本文選題：微囊藻毒素-LR(MC-LR) + 噬菌體隨機(jī)展示肽庫��； 參考：《長春理工大學(xué)》2013年碩士論文

【摘要】：隨著環(huán)境污染的加劇,淡水水體富營養(yǎng)化的狀態(tài)越來越嚴(yán)重,有毒藍(lán)藻水華在各地頻繁發(fā)生,其產(chǎn)生的毒素對淡水水體尤其是水源水及飲用水的污染也越來越嚴(yán)重,對人類及動物的健康造成了極大威脅。微囊藻毒素是一大類藍(lán)藻毒素,其中,最常見毒性最強(qiáng)的藍(lán)藻毒素是微囊藻毒素-LR (MC-LR), MC-LR是一種低分子量的環(huán)狀多肽,對生物體具有多重毒性,已將其列《入國家生活飲用水衛(wèi)生標(biāo)準(zhǔn)》(GB5749-2006)之中,規(guī)定的界限為1μg/L。目前MC-LR檢測方法很多,但都存在價(jià)格昂貴、耗時(shí)費(fèi)力、操作復(fù)雜等問題,因此,建立一種快速、便捷、實(shí)時(shí)、特異性檢測微囊藻毒素-LR的檢測方法具有重要意義。 本文通過培養(yǎng)銅綠微囊藻,收集足夠多的藻細(xì)胞進(jìn)行MC-LR提取,經(jīng)超聲波破碎、溶劑萃取、高效液相色譜(HPLC)分離純化、冷凍干燥獲得MC-LR純品。以MC-LR為靶分子,利用噬菌體隨機(jī)展示十二肽庫進(jìn)行MC-LR特異性親和配體序列的篩選。經(jīng)過四輪篩選、測序及序列比對,最終獲得了一條MC-LR特異性親和配體序列。后以固相化學(xué)合成法合成該配體肽,并于N端進(jìn)行FITC熒光標(biāo)記制得MC-LR特異性親和配體肽探針,最后對制得探針進(jìn)行活性檢測與應(yīng)用,驗(yàn)證并建立生物學(xué)新方法。 實(shí)驗(yàn)結(jié)果顯示,提取獲得的MC-LR純度達(dá)94%。經(jīng)過噬菌體隨機(jī)展示十二肽庫的四輪篩選及測序和序列比對,得到MC-LR特異性親和配體序列：H-F-F-K-W-H-T-R-T-N-D-Q,經(jīng)人工合成獲得了有FITC熒光標(biāo)記的MC-LR特異性親和配體肽P1:FITC-Acp-H-F-F-K-W-H-T-R-T-N-D-Q-COOH,通過熒光ELISA檢測實(shí)驗(yàn)表明配體肽P1與MC-LR具有一定的結(jié)合活性。以配體肽P1為熒光探針檢測實(shí)際水樣,并與HPLC檢測結(jié)果進(jìn)行對比,結(jié)果表明該方法靈敏度較強(qiáng),特異性較高,且具有快速、操作簡單、成本低等特點(diǎn),可為水質(zhì)安全檢測提供新產(chǎn)品和生物學(xué)新方法。
[Abstract]:With the intensification of environmental pollution, the eutrophication of freshwater water is becoming more and more serious. The toxic cyanobacteria Shui Hua occurs frequently in various places, and the toxin produced by it is more and more serious to the freshwater water, especially to the source water and drinking water. It poses a great threat to human and animal health. Microcystins are a large class of cyanobacterial toxins, of which the most common and most toxic is microcystin-LR MC-LRN. MC-LR is a low molecular weight cyclic polypeptide, which has multiple toxicity to organisms. It has been listed in the National drinking Water Sanitation Standard (GB5749-2006), with a limit of 1 渭 g 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1). At present, there are many methods for MC-LR detection, but they are expensive, time-consuming and complicated. Therefore, it is of great significance to establish a rapid, convenient, real-time and specific detection method for microcystin-LR. In this paper, enough algal cells were collected for MC-LR extraction by cultured microcystis aeruginosa. The purified MC-LR was isolated and purified by ultrasonic wave, solvent extraction, high performance liquid chromatography (HPLC), and freeze-dried. Using MC-LR as the target molecule, the phage displayed dodecapeptide library was used to screen the MC-LR specific affinity ligand sequence. After four rounds of screening, sequencing and sequence alignment, a MC-LR specific affinity ligand sequence was obtained. The ligand peptide was synthesized by solid-state chemical synthesis and then labeled with FITC at the N-terminal to obtain the MC-LR specific affinity ligand peptide probe. Finally, the activity of the prepared probe was detected and applied, and a new biological method was established. The results showed that the purity of MC-LR extracted was 94%. Four rounds of screening, sequencing and sequence alignment of a phage display library of dodecapeptide were carried out at random. The specific affinity ligand sequence of MC-LR: H-F-F-K-W-H-T-R-T-N-N-D-Qwas synthesized. The ligand peptide P1: FITC-Acp-F-F-K-W-H-T-R-T-N-D-COOHwith FITC fluorescent labeling was synthesized. The binding activity of ligand P1 to MC-LR was confirmed by fluorescence ELISA assay. The ligand peptide P1 was used as fluorescence probe to detect the actual water sample and compared with the results of HPLC. The results showed that the method was sensitive, specific, rapid, easy to operate and low cost. It can provide new products and new biological methods for water quality safety detection.
【學(xué)位授予單位】：長春理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R123.1

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