| 猪粒细胞-巨噬细胞集落刺激因子（pGM-CSF)基因的克隆与原核表达 |
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| 引用本文： | 杨恒,文心田,曹三杰,黄小波.猪粒细胞-巨噬细胞集落刺激因子（pGM-CSF)基因的克隆与原核表达[J].农业生物技术学报,2009,17(2):206-211. |
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| 作者姓名： | 杨恒 文心田 曹三杰 黄小波 |
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| 作者单位： | 四川农业大学动物医学院动物传染病与基因芯片实验室,雅安,625014 |
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| 基金项目： | 教育部长江学者和创新团队发展计划，四川省重点公益项目 |
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| 摘 要： | 通过RT-PCR方法，从经ConA刺激的猪外周血淋巴细胞中扩增出猪粒细胞-巨噬细胞集落刺激因子（pGM-CSF）编码区的cDNA并将其克隆至pMD-19T载体，序列测定表明pGM-CSF基因的长度为435 bp，编码144个氨基酸。通过PCR方法获得缺失其N端17个氨基酸残基信号肽序列的成熟pGM-CSF蛋白基因，将其克隆至原核表达载体pET-32a（+），构建重组表达质粒pET-GM并转入大肠杆菌(Escherichia coli )BL21（DE3）中进行诱导表达。经SDS-PAGE电泳和Western blot分析表明，表达的重组蛋白分子量约31 kD，主要以包涵体形式存在，表达量占菌体总蛋白的30.4% 。采用Ni2+-NTA亲和层析柱对经稀释复性的重组蛋白进行纯化，并采用MTT法以TF-1细胞检测纯化产物的生物学活性。结果表明，重组蛋白可有效刺激TF-1细胞增殖，其活性达到3.43×105 IU/mg。
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| 关 键 词： | 猪粒细胞-巨噬细胞集落刺激因子 基因克隆 原核表达 生物活性测定 |
| 收稿时间： | 2008-6-23 |
| 修稿时间： | 2008-7-23 |
Cloning of Porcine Granulocyte-macrophage Colony Stimulating Factor (pGM-CSF) Gene and Its Prokaryotic Expression |
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| YANG Heng,WEN Xin-tian,CAO San-jie,HUANG Xiao-bo.Cloning of Porcine Granulocyte-macrophage Colony Stimulating Factor (pGM-CSF) Gene and Its Prokaryotic Expression[J].Journal of Agricultural Biotechnology,2009,17(2):206-211. |
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| Authors: | YANG Heng WEN Xin-tian CAO San-jie HUANG Xiao-bo |
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| Abstract: | The porcine granulocyte-macrophage colony stimulating factor (pGM-CSF) full-length gene was cloned from porcine peripheral blood lymphocyte using RT-PCR and ligated into pMD19-T vector. Sequence analysis showed that the cDNA of pGM-CSF was 435 bp in length and encodes 144 amino acids. The gene of mature pGM-CSF deleting a 17 aa signal sequence at N-terminus was cloned by PCR and inserted into pET-32a(+). The recombinant plasmid pET-GM was transformed into Escherichia coli BL21(DE3) and induced by IPTG. The results of SDS-PAGE and Western blot showed that the recombinant protein was about 31 kD, which existed mainly in inclusion body, accounts 30.4% of total bacterium proteins. After dilution renaturation and purification procedure in Ni2+-NTA affinity chromatography column, the biological activity of purified protein was analyzed by MTT method using TF-1 cells. The result indicated that the recombinant protein could effectively stimulate the proliferation of TF-1 cells, with a specific bioactivity of 3.43×105 IU/mg. |
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