1 Screening And Validation Of MXRA7 Interacting Proteins 2 Preparation And Identification Of Recombinant ATP5B Epitope Polypeptide And Its Polyclonal Antibody

Objective: From our prior research on the mouse model of corneal neovascularization,we discovered that the Matrix-remodeling associated 7(Mxra7)gene was closely related to the reconstruction of extracellular mechanisms,however,there are few reports about the function and mechanism of this gene.The purpose of our study was aimed to elucidate the roles of Mxra7 gene in pathophysiology and the mechanisms of Mxra7 gene in this process.Methods:1.Prediction and screening of MXRA7 interacting proteins(1)Bioinfotmatid analysis of MXRA7 interacting proteins: bioinformatics predictions of MXRA7 interacting proteins were performed and analyzed using on-line analysis software and databases.(2)Screening of MXRA7 interacting proteins by yeast two hybrid Systems: the CDS sequence of mouse Mxra7 gene was cloned and the MXRA7 yeast expression vector was constructed.This protein was used as a "bait protein" to screen mouse c DNA library for MXRA7 interacting protein.Select a representative and potentially important research protein for subsequent validation.2.Bioinformatica analysis and identification of MXRA7 interacting proteins: the results of preliminary screening of the yeast two-hybrid system were validated using techniques such as protein co-immunoprecipitation and co-localization analysis.(1)Co-Immunoprecipitation in hepal-6 cells: Hepa1-6 cells(previously stably transfected with Mxra7-plasmid)transfected with p CDNA3.1-HA-MUP1 plasmid and performed separately by protein A/G-beads+Anti-HA and protein A/G-beads+ Anti-MXRA7(parallel experiments).Immune complexes were co-precipitated and Western blot analysis was performed using antibodies against MXRA7 and anti-HA,respectively,to determine whether there is an interaction between MXRA7 and MUP1 in the cells.(2)Co-location analysis: Hepa1-6 cells(previously stably transfected with Mxra7-plasmid)transfected with p CDNA3.1-HA-MUP1 plasmid.After 48 hours of transfection,double-labeled immunofluorescence was performed using antibody of different species and observed under Laser scanning confocal microscope.(3)His pull-down analysis: His-MUP1 and MXRA7 protein were inducible expression by prokaryotic,and His pull-down assay was performed in vitro.SDS-PAGE analysis was performed and anti-MUP1 antibody was used to detect the interaction between MXRA7 and MUP1 in vitro.3.The role of MXRA7 in blood glucose regulationConstructed Mxra7 knockout mice(controls were wild mice),and the other group was treated by tail vein injection of MXRA7 minicircol plasmids to construct Mxra7 overexpressing mice(the control group was tail vein minicircol plasmid).The effect of MXRA7 on blood glucose regulation was studied by comparing the changes in blood glucose levels after MXRA7 knockout and overexpression.Results:1.Screening and prediction of MXRA7 interacting proteins:In this study,we screened HDLBP,ALDH1L1,MUP1,GLUD1,MUP2,CYTB,TRF,CPT1 A,APOA2,RDH7,ALB,MAT1 A,SERPINA1A,MUP16,GNMT,etc.by the yeast two-hybrid system using MXRA7 protein as bait as Candidate protein.Among them,the MUP1 protein scored the highest in all candidate analyses,most likely the interaction protein of MXRA7 protein.Therefore,this part is now represented by MUP1 for follow up verification.2.Identification and bioinformatical analysis of MXRA7 interacting proteins: The proteins were extracted from liver of mice and used for Co-IP experiment.We found that MXRA7 protein could specifically pull-down MUP1 protein.Furthermore,we performed a His pull-down experiment using the purified His-MXRA7 proteins and MUP1 proteins,and found the bind between MXRA7 and MUP1 was specific and repeatable.Next,the colocalization of MXRA7 an HA-MUP1 was observed in intracellular of hepal1-6 cells.3.The MXRA7 gene can help mice remain blood glucose,and the absence or deficiency of MXRA7 could be more likely to lead to blood glucose upregulation.So it seems that MXRA7 plays a very important role in blood glucose regulation.Conclusion: MXRA7 and MUP1 can produce specific interaction effects in vitro and in vivo,and MXRA7 plays a very important role in blood glucose regulation.Objective: Prokaryotic induced expression of ATP5 B epitope concatenated peptides,immunized New Zealand white rabbits to obtain ATP5 B peptide-specific polyclonal antibodies for subsequent study of ectopic surface ATP5 B protein function.Methods: According to the ATP5 B 3D structure analysis,the epitope sequence located on the surface of the molecule was predicted and designed as a stranded peptide.The DNA sequence was synthesized and cloned into the p ET-24 a plasmid.The p ET24a-ATP5 B plasmid was transformed into E.coli,and the positive clone was screened.IPTG induced the expression of ATP5 B peptide.The purified desalted ATP5 B peptide was used to immunize New Zealand white rabbits to prepare anti-ATP5 B rabbit polyclonal antibodies.Saturated ammonium sulfate crude ATP5 B antibody was detected by ELISA.Western blot,flow cytometry and immunofluorescence were used to verify its application.Results: Successfully obtained purified ATP5 B polypeptide and its rabbit polyclone antibody with high valence,which is also apply for flow cytometry,western blot and immunocytochemistry.For the follow up application of recombinant polypeptides and antibodies for related functional studies provides a useful tool.Conclusion: The higher purity of ATP5 B polypeptidewas obtained,and the rabbit was immunized with this protein.A higher titer antibody was obtained and the specificity of the antibody was verified by a series of methods.