The Construction, Expression And Purification Of Recombinant IL-4 Fusion Protein

IL-4 is a multi-functional cytokine that plays an important role in immune system. It is secreted by activated T cells and mast cells, and mature IL-4 with a molecular weight of 18～19kDa is a kind of glycoprotein. IL-4 can stimulate the differentiation of B cell and enhance its ability of antigen presentation. IL-4 is a major modulator of class switching of immunoglobulin heavy chain, and promotes B cells to express and secrete IgE. As an autocrine cytokine of CD4+T cells, IL-4 stimulates Th0 cells to differentiate into Th2 cells. Otherwise, IL-4 can modulate the proliferation and differentiation of hematopoietic lineage cells. Dendritic cells(DCs) derived from mononuclear cells can be induced in vitro by a combination of cytokines consisting of IL-4 and granulocyte macrophage colony-stimulating factor. As the most potent professional antigen presenting cells, DCs play an important role in antitumor immune responses of human. So IL-4 has a great potency in antitumor immunotherapy, as a tumor immuno-regulator, IL-4 has been applied into the second clinical trial.Factor Xa is a kind of glycoprotein that is essential for blood coagulation. Factor Xa preferentially cleaves after the arginine residue in the amino acid sequence Ile-Glu-Gly-Arg. So it's often used to cleave fusion protein that contains a Factor Xa recognition site to obtain the native protein.Cpn10 is a subclass of chaperonins, which is a protein family in molecular chaperone. Cpn10 is a highly conserved protein that exists in most animals; it facilitates the folding and assembly of newly synthesized proteins in cell. Cpn10 can assist in both the regenerating of degenerated protein and the degrading of abnormal protein unable to regenerate. Molecular chaperone can participate in some processing of protein, including the folding, secreting and forming of native structure, but it's not the cytokine that consists part of the target protein. Improving the expression level of molecular chaperone may promote the secreting and soluble expression of target protein.To solve the problem in purification of recombinant fusion protein, affinity tag(6×His tag) is constructed in recombinant fusion protein to purify it by nickel chelating affinity chromatography. Nickel chelating affinity chromatography is a routine method of protein purification, it utilize the affinity between Ni2+ and 6×His tag to purify protein that has 6×His tag by affinity chromatography. There are three positions of 6×His tag in recombinant fusion protein: amino-terminal, carboxyl-terminal and middle.The contents of this paper are as follows:1.Expression of recombinant IL–4 fusion protein in E.coli BL21(DE3)FIL-4 gene containing sequence coding Factor Xa protease recognition site was amplified by PCR, then cloned into pMD18-T vector and sequenced. FIL-4 gene was subcloned into pET28a plasmid, and recombinant pET28a-FIL-4 plasmid was constructed and transformed into E.coliBL21(DE3).There was an obvious protein band with a molecular weight of 19kDa in SDS-PAGE. The expression level of recombinant FIL-4 fusion protein was high; but like other eukaryote proteins, most of the expression products were inclusion bodies.2.Construction of two recombinant plasmids containing Cpn10.HisCpn10 gene containing sequence coding 6×His tag was amplified by PCR, then cloned into pMD18-T vector and sequenced. HisCpn10 gene was subcloned into pET28a plasmid, and recombinant pET28a-HisCpn10 plasmid was constructed and sequenced. Using the same method, Cpn10 gene was subcloned into pET28a plasmid, and recombinant pET28a-Cpn10 plasmid was constructed and sequenced. There are multiple cloning sites in the two recombinant plasmids—pET28a-HisCpn10 and pET28a-Cpn10 that we constructed, target gene could be subcloned into the downstream of Cpn10 gene to express fusion protein in E.coli BL21(DE3).3.Expression of recombinant fusion protein HisCpn10-FIL-4 and Cpn10-HisFIL-4 in E.coli BL21(DE3)IL-4 gene containing sequence coding Factor Xa protease recognition site and 6×His tag was...