Antigen Specific VNAR Screening in Whitespotted Bamboo Shark (Chiloscyllium plagiosum) with Next Generation Sequencing
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ABSTRACT: 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: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.Next-generation sequencing (NGS) will be another method for VNAR screening with a number of advantages.Sequencing the obtained VNAR libraries through NGS can quickly and efficiently obtain immune-specific sequence information, which provides a basis for further screening of specific high-affinity antibodies.
2024-12-28 | GSE285001 | GEO
Project description:Epitope mapping and antigen discovery by T7 phage display
Project description:Induction of adaptive immune responses to commensal microbes is critical for intestinal homeostasis, and perturbation of these responses is associated with multiple chronic inflammatory disorders. However, the mechanisms underlying the induction and regulation of mucosal B cells targeting commensal microbes remain poorly understood, in part due to a lack of tools to identify commensal-specific B cells ex vivo. To address this, we first sought to identify immunodominant protein epitopes recognized by Segmented Filamentous Bacteria (SFB) specific serum antibodies using a whole-genome phage display screen and identified immunogenic proteins engaging IgA, IgG1 and IgG2b responses. Using these antigens, we generated B cell tetramers to identify and track SFB-specific B cell responses in the gut associated lymphoid tissue during natural and de novo colonization. We identified a compartmentalized response in B cell activation between Peyer’s patches and mesenteric lymph nodes, with a gradient of IgA, IgG1 and IgG2b isotypes along the small intestine, and selective production of IgG2b with the mesenteric lymph node chain. VDJ sequencing analyses and generation of SFB-specific monoclonal antibodies identified that somatic hypermutation drives affinity maturation to SFB derived antigens under homeostatic conditions. By combining phage display screening and B cell tetramer technologies, we now enable antigen-level based studies of immunity to intestinal microbes, which will advance our understanding of the ontogeny and function of commensal-specific B cell responses in tissue immunity, inflammation and repair.
Project description:Phages are viruses that infect prokaryotes and can shape microbial communitiesby lysis, thus offering applications in various fields. However, challengesexist in sampling, isolation and accurate prediction of the host specificity ofphages as well as in the identification of newly replicated virions in response toenvironmental challenges. A new workflow using biorthogonal non-canonicalamino acid tagging (BONCAT) and click chemistry (CC) allowed combinedanalysis of phages and their hosts, the identification of newly replicated virions,and the specific tagging of phages with biotin for affinity chromatography.Replication of phage λ in Escherichia coli was selected as a model for workflowdevelopment. Specific labeling of phage λ proteins with the non-canonicalamino acid 4-azido-L-homoalanine (AHA) during phage development in E. coliwas confirmed by LC–MS/MS. Subsequent tagging of AHA with fluorescentdyes via CC allowed the visualization of phages adsorbed to the cell surfaceby fluorescence microscopy. Flow cytometry enabled the automated detectionof these fluorescent phage-host complexes. Alternatively, AHA-labeled phageswere tagged with biotin for purification by affinity chromatography. Despitebiotinylation the tagged phages could be purified and were infectious afterpurification. Applying this approach to environmental samples would enablehost screening without cultivation. A flexible and powerful workflow for thedetection and enrichment of phages and their hosts in pure cultures has beenestablished. The developed method lays the groundwork for future workflowsthat could enable the isolation of phage-host complexes from diverse complexmicrobial communities using fluorescence-activated cell sorting or biotinpurification. The ability to expand and customize the workflow through thegrowing range of compounds for CC offers the potential to develop a versatiletoolbox in phage research. This work provides a starting point for these furtherstudies by providing a comprehensive standard operating procedure.
Project description:High-density phage epitope microarray from 31 samples were used for unsupervised analysis (GSM36153...GSM36183). 129 samples from prostate cancer patients and controls were screened on small focused epitope chips, which contained 180 phage elements. These data were used to train GA/KNN program (GSM36184...GSM36312). 128 samples from localized prostate cancer patients and controls were screened on small focused epitope chips. These independent data were used to validate the epitomic profile (GSM36313...GSM36375, GSM40203...GSM40213, GSM40216, GSM40218, GSM40219, GSM40222, GSM40225, GSM40227, GSM40229, GSM40233, GSM40237, GSM40246...GSM40294). Three subgroups of samples were used as test sets to validate the specificity of epitomic profile (GSM36376...GSM36410, GSM40214, GSM40215, GSM40217, GSM40220, GSM40221, GSM40224, GSM40226, GSM40228, GSM40231, GSM40234...GSM40236, GSM40238...GSM40244). Project----Identification of humoral signature for prostate cancer diagnosis We constructed a prostate cancer cDNA phage display library. cDNAs were reverse-synthesized from mDNA pool isolated from prostate cancer tissues. Enzyme-digested cDNA fragments were then inserted into phage vector to make a whole prostate cancer phage expressed cDNA library. In order to select cancer specific phage epitope from this library, we performed several cycles of affinity enrichment. We used the bounded IgG pool isolated from prostate cancer patient sera to select the tumor specific phage epitope clones. Once we had the enriched phage epitope library, we cultured the phage library on LB-agar dish for individual phage colonies. About 2300 phage colonies from agar dish were picked up using toothstick and cultured in 96-well microtiter plates. Each clone was labeled as microtiter plate #, column #, row#, i.e. clone ID. These 2300 clones were then spotted on slides in single spot (no any duplicate), i.e. each spot (labeled by clone ID) represents a single phage clone. The phage epitope microarrays were then screened using cancer or control sera. We employed two color system. Cy5-anti human IgG was to detect human IgG. For green color, we used Cy3-labeled anti-phage capsid protein as internal reference to normalize the ammount difference of phage particles spotted on each spot. Thus the ratio of Cy5/Cy3 would count for the immune response in cancer or control sera. Once we identified humoral signature in prostate cancer patients, we could sequence the phage clone to characterize the nature of the genes or proteins.
Project description:This study demonstrates the dichotomous activities of APRIL and BAFF in MM and DLBCL, which can be safely targeted by an engineered fusion protein designed to trap both ligands with ultrahigh binding affinity.