本文選題：格拉利素 + 抗菌肽　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2007年碩士論文

【摘要】： 人Glyrichin基因(中文：人格拉利素，又稱HL52)是在本室首次發(fā)現(xiàn)并注冊的鼠Glyrichin基因(GenBank number：AY028425，又稱mL52)的基礎(chǔ)上，以22周人胎肝cDNA文庫為模板擴增出的新基因。本研究是在鼠.Glyrichin的基礎(chǔ)上，通過生物信息學(xué)和分子細胞生物學(xué)對人Glyrichin基因進行功能研究。 生物信息學(xué)分析顯示人Glyrichin基因核苷酸長度為240bp，具有完整的開放閱讀框架，編碼79個氨基酸。電子PCR染色體定位提示人Glyrichin基因定位于染色體的20q11.22，基因組結(jié)構(gòu)分析顯示Glyrichin由兩個外顯子和一個內(nèi)含子組成。蛋白序列同源性分析未發(fā)現(xiàn)有已知功能的同源蛋白，也未發(fā)現(xiàn)已知的功能域，是一個功能未知的蛋白，這對該基因功能的研究帶來了一定的困難。但其理化特點如富含甘氨酸(21.5％)，pI=9.58。在pH=7時帶正電荷，是小分子堿性蛋白，二級結(jié)構(gòu)使用DNAstar5.0軟件分析為集親水性與疏水性于一體，以β折疊為主，這與已知抗菌肽共有的結(jié)構(gòu)特點相似。 本研究對人Glyrichin的天然存在性進行了探討，以Northern雜交方法分析該基因在四種人源性腫瘤細胞中的表達及轉(zhuǎn)錄本大小，證實了人Glyrichin具有單一轉(zhuǎn)錄本，大小為600bp左右：以RT-PCR分析其在胎肝、胎腎、胎脾、胎肺等四種組織中的表達情況，以GFP為標(biāo)記蛋白對基因的亞細胞定位進行探索，證實了該蛋白可能分布于胞漿。 生物信息學(xué)分析顯示人Glyrichin理化性質(zhì)具有了抗菌肽的諸多特點，我們合成了代表人Glyrichin功能的肽段pCM-19進行抗菌功能分析，證實了pCM-19良好的抗菌功能，然后利用原核表達系統(tǒng)，構(gòu)建了含有格拉利素基因的系列缺失體的pET22b表達載體，并轉(zhuǎn)化到大腸桿菌BL21(DE3)中進行誘導(dǎo)表達。觀察到該基因誘導(dǎo)表達時對宿主菌菌體生長具有明顯的抑制作用，，初步證實了人Glyrichin具有明顯的抗菌活性，同時也證實了蛋白C端對其抗菌活性的發(fā)揮是必須的。人Glyrichin完整蛋白原核表達也證實了具有良好的抗菌活性。 我們選用了畢赤酵母表達系統(tǒng)對該基因進行功能分析，將人Glyrichin基因構(gòu)建到畢赤酵母表達載體pPIC9和pPIC9K中，鋰鹽法轉(zhuǎn)化畢赤酵母GS115中，G418抗生素篩選高拷貝轉(zhuǎn)化子，進行培養(yǎng)誘導(dǎo)，對表達上清通過“紙片法”、“試管法”、“瓊脂糖紙片擴散法”驗證表達產(chǎn)物的活性。證實了人Glyrichin具有抗菌活性。 通過生物信息學(xué)比較我們發(fā)現(xiàn)了有29個成員組成的高度同源的全新抗菌肽家系，通過代表家系九個成員的4個合成肽的抗菌活性檢測和線蟲源格拉利素基因原核表達分析，證實了它們具有良好的抗菌活性，強烈提示一個跨物種的抗菌肽家系的存在。 綜上所述，本研究獲得了人源性和線蟲源的格拉利素基因，確認了其天然存在性，通過原核表達和畢赤酵母表達系統(tǒng)證實了其具有明顯的抗菌活性。生物信息學(xué)分析及實驗驗證提示一個跨物種的有29個成員的新的抗菌肽家系的存在。
[Abstract]:The human Glyrichin gene ( also known as HL52 ) is a new gene amplified by human Glyrichin gene ( GenBank number : AY028425 , also referred to as mL52 ) , which is first discovered and registered in the lab . The study is based on the mouse . Glyrichin gene , the function of human Glyrichin gene is studied by bioinformatics and molecular cell biology .









Bioinformatic analysis showed that the human Glyrichin gene had a length of 240 bp , a complete open reading frame and 79 amino acids . The electron PCR chromosomal location suggested that the Glyrichin gene was located at 20q11.22 of the chromosome . The analysis of the genomic structure showed that Glyrichin was composed of two exons and an intron .









In this study , the native existence of human Glyrichin was discussed . The expression of the gene in four human tumor cells and its transcript size were analyzed by Northern blot . The expression of Glyrichin in four human tumor cells was confirmed . The expression of Glyrichin in fetal liver , fetal kidney , fetal spleen and fetal lung was analyzed by RT - PCR .









Bioinformatic analysis showed that the physical and chemical properties of human Glyrichin had many characteristics of antimicrobial peptide . We synthesized peptide fragment pCM - 19 with the function of Glyrichin , and then transformed into E . coli BL21 ( DE3 ) .









We selected the Pichia pastoris expression system to perform functional analysis on the gene . The human Glyrichin gene was constructed into Pichia pastoris expression vector , and then transformed into Pichia pastoris GS115 by the lithium salt method . The high copy transformants were screened by G418 antibiotics . The expression supernatant was verified by the " paper sheet method " , " test tube method " and " agarose paper diffusion method " . The human Glyrich in has antibacterial activity .









Through bioinformatic comparison , we have found that there are 29 members of highly homologous new antibacterial peptide family , through the analysis of the antibacterial activity of four synthetic peptides representing the nine members of the family family and the prokaryotic expression analysis of the nematode - derived glabridin gene , it is confirmed that they have good antibacterial activity and strongly suggest the existence of an antibacterial peptide family of a cross - species .









In conclusion , this study has obtained the humanized and nematode - derived glabridin gene , confirmed its natural existence , confirmed its obvious antibacterial activity through prokaryotic expression and Pichia pastoris expression system . Bioinformatic analysis and experimental verification suggested that there existed a new antibacterial peptide family of 29 members across species .
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R346

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1 楊超文;抗菌肽基因格拉利素的克隆及功能研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2007年

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