The porcine reproductive and respiratory syndrome (PRRS) caused the serious economic damage to swine breeding around the world. It is a viral infective disease against which live attenuated and inactivated vaccines are not always successful. Development of new types of drugs such as DNA vaccines is necessary for improving the protection against the virus. DNA vaccines induce the development of both a cellular and humoral immune response. Such vaccines consist of a plasmid or viral vector with genes of potentially immunogenic proteins. The expression of these genes realized in cells of the vaccinated animal. It leads to the synthesis of antigen proteins triggering the immune response. The purpose of this work is to create a genetic construction that can be used as DNA vaccine against PRRS virus. The construction consists of the commercial vector pVAX1 and open reading frame of two structural proteins of PRRS virus, a lysosomal localization signal sequence of the invariant chain gene and regulatory elements necessary for the expression of cloned genes in mammalian cells.
Like other proteins of the cytokine family, bovine α-interferon activates and modulates antiviral state of the target cells and inhibits division and growth of the infected cells which makes it an excellent candidate as a new antiviral therapeutic agent.This study is concerned with the determination of the optimal isolation, purification and refolding conditions of the recombinant bovine interferon-α (rbIFN-α) from inclusion bodies (IBs). Main methods used were UV/Visible spectroscopy, electrophoresis, liquid chromatography and refolding by dilution.It was found that two step IBs washing with solutions containing 50 mmol/l Tris, 50 mmol/l NaCl and 3.5 mol/l urea and their subsequent solubilization in 50 mmol/l Tris-HCl, pH 9.8 mol/l Urea and 20 mmol/l β-mercaptoethanol allow us to receive the target protein in monomeric form and 53.18 ± 9.3 % purity. Further application of the anion-exchange tandem chromatography on DE 52 cellulose and toyopearl DEAE-650 M gives a possibility to remove the major impurities and obtain rbIFN-α with 80.7 ± 8.6 % purity. Refolding by dilution in the buffer containing 20 mmol/l NaPB, рН 7.4, 0.4 mol/l sucrose, 1 mmol/l L-Cys, 0.1 mmol/l L-Cystine, 1 mmol/l EDTA, 0.05 % Kolliphor EL at 10 °C followed by the protein collection allows to get the recombinant rbIFN-α in homogeneous state, with 98.43 % purity and antiviral activity about (5 ± 3.6)•106 U/mg.
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