抗菌肽基因格拉利素的克隆及功能研究

发布时间：2018-06-22 21:27

本文选题：格拉利素 + 抗菌肽　；参考：《中国人民解放军军事医学科学院》2007年硕士论文

【摘要】： 人Glyrichin基因(中文：人格拉利素，又称HL52)是在本室首次发现并注册的鼠Glyrichin基因(GenBank number：AY028425，又称mL52)的基础上，以22周人胎肝cDNA文库为模板扩增出的新基因。本研究是在鼠.Glyrichin的基础上，通过生物信息学和分子细胞生物学对人Glyrichin基因进行功能研究。生物信息学分析显示人Glyrichin基因核苷酸长度为240bp，具有完整的开放阅读框架，编码79个氨基酸。电子PCR染色体定位提示人Glyrichin基因定位于染色体的20q11.22，基因组结构分析显示Glyrichin由两个外显子和一个内含子组成。蛋白序列同源性分析未发现有已知功能的同源蛋白，也未发现已知的功能域，是一个功能未知的蛋白，这对该基因功能的研究带来了一定的困难。但其理化特点如富含甘氨酸(21.5％)，pI=9.58。在pH=7时带正电荷，是小分子碱性蛋白，二级结构使用DNAstar5.0软件分析为集亲水性与疏水性于一体，以β折叠为主，这与已知抗菌肽共有的结构特点相似。本研究对人Glyrichin的天然存在性进行了探讨，以Northern杂交方法分析该基因在四种人源性肿瘤细胞中的表达及转录本大小，证实了人Glyrichin具有单一转录本，大小为600bp左右：以RT-PCR分析其在胎肝、胎肾、胎脾、胎肺等四种组织中的表达情况，以GFP为标记蛋白对基因的亚细胞定位进行探索，证实了该蛋白可能分布于胞浆。生物信息学分析显示人Glyrichin理化性质具有了抗菌肽的诸多特点，我们合成了代表人Glyrichin功能的肽段pCM-19进行抗菌功能分析，证实了pCM-19良好的抗菌功能，然后利用原核表达系统，构建了含有格拉利素基因的系列缺失体的pET22b表达载体，并转化到大肠杆菌BL21(DE3)中进行诱导表达。观察到该基因诱导表达时对宿主菌菌体生长具有明显的抑制作用，，初步证实了人Glyrichin具有明显的抗菌活性，同时也证实了蛋白C端对其抗菌活性的发挥是必须的。人Glyrichin完整蛋白原核表达也证实了具有良好的抗菌活性。我们选用了毕赤酵母表达系统对该基因进行功能分析，将人Glyrichin基因构建到毕赤酵母表达载体pPIC9和pPIC9K中，锂盐法转化毕赤酵母GS115中，G418抗生素筛选高拷贝转化子，进行培养诱导，对表达上清通过“纸片法”、“试管法”、“琼脂糖纸片扩散法”验证表达产物的活性。证实了人Glyrichin具有抗菌活性。通过生物信息学比较我们发现了有29个成员组成的高度同源的全新抗菌肽家系，通过代表家系九个成员的4个合成肽的抗菌活性检测和线虫源格拉利素基因原核表达分析，证实了它们具有良好的抗菌活性，强烈提示一个跨物种的抗菌肽家系的存在。综上所述，本研究获得了人源性和线虫源的格拉利素基因，确认了其天然存在性，通过原核表达和毕赤酵母表达系统证实了其具有明显的抗菌活性。生物信息学分析及实验验证提示一个跨物种的有29个成员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 .
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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