本文選題：產(chǎn)氣莢膜梭菌ι毒素　切入點(diǎn)：Ia　出處：《中國(guó)獸醫(yī)學(xué)報(bào)》2017年02期 　論文類(lèi)型：期刊論文

【摘要】：選取經(jīng)產(chǎn)氣莢膜梭菌ι毒素免疫且抗體效價(jià)較高的小鼠,提取脾臟mRNA,通過(guò)PT-PC技術(shù),分別獲得340,325bp的重鏈可變區(qū)基因(VH)和輕鏈可變區(qū)基因(VL)。利用重疊延伸PCR(SOE-PCR)獲得大小為750bp的VH-Linker-VL單鏈抗體基因(ScFv)。利用One Step Cloning Kit試劑盒將ScFv基因片段與噬菌粒載體pCANTAB5E連接,構(gòu)建庫(kù)容約為3.5×106抗ι毒素噬菌體初級(jí)抗體庫(kù)。以Ia、Ib為抗原進(jìn)行Phage-ScFv表面展示文庫(kù)的5輪親和富集篩選,最終陽(yáng)性率均可達(dá)70%。選取親和力最強(qiáng)的3株陽(yáng)性抗體(Ia-4、Ia-8和Ib1)進(jìn)行可溶性表達(dá),為ι毒素特異性抗體的大量制備奠定基礎(chǔ),并對(duì)產(chǎn)氣莢膜梭菌ι毒素感染病例的檢測(cè)和臨床治療研究具有一定的理論價(jià)值和實(shí)踐意義。
[Abstract]:Selection of Clostridium perfringens iota toxin in immune and high titers of mice spleen extract, mRNA, through PT-PC technology, heavy chain variable region gene were obtained with 340325bp (VH) gene and the variable region of light chain (VL). Using overlap extension PCR (SOE-PCR) to obtain the size of VH-Linker-VL scFv gene 750bp (ScFv) the use of One Step. Cloning Kit kit for connecting the ScFv gene fragment and phagemid vector pCANTAB5E, construct the capacity of about 3.5 x 106 l anti phage antibody library. The primary toxin Ia, Ib antigen in Phage-ScFv surface display library of 5 rounds of affinity enrichment screening, the final positive rate was up to 70%. from the highest affinity 3 strains of positive antibody (Ia-4, Ia-8 and Ib1) soluble expression system for a large number of iota toxin specific antibody preparation and lay the foundation for C.perfringens iota toxin infection detection and clinical research has certain The theoretical value and practical significance.

【作者單位】： 河北師范大學(xué)生命科學(xué)學(xué)院;
【基金】：河北省科技支撐計(jì)劃資助項(xiàng)目(13226603D)
【分類(lèi)號(hào)】：S852.61
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本文編號(hào)：1602068