Project description:Display technologies, e.g., phage, ribosome, mRNA, bacterial, and yeast-display, combine high content peptide libraries with appropriate screening strategies to identify functional peptide sequences. Construction of large peptide library and display-screen system in intact mammalian cells will facilitate the development of peptide therapeutics targeting transmembrane proteins. Our previous work established linear-double-stranded DNAs (ldsDNAs) as innovative biological parts to implement AND gate genetic circuits in mammalian cell line. In the current study, we employ ldsDNA with terminal NNK degenerate codons as AND gate input to build highly diverse peptide library in mammalian cells. Only PCR reaction and cell transfection experiments are needed to construct the library. High-throughput sequencing (HTS) results reveal that our new strategy could generate peptide library with both amino acid sequence and peptide length diversities. Our work establishes ldsDNA as biological parts for building highly diverse peptide library in mammalian cells, which shows great application potential in developing therapeutic peptides targeting transmembrane proteins.
2020-07-19 | GSE134671 | GEO
Project description:Phage display library for antibody screening
Project description:IgNAR exhibits significant promise in the fields of cancer and anti-virus biotherapies. Notably, the variable regions of IgNAR (VNAR) possess comparable antigen binding affinity with much smaller molecular weight (~12 kDa) compared to IgNAR. Antigen specific VNAR screening is a changeling work, which limits its application in medicine and therapy fields. Though phage display is a powerful tool for VNAR screening, it has a lot of drawbacks, such as small library coverage, low expression levels, unstable target protein, complicating and time-consuming procedures. Here we report VNAR screening with next generation sequencing (NGS) could effectively overcome the limitations of phage display, and we successfully identified approximately 3000 BAFF-specific VNARs in Chiloscyllium plagiosum vaccinated with the BAFF antigen. The results of modelling and molecular dynamics simulation and ELISA assay demonstrated that one out of the top five abundant specific VNARs exhibited higher binding affinity to the BAFF antigen than those obtained through phage display screening. Our data indicates NGS would be an alternative way for VNAR screening with plenty of advantages.
Project description:We present a target-unbiased approach for antibody discovery that relies on generating mAbs against native target cell surfaces via phage display. This method combines a previously reported method for improved whole-cell phage display selections with next-generation sequencing analysis to efficiently identify mAbs with the desired target cell reactivity. This approach enabled the identification of three multiple myeloma cell surface antigens, and cognate monoclonal antibody probes.
Project description:The maternal immune system must simultaneously defend against pathogens with 25 tolerance of the antigenically distinct fetus. Despite the central importance of immune modulation during pregnancy, epitope-level analyses of the maternal antibody repertoire remain lacking. Here, we performed high-throughput, multi-isotype longitudinal serology of 119 women from preconception through pregnancy, combining phage display of linear peptides with whole protein-based antigen assays to profile antibody responses to viral and self-antigens. We observed a broad 30 expansion of the maternal IgG repertoire and a relative increase in IgA titers, both tracking with pregnancy progression. These changes may augment transplacental and mucosal immunity in the fetus and newborn, respectively. Notably, pregnancies ending in miscarriage exhibited pronounced IgA titer spikes against both viral and self-antigens unrelated to active infection (sensitivity 14/19, 74%, specificity 95%), indicating an underlying immune perturbation may 35 precede pregnancy loss.