摘要
为了表达奶牛粒细胞-巨噬细胞集落刺激因子(GM-CSF)并对其活性进行检测。根据GenBank中登录的牛GM-CSF基因序列(U22385),设计一对特异性引物;采用RT-PCR方法,以LPS体外诱导的奶牛肺泡巨噬细胞为材料,从总RNA中扩增出奶牛GM-CSFcDNA基因,克隆到pGEM-T载体中,经酶切鉴定与序列测定,结果显示克隆的奶牛GM-CSF基因与GenBank中登录的牛GM-CSF基因序列的核苷酸和氨基酸的同源性分别为99.7%和99.3%。构建pET32a-GM-CSF原核表达重组质粒,经IPTG诱导表达,SDS-PAGE结果显示重组蛋白大小约为35ku。分别运用集落形成试验与MTT比色法测定重组蛋白活性,结果显示重组奶牛GM-CSF蛋白能够诱导粒细胞前体呈集落性生长并具有较强的体外增殖淋巴细胞的活性,为下一步临床试验奠定了基础。
Bovine Granulocutemacrophage colony stimulating factor （GM-CSF） gene was amplified by RT-PCR from total RNA isolated from LPS-stimulated peripheral blood T cells of cow. The PCR fragments was cloned into pGEM-T vector and sequenced. The gene contained about 430 bp and had 99.3 % （and 99.77 % at amino acid level） sequence identity with the published sequences. The gene was subcloned into pET32a vector and transformed into BL21 Escherichia coil cells. SDS-PAGE confirmed that the expressed protein was about 35 ku. MTT colorimetric assay and colony-forming test showed that the recombinant protein could induce bovine T lymphocytes to grow in vitro.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2009年第10期808-812,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
新疆兵团科技攻关计划项目(2006GG22)
关键词
奶牛
GM.CSF
克隆
表达
活性检测
cow
GM-CSF
cloning
gene expression
biological activity testing
