重组人淋巴细胞活化基因-3蛋白胞外段的真核表达及鉴定

目的真核表达重组人淋巴细胞活化基因-3(lymphocyte activation gene-3,LAG-3)蛋白胞外段,并进行鉴定。方法用植物血球凝集素(phytohaemagg lutinin,PHA)刺激Jurkat细胞,流式细胞术检测Jurkat细胞中LAG-3蛋白的表达;提取Jurkat细胞总mRNA,RT-PCR法扩增人LAG-3蛋白胞外段基因片段,同时在蛋白C-末端引入His标签,将其克隆入载体pcDNA3. 1+,构建重组质粒,转染Expi293F真核细胞,当细胞活率低于50%时收获细胞上清,经镍柱亲合层析纯化。纯化产物进行4%～20%SDS-PAGE、HPLC及Western blot分析,BCA法测定浓度。结果经菌液PCR、双酶切及测序鉴定,表明质粒构建正确。重组表达蛋白的相对分子质量约60 000,纯化后纯度达95%以上,与鼠抗LAG-3单克隆抗体可发生特异性结合,浓度为2. 4 mg/mL。结论成功构建了重组真核表达质粒LAG-3/pcDNA3. 1+,并于Expi293F细胞中表达,纯化获得了纯度较高的LAG-3蛋白,为后期LAG-3蛋白的相关研究及其单抗的制...

Eukaryotic expression and identification of extracellular domain of recombinant human lymphocyte activation gene-3 protein

Objective To express the extracellular domain of recombinant human lymphocyte activation gene-3(LAG-3,CD223) in eukaryotic cells and identify the expressed product. Methods Jurkat cells were stimulated with phytohaemagglutinin(PHA),and tested for the expression of LAG-3 protein by flow cytometry. Total mRNA of Jurkat cells was extracted,with which the extracellular domain of recombinant human LAG-3 was amplified by RT-PCR. The PCR product,with His-tag introoduced at C-terminus,was cloned into vector pcDNA3. 1+,and the constructed recombinant plasmid was transfected to eukaryotic Expi293 F cells. When the cell viability was less than 50%,the cell supernatant was harvested,purified by nickel column affinity chromatography,analyzed by 4% ~ 20% SDS-PAGE,HPLC and Western blot,and determined for concentration by BCA method. Results PCR,restriction analysis and sequencing proved that recombinant plasmid LAG-3/pcDNA3. 1+ was constructed correctly. The expressed protein,with a relative molecular mass of about 60 000,reached a purity of more than 95% after purification and showed specific binding to mouse monoclonal antibody against LAG-3,of which the concentration was 2. 4 mg/mL. Conclusion Recombinant eukaryotic expression vector LAG-3/pcDNA3. 1+ was successfully constructed,and highly purified LAG-3 protein was expressed in Expi293 F cells. This study laid a foundation of further research on LAG-3 protein and preparation of LAG-3 monoclonal antibody.