Ge Xiao-dong, Liu You-sheng, Wang Xiao-dong, Wang Chang-shong, Deng Jun, Li Hong
Department of Pathology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2005 Mar;21(2):180-4.
To construct human phage antibody library and to prepare phage antibodies against N terminal fragment of human lipopolysaccharide binding protein (NH-LBP).
NH-LBP was expressed in insect cells sf21 cultured with serum-free culture medium (SF-900 II) by BAC-TO-BAC baculovirus expression system. Human antibody library (Fab) was constructed by phage display system (pComb3H/VCSM13) and screened with NH-LBP.
NH-LBP was successfully expressed in insect cells sf21 cultured with serum-free culture and effectively purified via affinity chromatography column with TALON resin. About 8 mg NH-LBP was obtained. Human combinatorial phage display antibody library consisting of 5.0 x 10(8) CFU was constructed. After eight rounds of panning with NH-LBP we got 3 phage antibodies with high affinity for NH-LBP (DNA sequences and amino acid sequences of kappa chains of these antibodies were deposited in GenBank (AY337713 and AY337714).
Phage antibodies against NH-LBP were obtained successfully, which lays the foundation for preparing disulfide stabilized Fv fragments antibody (dsFv) against NH-LBP, understanding the function of LBP in vivo, and prevention and therapy of excessive inflammatory reaction.
构建人噬菌体抗体库并制备抗人脂多糖结合蛋白N端片段（NH-LBP）的噬菌体抗体。
采用BAC-TO-BAC杆状病毒表达系统，在无血清培养基（SF-900 II）培养的昆虫细胞sf21中表达NH-LBP。利用噬菌体展示系统（pComb3H/VCSM13）构建人抗体库（Fab），并用NH-LBP进行筛选。
NH-LBP在无血清培养的昆虫细胞sf21中成功表达，并通过TALON树脂亲和层析柱有效纯化，获得约8 mg NH-LBP。构建了由5.0×10(8) CFU组成的人组合噬菌体展示抗体库。用NH-LBP进行八轮淘选后，获得了3种对NH-LBP具有高亲和力的噬菌体抗体（这些抗体κ链的DNA序列和氨基酸序列已存入GenBank（AY337713和AY337714））。
成功获得了抗NH-LBP的噬菌体抗体，为制备抗NH-LBP的二硫键稳定Fv片段抗体（dsFv）、了解LBP在体内的功能以及过度炎症反应的防治奠定了基础。
