重组人LIF融合蛋白表达纯化及其活性鉴定

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中国生物工程杂志 ›› 2013, Vol. 33 ›› Issue (5) : 50-55.

研究报告

重组人LIF融合蛋白表达纯化及其活性鉴定

孙静, 王斌, 段志青, 胡凝珠, 李建芳, 李彦涵, 胡云章

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Expression, Purification and Bioactivity Identification of Recombinant Human Leukemia Inhibitory Factor (hLIF) Fusion Protein

SUN Jing, WANG Bin, DUAN Zhi-qing, HU Ning-zhu, LI Jian-fang, LI Yan-han, HU Yun-zhang

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摘要

目的: 原核表达重组hLIF融合蛋白并进行诱导表达、纯化及活性鉴定。方法: 将hLIF基因克隆至pThioHisA载体,构建融合表达载体pThioHisA-hLIF,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经亲和层析后,Western blot检测目的蛋白的特异性。用小鼠胚胎干细胞脱饲养层培养对纯化后的重组hLIF融合蛋白进行生物活性的鉴定。结果: 降低诱导温度和延长诱导时间能增加hLIF融合蛋白的可溶性表达,纯化后的重组蛋白纯度大于95%,Western blot检测显示了良好的特异性。在脱饲养层细胞培养条件下,添加纯化的hLIF融合蛋白能够有效的维持小鼠胚胎干细胞的未分化状态。结论: 重组hLIF融合蛋白可在大肠杆菌中高效表达,具有良好的特异性,为干细胞研究及hLIF蛋白的其他功能研究奠定了基础。

Abstract

Objective: To express recombinant human Leukemia inhibitory factor (hLIF) fusion protein in prokaryotic cells, purify and identify the bioactivity of expressed product. Methods: The hLIF gene was cloned into vector pThioHisA, and the constructed recombinant plasmid pThioHisA-hLIF was transformed to E.coli BL21(DE3) for expression under induction of IPTG. After the expression product waspurified by affinity chromatography, the fusion protein was tested for its specificity by western blot detection. To assess the bioactivity of the hLIF fusion protein expressed in E.coli, it was tested to maintain the pluripotency of the mouse embryonic stem cells in feeder-independent culture system. Results: Lower the induction temperature and prolong the induction time can increase soluble hLIF fusion protein expression. The recombinant protein reached a purity of more than 95% after purification, and its specificity was successfully proved by western blot detection. In feeder-independent culture system, adding hLIF fusion protein can effectively maintain the mouse embryonic stem cells in an undifferentiated state. Conclusion: The recombinant hLIF fusion protein was highly expressed in E.coli and showed good specificity, which lay the foundation for stem cells research and further research on hLIF biological functions.

导出引用

孙静, 王斌, 段志青, 胡凝珠, 李建芳, 李彦涵, 胡云章. 重组人LIF融合蛋白表达纯化及其活性鉴定[J]. 中国生物工程杂志, 2013, 33(5): 50-55

SUN Jing, WANG Bin, DUAN Zhi-qing, HU Ning-zhu, LI Jian-fang, LI Yan-han, HU Yun-zhang. Expression, Purification and Bioactivity Identification of Recombinant Human Leukemia Inhibitory Factor (hLIF) Fusion Protein[J]. China Biotechnology, 2013, 33(5): 50-55

中图分类号： Q786

参考文献

[1] 葛秀国, 徐小明, 李勇,等.白血病抑制因子与胚胎干细胞. 中国生物工程杂志, 2003, 23(12):22-30. Ge XG, Xu XM, Li Y, et al. Leukemia inhibitory factor and embryonic stem cells. China Biotechnology, 2003, 23(12):22-30.
[2] Cartwright P, McLeanC, Sheppard A, et al. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development, 2005, 132(5): 885-896.
[3] Guasch G, Fuchs E. Mice in the world of stem cell biology. Nat Genet, 2005, 37(11):1201–1206.
[4] Smith A G, Heath J K, Donaldson D D, et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature, 1988,336(6200):688-90.
[5] Williams R L, Hilton D J, Pease S, et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature, 1988, 336(6200):684-687.
[6] Hanna J, Cheng A W, Saha K, et al. Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc Natl Acad Sci USA, 2010, 107(20):9222–9227.
[7] Tomala M, Lavrentieva A, Moretti P, et al. Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner. Protein Expr Purif, 2010, 73(1):51-57.
[8] Imsoonthornruksa S, Noisa P, Parnpai R, et al. A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli. J Biotechnol, 2011, 151(4):295-302.
[9] 韩甫,叶荣,鲍柳君,等.小鼠胚胎干细胞无饲养层培养及其生物学特性. 苏州大学学报(医学版), 2009, 29(2):204-206. Han F,Ye R, Bao L J, et al. Culture of mouse embryonic stem cells and their biological features without feeder layer cells. Suzhou University Journal of Medical Science, 2009, 29(2):204-206.
[10] 刘登华,张苏明,闫秋月,等.无饲养层条件原代培养小鼠胚胎干细胞的实验研究. 神经损伤与功能重建, 2011, 6(3):157-160. Liu D H, Zhang S M, Yan Q Y, et al. Culture of mural embryonic stem cells under feeder-free condition. Neural Injury and Functional Reconstruction, 2011, 6(3):157-160.
[11] 赵政凯,刘畅,刘福强,等. 饲养层细胞体系及白血病抑制因子对维持小鼠胚胎干细胞未分化状态的作用. 解剖科学进展, 2009, 15(3):283-285,289. Zhao Z K, Liu C, Liu F Q, et al. Effect of feeder layer culture system and leukemia inhibitory factor on keeping mouse embryonic stem cells in undifferentiation. Progress of Anatomical Sciences, 2009, 15(3):283-285,289.