The Screening Of Single-domain Antibodies Of Chiloscyllium Plagiosum

Background The Chiloscyllium plagiosum single-domain antibodies(sdAbs)is the smallest and complete antigen-binding fragment currently found with a molecular weight of around 12 kDa.sdAbs have a series of characteristics such as small molecular weight,good stability,and strong antigen binding ability,so that they can cross the blood-brain barrier,serve as sdAbs molecular probes,and even have application advantages in cancer treatment,molecular diagnosis and development.Moreover,the recombinant expression can be realized,thus greatly shortening the production cycle and production cost,so it is a hot spot of research and development at home and abroad.At present,sdAbs of nurse shark are mostly studied in cartilage fish,while there are few studies on sdAbs of Chiloscyllium plagiosum.Objective At least one sdAb of Chiloscyllium plagiosum is screened.Then the bioinformatics method is used to predict its structure and analyze its potential functions.Recombinant expression experiments of sdAbs obtained by screening were performed,and preliminary study on the activity of recombinant sdAbs were conducted which can provide new technical means for drug research and development of sdAbs or nanobodies(Nbs).Methods In order to increase the diversity and richness of screening antibodies,Vibrio parahaemolyticus(VP),which is typical pathogens commonly found in seafood,and Escherichia coli TG1,a commonly used strain in the laboratory,were used as the source of immunity to inoculate Chiloscyllium plagiosum.The inoculum was resuspended in PBS buffer and the final concentration was adjusted to 5.0 x 107 cfu/mL.Six Chiloscyllium plagiosums.Group 1 was inoculated with 200 ?L of PBS buffer as control,and the second group was inoculated with 200 ?L of Vibrio parahaemolyticus(VP)and 200 ?L of inactivated Vibrio parahaemolyticus(VP(D)),respectively.The third group was inoculated 200?L of E.coli TG1 and 200 ?L of inactivated E.coli TG1(TG1(D))respectively.Then the growth state and physiological response of sharks after inoculated were observed.On the 30th day after inoculation,a second inoculation was performed,and on the 60th day,the shark was dissected,and the lesions of the spleen and liver were observed,followed by pathological section analysis of the spleen and liver.At the same time,spleen cells and blood cells were subjected to transcriptome sequencing analysis.Subsequently,quantitative proteomics analysis was performed on shark serum,and functional enrichment analysis was performed on differentially expressed proteins,so as to preliminarily determine the protein subcellular localization and involvement of cellular immune signaling pathways related to shark immunity.After that,the first round screening was conducted.The shark serum protein spectrum sequencing results were used to compare with the protein sequences in the transcript library Summary-4 cds-Trinity_gene_pep to determine the corresponding gene names,deoxy nucleotide sequences,and then the serum proteins were comprehensively analyzed through gene analysis,functional annotation,BLAST and other methods,so as to screen out the antibody genes.The second round of screening:in terms of the analysis of shark antibody structure,the protein structure homology-modeling server SWISS-MODEL was used to predict the structure of the screened antibodies,and each simulated structure was evaluated for in-depth analysis,and the structure with the highest comprehensive evaluation was selected as a reference for subsequent studies.Finally,the selected shark sdAbs were further studied.The third round of screening:The shark sdAbs gene was cloned into pET-duet-His-SUMO vector,transformed into E.coli BL21(DE3)for expression and purification,and then quantitatively analyzed.At last,the active shark sdAbs were screened by antibacterial activity assay.Results A shark transcript library containing 53,585 genes were obtained,and 42%of the gene lengths in the library were distributed between 200-300 bp,indicating that shark genes were generally short.The quantitative information of 1466 proteins was obtained by analyzing the protein spectrum data of shark serum.51 shark antibodies were successfully screened by bioinformatics analysis.The nucleic acid sequence,amino acid sequence and molecular weight of these antibodies were clarified.In SWISS-MODEL modeling server,the structure model of 16 target shark sdAbs were successfully predicted and evaluated.A recombinant shark sdAb,Comp39220_c0-sdAb,was successfully expressed and purified.It is found that this sdAb can inhibit the growth of VP obviously and there is also a certain degree of inhibition on TGI.Conclusions A shark single domain antibody,Comp39220_c0-sdAb,was screened and expressed recombinantly in vitro.The recombinant antibody has obvious antibacterial activity against VP,suggesting that this sdAb may be the specific antibody produced after immunizing VP.