基于噬菌体展示技术筛选内毒素结合肽并建立内毒素检测新方法

发布时间：2018-09-19 11:43

【摘要】：内毒素又被称作为脂多糖是革兰氏阴性细菌的细胞壁中的一种重要的组成部分。内毒素进入血液中,将会出现发热、血压降低、弥散性血管内凝血、内毒素败血症等一系列临床反应,严重时会导致休克、甚至是死亡。所以一些食品、药品、医疗器械在出厂前都要进行内毒素的检测,以避免内毒素对人体造成的危害。中国药典中已经收录了光度法和凝胶法这两种用于测定细菌内毒素含量的的检查方法,这两种方法都是在利用鲎血提取物的基础上对内毒素的含量进行的定性甚至定量的测定。鲎试剂是从鲎血中提取的,原材料稀缺、价格昂贵、不稳定。本文利用噬菌体展示技术从构建好的十二肽库中经过了三轮筛选得到能够与内毒素结合的噬菌体克隆,再提取噬菌体ssDNA,运用DNASTAR和BLAST软件对序列进行分析,最后用固相合成法合成十二肽序列,利用分子互作技术鉴定十二肽与内毒素的结合能力,并建立增强比浊法检测内毒素。利用噬菌体展示技术,以内毒素做为目标靶分子并将其包被在聚丙烯平板上,然后将噬菌体展示出的肽库与已经预先包被好靶分子的平板共温育,经过两轮非特异性洗脱筛选及扩增和一轮特异性洗脱筛选,最后成功筛选出能够与内毒素高亲和性结合的噬菌体克隆。将筛选得到的结合噬菌体克隆进行培养,提取噬菌体ssDNA,其大小为6400bp,经测序及软件分析得到十二肽序列:QVTPQVPRSTQM和QVNGLGERSQ QM。运用固相合成法合成十二肽各1mg,纯度达95%。运用分子互作技术对合成的十二肽进行亲和性分析,其解离平衡常数(KD)分别达到3.52×10-9和8.012×10-9,Full^R2分别达到0.9979和0.9969,表明合成的十二肽与内毒素具有良好的亲和性。利用所合成的十二肽建立内毒素检测-增强比浊法,获得增强比浊检测内毒素标准曲线其线性范围是0.03~0.48EU/mL,R2达到0.9925。该方法能够简便、准确的检测内毒素,为临床快速检测内毒素奠定基础。
[Abstract]:Endotoxin, also known as lipopolysaccharide, is an important component of the cell wall of Gram-negative bacteria. A series of clinical reactions such as fever, decreased blood pressure, disseminated intravascular coagulation, endotoxemia and so on will occur when endotoxin enters the blood, which can lead to shock or even death. Therefore, some foods, drugs and medical devices should be tested for endotoxin before they leave the factory to avoid the harm of endotoxin to human body. Photometric and gel methods have been included in the Chinese pharmacopoeia for the determination of bacterial endotoxin. These two methods are qualitative and even quantitative determination of endotoxin on the basis of Limulus blood extract. Limulus reagent is extracted from Limulus blood, raw materials are scarce, expensive, unstable. In this paper, phage display technology was used to obtain phage clones which could bind with endotoxin from the constructed dodecapeptide library. The phage ssDNA, was extracted and sequenced by DNASTAR and BLAST software. Finally, the dodeceptide sequence was synthesized by solid state synthesis, the binding ability of dodecapeptide to endotoxin was identified by molecular interaction technique, and an enhanced turbidimetric method was established for the detection of endotoxin. Using phage display technology, endotoxin was used as target molecule and coated on polypropylene plate, then the peptide library was incubated with the plate which had been precoated with target molecule. After two rounds of non-specific elution screening and amplification and one specific elution screening, the phage clones which could bind to endotoxin with high affinity were successfully screened. The phage ssDNA, was extracted from the screened phage clone. The size of phage ssDNA, was 6400bp. the sequence of dodecapeptide was obtained by sequencing and software analysis. The sequence of dodecapeptide: QVTPQVPRSTQM and QVNGLGERSQ QM. were obtained. The dodecapeptide was synthesized by solid state synthesis with a purity of 95 mg each. The molecular interaction technique was used to analyze the affinity of the synthesized dodeceptide. The dissociation equilibrium constant (KD) reached 3.52 脳 10 ~ (-9) and 8.012 脳 10 ~ (-9) full ^ R2, respectively, 0.9979 and 0.9969, respectively, which indicated that the synthesized dodeceptide had good affinity with endotoxin. The standard curve of enhanced turbidity for endotoxin detection was obtained by using the synthesized dodeceptide to establish endotoxin-enhanced turbidimetry. The linear range was 0.03 ~ 0.48 EUU / mL ~ (2) ~ (2) up to 0.9925. This method is simple and accurate for the detection of endotoxin, and lays a foundation for rapid detection of endotoxin in clinic.
【学位授予单位】：长春理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5;O652

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