Eukaryotic Expression And Purification Of Recombinant Porcine Interferon-α And λ And Preparation Of Their Monoclonal Antibodies

Object:African swine fever virus has seriously affected the safety of Chinese pigs,and the antiviral function of interferon is very important to inhibit the infection of African swine fever.Interferon(IFN)plays an important role in antiviral,tumor inhibition and immune regulation.Interferon alpha(IFN-α),as a type I IFN,is mainly involved in the antiviral immune process.Interferon λ(IFN-λ)is type III IFN whose effect against the viral immune response is limited to epithelial tissues at higher risk of exposure and infection,in mucosal infections.Moreover,the recombinant proteins expressed through the expression system of Chinese Hamster Ovary(CHO)show that their molecular structure and biological function are most similar to those of natural proteins,and at the same time they have the advantages of high exogenous protein expression.Monoclonal antibodies play an important role in the field of immunoassay,greatly improving the specificity and sensitivity of immunological detection.This study intends to construct porcine IFN recombinant plasmid and express it efficiently through CHO expression system,so as to prepare monoclonal antibodies with high sensitivity and specificity,which will lay a foundation for future studies to better establish detection methods for different IFN molecules and establish a key technical platform to study the secretion of different cytokines in the process of porcine virus infection and immunity.Methods:(1)Construction of recombinant pig interferon(r Po IFN)plasmid: Total RNA was extracted from pig peripheral blood lymphocytes and reverse transcribed into c DNA.Primers were designed based on the sequence of the target gene in Gen Bank(α: NM＿214393.1;λ: NM＿001142837.1),and the Po IFN-α and Po IFN-λ genes were amplified by polymerase chain reaction(PCR).The pc DNA3.1 eukaryotic expression vector was digested with enzymes of Hind Ⅲ and Xho Ⅰ,purified,and then connected by T4 ligase to obtain the recombinant plasmid,which was then sequenced.(2)CHO cell expression and purification of IFN protein: Following the instructions of the Expi CHOTM expression system,the cells were revived,passaged,transfected,and the protein was collected and purified.The purified protein was then identified by SDS-PAGE and Western-blot.(3)Preparation of monoclonal antibody: immunize mice with protein emulsion,detect the titer of positive serum after three times of immunization,fuse spleen cells with SP2/0cells,establish an indirect enzyme-linked immunosorbent assay(ELISA)to screen the required positive hybridoma cells,prepare ascites after subclone expansion culture to obtain monoclonal antibody,purify the prepared antibody and appraisal it.(4)The production and distribution of IFN molecules in different parts of pigs were detected by immunohistochemistry(IHC)technique.Results:(1)Specific bands of Po IFN-α and Po IFN-λ were amplified by PCR,with sizes of 612 bp and 630 bp,respectively.Recombinant plasmids pc DNA3.1-Po IFN-α and pc DNA3.1-Po IFN-λ were successfully constructed by T4 ligase.(2)The correctly sized soluble proteins were successfully expressed and purified using the CHO eukaryotic expression system.The size of IFN-α was 20 k Da with a concentration of 1.2mg/m L,and the size of IFN-λ was 25 k Da with a concentration of 0.86 mg/m L.(3)Serum titer detection met the requirements of cell fusion.A single clone cell line(3c6)stably secreting anti-IFN-α protein and another single clone cell line(3e8)stably secreting anti-IFN-λ protein were obtained by indirect ELISA screening and 5 rounds of subcloning for both isotypes,which were identified as Ig G1 subtype and Ig Gκlight chain.Western-blotting experiments showed that the antibodies could specifically recognize IFN-αand IFN-λ,and the purified antibodies had an IFN-α titer of 1:256000 and an IFN-λ titer of 1:32000.(4)IHC revealed a widespread presence of IFN-positive cells in the spleen,with more in the red pulp than in the white pulp and marginal zone.Positive cells in the liver were mainly concentrated around the central veins.Conclusion:(1)Recombinant plasmids pc DNA3.1-Po IFN-α and pc DNA3.1-Po IFN-λ of porcine interferons were successfully constructed.(2)Soluble recombinant proteins of correct size were successfully expressed and purified,with high purity and concentration.(3)Monoclonal antibodies against r Po IFN-α and r Po IFN-λwere successfully prepared,with high sensitivity and specificity.(4)IFN-α and IFN-λ positive cells and their distribution in liver and spleen tissues were detected in ASFV-challenged pigs by using the monoclonal antibodies.