微囊藻毒素-LR快速检测生物学新方法的建立

发布时间：2018-05-18 21:41

本文选题：微囊藻毒素-LR(MC-LR) + 噬菌体随机展示肽库　；参考：《长春理工大学》2013年硕士论文

【摘要】：随着环境污染的加剧,淡水水体富营养化的状态越来越严重,有毒蓝藻水华在各地频繁发生,其产生的毒素对淡水水体尤其是水源水及饮用水的污染也越来越严重,对人类及动物的健康造成了极大威胁。微囊藻毒素是一大类蓝藻毒素,其中,最常见毒性最强的蓝藻毒素是微囊藻毒素-LR (MC-LR), MC-LR是一种低分子量的环状多肽,对生物体具有多重毒性,已将其列《入国家生活饮用水卫生标准》(GB5749-2006)之中,规定的界限为1μg/L。目前MC-LR检测方法很多,但都存在价格昂贵、耗时费力、操作复杂等问题,因此,建立一种快速、便捷、实时、特异性检测微囊藻毒素-LR的检测方法具有重要意义。本文通过培养铜绿微囊藻,收集足够多的藻细胞进行MC-LR提取,经超声波破碎、溶剂萃取、高效液相色谱(HPLC)分离纯化、冷冻干燥获得MC-LR纯品。以MC-LR为靶分子,利用噬菌体随机展示十二肽库进行MC-LR特异性亲和配体序列的筛选。经过四轮筛选、测序及序列比对,最终获得了一条MC-LR特异性亲和配体序列。后以固相化学合成法合成该配体肽,并于N端进行FITC荧光标记制得MC-LR特异性亲和配体肽探针,最后对制得探针进行活性检测与应用,验证并建立生物学新方法。实验结果显示,提取获得的MC-LR纯度达94%。经过噬菌体随机展示十二肽库的四轮筛选及测序和序列比对,得到MC-LR特异性亲和配体序列：H-F-F-K-W-H-T-R-T-N-D-Q,经人工合成获得了有FITC荧光标记的MC-LR特异性亲和配体肽P1:FITC-Acp-H-F-F-K-W-H-T-R-T-N-D-Q-COOH,通过荧光ELISA检测实验表明配体肽P1与MC-LR具有一定的结合活性。以配体肽P1为荧光探针检测实际水样,并与HPLC检测结果进行对比,结果表明该方法灵敏度较强,特异性较高,且具有快速、操作简单、成本低等特点,可为水质安全检测提供新产品和生物学新方法。
[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.
【学位授予单位】：长春理工大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R123.1

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