| Swine influenza virus(SIV),a detrimental pathogen that greatly challenges swine production,is composed of three influenza A virus(IAV)subtypes(H1N1,H1N2,and H3N2).Both swine and humans can be infected with IAV and exhibit similar clinical symptoms,which could manifest as acute upper respiratory disease characterized by fever,lethargy,anorexia and nasal congestion.The serological detection methods for influenza in swine include the hemagglutination inhibition(HI)test and enzyme-linked immunosorbent assay(ELISA).Although the HI test is the standard assay for detecting antibodies against SIV,it presents some defects,such as it is subjective and may not be suitable for testing large numbers of samples.In the traditional ELISA method,polyclonal or monoclonal antibodies are commonly used,but polyclonal antibodies result in low specificity of the assay,and the production of monoclonal antibodies is time-consuming and costly.Nanobodies have several advantages,such as small size,good stability,and high affinity and specificity.More importantly,nanobodies are suitable to genetic manipulation and could be obtained in different expression systems,adding to their great potential as diagnostic and therapeutic tools.In this study,the NP protein of swine influenza virus was expressed in E.coli,and the specific nanobodies of NP protein were screened by phage display technology.A nanobody linked with a biotin acceptor peptide(BAP)was expressed and purified,which was biotinylated by the interactions between biotin and BAP.Further,a specific,sensitive,and reproducible bELISA was successfully developed based on biotinylated nanobody,which offers a new,promising method to detect anti-SIV antibodies in swine serum.The main results are as follows:(1)Expression and purification of SIV-NP protein.The recombinant plasmid pET-32a-NP was transformed into E.coli Rosetta(DE3)competent cells.The expression of the soluble SIV-NP recombinant protein was induced with IPTG.After purification,a mixture containing Freund's adjuvant and SIV-NP recombinant protein was prepared to immunize camels.(2)Screening of specific nanobodies against SIV-NP protein.A week after the fifth immunization,peripheral blood mononuclear cells(PBMCs)were isolated from peripheral blood of camel.Total RNA was extracted from PBMCs and used to synthesize the cDNA strand.The VHH gene was amplified from synthesized cDNA and cloned into the phage vector pCANTAB 5E.Finally,the NP protein specific nanobodies were screened by phage display technique.After three rounds of selection,six SIV-NP specific nanobodies were isolated.The affinity test results showed that Nb5 had higher affinity.(3)Biotinization of nanobodyThe Nb5 gene-conjugated BAP was cloned into the vector pET-21 b and transformed into E.coli BL21(DE3).The Nb5-BAP fusion protein was induced with IPTG.The nanobody was purified by a Ni-NTA column and biotinylated with a Biotin-Protein Ligase/BirA Enzyme kit.The result of indirect ELISA showed that the titer of biotinylated Nb5 was above 1:100 000.(4)Establishment and evaluation of blocking ELISA method.The results of the checkerboard titration assay showed that 1 ?g/mL of SIV-NP protein was the optimal concentration for use as the coated antigen,the optimum dilution ratio of biotinylated Nb5(1 mg/mL)was identified as 1:30000,while the optimal serum dilution was 1:10.The 109 negative swine serum samples confirm a cut-off value of 29.8% for the bELISA.In addition,the developed blocking ELISA offers good sensitivity,specificity and reproducibility.In comparison,the results of bELISA are in close agreement with the measurements obtained from a commercial ELISA kit and Western blot.In conclusion,the specific nanobodies against SIV-NP protein-were screened.Based on biotinylated Nb5,a novel bELISA was developed for detecting anti-SIV antibodies in swine serum.The developed blocking can be used for detecting anti-SIV antibody levelsand evaluating the effects of vaccine immunisation in swine serum. |
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