| Background: Single-domain antibodies(Sd Ab)are a class of small molecule antibody with good tissue penetrability,stability and affinity,which can realize the combinatio of targeted drug brain transportation and antigen hiding epitopes.Initially,the Sd Ab were modified from the heavy-chain antibodies(HCAbs)in camel serum.Whereafter the discovery of Ig new antigen receptor(Ig NAR)in cartilaginous fishes such as nurse shark,large star shark and striped bamboo shark,a new shark-derived monodomain antibody VNAR(Variable domain of the heavy chain of the heavy-chain antibody)was obtained.As a new antibody in scientific research,the current research on VNAR shows that it has shown significant advantages in medical imaging,targeted therapy,rapid diagnosis and other fields,and has broad application potential and great economic value.In recent years,advances in phage display libraries and high-throughput sequencing technologies have led to shorter production cycles and lower production costs for VNAR,leading to a boom in VNAR research.PCV2(porcine circovirus type 2,PCV2)can induce diseases such as postweaning multisystemic wasting syndrome(PMWS),enteritis and porcine dermatitis nephropathy syndrome(PDNS)in weaning piglets,The mortality rate of these diseases can reach 40%,which will bring huge economic loss to the pig industry.Capsid protein(Cap),a structural protein encoded by the open reading frame 2 of the PCV2 genome,has major neutralizing antigen epitopes,which can induce immune protection in the body.Thus,there is a practical significance to develop monodomain antibodies against Cap proteins.Purpose: The purpose of this project is to immunize sharks with Cap proteins using striped bamboo sharks as the model organism in VNAR research,so as to obtain monodomain antibodies against Cap proteins and provide a new scheme and material basis for the rapid detection of PCV2 infection.Ultimately,it is expected to establish a convenient and rapid technology platform for screening specific VNAR by using bioinformatics methods,which provided new technical ideas for screening VNAR,and explored a method for efficient expression and rapid purification of VNAR in escherichia coli,so as to provide support for the application of VNAR.Methods:Wild striped bamboo sharks off the coast of zhangzhou city,fujian province,were domesticated in the laboratory for 1 week,and then randomly divided into experimental group and control group.1 mg/m L Cap protein(Capsid protein;Cap)and freund’s adjuvant were used to immunize the sharks in the experimental group,and normal saline and freund’s adjuvant were used to immunize the sharks in the control group,for a total of 5 immunization times,with each interval of 2 weeks,in order to induce the sharks to produce specific antibodies against Cap proteins.After immunization,the sharks were dissected and the spleen and whole blood samples were collected for transcriptome sequencing.Transcriptome data were analyzed by using gene expression differences,and genes with up-regulated expression were screened out.Then,genes related to immunoglobulin in up-regulated genes were enriched through gene annotation and functional enrichment.Finally,91 immunoglobulin related genes with up-regulated expression after Cap immunization were obtained.According to the Ig NAR sequences in immunoglobulin related genes,the universal primer of(VNAR)sequences in Ig NAR variable region was designed,and the spleen and VNAR in whole blood was amplified.Furthermore,High-throughput sequencing was performed on the amplification products to construct the VNAR library.The antibody in the antiserum was enriched by the affinity purification column prepared by antigen,and the enriched antibody mass spectrometry analyzer was used to obtain the peptide information of the antibody.The sequence alignment software was used to match the previously constructed VNAR library and the up-regulated immunoglobulin related gene sequence to screen the target antibody.The expression vector PET-22 b was used to construct recombinant plasmid to induce the target antibody to express in escherichia coli,and recombinant antibody were obtained through the Ni column purification,with double clamp method to detect resistance of recombinant antibody binding activity.Result: 1)The transcriptomic library of 143,537 unique genes and VNAR library of 3822 genes were successfully constructed,which can lay a foundation for the immune research of striped bamboo shark.2)Cap-specific related antiserum was successfully purified from shark serum which immunized with Cap protein by using Cap antigen to prepare Cap-specific antibody affinity purification column anti-serum,which established a purification method of sharkanti-serum to provide technical support for the research of VNAR.3)Eight single-domain antibodies that can specifically bind to Cap proteins were screened out.And finally,four VNAR sequences with strong antigen affinity were successfully purified and obtained,namely,shark-1171-sdab,shark-4598-sdab,shark-12458-sda and shark-22244-sdab.Conclusion:After Cap protein immunization in Striped bamboo shark,the effective VNAR sequence could be screened by high-throughput sequencing combined with mass spectrometry,which could be highly expressed in escherichia coli,and the obtained recombinant single-domain antibody had a strong antigen affinity. |
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