防御素基因克隆和表达载体构建

人防御素(HNP)是一类广谱抗菌阳离子小肽的总称,一般具有3个分子内二硫键。防御素对于细菌、真菌乃至某些被膜病毒有广谱杀伤作用,是免疫系统中的重要组分。实验根据已发表的HNP-1cDNA序列,设计并合成了一对引物。以HL-60细胞总RNA为模板,利用RT-PCR技术扩增目的片段并克隆到pUC18载体上,经筛选获得阳性重组子pUC18-HNP-1(pDFS1)。测序分析证实,扩增片段即为HNP-1cDNA。pDFS1再经SmaI与SalI酶切、插入pGEX-6p-1质粒构建HNP-1表达载体。利用PCR技术和酶切反应筛选转化子最终获得pGEX-6p-1-HNP-1(pDFS2)重组质粒。

The cloning of human defensin-1 (HNP-1) and construction of expression vector

Defensins, a family of small cationic peptide with three to four intramolecular cysteine disulfide bonds, have a broad spectrum of antimicrobial activity against bacterial, fungiand even some enveloped viruses. As multifunctional effector molecules of innate immunity, defensins are the key components in immune system. In this report, a pair of primers were designed and synthesized according to published HNP-1 cDNA nucleotide sequence. Total HL-60 cells RNA was isolated from cultured HL-60 cells. Then the target fragment was amplified by RT-PCR and cloned into pUC18 vector between two restriction enzyme sites(BamH I and Xal I). The sequence analysis showed the cloned fragment was HNP-1 cDNA. Furthermore, the recombinant was digested by Sma I and Sal I and inserted into pGEX-6p-1 expression vector. After screening by PCR and restriction analysis, we concluded HNP-1 expression vector was constructed. The achievement of this study established the foundation for the further research on expressing HNP-1.