目的:构建pFUSE-hIgG2-Fc2-BRBP1真核表达质粒表达BRBP1-Fc肽体,并验证其与乳腺癌脑转移细胞系(231-BR)的结合特异性。方法:合成带有EcoRⅠ、NcoⅠ酶切位点的脑转移性乳腺癌特异性肽BRBP1片段,利用该双酶切位点将其定向插入pFUSE-hIgG2-Fc2质粒,构建真核表达质粒pFUSE-hIgG2-Fc2-BRBP1。将测序正确的重组质粒转染至293T细胞真核表达BRBP1-Fc肽体,用蛋白质印迹实验验证蛋白表达,酶联免疫吸附试验、细胞免疫荧光等方法证实BRBP1-Fc肽体与231-BR细胞结合特异性。结果:成功构建重组质粒pFUSE-hIgG2-Fc2-BRBP1,蛋白质印迹法检测到BRBP1-Fc肽体的表达,分子质量约为37 kDa,并且证实BRBP1-Fc肽体与231-BR细胞特异性结合。结论:成功构建了真核表达重组质粒pFUSE-hIgG2-Fc2-BRBP1,表达了BRBP1-Fc肽体,并鉴定了其与231-BR细胞的结合特异性。

[1] SHIMAMOTO G,GEGG C,BOONE T,et al.Peptibodies a flexible alternative format to antibodies[J].mAbs,2012,5(4):586-591.
[2] YONEDA T,WILLIAMS PJ,HIRAGA T,et al.A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro[J].J Bone Miner Res,2001,16(8):1486-1495.
[3] FU B,ZHANG Y,LONG W,et al.Identification and characterization of a novel phage display-derived peptide with affinity for human brain metastatic breast cancer[J].Biotechnol Lett,2014,36(11):2291-2301.
[4] FU B,LONG W,ZHANG Y,et al.Enhanced antitumor effects of the BRBP1 compound peptide BRBP1-TAT-KLA on human brain metastatic breast cancer[J].Sci Rep,2015,5:8029.
[5] 齐斌,任威,李春江,等.NLRP3基因真核表达载体的构建和定位[J].现代医学,2014,42(4):389-392.
[6] STURM M B,JPSHI B P,LU S,et al.Targeted imaging of esophageal neoplasia with a fluorescently labeled peptide:first-in-human results[J].Sci Transl Med,2013,5(184):184-203.
[7] LARIMER B M,DEUTSCHER S L.Development of a peptide by phage display for SPECT imaging of resistance-susceptible breast cancer[J].Am J Nucl Med Mol Imaging,2014,4(5):435-447.
[8] KONTEMANN R E.Strategies to extend plasma halflives of recombinant antibodies[J].Bio Drugs,2009,23(2):93-109.
[9] ROOPENIAN D C,AKILESH S.FcRn:the neonatal Fc receptor comes of age[J].Nat Rev Immunol,2007,7(9):715-725.
[10] NIMMERJAHN F,RAVETCH J V.Fc gamma receptors as regulators of immune responses[J].Nat Rev Immunol,2008,8(1):34-47.
[11] DANIDL M,CZAJKOWSK Y,RICHARD J.et al.Fc-fusion proteins:new developments and future perspectives[J].EMBO Mol Med,2012,4(10):1015-1028.
[12] ECKHARDT B L,FRANCIS P A,PARKER B S,et al.Strategies for the discovery and development of therapies for metastatic breast cancer[J].Nat Rev Drug Discov,2012,11(6):479-497.