抗人肺泡表面活性蛋白A纳米抗体的制备及其靶向性的检测

发布时间：2018-05-10 05:01

本文选题：纳米抗体 + 单域抗体　；参考：《苏州大学》2015年硕士论文

【摘要】：目的:本论文以人肺表面活性蛋白A(SP-A)为靶点,制备针对人肺SP-A的纳米抗体,为后期临床带药提供理想的靶向配体。方法:1.人肺泡表面活性蛋白A(h SP-A)的获取及鉴定:从新鲜人肺组织中利用动物蛋白提取液提取的人肺组织总蛋白,Western blot、ELISA验证蛋白特异性。2.人肺组织SP-A特异性纳米抗体(h SPA-Nb)的筛选:用固相法对噬菌体纳米抗体库进行三轮淘筛,获得富集的三级筛选抗体库,并采用间接phage-ELISA从三级抗体库中挑出高特异性基因序列。3.特异性h SPA-Nb的表达与纯化:测序分析阳性菌株并构建重组质粒PET-26b(+)/Nb4-VHH,进一步表达纯化。4.h SPA-Nb4的肺靶向性及安全性检测:用Western Blot、El ISA检测蛋白特异性,细胞荧光实验,免疫组化及小鼠活体内成像中肺靶向性和安全性。结果:本研究首次成功制备出与人肺组织h SP-A存在特异性结合的抗h SP-A纳米抗体Nb4。结论:该纳米抗体不仅能特异性结合h SP-A,还可通过靶向天然的h SP-A而实现高度特异性肺靶向。关键词:纳米抗体、单域抗体、人肺泡表面活性蛋白A、噬菌体抗体库、靶向性。
[Abstract]:Aim: in this paper, the human pulmonary surfactant protein (ANAS SP-A) was used as the target for the preparation of human lung SP-A nanoparticles, and to provide an ideal target ligand for late clinical drug delivery. Method 1: 1. Preparation and identification of human alveolar surfactant protein (AHSP-A): the protein specificity of human lung tissue total protein extracted from fresh human lung tissue was confirmed by Western blottmeter Elisa. Screening of human lung SP-A specific nano-antibody (h SPA-Nb): the phage antibody library was screened for three rounds by solid phase method to obtain the enriched antibody library, and the highly specific gene sequence. 3 was selected from the third class antibody library by indirect phage-ELISA. Expression and purification of specific h SPA-Nb: the positive strain was sequenced and the recombinant plasmid PET-26b (P / Nb4-VHHH) was constructed. The lung targeting and safety of purified SPA-Nb4 were further detected. The protein specificity and cell fluorescence were detected by Western blott El ISA. Immunohistochemistry and lung targeting and safety in vivo imaging of mice. Results: in this study, we successfully prepared the anti h SP-A nano antibody Nb4 which has specific binding with human lung tissue h SP-A for the first time. Conclusion: this antibody can not only specifically bind to h SP-A, but also achieve high specific lung targeting by targeting natural h SP-A. Keywords: nanometer antibody, single domain antibody, human alveolar surfactant protein A, phage antibody library, targeting.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R943

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