| Stem cells are a type of cells that have multiple functions such as self-renewal,proliferation,and differentiation under specific conditions.They produce more specific cell types according to the needs of the body,and achieve the purpose of tissue regeneration.Mesenchymal stem cells(MSCs)is one of the stem cell types that are studied most in research and clinical.CD271 is a specific marker expressed on the surface of MSCs and multiple tumor stem cells(CSCs),and its antibody can be used for the isolation,enrichment,and targeting of MSCs and CSCs.However,there is still no widely-accepted anti-human CD271 monoclonal antibodies in pre-clinical or clinical research,it’s of great significance to develop a novel monoclonal antibody against human CD271 with high affinity and binding ability for MSCs.The antigenicity of the protein is determined by the hydrophilicity,flexibility,secondary structure and post-translational modification of amino acid sequences.The hydrophobic residues of most protein molecules are buried inside the molecules,while the n-terminal and c-terminal peptides exposed on the surface of the molecules have good flexibility,charge and hydrophilicity,so they become better antigen selection.The extracellular region of CD271 is complex in structure with N-and O-glycosylation and phosphorylation modifications,4 repeated cysteine rich domains(CRD1-4)which requires higher spatial structure and post-translational modification of immune antigens,but the stalk region near the cell membrane has more stable structure and no glycosylation modification.Theoretically,antibodies targeting it will be more versatile.However,there are few studies on the CD271 monoclonal antibody,and it has been reported that it recognizes the CRD1 region.In this work,CD271 antigens of full-length or truncated stalk region were designed and prepared from eukaryotic expression system and prokaryotic expression system respectively.Further,two antigens were alternately used in the stages of animal immunity,serum titer detection and hybridoma screening and detection.Finally 7 anti-human CD271 hybridoma cell lines were obtained,which were identified to be with high-affinity to CD271 antigen.Flow cytometry showed that all 7 antibodies could bind to MSC and CD271-2 antibody could bind to A375-S2 and SGC expressing human CD271 molecule on the cell surface.We selected the optimal hybridoma cell line CD271-2 to analyse the sequence of the variable regions by PCR and sequencing.Mammalian cells express recombinant protein and monoclonal antibody mainly by transient expression and stable expression,in which stable expression can achieve sustained and stable quality and yield,but the construction of stable cell line is difficult.Traditional stable cell lines are mainly developed through random integration of transient gene expression plasmids and subsequent screening.Conventionally,recombinant cell lines were developed by random integration of transient GOI expression,cell lines show heterogeneity in the long-term culture process for its uncontrollability of gene insertion site which have great impact on the quality and yield in the production of biopharmaceuticals.HEK293 cells are one of the most commonly used mammalian cell expression hosts and are widely used in the field of transient transfection.This study combined CRISPR/Cas9-mediated gene site-specific integration method to establish a highly efficient stable cell line construction technology for HEK293 cells and improve the application level of HEK293 cells in the field of biotechnology drug production.Using AAVS1 as the integration site,we first verified the feasibility of site-specific integration of HEK293 T cell lines with EGFP as the model protein,and further established multiple stable expression cell lines with CTLA4 Ig as the model protein.We evaluated the consistency of CTLA4 Ig stable cell line construction process and the expression capacity of CTLA4 Ig screened and cloned,and confirmed that CRISPR/Cas9 technology mediated site-specific integration of AAVS1 gene in HEK293 T cells could achieve efficient stable cell line construction,and the yield consistency between cell lines reached more than 83%.Our method greatly simplifies the construction process of stable cell lines and has good application potential.In summary,this study successfully obtained anti-human CD271 hybridoma cell lines,which produced monoclonal antibodies were of high-affinity and recognizing ability for MSCs.In addition,we used CRISPR/Cas9-mediated gene site-integration technology to develop recombinant HEK293 T cell lines for the production of recombinant proteins and antibodies,and the process was of high efficiency and reliability,and of great potential to be applied in the production of recombinant protein and antibodies. |
PDF Full Text Request