[人粒细胞-巨噬细胞集落刺激因子（9-127）-白细胞介素-6（29-184）融合蛋白基因的构建与表达]

[Construction and expression of huGM-CSF (9-127)-IL-6(29-184) fusion protein gene].

作者信息

Sun Q M, Xu W M, Ma Y B, Yang X, Liu H Y, Sun M S, Dai C B

机构信息

Department of Molecular Biology, Institute of Medical Biology, CAMS, PUMC, Kunming 650118, China.

出版信息

Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2001 Dec;23(6):603-8.

PMID:12901107

Abstract

OBJECTIVE

To construct and express huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity and both huGM-CSF and huIL-6 biologic activities.

METHODS

The novel gene coding for the fusion protein of huGM-CSF(9-127)-IL-6(29-184) was constructed by strategy of step by step cloning in pBV220 expression vector. The amino acids 1-8 of huGM-CSF and the amino acids 1-28 of huIL-6 were deleted by PCR technique. The mutant huGM-CSF (9-127) and huIL-6 (29-184) cDNAs were linked via a linker sequence coding 15 amino acid residues (G-G-S-G-S)3. Fusion protein was expressed in E.coli host strain DH5 alpha. To obtain the fusion protein, Q Sepharose H.P. ion exchange chromatography and Sephacryl S-200 gel filtration were performed. The biologic activities were detected by MTT method.

RESULTS

Fusion protein was expressed in E.coli host strain DH5 alpha in the form of inclusion body. The expression level was more than 25% of the total cell lysate. Through Q Sepharose H.P. ion exchange chromatography and Sephacryl S-200 gel filtration, huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity was obtained. The protein showed both huGM-CSF and huIL-6 biologic activities. The specific activity of huGM-CSF was 1.08 x 10(8) U/mg, and for huIL-6, it reached 1.95 x 10(7) U/mg.

CONCLUSION

huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity and both huGM-CSF and huIL-6 biologic activities was obtained.

摘要

目的

构建并表达具有高纯度以及人粒细胞-巨噬细胞集落刺激因子（huGM-CSF）和人白细胞介素-6（huIL-6）生物学活性的huGM-CSF(9-127)-IL-6(29-184)融合蛋白。

方法

采用逐步克隆策略，在pBV220表达载体中构建编码huGM-CSF(9-127)-IL-6(29-184)融合蛋白的新基因。通过PCR技术缺失huGM-CSF的第1-8位氨基酸和huIL-6的第1-28位氨基酸。突变型huGM-CSF (9-127)和huIL-6 (29-184) cDNA通过编码15个氨基酸残基（G-G-S-G-S）3的接头序列连接。融合蛋白在大肠杆菌宿主菌株DH5α中表达。为获得融合蛋白，进行了Q Sepharose H.P.离子交换层析和Sephacryl S-200凝胶过滤。采用MTT法检测生物学活性。

结果

融合蛋白在大肠杆菌宿主菌株DH5α中以包涵体形式表达。表达水平超过总细胞裂解物的25%。通过Q Sepharose H.P.离子交换层析和Sephacryl S-200凝胶过滤，获得了高纯度的huGM-CSF(9-127)-IL-6(29-184)融合蛋白。该蛋白具有huGM-CSF和huIL-6的生物学活性。huGM-CSF的比活性为1.08×10(8) U/mg，huIL-6的比活性达到1.95×10(7) U/mg。