一种新型东亚钳蝎钠通道毒素BmKNaTx12的基因克

发布时间：2018-06-08 19:37

本文选题：东亚钳蝎 + 转录组学　；参考：《南京中医药大学》2017年硕士论文

【摘要】：目的:利用毒素组学(转录组、蛋白组)和分离鉴定技术(分子筛、高效液相、质谱)构建陕西来源的野生东亚钳蝎毒素多肽信息资源库;并基于该资源库结合东亚钳蝎天然蝎毒多肽分离纯化的结果,筛选出新型东亚钳蝎Na+通道毒素;对发现的新型东亚钳蝎Na+通道毒素进行性质鉴定和初步活性研究。方法:(1)构建陕西来源的东亚钳蝎的转录组和cDNA文库,得到的unigene经识别、拼接、比对后得到同源基因,再利用Blast进行数据库比对,进行基因功能分类注释,分析其生物学功能,筛选出可能为毒素的基因。(2)构建并分析陕西来源的东亚钳蝎蛋白组学数据,利用iTRAQ技术对东亚钳蝎蝎毒素进行标记和定量,通过SCX分离、液相串联质谱进行蛋白质鉴定。(3)利用Sephadex G50凝胶过滤色谱和高效液相仪进行东亚钳蝎天然蝎毒多肽的逐级分离,结合电生理活性筛选具有活性的毒素蛋白单组份,并对其进行质谱鉴定,利用组学数据结合东亚钳蝎天然蝎毒多肽的分离鉴定结果筛选得到新Na+通道毒素BmKNaTx12。(4)利用与BmKNaTx12具有较高同源性的Cn12进行同源建模,选取打分最高的模型得到BmKNaTx12的结构,并进行分子动力学模拟。构建BmKNaTx12重组表达系统,通过Western blot技术确定重组蛋白最佳表达时间,并在HEK293细胞中表达,经蛋白A亲和层析后获得重组蛋白,利用SDS-PAGE凝胶电泳和高效液相进行鉴定。(5)利用全细胞膜片钳检测rBmKNaTx12对hNav1.7通道和rNav1.8通道电流的影响。结果:(1)构建的均一化cDNA文库容量为1.6×106cfu/ml,重组率为93.75%,插入序列平均长度大于750bp,得到36665条unigene,经数据库比对得到2231条同源基因,其中毒素相关基因超过50%。转录本数据进行基因功能注释后,约有8%的序列疑似新Na+通道毒素。(2)蛋白质组通过液相串联质谱经Mascot分析,得到3382张unique谱图,鉴定到245个蛋白。(3)陕西来源的东亚钳蝎天然蝎毒多肽经凝胶过滤色谱和高效液相分离后共鉴定出49个单组份蛋白。经组学数据筛选鉴定出19条全新的毒素多肽序列,其中有9条疑似新型东亚钳蝎Na+通道毒素,结合组学数据和天然蝎毒多肽的分离鉴定结果筛选得到新型东亚钳蝎Na+通道毒素BmKNaTx12。(4)BmKNaTx12与Cn12的同源相似性为35%,利用Cn12的结构为模板进行同源建模,并对BmKNaTx12进行分子动力学模拟。成功构建BmKNaTx12的重组表达载体,并通过HEK293细胞成功表达,ProteinA亲和纯化柱纯化后得到浓度为0.12mg/ml的重组蛋白溶液共30ml。SDS-PAGE凝胶电泳显示其大小约40kD,高效液相峰图单一,说明重组蛋白的纯度较高。(5)1μmol/LrBmKNaTx12能增大20%hNav1.7峰电流,不影响TTX-R的rNav1.8电流,结果显示rBmKNaTx12能够显著激活hNav1.7电流。结论:利用毒素组学(转录组、蛋白组)和分离鉴定技术构建陕西来源的野生东亚钳蝎毒素多肽信息资源库;发现并鉴定了以BmKNaTx12为代表的几种新型Na+通道毒素;成功对BmKNaTx12进行了重组表达,BmKNaTx12明显增大hNav1.7电流。以BmKNaTx12为模板建立新型Na+通道毒素的从分离鉴定到重组制备的工艺,为其后续的生物学功能研究奠定了基础。
[Abstract]:Objective: to construct a Shaanxi wild scorpion toxin polypeptide information resource library from Shaanxi, using the toxin group (transcriptional group, protein group) and separation identification technique (molecular sieve, high performance liquid phase, mass spectrometry), and to screen out the new Na+ channel toxin of the new East Asian scorpion scorpion, based on the result of the separation and purification of the natural scorpion venom polypeptide of East Asia scorpion. The properties identification and preliminary activity study of the new Na+ channel toxin of the new East Asian scorpion. Methods: (1) to construct the transcriptional group and cDNA Library of the Shaanxi source of East Asia pincer scorpion, the obtained UniGene was identified, spliced, and the homologous genes were obtained after comparison. Then Blast was used to compare the database, and the gene function was classified and annotated, and the biological function was analyzed. The gene of possible toxin was selected. (2) construct and analyze the data of the protein composition of the scorpion scorpion in Shaanxi, using the iTRAQ technique to mark and quantify the scorpion scorpion toxin in East Asia, and to identify the protein by SCX separation and liquid phase tandem mass spectrometry. (3) the scorpion scorpion natural Scorpion was carried out by Sephadex G50 gel filtration chromatography and high performance liquid chromatography. The single component of the active toxin protein was screened by the isolation of the toxic peptide, combined with the electrophysiological activity, and was identified by mass spectrometry. The new Na+ channel toxin BmKNaTx12. (4) was screened by the combination of the group data and the isolation and identification results of the natural scorpion venom polypeptide of East Asia. The homologous modeling of Cn12 with high homology with BmKNaTx12 was used. The highest scoring model was selected to obtain the structure of BmKNaTx12 and to simulate the molecular dynamics. The BmKNaTx12 recombinant expression system was constructed. The optimal expression time of the recombinant protein was determined by Western blot technology, and the recombinant protein was expressed in HEK293 cells. The recombinant protein was obtained after protein A affinity chromatography, and was carried out by SDS-PAGE gel electrophoresis and high performance liquid phase. (5) the effect of rBmKNaTx12 on the current of hNav1.7 channel and rNav1.8 channel was detected by whole cell patch clamp. Results: (1) the capacity of the homogenized cDNA library was 1.6 * 106cfu/ml, the recombination rate was 93.75%, the average length of the insertion sequence was greater than 750bp, and 36665 UniGene were obtained, and 2231 homologous genes were obtained by database comparison, in which the toxin was related. After gene function annotation of gene over 50%., about 8% of the sequence suspected new Na+ channel toxin. (2) 3382 unique spectra were obtained by Mascot analysis by liquid phase tandem mass spectrometry, and 245 proteins were identified. (3) the natural scorpion polypeptide from Shaanxi source scorpion venom was separated by gel filtration chromatography and high performance liquid phase separation. A total of 49 single component proteins were identified. 19 new toxin polypeptide sequences were screened and identified by the data of the group. There were 9 suspected new Na+ channel toxin of the new East Asian pincer scorpion. The homologous similarity of the new East Asian scorpion Na+ channel venom BmKNaTx12. (4) BmKNaTx12 and Cn12 was obtained by combining the data of the group and the isolation and identification results of the natural scorpion venom polypeptide. 35%, using the structure of Cn12 as a template for homologous modeling and molecular dynamics simulation of BmKNaTx12. The recombinant expression vector of BmKNaTx12 was successfully constructed and expressed successfully through HEK293 cells. After purification of ProteinA affinity purification column, a recombinant egg white solution with a concentration of 0.12mg/ml was obtained by 30ml.SDS-PAGE gel electrophoresis. About 40kD, the high performance liquid peak map is single, indicating that the purity of the recombinant protein is high. (5) 1 mu mol/LrBmKNaTx12 can increase the 20%hNav1.7 peak current and do not affect the rNav1.8 current of TTX-R. The result shows that rBmKNaTx12 can activate the hNav1.7 current significantly. Conclusion: using the toxin omics (transcriptional group, protein group) and the isolation and identification technology to construct the wild east of Shaanxi origin. Several new Na+ channel toxins, represented by BmKNaTx12, were found and identified. The recombinant expression of BmKNaTx12 was successfully expressed, and the hNav1.7 current was significantly increased by BmKNaTx12. The process of isolation and identification of a new Na+ channel toxin was established by BmKNaTx12 as a template, and it was a subsequent biology. It lays the foundation for the study of function.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q51;Q78

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