摘要
为了探讨天蚕类抗菌肽在动物早期抗感染过程中的作用机理,本研究以Shiva1a基因的成熟肽为模板设计4条引物,利用重叠延伸PCR技术获得目的基因,并在C端添加6×His的标签。将此序列与真核表达载体pIRES2-EGFP进行重组,构建pIRES2-EGFP-Shiva 1a重组表达质粒,对重组质粒进行酶切和测序鉴定后,采用阳离子脂质体转染将重组质粒转染到CHO-K1细胞,荧光显微镜观察其表达情况。通过生物信息学软件对抗菌肽Shiva 1a的二级结构和三级结构进行预测分析。其结果为进一步研究Shiva 1a的抗菌活性和在动物抗病育种方面的应用奠定了基础。
To explore the effect mechanism of cecropin-class lytic peptide at early stage of infection.The mat peptide of Shiva 1a was amplified by overlap extension PCR and the C-terminus contained 6×His-marker.The gene sequence were recombinant with eukaryotic expression vector pIRES2-EGFP.After being identified by restriction enzyme digestion and sequencing,the recombinant plasmid pIRES2-EGFP-Shiva 1a was transfected into CHO-K1 cells by liposomes.The expression of the the recombinant plasmid pIRES2-EGFP-Shiva 1a was observed by fluorescence microscope.At the same time,the secondary structure and 3D structure were predicted by bioinformatics tools.The results lay the foundation in research of antimicrobial activities and applications of the antimicrobial peptide Shiva 1a in breeding for disease resistance of animals.
出处
《中国畜牧兽医》
CAS
北大核心
2011年第12期96-100,共5页
China Animal Husbandry & Veterinary Medicine
基金
转基因生物新品种培育科技重大专项(2009ZX08008-010B)
关键词
抗菌肽
Shiva
1a
真核表达
序列分析
antimicrobial peptide
Shiva 1a
eukaryotic expression
sequence analysis
