Establishment Of BHK-21Cell Line Stably Expressing Bovine Mx1Protein And Its Anti-FMDV Activity

Mx proteins are one of the type I interferon-inducible antiviral proteins produced by host cells. They are the members of the dynamin superfamily that are known to block the replication of a wide range of viruses. To date, Mx proteins have been discovered in mouse, human, cattle, sheep, fish and other species. They have biological activity to inhibit replication of a variety of RNA viruses and some DNA viruses, such as influenza A, Vesicular stomatitis virus, Thogoto virus, Hepatitis B vims (HBV) and so on.Foot-and-mouth disease virus (FMDV) is one of the list A diseases notifiable to the International Office of Epizootic Disease (OIE). It is the most important veterinary pathogen that causes foot-and-mouth disease (FMD) of cloven hoofed animals, which is a serious threat to the development of animal husbandry because of its highly infectious nature, ability to cause persistent infections and long term effects on the condition and productivity. It was demonstrated that IFN-α/β could inhibit the replication of a variety of viruses, it is an important line of defense against virus in vertebrates. Studies have demonstrated that IFN-α/β could inhibit the replication of FMDV, and the inhibition of FMDV replication involves two IFN-stimulated-gene products:OAS and PKR. Mx proteins are one of antiviral proteins that are synthesized in response to interferon (IFN) stimulation. The action of Mx proteins in the prosess of anti-FMDV infection is not clear. In order to demonstrate whether the bovine Mxl protein could interfere with the replication of FMDV, bovine Mxl gene was subcloned, the prokaryotic expression vector of pET-32a/bMxl and eukaryotic expression vector of pcDNA3.1/bMxl were constructed, and the recombinant BHK-21cell clone which constitutively express bovine Mxl were successfully established by G418resistant selection. The antiviral activity of bovine Mxl against FMDV at cell level was accessed. Research methods and the results are as follows:1. Cloning and construction of prokaryotic expression vector of bovine Mxl gene and preparation of its polyclonal antibody:cDNA of bovine Mxl gene was obtained using reverse transcription polymerase chain reaction(RT-PCR) amplification of total RNA extracted from Chinese Holstein Cows peripheral blood lymphocytes. The recombinant plasmid pET-32a/bMxl was constructed by inserting the amplified products into pET-32a(+) which were both digested by Kpn I and Xho I. The recombinant plasmid was identified by PCR, restriction endonuclease reaction and sequencing. Then the eukaryotic expression recombinant pET-32a/bMxl plasmid was transformed into the engineering bacterium Rossetta-gami (DE3), induced by IPTG, fusion protein was analysed by SDS-PAGE and Western blot; fusion protein was purified by passing extractover a Ni-agarose column; The Laboratory mice were immunized by purified fusion protein for four times and antiserum were obtained, the titer of antibody serum was estimated by agar diffusion test. The results implied that the bovine Mxl gene was cloned correctly, the prokaryotic expression vector pET-32a/bMxl was successfully constructed, the recombinant bovine Mxl protein was highly expressed under conditions which was kept for6hours in28℃and1.0mmol/L IPTG. Trough SDS-PAGE, indicated that the fusion protein of bovine Mxl was highly expressed in E.coli BL21(DE3) which accounted for28%of total bacterial protein; The Western blot showed that bovine Mxl fusion protein could be combined with the bovine Mxl antibody specifically; The agar diffusion test showed that the antibody titer was1:32.2. Construction and identification of recombinant BKH-21cells that expressing bovine Mxl protein: The eukaryotic expression vector pcDNA3.1/bMxl was constructed firstly. Then BHK-21cells were transfected by the Lipofectamine2000procedure with the vector pcDNA3.1/bMxl and pcDNA3.1(+). Cells were selected by G418, and the bovine Mxl protein steady expression pattern was confirmed by PCR, RT-PCR, Western blot and indirect immunofluorescence assay (IFA); The genetic stability of bovine Mxl protein was detected by PCR, RT-PCR after20times of cell passages. The results of PCR and RT-PCR showed that a specific band about1947bp were amplified; the result of western blot showed that the dominant band had an apparent molecular mass of75kDa consistent with the predicted size of bovine Mxl protein; The result of IF A showed that the bovine Mxl protein in cells was expressed; The results of genetic stability showed that a specific1947bp band was amplified from the recombinant BHK-21cells after20times of cell passages. These results demonstrated that the recombinant BHK-21cells which constitutively express bovine Mxl were successfully established, and its genetic stability was maintained3. The antiviral activity against FMDV of bovine Mxl protein:100TCID50FMDV serotype O was inoculated onto bovine Mxl expressing BHK-21cell clone. The relative copy number of FMDV VP1gene were detected by real-time reverse transcriptase PCR at12h、24h、36h and48h post infection, cultures were harvested at different time points post infection and virus titers were further determined onto BHK-21cell monolayers by the50%tissue culture infective dose method (TCID50), to confirm these results, indirect immunofluorescence(IFA) was then performed20h after infection. The results showed that the presence of CPE in bovine Mxl expressing BHK-21cells infected with FMDV were delayed compared with that of the control, and the typical CPEs appeared in bovine Mxl expressing BHK-21cells were more severe than that of the control; The relative copies number of FMDV VP1gene were profoundly reduced in bovine Mxl expressed clones by45%,60%,25%and10%respectively; the TCID50in recombinant BHK-21cells were lower than that of empty voctor control, viral yields were reduced about10fold by bovine Mxl expression at24hours post infection; the result of IFA showed that the intensity of fluorescence in the bovine Mxl recombinant BHK-21cells was lower the control. These results demonstrated that the bovine Mxl protein could effectively interfere with the replication of FMDV.The bovine Mxl recombinant BKH-21cells that constitutively express bovine Mxl protein was constructed, and its anti-FMDV activity was preliminarily examined. This study provided basic data for further studying on the anti-FMDV molecular mechanisms of bovine Mxl and molecular breeding of domestic animal.