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| 小鼠可溶性PD1的克隆、真核表达及生物学活性检测 | |
| 引用本文： | 覃晓琳,刘朝奇,唐国慧,金羽,孙俪.小鼠可溶性PD1的克隆、真核表达及生物学活性检测[J].生物技术通报,2011(2). |
| 作者姓名： | 覃晓琳 刘朝奇 唐国慧 金羽 孙俪 |
| 作者单位： | 1. 三峡大学分子生物学研究所,宜昌,443002 2. 三峡大学医学院,宜昌,443002 3. 三峡大学化学与生命科学学院,宜昌,443002 |
| 摘 要： | 旨在克隆小鼠PD1胞外区(简称mPD-1)基因,利用真核表达系统表达有活性的分泌型mPD-1蛋白,初步研究其生物学活性。克隆mPD-1基因,将其连入pcDNA3.1(+)/Fc中获得pcDNA3.1(+)-Fc/mPD-1重组表达质粒,转化至大肠杆菌DH5α,进行PCR和双酶切鉴定,并送测序。将阳性质粒转染L929细胞,利用RT-PCR和Western blotting方法鉴定mPD-1/L929稳定表达株。利用Alamar Blue法检测分泌的mPD-l蛋白对淋巴细胞增殖的影响,评价其生物学活性。结果显示,成功构建重组质粒pcDNA3.1(+)-Fc/mPD-1,转染了pcDNA3.1(+)-Fc/mPD-1的L929细胞可将mPD-l蛋白分泌至胞外。A lamar Blue检测结果显示,真核细胞分泌的mPD-1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。本试验成功地克隆mPD-1胞外区蛋白,并在L929细胞中得到了分泌型表达。分泌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。 |
| 关 键 词： | Fc-PD-1 真核表达 淋巴细胞增殖 |
Cloning,Eukaryotic Expression and Biologic Characteristics of Mouse PD-1 Extracellular Region |
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| Qin Xiaolin,Liu Chaoqi,Tang Guohui,Jin Yu,Sun Li.Cloning,Eukaryotic Expression and Biologic Characteristics of Mouse PD-1 Extracellular Region[J].Biotechnology Bulletin,2011(2). | |
| Authors: | Qin Xiaolin Liu Chaoqi Tang Guohui Jin Yu Sun Li |
| Institution: | Qin Xiaolin1 Liu Chaoqi1 Tang Guohui2 Jin Yu3 Sun Li2(1Institute of Molecular Biology,Three Gorges University,Yichang 443002,2Medical Science College of China Three Gorges University,3College of Chemistry and Life Science,Yichang 443002) |
| Abstract: | The study was to construct eukaryotic expression plasmid of murine PD-1 extracellular region(mPD-1) eukaryotic expression vector,and express the functional fusion protein in L929 cells.Murine PD-1 was cloned into eukaryotic expression vector pcDNA3.1(+)-Fc and positive plasmid identified.The pcDNA3.1(+)-Fc/mPD-1 plasmid was transfected into L929 cell and analyzed by Western blotting.The lymphocyte proliferation activity of the mPD-1-Fc supernatant was measured by Alamar Blue.Results showed that the extracel... |
| Keywords: | Fc-PD-1 Eukaryotic expression Lymphocyte proliferation |
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