The Expression Of Human FSHR Protein And The Study On The Binding Effect Of Its Ligand

FSHR(follicle-stimulating hormone receptor)is a seven-fold transmembrane receptor,belonging to the G protein-coupled receptor family(GPCRs)members.so far GPCRs are the most receptive superfamily of the largest multi-drug target found,and their agonists or antagonists are often used to treat various diseases and play an important role in drug development.The binding assay of ligand and receptor affinity is an indispensable part of the study of drug evaluation.The combination of hormones from the structure,the change in the receptor or ligand on some of the peptide will affect the binding of hormones and downstream signal transduction,from the function to change a binding site of the amino acid,it will affect Physiological function of the normal play.Structural changes affect normal functioning.Through the analysis of its structure,we can more accurately understand the abnormal physiological function and to develop a more effective treatment。FSHR consists of 695 amino acids,consisting of extracellular domain(ECD),intracellular domain(ICD)and transmembrane domain(TMD),respectively.The ECD at the amino terminus is composed of a hydrophilic domain consisting of 349 amino residues,horseshoe or U-shaped from the three-dimensional space,and is rich in leucine-rich repeats(LRR)The combination of hormones.Previous studies of receptor FSHR have found that the extracellular domain(ECD)of FSHR is considered to be the primary determinant of hormone selective binding.The ECD contains discrete contiguous regions,which have been considered essential for hormone binding.The LRR region of the ECD contains a Leu repeating sequence(LRR)of 241 amino acids,which is confirmed to be a critical site of binding,with LRR having 12 beta folds,each with about 24 amino acid residues that specifically bind to FSH Points are located between LRR5 and LRR10,and each fold of LRR is important for the combination of hormones.By screening for synthetic peptides and FSHR structural epitopes,it was found that the 9-30 peptides linked to the signal peptide and the extracellular domain in the FSHR structure played a key role in hormone binding and signal transduction.The N-terminal region 9-30 is considered to be its unique sequence,and the 9-30 amino acid region is considered to be the neutralizing epitope of the receptor.In the crystal structure shows that a part of its amino acids are also involved in the combination of hormones.In a previous study,Dattatreyamurty etal found that FSHR9-30 was statistically dependent on the binding of hormones.Based on this,FSHR peptide antibodies were used as probes for understanding structures and functions that blocked binding or signal transduction of receptors to ligands.The currently studied FSH fragments were 1-15,33-53,51-65 and 81-95,with 33-53 and 81-95 peptides involved in receptor recognition and binding.It has recently been reported that FSH33-53 peptide binds to the targeting effect of dendrimers on ovarian cancer cells,and FSH33-53 peptides can silence ovarian cancer cells.However,with the FSH33-53 peptide binding hormone sites have not yet been reported,this study selected FSH33-53 peptide,to study its binding effect.In view of the above study,we selected the FSH33-53 peptide and ECD,LRR and 9-30 as the research materials.The ECD,LRR,9-30,29-173 and 174-331 of FSHRN were obtained in the experiment.5 different peptides of the protein,for the subsequent determination of FSH33-53 peptide binding capacity to prepare.Immunization of New Zealand white rabbits and ICR mice were used to prepare anti-ECD,LRR and 9-30 polyclonal antibodies,and the blocking effect of the polyclonal antibody was tested.The binding force of FSHR was identified from the side.Thus providing a reference for the future design of drugs and vaccines targeting FSHR.The research includes the following two aspects: 1、 FSHR protein expression and polyclonal antibody preparationFirst,different fragments of FSHR extracellular domain were prepared by constructing recombinant plasmids.The ECD,LRR,29-173 and 174-331 gene fragments of h FSHR were amplified by PCR with FSHR as template.The recombinant plasmid was constructed on pET22 b vector.The recombinant plasmid was correctly constructed by digestion and sequencing.And the protein size was 43 kDa,32 kDa,22 kDa and 20 kDa,respectively,and the protein size was consistent with the expected result.The protein was transfected into BL21 E.coli and purified by Ni ion purification column.The antigenic activity of prokaryotic proteins was detected by western blot for the preparation of polyclonal antibodies.In this case,the 9-30 peptide amino acid was less,and we obtained the 9-30-KLH protein by peptide synthesis.It is well known that prokaryotic proteins have some defects in structure and function.Therefore,we designed the eukaryotic protein expression of FSHR.First,we constructed eukaryotic expression vector pMT-FSHR,and the digestion and sequencing showed that the eukaryotic expression was correct.After transfection of Drosophila S2 cells,the transfection was successfully carried out by fluorescence microscopy.The supernatant was transfected for 5 days.The eukaryotic expression of FSHR was detected by SDS-PAGE.However,the eukaryotic protein was not obtained.Protozoin ECD,LRR and 9-30 peptides were immunized with New Zealand white rabbits and ICR mice.Four sera were collected and the antiserum titers were 1: 128000,1: 64000 and 1: 64000,respectively.The results of immunoprecipitation test showed that polyclonal antibody could bind to FSHR on the surface of Caov-3 cells.Crossover experiments with polyclonal antibodies showed that there was a cross reaction between anti-ECD polyclonal antibody and anti-LRR polyclonal antibody,whereas anti-9-30 polyclonal antibody did not cross-react with the two polyclonal antibodies.2、 FSHR combined with blocking experimentsIn the previous experiments,we obtained the ECD,LRR,29-173 and 174-331 proteins of hFSHR,and synthesized the short peptide protein hFSHR9-30-KLH.A polyclonal antibody against ECD,LRR and 9-30 was also obtained.The affinity of the receptor and ligand was 0.21 × 10-6,0.45 × 10-6 and 0.056 × 10-6 mol / L,0.43 × 10-6 and 1.1 × 10-6,respectively.The polyclonal antibody was tested by ELISA.Combined with FSHR.The results of this study show that human FSHR recombinant proteins ECD,LRR,FSHR9-30,29-173 and 174-331 have been successfully obtained from the experimental results,and ECD,LRR,9-30 proteins have good antigen specificity,In the expression of human FSHR eukaryotic protein,but failed to obtain eukaryotic protein.In the preparation of polyclonal antibody,we obtained polyclonal antibodies against ECD,LRR and 9-30,and the antibody titers were higher.In the antibody cross-experiments,anti-ECD polyclonal antibodies were found to cross-bind with anti-LRR polyclonal antibodies,and there was no cross between the anti-9-30 polyclonal antibody and the two polyclonal antibodies.Finally,the affinity of ECD,LRR,9-30,29-173 and 174-331 and FSH 33-53 peptides showed that the affinity of 9-30 was higher,and the blocking effect of anti-ECD was higher than others.May be long peptide length,spatial structure is relatively complete,so the blocking effect is strong.In this study,by analyzing the binding and blocking of different functional fragments of FSHR,it was suggested that the higher binding sites of FSH33-53 peptide and its receptor were located in the 9-30 region of the amino group,but the LRR region was also in the process of FSH binding Play a role.The results provide a reference for the future design of drugs and vaccines targeting FSHR.