The Construction of Prokaryotic Expression Vector for Human Gene IL-24 and Expression and Purification of Its Protein

doi:10.13560/j.cnki.biotech.bull.1985.2016.02.011

Abstract

Abstract: This work aims to construct a prokaryotic expression vector for human gene IL-24, and express and purify soluble IL-24 protein via ELP-Intein system.The human gene IL-24 without signal peptide was amplified by PCR and cloned into vector pET-ELP-Intein, and the recombinant expression vector pET-ELP-Intein-IL-24 was constructed.The recombinant plasmid was transformed to Escherichia coli BLR(DE3), and the expression was induced at 20℃ by IPTG.The soluble IL-24 protein was purified based on the transformation of ELP protein at different temperatures and the self-cleavage reaction of Intein protein.The purified protein was identified by Western blot.The bioactivity of IL-24 protein was measured by Annexin V-FITC/PI apoptosis detection kit.The recombinant expression vector pET-ELP-Intein-IL-24 was successfully constructed, and the soluble IL-24 protein was expressed for the first time by prokaryotic vector and purified.Western blot analysis showed the target protein specifically bound with IL-24 antibody, which proved that the purified protein was indeed IL-24 protein.Apoptosis detection experiment confirmed that IL-24 protein significantly induced the apoptosis of hepG2 cells.

Key words: IL-24, recombinant plasmid construction, prokaryotic expression, protein purification

YU Fang, YANG Zhong-hua, FAN Han-dong, ZUO Zhen-yu. The Construction of Prokaryotic Expression Vector for Human Gene IL-24 and Expression and Purification of Its Protein[J]. Biotechnology Bulletin, 2016, 32(2): 84-89.

References

[1]Jiang H, Lin JJ, Su ZZ, et al.Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression[J].Oncogene, 1995, 11(12)：2477-2486.
[2]Dent P, Yacoub A, Hamed HA, et al.The development of MDA-7/IL-24 as a cancer therapeutic[J].Pharmacol Ther, 2010, 128(2)：375-384.
[3]兰观华, 邵增务.癌特异性的细胞凋亡诱导因子MDA-7/IL-24的研究进展[J].中国骨肿瘤疾病, 2008, 7(1)：50-53.
[4]王少慧, 郑锐青, 孙万邦.mda-7/IL-24及其抗肿瘤机理研究进展[J].国际免疫学杂志, 2013, 36(1)：36-39.
[5]Panneerselvam J, Munshi A, Ramesh R.Molecular targets and signaling pathways regulated by interleukin(IL)-24 in mediating its antitumor activities[J].Journal of Molecular Signaling, 2013, 8(15)：1-14.
[6]Lee KM, Kang HA, Park M, et al.Interleukin-24 attenuates beta-glycerophosphate-induced calcification of vascular smooth muscle cells by inhibiting apoptosis, the expression of calcification and osteoblastic markers, and the Wnt/beta-catenin pathway[J].Biochemical and Biophysical Research Communications, 2012, 428(6)：50-52.
[7] Bhutia SK, Das SK, Azab B, et al.Targeting breast cancer-initiating/stem cells with melanoma differentiation-associated gene-7/interleu-kin-24[J].International Journal of Cancer, 2013, 133(11)：2726-2736.
[8]梁光辉, 赵军.白细胞介素24抗肿瘤治疗的研究进展[J].中国血液流变学杂志, 2011, 21(2)：375-377.
[9]Whitaker EL, Filippov VA, Duerksen-Huqhes PJ.Interleukin 24：Mechanisms and therapeutic potential of an anti-cancer gene[J].Cytokine & Growth Factor Reviews, 2012, 23(6)：323-331.
[10]Banki MR, Feng LA, Wood DW.Simple bioseparations using self-cleaving elastin-like polypeptide tags[J].Nature Methods, 2005, 2(9)：659-661.
[11]Cheng H, Zou L.IL-24 expression at maternal-fetal interface and its roles in trophoblast invasion[J].Huazhong Univ Sci Technolog Med Sci, 2008, 28(4)：456-459.
[12]卫裴, 梁杰.IL-24抗肿瘤机制及其对瘫痕疙瘩作用研究进展[J].齐齐哈尔医学院报, 2013, 34(1)：83-85.
[13] 包红, 崔逢德.白细胞介素24及其抗肿瘤特性的研究进展[J].延边大学医学学报, 2010, 33(2)：148-151.
[14]Gupta P, Walter MR, Su ZZ, et al.Bip/GRP78 is an intracellular target for MDA-7/IL-24 induction of cancer-specific apoptosis[J].Cancer Res, 2006, 66(16)：8187-8189.
[15]Wang Z, Lv J, Zhang T.Combination ofIL-24and cisplatin inhibits angiogenesis and lymphangiogenesis of cervical cancer xenografts in a nude mouse model by inhibiting VEGF, VEGF-C and PDGF-B[J].Oncol Rep, 2015, 33(5)：2468-2476.
[16]Emdad L, Lebedeva IV, Su ZZ, et al.Melanoma differentiation associated gene-7/interleukin-24 reverses multidrug resistance in human colorectal cancer cells[J].Mol Cancer Ther, 2007, 6(11)：2985-2994.
[17]Inoue S, Branch CD, Gallick GE, et al.Inhibition of Src kinase activity by Ad-mda7 suppresses vascular endothelial growth factor expression in prostate carcinoma cells[J].Mol Ther, 2005, 12(4)：707-715.
[18]Pan XT, Zhu QY, Li DC, et al.Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth[J].Chin Med J(Engl), 2008, 121(20)：2031-2036.
[19]刘朝阳, 高丹, 徐帆洪.TRAIL114～281-IL-24融合蛋白的原核表达及其体外抗肿瘤细胞活性[J].中国生物制品学杂志, 2010, 23(9)：1-4.
[20]刘志刚, 朱健琦, 黄海珍, 等.真核表达与原核表达的德国小蠊变应原Blag2蛋白结构的光谱学研究[J].光谱学与光谱分析, 2006, 26(5)：879-881.
[21]林衡, 许崇波, 孙利慧, 等.类弹性蛋白标签在重组蛋白分离纯化中的应用[J].生物技术通报, 2012(12)：40-44.