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  

Cloning of Porcine Granulocyte-macrophage Colony Stimulating Factor(pGM-CSF)Gene and Its Prokaryotic Expression

作　　者：杨恒[1] 文心田[1] 曹三杰[1] 黄小波[1] 

机构地区：[1]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

出　　处：《农业生物技术学报》2009年第2期206-211,共6页Journal of Agricultural Biotechnology

基　　金：教育部长江学者和创新团队发展计划项目(No.IRT0555-9);四川省重点公益项目(No.2007NGY007)资助

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435bp,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31kD,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3.43×105IU/mg。The porcine granulocylc-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 ofpGM-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 Ni^2＋-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× 10^5 IU/mg.

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 

分 类 号：S188[农业科学—农业基础科学]