摘要
目的人工合成恒河猴白细胞介素-4(Macaca mulatta Interleukin-4,Mamu IL-4)基因,并在大肠杆菌中高效表达及纯化。方法根据大肠杆菌遗传密码偏爱性优化设计并人工合成Mamu IL-4(mIL-4)基因,克隆入pET43.1a(+)表达载体,构建重组表达质粒pET43.1a-mIL-4,转化大肠杆菌BL21-CodonPlus(DE3)-RIPL,经IPTG诱导表达。通过阳离子交换层析及稀释复性等步骤对表达的重组mIL-4蛋白进行纯化,纯化产物经Western blot进行鉴定。结果双酶切及测序证实重组表达质粒pET43.1a-mIL-4构建正确;表达的重组蛋白相对分子质量约为15 000,表达量约占菌体总蛋白的40%,主要以包涵体形式存在,经纯化复性后纯度可达95%以上;Western blot检测其能与鼠抗mIL-4单克隆抗体特异性结合。结论 在大肠杆菌中高效表达了重组mIL-4蛋白,纯化的蛋白纯度较高,为其生物学活性及佐剂的相关研究奠定了基础。
Objective To synthesize Macaca mulatta interleukin-4（Mamu IL-4） gene,highly express in E.coli and purify the expressed product.Methods Mamu IL-4（mIL-4） gene was designed and synthesized according to the preference of codon of E.coli,then cloned into expression vector pET43.1a（＋）.The constructed recombinant plasmid pET43.1a-mIL-4 was transformed to E.coli BL21-CodonPlus（DE3）-RIPL for expression under induction of IPTG.The expressed recombinant mIL-4 protein was purified by cation exchange chromatography followed by dilution refolding,then identified by Western blot.Results Restriction analysis and DNA sequencing confirmed that the recombinant plasmid pET43.1a-mIL-4 was constructed correctly.The expressed recombinant mIL-4 protein,with a relative molecular mass of about 15 000,contained about 40% of total somatic protein and mainly existed in a form of inclusion body,which reached a purity of more than 95% after purification and refolding.Western blot showed specific binding of the purified protein to anti-mIL-4 monoclonal antibody.Conclusion Recombinant mIL-4 was successfully expressed in E.coli and reached a high purity after purification,which laid a foundation of further study on bioactivity and adjuvant of mIL-4.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第9期1046-1049,1053,共5页
Chinese Journal of Biologicals
基金
"重大新药创制"科技重大专项"十一五"计划(2009ZX09103-741)
关键词
恒河猴
白细胞介素-4
基因合成
原核细胞
基因表达
纯化
Macaca mulatta
Interleukin-4
Gene synthesis
Prokaryotic cells
Gene expression
Purification
