Preparation Of Single Chain Antibody Against Spring Viraemia Of Carp Virus Establishment Of Immunochromatographic Detection Methods

Cyprinidae is the largest family of fish,with a wide distribution and many species.Currently,there are over 200 genera and over 2000 species.The Ministry of Agriculture and Rural Affairs listed Spring viremia of carp（SVC）as a national Class II animal epidemic in the"List of Class I,II,and III Animal Epidemic Diseases"（2022）,characterized by acute onset and high mortality.The mortality rate of young fish infected with SVC is as high as90%,and the harm to Cyprinidae fish is becoming increasingly serious,causing huge economic losses.The prevention and control measures for highly pathogenic infectious diseases in aquatic animals mainly focus on prevention,and virus detection is an important component of them.Currently,the commonly used detection technology cannot meet the requirements of intensive and high-density aquaculture of aquatic animals.However,colloidal gold immunochromatography technology has the advantages of speed,convenience,and economy,which can be used for timely detection by customs,real-time monitoring of free-range farmers,and efficient prevention and control of standardized aquaculture farms.In this study,a single chain antibody fragment（Sc Fv）against Spring viremia of carp virus（SVCV）was prepared,and an immunochromatographic test strip for detecting SVCV was developed based on this.The main contents of this study are as follows:1.Construction of rabbit derived SVCV single chain antibody libraryNew Zealand white rabbits were immunized with SVCV as an immunogen by multiple booster immunizations,a total of 4 times.During the first immunization,SVCV was mixed with an equal volume of complete Freund’s adjuvant as an immunogen.After the booster immunization,SVCV was mixed with an equal volume of incomplete Freund’s adjuvant as an immunogen.After the fourth immunization（at this time,the titer of the polyclonal antibody reached 1:204,800 or more）,blood was collected from the heart to collect serum for standby.The spleen cells were isolated,and primers were designed using the framework region of rabbit derived embryonic line antibodies included on NCBI to amplify the variable region of heavy chain（VH）and variable region of light chain（VL）genes of the antibody.Then,VH and VL were spliced together with a flexible short peptide and connected to the phage vector p CANTAB 5E.Subsequently,the connecting product was transformed into Escherichia coli TG1,After 25 repetitions of transformation,a library capacity of 2.1×10⁵anti SVCV single chain antibody library.2.Screening and identification of bacteriophage librariesAfter infecting the constructed single chain antibody library with the helper phage M13K07 virus,a phage single chain antibody library was constructed.Then,using SVCV with a virus concentration of 1×10⁶TCID₅₀/m L as the target,Sc Fv was screened by biological elutriation.After eliminating Sc Fv with weak affinity,the antibody with strong affinity was eluted with glycine hydrochloric acid buffer（GLy-HCl）,and five rounds of biological elutriation were conducted.The enrichment factor of the phage reached 132 times.After that,enzyme-linked immunosorbent assay（ELISA）was used to further screen antibodies,and 10 phages with high binding ability were selected and sent to the company for sequencing.Two strains of Sc Fv with different amino acid sequences were obtained,and their sequences shared more than 90%homology with the frame region sequence of rabbit derived embryonic line antibodies included on NCBI.3.Prokaryotic expression of single chain antibodiesProkaryotic expression of the two Sc Fv strains obtained from the above screening,design specific primers containing restriction endonuclease sites to amplify the target gene,recover and purify the target gene,connect it to the PMD-19T vector,and transform it into a cloned strain DH5αA large number of target genes were cloned in.The transformed clone plasmid and expression vector p ET-32a were digested with two enzymes,then linked to the constructed recombinant plasmid and transformed into the expression strain BL21（DE3）for prokaryotic expression.After fermentation and purification,the protein expression of the two Sc Fv strains was verified by SDS-PAGE electrophoresis and Western blotting,and the two antibody proteins were labeled S1 and S2.The antibody titer of the purified antibody protein measured by ELISA is:when the original concentration of the antibody protein is 500μg/m L,the antibody titer of S1 is 1:512,and the antibody titer of S2 is 1:256.Therefore,this study selected S1 antibody as a gold labeled antibody to prepare a SVCV test strip.4.Preparation of colloidal gold immunochromatographic test stripsUsing S1 antibody protein as a gold labeled antibody,rabbit derived anti SVCV polyclonal antibody as a detection antibody,and mouse anti His monoclonal antibody as a quality control antibody,7 mm×6 cm test strip.The optimal concentration of gold labeled antibody is 50μg/m L per 200μL colloidal gold solution added 8μL K₂CO₃（0.2 mol/L）solution is the optimal labeling p H.After testing,this test strip has specificity compared to common fish viruses in detecting SVCV,and its sensitivity is:when the concentration of SVCV virus solution is≥10 TCID₅₀/m L（copy number≥7/μL）Can effectively detect the virus;The virus can be detected in the intestines and gills of zebrafish infected with SVCV,and can be effectively detected when the concentration of intestinal tissue suspension reaches 1mg/m L.Therefore,this test strip has high sensitivity,good specificity,strong stability,and has the universality of SVCV detection.In summary,the single chain antibodies S1 and S2 prepared in this study have a good ability to bind SVCV,with S1 antibody having a stronger binding ability.Immunochromatographic strips prepared using S1 as a gold labeled antibody have good specificity,repeatability,stability,and sensitivity,and can be used to detect viruses in the gills and intestines of infected fish.Therefore,the virus detection test strip with Sc Fv as the core has the advantages of economy,convenience,and efficiency,and is more suitable for virus detection of aquatic animals.It is hoped that the immunochromatographic test strip can be commercialized and industrialized as soon as possible for virus detection,achieving early detection and control of epidemics,and promoting the aquaculture industry to move towards a high-quality,green,and healthy development model.