本文選題：東亞鉗蝎 + 轉(zhuǎn)錄組學(xué)　； 參考：《南京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的:利用毒素組學(xué)(轉(zhuǎn)錄組、蛋白組)和分離鑒定技術(shù)(分子篩、高效液相、質(zhì)譜)構(gòu)建陜西來源的野生東亞鉗蝎毒素多肽信息資源庫;并基于該資源庫結(jié)合東亞鉗蝎天然蝎毒多肽分離純化的結(jié)果,篩選出新型東亞鉗蝎Na+通道毒素;對發(fā)現(xiàn)的新型東亞鉗蝎Na+通道毒素進(jìn)行性質(zhì)鑒定和初步活性研究。方法:(1)構(gòu)建陜西來源的東亞鉗蝎的轉(zhuǎn)錄組和cDNA文庫,得到的unigene經(jīng)識別、拼接、比對后得到同源基因,再利用Blast進(jìn)行數(shù)據(jù)庫比對,進(jìn)行基因功能分類注釋,分析其生物學(xué)功能,篩選出可能為毒素的基因。(2)構(gòu)建并分析陜西來源的東亞鉗蝎蛋白組學(xué)數(shù)據(jù),利用iTRAQ技術(shù)對東亞鉗蝎蝎毒素進(jìn)行標(biāo)記和定量,通過SCX分離、液相串聯(lián)質(zhì)譜進(jìn)行蛋白質(zhì)鑒定。(3)利用Sephadex G50凝膠過濾色譜和高效液相儀進(jìn)行東亞鉗蝎天然蝎毒多肽的逐級分離,結(jié)合電生理活性篩選具有活性的毒素蛋白單組份,并對其進(jìn)行質(zhì)譜鑒定,利用組學(xué)數(shù)據(jù)結(jié)合東亞鉗蝎天然蝎毒多肽的分離鑒定結(jié)果篩選得到新Na+通道毒素BmKNaTx12。(4)利用與BmKNaTx12具有較高同源性的Cn12進(jìn)行同源建模,選取打分最高的模型得到BmKNaTx12的結(jié)構(gòu),并進(jìn)行分子動力學(xué)模擬。構(gòu)建BmKNaTx12重組表達(dá)系統(tǒng),通過Western blot技術(shù)確定重組蛋白最佳表達(dá)時(shí)間,并在HEK293細(xì)胞中表達(dá),經(jīng)蛋白A親和層析后獲得重組蛋白,利用SDS-PAGE凝膠電泳和高效液相進(jìn)行鑒定。(5)利用全細(xì)胞膜片鉗檢測rBmKNaTx12對hNav1.7通道和rNav1.8通道電流的影響。結(jié)果:(1)構(gòu)建的均一化cDNA文庫容量為1.6×106cfu/ml,重組率為93.75%,插入序列平均長度大于750bp,得到36665條unigene,經(jīng)數(shù)據(jù)庫比對得到2231條同源基因,其中毒素相關(guān)基因超過50%。轉(zhuǎn)錄本數(shù)據(jù)進(jìn)行基因功能注釋后,約有8%的序列疑似新Na+通道毒素。(2)蛋白質(zhì)組通過液相串聯(lián)質(zhì)譜經(jīng)Mascot分析,得到3382張unique譜圖,鑒定到245個(gè)蛋白。(3)陜西來源的東亞鉗蝎天然蝎毒多肽經(jīng)凝膠過濾色譜和高效液相分離后共鑒定出49個(gè)單組份蛋白。經(jīng)組學(xué)數(shù)據(jù)篩選鑒定出19條全新的毒素多肽序列,其中有9條疑似新型東亞鉗蝎Na+通道毒素,結(jié)合組學(xué)數(shù)據(jù)和天然蝎毒多肽的分離鑒定結(jié)果篩選得到新型東亞鉗蝎Na+通道毒素BmKNaTx12。(4)BmKNaTx12與Cn12的同源相似性為35%,利用Cn12的結(jié)構(gòu)為模板進(jìn)行同源建模,并對BmKNaTx12進(jìn)行分子動力學(xué)模擬。成功構(gòu)建BmKNaTx12的重組表達(dá)載體,并通過HEK293細(xì)胞成功表達(dá),ProteinA親和純化柱純化后得到濃度為0.12mg/ml的重組蛋白溶液共30ml。SDS-PAGE凝膠電泳顯示其大小約40kD,高效液相峰圖單一,說明重組蛋白的純度較高。(5)1μmol/LrBmKNaTx12能增大20%hNav1.7峰電流,不影響TTX-R的rNav1.8電流,結(jié)果顯示rBmKNaTx12能夠顯著激活hNav1.7電流。結(jié)論:利用毒素組學(xué)(轉(zhuǎn)錄組、蛋白組)和分離鑒定技術(shù)構(gòu)建陜西來源的野生東亞鉗蝎毒素多肽信息資源庫;發(fā)現(xiàn)并鑒定了以BmKNaTx12為代表的幾種新型Na+通道毒素;成功對BmKNaTx12進(jìn)行了重組表達(dá),BmKNaTx12明顯增大hNav1.7電流。以BmKNaTx12為模板建立新型Na+通道毒素的從分離鑒定到重組制備的工藝,為其后續(xù)的生物學(xué)功能研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q51;Q78

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