,the precipitates were resuspended in RPMI-1640 medium with double antibodies and serum.After one day,the morphology of macrophages was observed under microscope and the photos were taken under electron microscope.Western blot,immunocytochemistry and flow cytometry were used to confirm that CD14 monoclonal antibody could bind to macrophages.