Preliminary Study On Expression,Purification And Biological Characteristics Of BRBP1-Fc Peptibody

In recent years,an antibody-like drug peptide has been rapidly developing.The peptibody belongs to the Fc fusion protein and consists of the Fc region of the IgG antibody(Hinge-CH2-CH3)and the desired protein or peptide.Since the first CD4-Fc fusion antagonists that inhibited the entry of human immunodeficiency virus into T cells in 1989,several peptibodies that have been repeatedly validated to effectively contain a series of pathological responses have successfully entered the market.Due to the fusion of the biologically functional polypeptide and the Fc region of the IgG antibody,the peptibody has the advantages of enhanced stability,affinity,ease of purification,and exerting ADCC/CDC action.The phage display technology was used in the laboratory to obtain the target binding polypeptide BRBP1 of breast cancer specific brain metastasis cell line,and it was confirmed to have high binding specificity at the cell and tissue level.The BRBP1 peptide can specifically bind to human breast cancer primary tumor cells,and exhibits a higher intensity of positive binding signal with lymph node metastasis tumor cells,further suggesting that BRBP1 peptide may bind to high metastatic potential breast cancer cells with clinical application potential.Combining the advantages of peptibodies with BRBP1 peptide,we previously expressed BRBP1-Fc peptibody by transient transfection using mammalian cell expression systems,but this expression is insufficient,and the biological function of BRBP1-Fc peptibody did not meet expectations..Therefore,the main research contents of this topic are as follows: 1.Expression and Binding Specificity Identification of BRBP1-Fc peptibody in Mammalian Cell Expression SystemPurpose: The BRBP1-Fc peptibody was successfully expressed in the laboratory using the transient transfection method of mammalian cell expression system and the binding specificity was verified.The peptibody expressed in this method have insufficient yield,purity and stability,and their binding specificity is not as expected.The protein expressed by the mammalian cell expression system has a glycosylation modification site,and the Fc segment of the peptoid can exert therapeutic effects such as ADCC/CDC.Therefore,in this chapter,we continue to select mammalian cell expression systems to express the stable cell line.The BRBP1-Fc peptibody enhances its yield,purity and expression stability and identifies its ability to specifically bind to 231-BR cells.Methods:The pFUSE-hIgG2-Fc2-BRBP1 eukaryotic expression vector constructed in th e previous stage was single-enzyme-removed,sequenced and analyzed,and then transfected i nto the tool cell CHO.After preliminary verification,the zeocin pressure screening was use d to screen about 20 Monoclonal cells were obtained by limiting dilution or limiting dilutio n.Western blot was used to identify the expression of BRBP1-Fc peptibodies in monoclona l cells.The positive monoclonal cell intracellular DNA was extracted for PCR amplification and sequenced to analyze whether pFUSE-hIgG2-Fc2-BRBP1 was correctly integrated into th e host genome,and finally stably transformed pFUSE-hIgG2-Fc2-BRBP1 CHO cells were o btained.One of the highest expression strains was selected for expansion culture,and the c ell culture supernatant was collected and concentrated,and purified by Protien G column to obtain BRBP1-Fc peptibodies.The binding specificity of BRBP1-Fc peptibody to 231-BR ce lls was verified by cell ELISA and flow cytometry.Results: After Western blot,PCR amplification and sequencing,we obtained five stable transfected pFUSE-hIgG2-Fc2-BRBP1 CHO cells,of which cell number 4 was the highest.Therefore,we selected cell No.4 for adherent expansion culture,and finally obtained a total of about 0.64 mg of BRBP1-Fc peptibody.The results of cell ELISA and flow cytometry showed that the BRBP1-Fc peptibody expressed by the stable cell line retains the binding specificity of the BRBP1 peptide,and its specificity and affinity are not comparable to those of the pre-fluorescent peptide.Conclusion: The stable pFUSE-hIgG2-Fc2-BRBP1 CHO cell line was successfully constructed and expressed in sufficient quantity and purity of the BRBP1-Fc peptibody.The binding specificity and affinity of the BRBP1-Fc peptibody to 231-BR cells were not as good as those of the prefluorescent peptides,provided that the experimental conditions were reasonable and the cell state was good.Therefore,subsequent optimization and modification of the spatial conformation of the BRBP1-Fc peptibody is required.2.Expression and Binding Specificity Identification of BRBP1-Fc peptibody in E.coli Expression SystemPurpose: The literature suggests that the length of the linker between the polypeptide and the Fc fragment in the Fc fusion protein affects its affinity,so we explored the extension of the linker length between BRBP1 and Fc,expressing BRBP1-Fc peptibody with two sets and three sets of repeater linker(GGGGS)to solve the problem of its binding specificity and poor affinity.It is known that human IgG1 is more valuable than other subtypes,and our transformation strategy selects the Fc segment of human IgG1 as a component of the peptibodies.Since this study aimed to explore the application of BRBP1-Fc peptibody in the detection of clinical specimens,it does not involve its therapeutic application,and the preliminary results indicate that non-glycosylation does not affect the binding ability of BRBP1,so we use aglycosylation modification but yield The high,time-consuming E.coli expression system performs peptibody expression and validates its biological function.Methods:In order to obtain the Fc segment of human IgG1 necessary for constructing the peptibody,the pFUSE-hIgG1-Fc2-BRBP1-Fc eukaryotic expression vector was first constructed,and then the BRBP1-Fc fragment was amplified by PCR using the eukaryotic expression vector as a template.Finally,it was ligated with pET-16 b prokaryotic expression vector to construct pET-16bBRBP1-Fc(L2),pET-16b-BRBP1-Fc(L3)prokaryotic expression vector.Subsequently,each vector was transformed into an expression strain,and the expression was induced by the inducer IPTG,the peptibody was purified by Protien G purification column,and finally the modified BRBP1-Fc peptibody with different length linker was re-verified by flow cytometry.Results : In this chapter,we successfully constructed pET-16b-BRBP1-Fc(L2),pET-16bBRBP1-Fc(L3)prokaryotic expression vector,and obtained sufficient amount of BRBP1-Fc(L2)after induction and Protien G purification.,BRBP1-Fc(L3)peptibodies.By flow cytometry,BRBP1-Fc(L2)and BRBP1-Fc(L3)peptibodies showed high binding specificity to 231-BR cells,and their binding rates reached 64% and 87%,respectively.The results of competitive inhibition also indicate that the affinity of the BRBP1-Fc peptibody is gradually increased as the linker length increases.Conclusion: The BRBP1-Fc(L3)peptibody with improved binding specificity and affinity was successfully obtained by transformation,which laid an important foundation for the clinical application of the next BRBP1-Fc peptibody.3.Specificity Identification of BRBP1-Fc peptibody and Human Breast Cancer Pathological Tissue SpecimensPurpose: The results of previous experiments in this laboratory showed that BRBP1 peptide can specifically bind to paraffin-embedded specimens of human breast invasive ductal carcinoma and is more likely to bind to primary tumors with lymph node metastasis,suggesting that BRBP1 peptide may be inclined to bind to high metastatic potential tumor cells.Since BRBP1-Fc(L3)peptibodies with three sets of GGGGS flexible linkers are known to exhibit optimal binding specificity and affinity to 231-BR cells,we selected BRBP1-Fc(L3)to verify this in this chapter to explore the potential of clinical application of BRBP1-Fc(L3).Methods:Seventeen cases of paraffin-embedded specimens from the invasive ductal carcinoma of the breast in the Zhongda Hospital affiliated to Southeast University were collected.The BRBP1-Fc(L3)peptibody and Fc control were incubated after dewaxing and antigen retrieval.HRP was incubated after washing.The labeled rabbit anti-human IgG antibody was visualized by DAB method to observe the binding of BRBP1-Fc(L3)peptibody.Results:The BRBP1-Fc(L3)peptibody can specifically bind to human breast cancer primary tumor cells,while the control Fc protein does not bind to human breast cancer primary tumor cells,indicating that BRBP1-Fc(L3)peptibody and human mammary gland Tumor-positive tumor cells specifically bind.In addition,in some specimens,the BRBP1-Fc(L3)peptibodies specifically bind to lymph node metastasis tumor cells while binding to human breast cancer primary tumor cells.Conclusion: Similar to the previous results of the laboratory,the BRBP1-Fc(L3)peptibody specifically binds to tumor cells of lymph node metastasis while binding to human breast cancer primary tumor cells.Since the peptibody is more stable than the polypeptide and the detection method is more practical for clinical use,the clinical sample volume can be expanded after the condition optimization to further explore the potential of clinical application of BRBP1-Fc(L3)peptibody.In summary,this study successfully used the E.coli expression system to express and purify the BRBP1-Fc(L3)peptibody with high binding specificity and high affinity to the breast cancer brain metastasis cell line 231-BR.Preliminary clinical pathological tissue samples showed that the BRBP1-Fc(L3)peptibody specifically binds to lymph node metastasis tumor cells while binding to human breast cancer primary tumor cells.This study laid an important experimental basis for the expression and application of BRBP1-Fc(L3)peptibody.