Molecular Cloning And Expression Of Porcine BST-2and Detection Of Its Biological Activity

Virus can complete infection and replication cycle after infecting host cells, dependingon the accessory factors of host cells. Meanwhile the host cells evolve many kinds of waysagainst the virus under the pressure of virus. The accessory factors as the components ofinnate immune system have antiviral activity. The accessory factors include theinterferon-inducible protein, such as TRIM-5a, APOBEC3G, APOBEC3F and BST-2(whichis also called Tetherin or CD317) and so on.Interferon was discovered by Britain virus biologists Alick Isaacs and Swiss researcherJean Lindenmann when they used chick chorioallantoic membrane to research the interferencephenomenon of influenza virus.Interferon is produced by the virus infected cells which caneffect the other cells and disturb the replication of virus. Interferon is resembled hormonalprotein which can induce human and animal cells producing many kinds of broad spectrumantiviral protein. Interferon has many kinds of biological activity, such as antiviral, antitumorand immunoregulation activity. Eukaryocyte can directly or indirectly produce a series ofproteins to resist the infection of virus.To express the fusion the antiviral protein, the sequence of porcine BST-2was amplifiedby PCR and expressed in HEK293T cells based on pcDNA3.1/V5-His vector. One of specificprimer for chicken BST-2cDNA was designed and synthesized according to the previouslypublished gene sequence of BST-2. BST-2was amplified by RT-PCR from the total RNAextracted from PK15cells. The recombinant expression plasmid pcDNA-BST-2wasconstructed and then was transfected into HEK293T cells to expresse the BST-2fusionprotein. Western blot and the expression of the recombinant protein was identified by indirectimmunof luorescence assay (IFA). The BST-2was purified and quantified by Coomassiebrilliant blue, and identified by SDS-PAGE and Western blot. The mices were immunized with the purified BST-2fusion protein to prepare the positive serum. Then its biologicalactivity was detected by the protein and the positive serum.The results showed that the construction of recombinant plasmid pcDNA-BST-2wasidentified by restriction enzyme digestion and sequencing. The expressed production hadantiviral activity against Vesicular stomatitis virus (VSV), Avian influenza virus (AIV) andPorcine reproductive and respiratory syndrome virus (PRRSV). BST-2also can restrain theproliferation of tumour cell. The research paves the way for further research on bioactivityassay and explores a new way for antiviral medication.