中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (19): 3476-3480.doi: 10.3969/j.issn.1673-8225.2011.19.013

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

Flt3配体胞外域的原核表达纯化及对脐血CD34+细胞的扩增作用

刘  强1，郑  瑾2，赵星成3，尹郸丹1，陈任安1，刘  利1，郝淼旺1，梁英民1   

  1. 解放军第四军医大学唐都医院，1血液内科，2中医科暨中西医结合肿瘤科，陕西省西安市   710038；3解放军第四军医大学医学遗传与发育生物学教研室，陕西省西安市   710032
  • 收稿日期:2010-11-10 修回日期:2010-12-13 出版日期:2011-05-07 发布日期:2011-05-07
  • 作者简介:刘强★，男，1972年生，辽宁省丹东市人，汉族，2003年解放军第四军医大学毕业，硕士，主治医师，主要从事造血干细胞研究。 liuqiang@fmmu.edu.cn
  • 基金资助:

    国家高技术研究发展计划(863计划)(2006AA02A111)。

Prokaryotic expression and purification in Flt3 ligand extracellular domain and effects on cord blood CD34+ cells amplification

Liu Qiang1, Zheng Jin2, Zhao Xing-cheng3, Yin Dan-dan1, Chen Ren-an1, Liu Li1, Hao Miao-wang1, Liang Ying-min1   

  1. 1Department of Hematology, 2Department of Traditional Chinese Medicine, Tangdu Hospital, Fourth Military Medical University of Chinese PLA, Xi’an  710038, Shaanxi Province, China; 3Department of Medical Genetics and Developmental Biology, Fourth Military Medical University of Chinese PLA, Xi’an  710032, Shaanxi Province, China
  • Received:2010-11-10 Revised:2010-12-13 Online:2011-05-07 Published:2011-05-07
  • About author:Liu Qiang★, Master, Attending physician, Department of Hematology, Tangdu Hospital, Fourth Military Medical University of Chinese PLA, Xi’an 710038, Shaanxi Province, China liuqiang@fmmu.edu.cn
  • Supported by:

    the National High-Tech Research and Development Plan of China (“863” Program), No. 2006AA02A111*

PDF

366

摘要：

背景：Fms 样酪氨酸激酶3配体(Flt3配体)是一种重要的生长因子，通过激活特定的酪氨酸激酶受体，调控造血细胞的生长、生存和/或分化，具有促进造血干细胞体外扩增的应用潜力。
目的：构建pET32a(+)-hFLext原核表达载体，表达、纯化hFLext蛋白，观察其对脐血CD34+细胞的扩增作用。
方法：克隆hFLext，构建pET32a(+)-hFLext重组表达载体。转化大肠杆菌 BL21，IPTG诱导蛋白表达，镍珠亲合层析纯化蛋白。磁珠分选脐血CD34+细胞，单独加入hFLext或联合干细胞因子、血小板生成素孵育1周，观察体外扩增作用。
结果与结论：成功克隆hFLext，并构建了pET32a(+)-hFLext重组表达载体。在大肠杆菌BL21成功表达Trx-hFLext融合蛋白，经8 mol/L尿素变性包涵体蛋白，逐步透析复性，镍珠亲合层析纯化蛋白，成功获得高纯度的Trx-hFLext融合蛋白。Trx-hFLext融合蛋白不仅具有维持及轻度刺激CD34+细胞体外扩增的作用，并且与干细胞因子及血小板生成素具有协同作用，为造血干/祖细胞体外扩增研究奠定了基础。

关键词: Flt3配体, CD34+, hFLext, Trx-hFLext, 载体

Abstract:

BACKGROUND: Fms-like tyrosine kinase 3 ligand (Flt3 ligand, FL) is a cytokine that affects the growth, survival and/or differentiation of hematopoietic cells through the activation of specific tyrosine kinase receptors, and it is potentially useful for in vitro hematopoietic stem cells (HSCs) amplification.
OBJECTIVE: To construct the recombinant prokaryotic expression vector pET32a(+)-hFLext, and to express and purify hFLext protein and investigate its effect on expansion of cord blood CD34+ cells.
METHODS: The hFLext gene was cloned and the recombinant expression vector pET32a-hFLext was constructed and transformed into E. coli BL21. The hFLext protein was expressed under IPTG induction, purified by affinity chromatography with Ni beads. The cord blood CD34+ cells were isolated using CD34 microbead kit and incubated with hFLext alone or in combination with SCF and TPO. The effect of hFLext on expansion of CD34+ cells was examined after 1-week incubation.
RESULTS AND CONCLUSIONS: The recombinant expression vector pET32a-hFLext was successfully constructed and the hFLext was successfully expressed as a Trx fusion protein (Trx-hFLext) in E. coli BL21. The Trx-hFLext with high purity was successfully obtained after degenerating proteins in inclusion bodies with 8M urea, renaturing by gradual dialysis, purifying by affinity chromatography with Ni beads. The purified hFLext can not only effectively maintain and slightly stimulate the proliferation of cord blood CD34+ cells, but also expand profoundly in combination with SCF and TPO, thus paving the way for its application in expanding human hematopoietic stem/progenitor cells in vitro further.

中图分类号: