【摘要】：目的構(gòu)建V-set and immunoglobulin domain containing 4(Vsig4)特異性納米抗體,以期作為巨噬細(xì)胞的分子探針。方法用Vsig4重組蛋白對(duì)羊駝進(jìn)行免疫,分離血液中的淋巴細(xì)胞,利用噬菌體展示技術(shù),構(gòu)建噬菌體展示文庫,經(jīng)過連續(xù)3次生物淘篩獲得與Vsig4蛋白結(jié)合的噬菌體,經(jīng)測(cè)序和基因比對(duì)所得VHH序列,用ELISA法篩選出抗Vsig4的高親和力納米抗體,并用Vsig4穩(wěn)定表達(dá)細(xì)胞系驗(yàn)證納米抗體的結(jié)合能力。結(jié)果成功構(gòu)建了插入率為70%、庫容量為7.27×107的噬菌體表達(dá)文庫,經(jīng)過克隆篩選獲得136個(gè)Vsig4陽性單克隆,經(jīng)測(cè)序獲得15個(gè)不同的VHH基因,將這些基因克隆至原核表達(dá)體系,表達(dá)和純化后獲得了高純度的Vsig4納米抗體,其中Nb119的親和力最高,并且可以與Vsig4穩(wěn)定表達(dá)細(xì)胞系結(jié)合。結(jié)論成功構(gòu)建并篩選了特異性、高親和力的Vsig4納米抗體,以期用于檢測(cè)巨噬細(xì)胞表面Vsig4的表達(dá)和構(gòu)建特異性分子探針。
[Abstract]:Objective to construct V-set and immunoglobulin domain containing-4 (Vsig4)-specific nano-antibody to serve as a molecular probe for macrophages. Methods Vsig4 recombinant protein was used to immunize alpaca, and lymphocytes were isolated from the blood. The phage display library was constructed by phage display technique, and the phage binding to Vsig4 protein was obtained by three successive biological screening. The VHH sequence was obtained by sequencing and gene alignment, and the high affinity nano-antibodies against Vsig4 were screened by ELISA method. The binding ability of nano-antibodies was verified by Vsig4 stable expression cell line. Results A bacteriophage expression library with 70% insertion rate and 7.27 脳 10 ~ 7 library capacity was successfully constructed. After cloning and screening, a total of 136 Vsig4 positive clones were obtained. 15 different VHH genes were obtained by sequencing, and these genes were cloned into prokaryotic expression system. After expression and purification, high purity Vsig4 nano-antibodies were obtained, among which Nb119 had the highest affinity and could bind to the stable expression cell line Vsig4. Conclusion the specific and high affinity Vsig4 nano-antibodies were successfully constructed and screened in order to detect the expression of Vsig4 on the surface of macrophages and construct specific molecular probes.
【作者單位】： 西安交通大學(xué)醫(yī)學(xué)部基礎(chǔ)醫(yī)學(xué)院 西安交通大學(xué)環(huán)境與疾病相關(guān)基因教育部重點(diǎn)實(shí)驗(yàn)室;西安交通大學(xué)生命科學(xué)與技術(shù)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(No.81501527) 陜西省自然科學(xué)基礎(chǔ)研究計(jì)劃項(xiàng)目(No.XJJ2015048)~~
【分類號(hào)】：R392.11
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本文編號(hào)：2454027