论文部分内容阅读
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 identify hGlyrichin, a novel homologous human gene of mL52 gene (GenBank: AY028425), and to study its function. Methods: The new gene was amplified from human fetal liver cDNA library by PCR. The gene was named hGlyrichin because of its high content of glycine, and its expression and transcript size were analyzed by RT-PCR and Northern blot. The gene was analyzed by bioinformatics. The recombinant plasmid pET22bh-Glyrichin was constructed by using pET22b expression vector and transformed into E. coli BL-21 (DE3) for IPTG induction. RESULTS AND CONCLUSION: Electron PCR confirmed that the gene was located on human chromosome 20q11.22 and contained 3 exons and 2 introns. Northern blot results showed a single transcript, approximately 600 bp in length, consistent with the full-length cDNA of mL52. The encoded protein is a small cationic protein with hydrophobic region, the relative molecular mass and isoelectric point are 8182.83 and 9.58, respectively. The secondary structure (Garnier-Robson model) predicts that β-sheet is the predominant type, The structure and partial physical and chemical properties of most of the known cationic antibacterial peptides are very similar. The growth of E.coli transformed with the gene is obviously inhibited when induced by IPTG, and the antibacterial activity of hGlyrichin is preliminarily confirmed.