海洋聚醚类毒素基于适配体的检测及生物合成的探索

发布时间：2018-05-15 13:42

本文选题：短裸甲藻毒素 + 岩沙海葵毒素　；参考：《第二军医大学》2016年博士论文

【摘要】：海洋聚醚类毒素结构新颖复杂,毒性强,稳定性好,在全球分布广泛。多来源于容易形成赤潮的有毒甲藻,通过食物链在贝类、鱼类等海产品体内富集,对于人类健康和海洋渔业造成了严重威胁。现有的毒素检测方法都存在各种各样的不足,因此急需新型的快速检测聚醚类毒素的方法。此外,由于天然海洋聚醚类毒素提取困难,甲藻基因组又很难解析,因此目前对于毒素的生物合成了解的比较匮乏。本课题以海洋聚醚类毒素中代表性的短裸甲藻毒素BTX、岩沙海葵毒素PLTX和大田软海绵酸OA为研究对象,从毒素的检测和生物合成两方面展开研究,主要研究内容及结果如下:1.海洋聚醚类毒素BTX适配体的筛选与应用。采用体外SELEX技术经过18轮的筛选富集,分别获得了与海洋聚醚类毒素BTX-A和BTX-B特异性结合的高亲和力适配体A5和B2。通过先混合靶标筛选后分开次级文库筛选的方式,一次性获得了两种毒素同系物的适配体,建立了一种高效筛选同系物适配体的SELEX方法。在此基础上A5和B2这两个适配体序列进行了截短优化和突变改造。基于mfold软件对该适配体的二级结构进行分析,截去A5引物区及5’端第一个茎环,并进一步进行了非必须核苷酸的去除和突变,获得仅含有32个核苷酸的核心序列A5-S3G(Kd=72n M)。且不与STX、GTX、BTX-B、PLTX、OA等毒素结合,具有较好的特异性。通过生物素-链霉亲合素耦合在线制备了基于SSA芯片和BLI技术的适配体传感器,建立了基于生物膜干涉适配体传感器检测BTX的方法。最佳检测时间250 s,检测范围为100~2000 n M,最低检测限4.5 n M(4 ng/ml),且无交叉反应,同时具有较低的变异系数,可用于BTX-A的实验室检测。2.海洋聚醚类毒素PLTX适配体的筛选与优化。采用体外SELEX技术经过10轮的筛选富集,通过序列同源比对聚类分析,结合生物膜干涉技术对筛选得到的若干适配体序列进行亲和力表征,获得了6条与海洋聚醚类毒素PLTX特异性结合的高亲和力适配体,适配体P-13亲和力最高(Kd=0.9 n M)。在此基础上,对P-5、P-13和P-18三条适配体序列进行了截短优化。基于mfold二级结构的分析,截去引物区及非必须核苷酸,获得了2段与PLTX高亲和力结合的核心序列,P-13S2(Kd=0.56 n M)和P-18S2(Kd=0.93n M),为PLTX生物传感器的制备奠定了基础,提供了识别元件。将PLTX通过氨基偶联到SPR CM5生物芯片表面,并用该芯片对优化后的序列进行亲和力表征,结合优化后的序列及SPR技术,该芯片可用于制备高灵敏的竞争法检测PLTX的生物传感器。3.基于转录组测序的OA生物合成相关基因的挖掘。选取了初始氮浓度:磷浓度分别为15:1和30:1的培养条件培养利玛原甲藻P.lima,培养不同时间收集藻细胞进行转录组测序和de novo拼接,得到了98594个Unigene,其平均长度为1319 nt,N50为1856 nt。得到的转录本通过Nr数据库、Uni Prot KB/Swiss-Prot数据库、COG数据库进行功能注释及分类,通过KEGG数据库进行相关代谢途径注释,其中73289个Unigene成功注释到功能,注释到333条代谢途径。在此基础上初步确定了代谢途径中基因的上调及下调水平。最后利用RSEM计算每个转录本的RPKM值,并对相关代谢途径中的转录本的表达差异进行了初步分析。在此基础上初步解析了OA生物合成相关的重要基因,如编码PKS、硫脂酶、黄素单加氧酶、环氧化酶、环氧化物水解酶、细胞色素P450等酶的基因在不同样品中表达量的差异。综上,本研究分别筛选出了能与BTX和PLTX高亲和力、特异性结合的适配体,并对其进行优化改造,成功制备了基于生物膜干涉技术的快速检测BTX的适配体传感器,获得了能够开发用于PLTX检测的SPR芯片和适配体识别元件,对于聚醚类毒素检测提供了新的方法和工具。此外,实现了对利玛原甲藻P.lima的转录组解析,并比较了不同营养条件下培养不同时间利玛原甲藻的基因的表达差异,在此基础上初步解析了OA生物合成相关的重要基因的差异表达,对进一步阐明OA的生物合成途径和表达调控以及对利玛原甲藻的产毒进行遗传改造奠定基础。
[Abstract]:Marine polyether toxoid has a complex structure, strong toxicity, good stability and wide distribution in the world. It comes from toxic methania which is easy to form red tide, and is enriched in marine products such as shellfish and fish by food chain. There are serious threats to human health and marine fishery. There are all kinds of deficiencies in the existing methods of detection of toxins. Therefore, a new method for rapid detection of polyether toxoid is urgently needed. In addition, because the extraction of the natural marine polyether toxoid is difficult, the genome of the methatom is difficult to be analyzed. Therefore, the biosynthesis of the toxin is scarce. This topic is based on the representative short methanobacteria toxin BTX of the marine polyether toxoid, the sands sea anemone toxin PLTX and Soft cavernic acid OA is the research object, which is studied from two aspects of detection and biosynthesis of toxin. The main contents and results are as follows: 1. screening and application of marine polyether toxoid BTX aptamers. Using in vitro SELEX technology after 18 rounds of screening and enrichment, the specific binding of marine polyether toxoid BTX-A and BTX-B was obtained. High affinity ligands, A5 and B2., were selected by the screening of separate secondary libraries after the first mixed target screening. The aptamers of two toxin homologues were obtained at one time, and a SELEX method to efficiently screen the homologue aptamers was established. On this basis, the two aptamer sequences of A5 and B2 were truncated and mutated. Based on mfold The two stage structure of the aptamer was analyzed by the software, the A5 primer area and the first stem ring at the 5 'end were truncated, and the removal and mutation of non essential nucleotides were further carried out. The core sequence of only 32 nucleotides, A5-S3G (Kd=72n M), was obtained. It was not combined with STX, GTX, BTX-B, PLTX, OA and other toxins, which had better specificity. Through biotin - The aptamer sensor based on SSA chip and BLI technology is prepared online by streptavidin coupling. The method of detecting BTX based on the biofilm interferometric aptamer sensor is established. The optimum detection time is 250 s, the detection range is 100~2000 n M, the minimum detection limit is 4.5 n M (4 ng/ml), and there is no cross reaction, and has a low coefficient of variation, which can be used in B. The TX-A laboratory tests the screening and optimization of the.2. marine polyether toxoid PLTX aptamers. After 10 rounds of screening and enrichment by the SELEX technique in vitro, the affinity sequence of selected aptamers was obtained through the sequence homologous alignment cluster analysis and the biofilm interference technique, and 6 marine polyether toxoid PLTX was obtained. High affinity aptamers of heterosexual binding and aptamer P-13 have the highest affinity (Kd=0.9 n M). On this basis, the three aptamers of P-5, P-13 and P-18 are truncated and optimized. Based on the analysis of mfold two structure, the primer region and non essential nucleotides are truncated, and the core sequence of 2 segments with high affinity to PLTX is obtained, P-13S2 (Kd=0.56 n). And P-18S2 (Kd=0.93n M), which lays the foundation for the preparation of PLTX biosensors, and provides a recognition element. PLTX is coupled to the surface of the SPR CM5 on the surface of the biochip of the SPR CM5, and the affinity characterization of the optimized sequence is carried out with this chip. Combined with the optimized sequence and SPR technology, the chip can be used to prepare a highly sensitive competitive method for detecting PLTX. The biosensor.3. was based on the mining of OA biosynthesis related genes of the transcriptional sequence. The initial nitrogen concentration was selected: the phosphorus concentration was cultured in 15:1 and 30:1, respectively, to train the P.lima of OA, and to collect the algal cells for sequencing and de novo splicing at different time. 98594 Unigene were obtained, the average length was 1319 NT, N5. The transcriptional transcript obtained by 0 for 1856 nt. is annotated and classified through the Nr database, the Uni Prot KB/Swiss-Prot database, the COG database, and the related metabolic pathway annotations through the KEGG database, of which 73289 Unigene are successfully annotated to the function, annotated to 333 metabolic pathways. On this basis, the gene in the metabolic pathway is preliminarily identified. At last, the RPKM value of each transcript was calculated by RSEM and the difference in the expression of the transcriptional transcript in the related metabolic pathways was preliminarily analyzed. On the basis of this, the important genes related to OA biosynthesis, such as encoding PKS, sulfur lipase, flavin monooxygenase, cyclooxygenase, epoxide hydrolase, cytochrome P, were preliminarily analyzed. The difference in the expression of 450 isozyme genes in different samples. In this study, we screened out the aptamers with high affinity and specific binding to BTX and PLTX, and optimized them, and successfully prepared the aptamer sensor for rapid detection of BTX based on biofilm interference technology, and obtained the SPR that can be developed for PLTX detection. The microarray and aptamer identification elements provide new methods and tools for the detection of polyether toxoid. In addition, the transcriptional analysis of P.lima is realized and the differences in the expression of gene in different nutrition conditions at different time are compared. On the basis of this, the importance of OA biosynthesis is preliminarily analyzed. The differential expression of genes lays the foundation for further elucidate the biosynthetic pathway and expression regulation of OA and the genetic transformation of the toxin produced by dinoflagellate Lima.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78

【参考文献】