Preparation Of Canine Interferon ?/RTB Recombinant Fusion Protein And Its Antiviral Activity Study

Interferon(IFN)is the first immune barrier of antiviral infection.Its application field has gradually developed to antiviral and anti-tumor aspects,Canine interferon alpha(CaIFN?)is one of cytokines secreted by immune cells in dogs after being stimulated by the external specific environment.It has a series of characteristics such as broad-spectrum antiviral,increasing immune response and weak antigenicity.The antiviral effect of CaIFN? is mainly related to interferon stimulated genes(ISGs),which play an antiviral role by inhibiting the transcription,translation and protein synthesis of viral nucleic acids.However,the short half-life of CaIFN? limits its clinical application.Ricin is a potent toxic plant protein,which mainly exists in castor seeds.It consists of RTA and RTB chains which of them are linked by disulfide bonds.RTA is a toxic chain and RTB is a transport vector.The RTB chain acts as a lectin which could bind to mannose receptor on the surface of mammalian cell membrane,and transports RTA subunit into cells to exert toxicity.Previously our studies have found that the recombinant RTB could be used as an immunoenhancer to enhance the cellular and humoral immune responses.Moreover,RTB could activate macrophage and increase the phosphorylation level of JAK1,TYK2,STAT1 and other cytokines.In this study,CaIFN? and RTB were fused,and a novel interferon fusion protein was expressed.The protein was stable and could play an antiviral role in vivo and in vitro.After optimizing the dosage form,a nano preparation of rCaIFN?/RTB was developed,and the rCaIFN?/RTB nanoparticles could significantly improve the antiviral-stability of the fusion protein.Our research is divided into the following four aspects:1.Prokaryotic expression of rCaIFN?/RTB fusion proteinThe recombinant plasmid pET28a-CaIFN?/RTB containing rCaIFN?/RTB coding sequence was obtained by overlapping PCR from CaIFN?,(G4S)3Linker and RTB respectively.The recombinant expression plasmid pET28a-CaIFN?/RTB was transformed into E.coli host strain BL21(DE3).The rCaIFN?/RTB fusion protein was mainly expressed in the form of inclusion body after induction conditions as follows:IPTG concentration 0.8 mmol/L,37?,8 h.The inclusion body form of rCaIFN?/RTB was denatured and purified by ion exchange chromatography and metal chelating affinity chromatography,then renatured to obtain soluable rCaIFN?/RTB fusion protein.2.Antiviral Activity of rCaIFN?/RTB Fusion Protein in VitroThe antiviral activity of rCaIFN?/RTB was detected by MDCK-VSV method.The specific activity of rCaIFN?/RTB was found to be 1.04×109 IU/mg,which proved that the fusion expression of CaIFN? and RTB did not affect the antiviral activity of CaIFN?It was also found that rCaIFN?/RTB could inhibit the replication of VSV in MDCK cells but not in WISH cells,which proved that rCaIFN?/RTB had species specificity.3.Pharmacokinetics of rCaIFN?/RTB fusion protein in dogs and its antiviral activity against canine parvovirusThe pharmacokinetics of rCaIFN?/RTB showed that the half-life(t1/2)of rCaIFN?/RTB was 49.65 h and increased 9.5 times compared with rCaIFN?after single intramuscular injection.The peak time of the drug was shortened and the bioavailability of rCaIFN?/RTB was also increased.The anti-canine parvovirus test in dogs was executed,the rCaIFN?/RTB could effectively decrease the lethal rate of canine parvovirs and prevent the canine parvovirus diseases,prevention rate was 100%.The blood routine analysis also.4.Study on nano-formulation of rCaIFN?/RTB fusion protein and its antiviral activityThe dosage form of rCaIFN?/RTB was studied.The protein drug was nanomerised by PEI10000.It was confirmed that rCaIFN?/RTB@PEI10000 nanoparticles had nanometer size(?300 nm).The antiviral activity of rCaIFN?/RTB@PEI10000 nanoparticles was detected by MDCK-VSV system in vitro.It was found that there was no decrease in the antiviral activity of protein drugs during the process of nanocrystallization,and rCaIFN?/RTB@PEI 10000 nanoparticles had the same antiviral activity.It has tolerance to pH and temperature,and can still have significant antiviral activity in extremely acidic conditions:pH 2.0,dialysis 4 hours,incubation 4 hours at 65?.This work provides a new idea and developed direction for the biological modification of canine interferon-?,and provides a new proof that biotoxin subunits can be used as an excellent carrier protein.It provides a new idea for the study of new dosage forms of canine interferon ?,and shows the great potential of PEI 10000 as a simple and effective nano-drug carrier in the study of drug stability.