论文部分内容阅读
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。
Objective To construct, express and purify eukaryotic expression vector of mouse PD-1 extracellular domain (ePD-1) and study its biological effects. Methods The total extracellular domain of PD-1 gene was cloned by RT-PCR and then cloned into prokaryotic expression vector pGEX-4T-1. The prokaryotic expression vector pGEX-4T-1-ePD- Transformed into E. coli DH5α, screening positive colonies, digestion and sequencing identification. The recombinant plasmid pGEX-4T-1-ePD-1 was transformed into E.coli BL21 (DE3). After induced by IPTG, the expressed product was detected by SDS-PAGE and Western blot, and purified by protein purification GST-ePD-1 fusion protein was purified. The effects of purified PD-1 extracellular fusion protein on the proliferation of lymphocytes were assayed by flow cytometry and Alamar Blue method, and their biological activities were evaluated. Results The recombinant plasmid pGEX-4T-1-ePD-1 was successfully constructed and successfully expressed in E. coli BL21 (DE3). The purified GST-ePD-1 fusion protein was obtained by using a protein purification instrument. Flow cytometry and Alamar Blue results showed that different concentrations of fusion protein on mixed lymphocytes, compared with the negative control, can significantly promote lymphocyte proliferation. Conclusion The mouse ePD-1 protein was successfully cloned, expressed and purified. The purified recombinant protein can effectively promote the proliferation of lymphocytes, which provided the conditions for further study of its function and clinical application.