Clone And Expression Of Fusion Gene Of THYα1-IFNα2b And Activity Analysis Of Fusion Protein

Thymosin alpha1 (Talpha1) is a 28-amino acid peptide, a well-known immune system enhancer for the treatment of various diseases. In the present investigation, the effects of Talpha1 on the proliferation and apoptosis of human leukemia cell lines (HL-60, K562 and K562/ADM). Interferons (DFNs) are a family of cytokines, or protein hormones, which modulate the immune response and provide resistance to viral infection. There are multiple members of the type-I IFN family, including IFN-a .Many studies have explored the effects of immunotherapy, alone or in combination with thymosin alpha 1 and interferon (IFN). Evidence has been provided that combined treatments with thymosin alpha 1 (T alpha 1) and interferon (IFN) are highly effective in restoring several immune responses depressed by tumor growth and/or cytostatic drugs. For this reason, when combined with T alpha1and IFN, they are able to increase the anti-tumor effect of chemotherapy while markedly reducing the general toxicity of the treatment.At present, there are mainly combination with thymosin alpha 1 and interferon (IFN). But there were few reports about expression thymosin alpha 1 and interferon as a integrated protein.In this study, we expression thymosin alpha 1 and interferon recombinant gene, and get the fusional protein. First, The expression plasmid pET28a Vector was used as the vector to construct the expression plasmids pEToy1 Vector by technology of gene engineering.We design a linker contain 3 amino acids, THYa1 and IFNα2bwas conjunction together by this linker. And two enzyme cutting sited and initiator/ terminal codons were add to this fusion genes.we obtain the fusion gene by PCR. We sequencing the pGEM-T vector which contain the fusion gene,obtain the interest fragment ,608bp.We used the expressional vector pEToy1,constructed a expressional plasmid contained the fusion gene.Through enzyme cutting, after sequencing ,screening and characterization, The consult indicated that the insertion sequences was accord with the fusion gene that designed. And it indicated that we constructed pEToy1 plasmid which contained the fusion gene of thymosin alpha 1 and interferon succeed .The recombinant plasmids were transfected into Bacterium coli BL21(DE3)cells, induced by IPTG at 37℃, transcript and expression. And there was a 22KDa protein in polyacrylamide gel electrophoresis. The results showed that the recombinant plasmids could express the interest proteins. We obtain the pure protein through inclusion by degeneration, renaturation, and DEAE-Sepharose–FF ion exchange chromatography.We have detect the active of the pure protein,The results showed that this fusional protein has anti-virus activity of IFN, the specific activity about 9.3×106U/mg.and it also has the activity of thymosin alpha 1,about 12.2%.