重组人PD-L1在大肠杆菌BL-21中的表达、纯化和鉴定
Expression and identification of rhPD-L1 in E. coli BL-21(DE3)
作者：陈娇;张金鸽;王辉;计宝辉;孔祥丽;张平;
Author：
收稿日期： 年卷（期）页码：2009,24(05):-487-490
期刊名称：华西药学杂志
Journal Name：WEST CHINA JOURNAL OF PHARMACEUTICAL SCIENCES
关键字：程序死亡蛋白-1;原核表达;纯化和鉴定;大肠杆菌BL-21
Key words：
基金项目：
中文摘要
目的分离纯化人程序死亡蛋白-1(PD-L1)基因,制备重组人PD-L1蛋白,为进一步研究PD-L1在肿瘤免疫逃逸中的作用和机制建立基础。方法通过RT-PCR分离纯化人PD-L1基因,并将其克隆到原核表达质粒pGEX-4T-1中,经酶切,测序鉴定分析后,转化大肠杆菌BL21(DE3),经IPTG诱导表达。产物经GST蛋白纯化系统进行纯化后,用10%SDS-PAGE及Western Blot分析鉴定。结果经RT-PCR分离纯化的PD-L1 cDNA分子由645 bp构成,编码相对分子量约25 KD的蛋白,并与GST形成融合蛋白。GST-PD-L1融合蛋白相对分子量约46 KD。Western Blot结果显示,该蛋白能被兔抗GST和抗PD-L1抗体识别。结论成功构建了PD-L1原核表达载体,并在大肠杆菌BL21中获得高效表达。
参考文献
[1]Ravetch JV,Lanier LL.Immune inhibitory receptors[J].Sci,2000,290(5489):84-89.
[2]Freeman GJ,Long AJ,Iwai Y,et al.Engagement of the PD21immunoinhibitory receptor by a novel B7 family member leads tonegative regulation of lymphocyte activation[J].J Exp Med,2000,192(7):1027-1034.
[3]Carreno BM,Collins M.The B7 family of ligands and its recep-tors:new pathways for costimulation and inhibition of immune re-sponses[J].Ann Rev Immunol,2002,20:29-53.
[4]Gao W,Demirci G,Strom TB,et al.Stimulating PD212negativesignals concurrent with blocking CD154 co2stimulation induceslong-term isl et al lograft survival[J].Transplantation,2003,76(6):994-999.
[5]Dong H,Zhu G,Tamada K,et al.B7-H1,a third member ofthe B7 family,co-stimulates T-cell proliferation and interleu-kin-10 secretion[J].Nat Med,1999,5:1365-1369.
[6]LIM DY,TANAKA Y,IWASA KI M,et al.ThePD-1/PD-L1complex resembles the antigen-binding Fv domains of antibodiesand Tcell receptors[J].Proc Natl Acad Sci USA,2008,105:3011-3016.
[7]Iwai Y,Ishida M,Tanaka Y,et al.Involvement of PD-L1 ontumor cells in the escape from host immune system and tumor im-munotherapy by PD-L1 blockade[J].Proc Natl Acad Sci USA,2002,99(19):12293-12 297.
[8]Youngnak-Piboonratanakit P,Tsushima F,Otsuki N,et al.Theexpression of B7-H1 on keratinocytes in chronic inflammatorymucocutaneous disease and its regulatory role[J].Immunol Lett,2004,94(3):215-222.
[9]萨姆布鲁克J,拉塞尔著DW,黄培堂译.分子克隆试验指南[M].北京:科学出版社,2002.1217-1231.
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