摘要
目的小鼠PD-1胞外段(ePD-1)原核表达载体的构建、表达、纯化及其生物学效应初步研究。方法应用小鼠脾细胞总RNA,采用RT-PCR克隆PD-1胞外段基因,将其重组到原核表达载体pGEX-4T-1中,构建原核表达载体pGEX-4T-1-ePD-1,转化至E.coli DH5α,筛选阳性菌落,进行酶切及测序鉴定。将测序正确的重组表达质粒pGEX-4T-1-ePD-1转化至E.coli BL21(DE3)中,经IPTG诱导表达后,对表达产物进行SDS-PAGE和Western blot检测,同时通过蛋白质纯化仪纯化GST-ePD-1融合蛋白。利用流式细胞术和Alamar Blue法,检测纯化的PD-1胞外段融合蛋白对淋巴细胞增殖的影响,评价其生物学活性。结果成功构建重组质粒pGEX-4T-1-ePD-1,并在E.coli BL21(DE3)中获得了成功表达,利用蛋白质纯化仪得到纯化的GST-ePD-1融合蛋白。流式细胞术和Alamar Blue法结果均显示,不同浓度的融合蛋白作用于混合淋巴细胞,与阴性对照相比,可明显促进淋巴细胞的增殖。结论成功地克隆、表达和纯化了小鼠ePD-1蛋白,纯化的重组蛋白可有效促进淋巴细胞增殖,为进一步研究其功能和临床应用提供了条件。
To investigate the biological activity of mouse PD-1 ectodomain（ePD-1）,we cloned and expressed ePD-1 gene. ePD-1 gene was amplified from mouse spleen cell mRNA by RT-PCR,and then cloned into prokaryotic expression vector pGEX-4T- 1,for screening positive recombinant plasmid pGEX-4T-1-ePD-1 by enzyme digestion and sequencing.Then the correct sequence of the recombinant plasmid was transformed into E.coli BL21,and induced by IPTG.The expressed recombinant protein was analyzed by SDS-PAGE and Western blotting;the fusion protein was purified by GSTrap FF affinity chromatograph on the Protein Purification System, and then analyzed by Alamar Blue assay and flow cytometry（FCM） for evaluating its biological activity.While the results of Alamar Blue assay and Flow cytometry showed that the fusion protein added to the mixed lymphocytes could significantly promote the proliferation of lymphocytes,as compared with the negative control.All these data mean that the purified recombinant protein can be effective in promoting lymphocyte proliferation,which can be used for further research in the function and clinical application of ePD- 1.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2010年第5期403-407,共5页
Immunological Journal
基金
三峡大学研究生科研创新基金项目(2009-36)
