Preparation,Identification And Application Of Monoclonal Antibodies Against Porcine IFN-γ

Interferon-gamma(IFN-γ),also termed as immune interferon or type Ⅱ interferon.It can be secreted by a variety of immune cells,including activated T cells,NK cells,macrophages,dendritic cells and B cells,etc.IFN-γ belongs to Th1 cytokine,which can promote the development of cellular immune response.In addition,IFN-γ has antiviral,immunoregulatory,and anti-tumor functions.Since porcine IFN-γ(PoIFN-γ)was cloned for the first time in 1990,it has been proved that it can not only effectively inhibit african swine fever virus(ASFV),porcine reproductive and respiratory syndrome virus(PRRSV),etc.,but also has immunomodulatory effects.The level of endogenous PoIFN-γ can reflect the cellular immune response.The qualitative and quantitative detection of PoIFN-γ is important for the study of porcine immune mechanism and function,the evaluation of vaccine effect and the diagnosis of pathogenic infection.Although commercial PoIFN-γ detection reagents based on monoclonal antibodies(McAbs)are available on the market,the quantity and variety are lacking,and they all rely on imports,which are expensive and difficult to meet the demand for PoIFN-γ detection.The preparation of highly specific monoclonal antibodies against PoIFN-γ is an important prerequisite to meet its immunological detection.The aim of this study is to prepare highly specific McAbs against PoIFN-γ and to establish a preliminary flow cytometry based on McAbs for the detection of PoIFN-γ.This study will provide biomaterials and detection methods for the study of porcine cellular immune mechanism,diseases diagnosis and vaccine evaluation.1 Prokaryotic expression and bioactivity determination of Porcine IFN-γ.The total RNA was extracted from stimulated porcine peripheral blood mononuclear cells(PBMCs),and then reverse-transcribed into cDNA.The sequence of the PoIFN-γ gene coding sequences(CDs)was amplified with PCR using the cDNA as a template.The nucleotide and amino acid sequences of cloned PoIFN-γ CDs were compared with different species published in GenBanK.The similarity between the nucleotide sequences of PoIFN-γ and human,mouse,domestic dog,cow,horse,sheep and chicken were 74.9%,62.5%,82.3%,86.7%,81.1%,86.5%,and 51.4%,respectively.The similarity of amino acid sequences was 60.1%,44.6%,72.6%,77.4%,70.8%,78.6%,33.8%,respectively.The prokaryotic recombinant expression plasmid pCold-PoIFN-γ was constructed to express recombinant His-PoIFN-γ protein carrying His tag in E.coli.At the same time,different molecular chaperones were added to promote the soluble expression of His-PoIFN-γ,respectively.The SDS-PAGE result showed that the lysate supernatants of all expressed bacteria showed different levels of expected size target bands at 16 kDa,and the chaperone plasmid pTfl6 could significantly increase the soluble expression of His-PoIFN-γ.The prokaryotic recombinant expression plasmid pGEX6p-1-PoIFN-γ was constructed to express GST-PoIFN-γ protein carrying with GST tag in E.coli.The SDS-PAGE result showed that the expected size target band appeared at the 42 kDa in the lysate supernatant of the expressed bacteria.His-PoIFN-γ and GST-PoIFN-γproteins were purified respectively.SDS-PAGE and Western blot showed that the recombinant target proteins with molecular weights of 16 kDa and 42 kDa were successfully obtained with high purity.Using Vero cells as a model,the anti-VSV-GFP virus activity of His-PoIFN-γ was evaulated.The result showed that His-PoIFN-γ could inhibit the fluorescence intensity after virus infection,indicating that His-PoIFN-γ could inhibit virus replication and had good antiviral activity.2 Preparation,identification and application of monoclonal antibodies against Porcine IFN-γ.BALB/c mice aged 6-8 weeks were immunized with purified His-PoIFN-γ protein for 3 times.Adjuvants were Freund’s complete adjuvant and Freund’s incomplete adjuvant.Blood was collected from the eye frame and the antibody titers were measured.The spleen cells from mice with highest antibody titers were fused with SP2/0 cells using the hybridoma technique.The purified GST-PoIFN-γ protein was used as the detection antigen,and the positive hybridoma cells were screened by indirect ELISA.After three rounds of subcloning,a total of 25 hybridoma cell lines stably secreting monoclonal antibodies against PoIFN-γ were obtained,and named 1B6,1C12,1D3,1D11,1H3,1H4,2H1,2E10,2E12,2F2,3A10,3A11,3B7,3B9,3C7,3D6,3E6,3F1,3G8,4H3,5B3,5B6,5C4,6A4,6D7,respectively.The subtypes of 25 McAbs were identified,and results showed that 7 strains were IgGl type,14 strains were IgG2b type and 4 strains were IgM type.The titer of culture supernatant and ascites of hybridoma cells were detected by indirect ELISA,and the results showed that the titer of culture supernatant were between 1:12800 and 1:204800,and the titer of ascites were between 1:128000 and 1:65536000.The reactivity of the monoclonal antibodies with the prokaryotic expressed proteins was detected by Western blot,and the results showed that all of 25 monoclonal antibodies could react specifically with the prokaryotic expressed His-PoIFN-γand GST-PoIFN-γ proteins,showing specific target bands at 16 kDa and 42 kDa,respectively.The reactivity of the monoclonal antibody with eukaryotic PoIFN-γ protein expressed by HEK293T cells was identified by indirect immunofluorescence.And the results showed that all the McAbs could react with eukaryotic expressed PoIFN-γ protein,among which 1B6,1D3,1D11,1H3,3A11,3B9,3D6,5C4,6A4 and 6D7,total of 10 McAbs had highly reactivity and showed obvious specific green fluorescence.The reactivity of McAbs to natural PoIFN-γ was identified by flow cytometry.And the results showed that 9 McAbs,including 1B6,1H4,2E10,3C7,3E6,4H3,5B3,6A4 and 6D7,could recognize natural PoIFN-γ,among which 6A4 monoclonal antibody had the strongest reactivity.Based on 6A4 monoclonal antibody,a preliminary PoIFN-γ detection method based on flow cytometry was established by exploring the concentration of stimulant and the concentration of antibody.And the results showed that the optimal condition for detecting the secreted PoIFN-γ in porcine PBMCs by flow cytometry was to use PMA combined with ionomycin as stimulant,and the optimal stimulation concentration of PMA and ionomycin were 100 ng/mL and 500 ng/mL,respectively,and the optimal concentration of antibody 6A4-FITC was 5 μg/mL.