重组大鼠睫状神经营养因子在大肠杆菌中的表达及其与碱性成纤维细胞生长因子的协同作用。

Expression of recombinant rat ciliary neurotrophic factor in Escherichia coli and its synergy with basic fibroblast growth factor.

机构信息

Department of Cell, Molecular and Structural Biology, Northwestern University Medical and Dental Schools, 303 East Chicago Avenue, Chicago, Illinois 60611, USA.

出版信息

Mol Cell Neurosci. 1991 Oct;2(5):369-76. doi: 10.1016/1044-7431(91)90023-h.

DOI:10.1016/1044-7431(91)90023-h

PMID:19912821

Abstract

Rat CNTF was expressed in Escherichia coli using a T7 RNA polymerase vector system. rCNTF was obtained as an insoluble aggregate at levels approaching 70% of total E. coli protein. After extraction, renaturation, and purification by anion-exchange chromatography and gel filtration the yield of biologically active rCNTF was nearly 0.5 mg/ml of E. coli culture broth. The effects of rCNTF and bFGF on cultured chick ciliary neurons were compared. bFGF was primarily neuritogenic and did not support ciliary neuron survival beyond 2 days in culture. rCNTF induced neuritogenesis more slowly and supported ciliary neuron survival in culture for a longer period. bFGF strongly potentiated the effect of rCNTF. Without bFGF, the ED(50) for rCNTF was 102 +/- 25 pg/ml. With bFGF, the potency of rCNTF increased to an ED(50) of 42 +/- 6 pg/ml. Chick nodose neurons also responded to rCNTF and their response was potentiated by bFGF. Thus, bFGF modulates the response of ciliary and nodose neurons to CNTF in vitro and may have a similar effect in vivo.

摘要

大鼠 CNTF 采用 T7 RNA 聚合酶载体系统在大肠杆菌中表达。rCNTF 作为不溶性聚集体获得，接近大肠杆菌蛋白总量的 70%。经过提取、复性、阴离子交换层析和凝胶过滤纯化，具有生物活性的 rCNTF 的产率接近大肠杆菌培养液的 0.5 毫克/毫升。比较了 rCNTF 和 bFGF 对培养的鸡睫状神经元的影响。bFGF 主要具有神经突生成作用，在培养物中不能支持睫状神经元存活超过 2 天。rCNTF 诱导神经突生成的速度较慢，在培养物中支持睫状神经元存活的时间更长。bFGF 强烈增强了 rCNTF 的作用。没有 bFGF 时，rCNTF 的 ED（50）为 102 ± 25 pg/ml。有 bFGF 时，rCNTF 的效力增加到 ED（50）为 42 ± 6 pg/ml。鸡结状神经节神经元也对 rCNTF 有反应，bFGF 增强了它们的反应。因此，bFGF 在体外调节睫状神经元和结状神经元对 CNTF 的反应，在体内可能具有类似的作用。