Establishment Of Prokaryotic Expression System Of Feline IFNω3-THYα1 Fusion Protein

In recent years,The keeping Cats,dogs and other pets developed rapidly in our country.However,diseases particularly virus serious harm the health of the pet.Since entering the twentieth century,with the development of science and technology, antibiotic treatment and the wider use of feed additives,the constant drift and mutation of gene,growing resistance of pathogenic bacteria,environmental pollution,these factors make animals in stringent state,so that the overall immune defense of animals decreased,inhibition or impairment immune response of inactivated vaccine inoculation,and the immune protection rate will be reduced,caused development or popular of infectious diseases,animals prone to other complications or secondary infection,lead to substantial losses.Interferon(IFN) as an antiviral drug have been used in clinical practice for more than three decades,But it was not until the 80's on the records it was approved by regulatory agencies.Interferon(IFN) is the first drug of gene to market in our country.Since 1989,Interferon(IFN) has been widespread used.Approved by the international community about the treatment of dozens in indications.Interferon(IFN) is a small peptide secreted by cells With anti-virus,anti-proliferative and immune regulation,such as a wide range of biological activity.IFN-αdisplay the antiviral activity has been confirmed in xenograft animal cells.Normal cells do not spontaneously produce interferon generally,gene was inhibited in the state.Interferon gene expression and the lifting of suppression in conditions of antileptic.Although the interferon antiviral research have several decades of history,however,the research of source of cat combined with interferon and thymosin,is still not been reported in our country.IFN-ω3 Feline interferon is an typeⅠinterferon,With anti-virus,anti-proliferation and the immune regulatory function.The biological activity of interferon has a genera restriction.But there are also reports,people IFN-ω3 of other species also have the effection.IFN-ω3 has a more broad-spectrum of biological activity and higher capacity of anti-virus than IFN-α.IFN-ω3 on the immune system plays a key regulatory role.The results showed that IFN-ω3 in improving the clinical symptoms and mortality has obvious therapeutic effect.In particular,the inhibitory effect of parvovirus with immediate effect.Thymosin(thymic peptide) is a class of peptide hormones secreted by the organization of thymic epithelial cells,Thymosin can promote maturation of T cells and to participate in the neuroendocrine system and immune system activation of cellular immune interactions at the same time.Since 1949,Roberts extract preparation thymopeptide with calf thymus,Many scholars dedicated to research work in this area domestic and abroad,They has been made a great deal of progress in the extraction and isolation, physical and chemical nature of structure and function,clinical pharmacology.In recent years,thymopeptide as an immunomodulatory agent used in clinical medicine widely,Achieved satisfactory results in prevention and treatment in veterinary clinical trials,and brings new hope for viral diseases and cancer treatment.But there is excessive consumption of raw materials,and have the problem of low extraction.Interferon and thymosin with the similar and complementary in the biological activity,Such as in immune regulation,the treatment of viral hepatitis and cancer or other diseases.In recent years,interferon and thymosin on a joint applied research confirmed that their joint application,with the promotion of T cell growth, proliferation,differentiation and activation of NK cell activity and other functions.It can be synergy to enhance ability of the immune and have role in the treatment of viral diseases than their more stand-alone applications.Fusion gene is the mutation through the formation of spontaneous,or application of recombinant DNA technology in vitro constructed a class of two or more different gene nucleotide acid sequence of the new gene.IFN of human combined with THY-α1 achieved remarkable results in clinically.Particularly in the treatment of viral diseases,is currently one of the better treatment.In this paper,construct the two recombinant DNA with recombinant DNA technology into a prokaryotic fusion protein expression vector.In conditions of 42℃, induced high level expression of pBVIL-IFNω3,pBVIL-IFNω3-THYα1 which are prokaryotic expression genetically engineered bacteria.Fusion protein existed in the form of inclusion bodies in the intracellular in the intracellular.At about 38KDa,43KDa,shows that a obvious characteristics bands of the protein.Consistent with the theoretical molecular weight.Identified by Western blot is fusion protein which with His tag.Carried out on interest protein with denaturation,renaturation in inclusion bodies.Use the His affinity column laminar analysis the protein,The results showed that the effect of purification was a well purification,Use the thrombin digest the fusion proteins after purification,than use the affinity column laminar analysis the protein.Finally,obtain a purity of 80%of the fusion protein.IFN-ω3,IFNω3-THYα1 protein concentration were 1.6 mg/mL,0.98 mg/mL determinate by BAC kit.interest Protein concentrations were 1.28 mg/mL,0.78 mg/mL.After cytopathic effect inhibition assay proved that the interest protein of IFNω3-THYα1 with antiviral activity of the contrast stronger than the separate activity of IFN-ω3.After the virus inhibition test,determin potency according to anti-Vesicular stomatitis virus(VSV) by interferon.Use trace inhibition assay which based on cells inhibit pathogenic effect.Determination of IFNω3-THYα1 with stronger activity than single IFN-ω3 by measuring the endothelial cell of cat CRFK kidney anti-VSVanti-viral activity.In this process,Polypeptide chain unceasing to form the folding intermediates and retain partial correct folding.Final form the correct protein folding conformation.Under the vitro conditions may be to slow the pace of change in complex environmental conditions provided sufficient time for polypeptide chain to form the correct folding intermediates.Thus to promote the renaturation of protein.The experimental results of these studies will provide the useful experience for study after. In this study,has provided an important theoretical basis for the next phase of clinical research,Make a theoretical basis and research tools for to find the animals new and effective antiviral drugs and construct the high-performance products of genetic engineering interferon.