作　　者： ; ; ; ; ;

机构地区： 成都军区总医院

出　　处： 《中华实验外科杂志》 2003年第10期884-885,共2页

摘　　要： 目的　克隆人防御素 5基因 (HD 5cDNA) ,构建其真核表达质粒载体。方法　从人小肠粘膜中提取总RNA ,并分离mRNA ,经过逆转录 聚合酶链反应 (RT PCR)扩增出HD 5cDNA ,并将其插入经BamHⅠ和EcoRⅠ双酶切的真核表达质粒载体 pcDNA3 .1(+ )中 ,构建重组质粒载体 pcDNA3 .1(+ ) HD 5cDNA。 结果　经过限制性酶切鉴定和测序确定 ,HD 5cDNA全长 2 82bp ,编码 94个氨基酸 ,由编码信号肽、前片段和成熟肽的 3部分序列组成。理论推导的氨基酸序列中 ,决定HD 5活性的 6个半胱氨酸残基和 2个保守的精氨酸残基处于正确的位置。结论　成功获得了HD 5cDNA ,构建了含HD 5cDNA的真核表达质粒载体 ,为研究重组HD Objective To construct the recombinant eukaryotic plasmid of human defensin 5 (HD 5) and prepare for expressing in eukaryotic cells.Methods Total RNA as well as mRNA were isolated from human small intestinal mucosa and cDNA encoding HD 5 was amplified by reverse transcription PCR (RT PCR).The cDNA was inserted into the vector pcDNA3.1(+).The constructed vector pcDNA3.1(+) HD 5 cDNA was identified by restriction endonucleases and sequenced.Results No discrepancy was found in nucleotide as compared with standard sequence of HD 5 from GenBank.It encoded 94 amino acids,whose length was 282 bp,including three pieces of sequence encoding signal peptide,propiece and mature peptide.Conclusion We successfully obtained the cDNA of HD 5 and constructed the recombinant plasmid correctly.It will be helpful for analyzing the bactericidal function of recombinant HD 5.

关 键 词： 人源性空回肠粘膜 防御素 基因克隆 基因表达 聚合酶链式反应 真核表达质粒载体

领　　域： [生物学]