重组人干细胞因子-血小板生成素融合蛋白表达及生物学功能的初步研究

[A preliminary study on the expression and biological function of recombinant human SCF-TPO fusion protein].

作者信息

Liu Nan, Xi Yong-zhi, Guo Si-qi, Sun Yu-ying, Yuan Zhi-hong, Cui Jian-wu, Xi Cai-xia, Liang Fei, Kong Fan-hua

机构信息

Department of Immunology, Affiliated Hospital of AMMS. Laboratory of Immunoassary of NCBA, Beijing 100039, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2005 Jan;26(1):19-22.

PMID:15946503

Abstract

OBJECTIVE

To study the expression of recombinant human SCF-TPO fusion protein and its biological function.

METHODS

Four primers were designed according to known sequences of TPO and SCF. The functional amino acid domains of TPO and SCF were amplified by RT-PCR from fetus hepatocytes, respectively. The expression plasmid pET32a/SCF-TPO was constructed by VOE gene fusion technique and expressed in E. coli BL21(DE3) plysS as inclusion body after isopropyl-beta-D-1-thiogalactopyranoside (IPTG) induction. The fusion protein was tested by SDS-PAGE and Western blot. The biological functions of SCF-TPO fusion protein in MO7e cells was investigated by MTT method after purification with metal chelating chromatography.

RESULTS

The high expression SCF-TPO fusion protein was obtained, reaching up to 30% of the total cellular protein. Western blot verified the correct fusion expression and MTT results showed the growth promoting effect of the SCF-TPO fusion protein on MO7e cells, with a higher promoting activity at 100 ng/ml.

CONCLUSIONS

Expressed SCF-TPO fusion protein after renaturation has biological activity in promoting the proliferation of MO7e cells.

摘要

目的

研究重组人干细胞因子-血小板生成素（SCF-TPO）融合蛋白的表达及其生物学功能。

方法

根据血小板生成素（TPO）和干细胞因子（SCF）的已知序列设计4条引物，分别从胎儿肝细胞中通过逆转录聚合酶链反应（RT-PCR）扩增TPO和SCF的功能氨基酸结构域。采用VOE基因融合技术构建表达质粒pET32a/SCF-TPO，经异丙基-β-D-1-硫代半乳糖苷（IPTG）诱导后在大肠杆菌BL21(DE3)plysS中表达，以包涵体形式存在。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blot）对融合蛋白进行检测。用金属螯合层析法纯化后，采用噻唑蓝比色法（MTT法）研究SCF-TPO融合蛋白在MO7e细胞中的生物学功能。