鸡β-防御素-2基因的克隆及原核表达载体的构建

鸡β-防御素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。防御素对细菌、真菌乃至某些被膜病毒具有广谱杀伤作用。试验根据已发表的Gal-2基因序列,设计并合成了一对引物。以骨髓中的总RNA为模板,利用RT-PCR技术扩增目的片段并克隆到pGEM-TEasy载体上,经筛选鉴定获得阳性重组子pGEM-T-Gal-2。再经EcoRI、XbaI双酶切,插入pMAL-c2x质粒构建表达载体。利用PCR技术和酶切反应筛选转化子成功获得pMAL-c2x-Gal-2重组质粒。

The cloning of Gallinacin-2 and construction of expression vector

Defensins,a family of small cationic peptide with three 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 Gal-2 nucleotide sequence.Total RNA was isolated from marrow.Then the target fragment was amplified by RT-PCR and cloned into pGEM-T Eazy vector between two restriction enzyme sites(EcoR I and Xba I). The sequence analysis showed the cloned fragment was Gal-2.Furthermore, the recombinant was digested by EcoRI and XbaI and inserted into pMAL-c2x expression vector.After screening by PCR and restriction analysis, we concluded pMAL-c2x-Gal-2 expression vector was constructed.