本文選題：納米抗體 + 單域抗體��； 參考：《蘇州大學》2015年碩士論文

【摘要】：目的:本論文以人肺表面活性蛋白A(SP-A)為靶點,制備針對人肺SP-A的納米抗體,為后期臨床帶藥提供理想的靶向配體。方法:1.人肺泡表面活性蛋白A(h SP-A)的獲取及鑒定:從新鮮人肺組織中利用動物蛋白提取液提取的人肺組織總蛋白,Western blot、ELISA驗證蛋白特異性。2.人肺組織SP-A特異性納米抗體(h SPA-Nb)的篩選:用固相法對噬菌體納米抗體庫進行三輪淘篩,獲得富集的三級篩選抗體庫,并采用間接phage-ELISA從三級抗體庫中挑出高特異性基因序列。3.特異性h SPA-Nb的表達與純化:測序分析陽性菌株并構建重組質(zhì)粒PET-26b(+)/Nb4-VHH,進一步表達純化。4.h SPA-Nb4的肺靶向性及安全性檢測:用Western Blot、El ISA檢測蛋白特異性,細胞熒光實驗,免疫組化及小鼠活體內(nèi)成像中肺靶向性和安全性。結(jié)果:本研究首次成功制備出與人肺組織h SP-A存在特異性結(jié)合的抗h SP-A納米抗體Nb4。結(jié)論:該納米抗體不僅能特異性結(jié)合h SP-A,還可通過靶向天然的h SP-A而實現(xiàn)高度特異性肺靶向。關鍵詞:納米抗體、單域抗體、人肺泡表面活性蛋白A、噬菌體抗體庫、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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R943

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本文編號：1867889