การสร้างรีคอมพ์บิแนนท์โปรตีนสำหรับอินเตอร์ลูคินทูของมนุษย์ เพื่อประยุกต์ใช้สำหรับการรักษามะเร็ง

บทคัดย่อ

Interlukin-2 (IL-2) is an important cytokine signaling molecule for the growth and function of T cells in the immune system. It also enhances various T-cell functions, natural killer cells (NK cells) and lymphokine-activate killer (LAK) cells. IL-2 uses as a biological response modifier to boost the immune system in human cancer therapy. The purpose of this study is to clone the gene encoding human interleukin-2, express and purify the recombinant human interleukin-2 using the recombinant DNA technology. A gene which is specific for human IL-2 was isolated from peripheral blood mononuclear cells which stimulated with lipopolysaccharides. Total RNA was extracted and prepared the complementary DNA by using reverse transcriptase and oligo dT primer. A gene encoding the full-length mature human IL-2 (133 amino acids, 399 bp) was amplified by polymerase chain reaction (PCR). A single band at about 400 bp in length was shown on agarose gel electrophoresis and purified. The secondary PCR was performed to add the nucleotide sequences coding for the restriction sites of EcoR I, Nco I, the six histidine residues and factor Xa at 5? end, whereas BamH I, HindIII was incorporated at its 3? end. The estimated size of a 450 bp in length was observed, purified and ligated into the pJET1.2/blunt cloning vector and transformed into E.coli XL1-Blue. Recombinant plasmids were randomly selected, characterized by PCR and restriction analysis. The nucleotide sequences were verified by using automated DNA sequencing. Analysis of insert nucleotide sequences showed the perfect similarity with human IL-2 gene using GenBank database. Recombinant plasmids harboring the mature human IL-2 gene were subcloned into the Nco I and BamH I sites of the expression vector and transformed into E.coli cells. The recombinant human IL-2 was expressed under the control of T7 promoter in E.coli BL21(DE3) with IPTG induction. Western blot analysis using the antibody specific to human interleukin-2 confirmed the expression of recombinant hIL-2. The recombinant human interleukin-2 after expression and purification will be a source of application for human cancer treatment.

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