Eukaryotic Expression And Purification Of Classical Swine Fever Virus E2 Protein And Preparation Of Monoclonal Antibody

Classical swine fever(CSF)is a highly contagious disease of swines.The World Organisation for Animal Health(OIE)stipulates that swine fever is an animal epidemic that must be reported as required by the Animal Health Code.In China,swine fever is Class I animal epidemics on the animal disease list.At present,the swine fever vaccine has been widely used in the pig industry.The main application method of swine fever prevention and control is enzyme-linked immunosorbent assay(Elisa),which detects whether pigs are produced after vaccination.However,IDEXX has monopolized competition method Elisa kit market,the domestic raw material preparation of the kit can not compete with the company.The etiological agent of CSF virus(CSFV)is an enveloped virus with a positive-sense,single-stranded RNA genome,classified as a member of the genus Pestivirus within the Flaviviridae family.The 12.5 kb CSFV genome contains a single open reading frame that encodes a 3898-amino-acid polyprotein that is ultimately yields to 11–12 final cleavage products(NH2-Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5BCOOH)through co-and post-translational processing of the polyprotein by cellular and viralproteases.The structural components of CSFV virion include the Core(C)protein and glycoproteins Erns,E1 and E2.E1 and E2 are type I transmembrane proteins with an N-terminal ectodomain and a C-terminal hydrophobic anchor.E2 is the most immunogenic of CSFV glycoproteins that induce neutralizing antibodies,providing protection against lethal CSFV challenge.Therefore,E2 protein is a key research target for CSF prevention and control.Production of E2 protein and preparation of E2 related antibodies as Elisa detection materials are of great significance for monitoring CSF.In this study,the CSF virus strain Shimen/HVRI E2,with a NCBI accession number AY775178.2:2441-3559,was studied.Through bioinformatics prediction analysis,we cut off the C-terminal 341-373 hydrophobic group amino acid and loaded a 6×His tag.Nterminal loading of melittin peptides as signal peptides,the E2 codon was optimized and was then loaded on the pUC57.The resulted pUC57-E2/pFastBac1 was digested with BamHI/XhoI and ligated.The resulted construct was recovered and transformed into DH5?.The cloned plasmid was digested and identified by sequencing.After identification,the recombinant plasmid was transferred into DH10 b for two rounds of blue and white screening under kanamycin,gentamicin,tetracycline,IPTG and X-Gal.The positive clones were identified by M13 colony PCR and the verified clones were harvested.The pFastBac1-E2 secretes and expresses the E2 protein in the insect expression system.The liposome was first transfected into the insect sf9 cells.By using P1 generation to infect P2 generation,then to P4 representing the E2 protein.We collected the culture medium and performed ultrafiltration after centrifugation.After concentration,the E2 protein was purified using an AKTA protein purification system and Ni resin.The protein was eluted with 200 mM imidazole,then desalted with G25 and concentrated to obtain a amount of 4.42 mg,with 0.5 mg/ml total protein.Furthermore,Balb/c mice were immunized with the prepared E2 protein for the first time at 60 ?g/mouse.The immuninity was boosted once every two weeks,and lasted for four times with 30 ?g/each time.The optimal antigen coating amount was determined by indirect ELISA using commercialized E2 antibody(1 ?g/mL).Indirect ELISA was performed using a checkerboard titration to determine the optimal antigen coating amount as 1 ?g/mL.After three weeks of boosting immunization,tail-tailing was performed and indirect ELISA serum titer was performed.When the immune dilution of the mouse 1# serum was detected,the P/N value was found 3 or more.Mouse 1# was selected for cell fusion.After fusion,subcloning was performed for three times.Positive clones were selected for ascites preparation and purification.Finally,antibody isotyping,antibody concentration and purity were detected.Finally,6 hybridomas were obtained,in which 2H4 was identified as heavy chain subtype IgG1,light chain subtype was Kappa,3.5 mg/mL,7 mL,total antibody mass was 24.5 mg,and purity was about 95%.Collectively,the development of the CSFV antibody ELISA kit and the application of microfluidic technology laid a solid foundation for CSFV prevention and control.