Cloning, Expression And Identification Of Recombinant Sustained-Release Interleukin 4
Keywords
Slow-Release Collagen Binding Domain Interleukin 4 Collagen I Slow-Release Collagen Binding Domain Interleukin 4 Collagen I
Abstract
Objective To recombinantly express the anti-inflammatory factor interleukin 4 (IL-4) that can be sustained-released. Methods Based on the amino acid sequence of mouse IL-4 and the gene for synthesizing IL-4 based on the codon preference of Escherichia coli, the collagen binding domain (CBD) was encoded by the polymerase chain reaction (PCR) method. The sequence and histidine tag sequence were connected to the IL-4 gene, and the above recombinant gene was inserted into the pET-30a(+) vector using seamless cloning technology. The recombinant protein was purified through histidine tags. The purified protein was used to induce the differentiation of M0 macrophages into M2 macrophages, while detecting the expression of genes related to M2 macrophages and the secretion of interleukin 10 (IL10). . Combined with type I collagen to verify the adsorption and sustained release functions of collagen to recombinant factors. Results IL-4 with CBD sequence and histidine purification tag was purified through nickel column and had similar biological activity to commercial IL-4. Functional experiments have confirmed that it can be adsorbed by collagen and can be released slowly from collagen. Conclusion The slow-release anti-inflammatory factor interleukin4(IL-4) gene was obtained through expression and purification in Escherichia coli. Objective To construct and identify the slow-release anti-inflammatory factor interleukin4(IL-4) gene. Methods The mouse IL-4 gene was synthesized according to its amino acid sequence(ACCESSION: NP_067258) and the E.coli codon usage preference. Collagen binding domain(CBD) and His Tag sequence were linked with IL-4 sequence by PCR amplification with primers containing CBD and His Tag sequence. PCR products were reassembled into the cloning vector pET-30a(+) by seamless cloning technique. After NI-sepharose purification, the bioactivity of fusion protein was tested by polarizing M0 macrophage to M2 macrophage through measuring the gene expression and interleukin10(IL10) secretion. In combination with collagen I, the adsorption and release of recombinant proteins by collagen I was tested. Results IL-4-CBD-His Tag was purified successfully by Nickel column and it had the similar bioactivity with commercial IL-4. The function assay indicated that IL-4-CBD-His Tag was able to bind to collagen I and to be slowly released from collagen. Conclusion The slow-release anti-inflammatory factor IL-4 gene has been successfully constructed through E.coli expression system
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Original research was done by Zhang Chong, Huang Zike, Li Siguang
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