人源新基因hGlyrichin的克隆及其功能的初步研究  

Molecular cloning and functional determination of a novel human glycine-rich gene

作　　者：杨超文[1] 尹丽莉[1] 张咏[1] 鱼咏涛[1] 赵彦林[1] 李红梅[1] 赵蕾[1] 侯冰[1] 赵光[1] 沙继斌[1] 赵士富[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《军事医学科学院院刊》2005年第6期501-505,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家"973"项目资助(G199053903)

摘　　要：目的:克隆获得与mL52新基因(GenBank:AY028425)同源人类新基因hGlyrichin,并研究其功能。方法:采用PCR法从人胎肝cDNA文库中扩增出该新基因,因富含甘氨酸而命名为hGlyrichin,以RT-PCR和Northern杂交分析在多种细胞株中的表达及转录本大小,并对该基因进行生物信息学分析,利用pET22b表达载体构建重组质粒pET22bh-Glyrichin,转化大肠杆菌BL-21(DE3)中进行IPTG诱导表达。结果与结论:电子PCR证实该基因定位于人染色体20q11.22,包含3个外显子和2个内含子。Northern杂交结果显示为单一转录本,大小约600 bp,与mL52全长cDNA长度相符。所编码的蛋白为含有疏水区的小分子阳离子蛋白,相对分子质量和等电点分别为8182.83和9.58,二级结构(Garn ier-Robson模型)预测是以β折叠型为主,这些特点与目前已知的大多数阳离子抗菌肽的结构及部分理化特性极为类似,该基因转化后的大肠杆菌在IPTG诱导表达时的生长受到明显抑制,初步证实了hGlyrichin具有明显的抗菌活性。Objective：To clone and investigate the function of a novel human glycine-rich gene highly homologous to previously cloned mouse gene mL52 （GenBank number： AY028425）. Methods：PCR was used to amplify the entire open reading frame of hGlyrichin from human fetal liver cDNA library. RT-PCR and Northern blot were used to test the expression and the size of the transcript in different human tumor cell lines. Public Microsoftwares were applied for the bioinformatic analysis. Transformed E. coli BL-21 （DE3） with constructed pET22b-hGlyrichin was induced with IPTG for the expression of the protein. Results and conclusions： Electronic PCR demonstrated that hGlyrichin gene was localized in human chromosome 20q1 1.22 containing 3 exons and 2 introns. About 600 bp single transcript was observed from Northern blot analysis, which was similar to the full-length cDNA of mL52 in size. The encoded protein containing a hydrophobic region was a cationic protein with relative molecular mass about 8 182.83 and pI 9.58. The secondary structure prediction showed that hGlyrichin was mainly a β-sheet type protein. These characterizations are quite similar to those of known cationic antimicrobial peptides and were functionally confirmed by the inhibition of the growth of E. coli BL-21 （DE3） after transformed with pET22b-hGlyrichin and induced expression with IPTG. All these results from bioinformatic analysis and experiments demonstrate that hGlyrichin is a novel human antimicrobial peptide.

关 键 词：hGlyrichin 抗生素类 肽 基因表达 聚合酶链反应 甘氨酸 

分 类 号：R346[医药卫生—基础医学]