猪杀菌/通透性增加蛋白基因siRNA载体构建及干扰效果评价  

Construction and Evaluation of Porcine Bactericidal/Permeability-increasing Protein Gene(BPI) siRNA Expression Vector

作　　者：吴正常[1] 殷学梅[1] 夏日炜 孙寿永[1] 朱国强[2] 吴圣龙[1] 包文斌[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009 [2]扬州大学兽医学院,扬州225009

出　　处：《畜牧兽医学报》2015年第3期491-496,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31172183;31140027);转基因生物新品种培育科技重大专项(2014ZX08006-001B);江苏省科技支撑计划(BE2012330;BE2013345;BE2014357);江苏高校优势学科建设工程资助项目(PAPD)

摘　　要：本研究旨在构建并筛选猪(Sus scrofa)BPI基因的高效siRNA干扰载体,为在细胞水平上研究猪BPI基因的功能和作用机制提供基础。参照猪BPI基因(GenBank登录号:EF436278)全长编码区序列,设计其特异性发夹siRNA干扰片段,并将其克隆插入pcDNA 6.2-GW/EmGFPmiR干扰载体中,构建猪BPI基因4个干扰siRNA表达载体RB1、RB2、RB3和RB4,1个阴性对照NC,并通过PCR和测序进行验证,构建成功后转染猪小肠上皮细胞IPEC-J2并检测其干扰效率。结果发现,所构建的4个特异性siRNA载体均可显著降低猪BPI基因mRNA表达(P<0.05),其中RB4载体干扰效果最好,其干扰效率达到69%。本研究成功筛选可靶向干扰猪BPI基因的高效siRNA,为今后在细胞水平进一步研究BPI基因对猪肠道革兰阴性菌感染抗性的作用及其机制奠定了试验基础。This study was conducted to construct and select the efficient siRNA interference vector for porcine BPI gene,which provided the foundation for studying the function and mechanism of porcine BPI gene at the cellular level.Referring to the whole coding sequence of porcine BPI gene(GenBank accession number:EF436278),porcine BPI specific hairpin siRNA fragments were designed and inserted into pcDNA 6.2-GW/EmGFPmiR vector.Four pairs of siRNA expression vectors(RB1,RB2,RB3,RB4)and one pair of negative control(NC)were constructed by PCR and sequencing verification.Then the above vectors were transfected into IPEC-J2 intestinal epithelial cell and further the interference effect was detected.The results showed that 4 BPI-specific siRNA vectors all reduced the BPI gene mRNA expression(P<0.05).Meanwhile the interference effect of RB4 vector was the best,whose efficiency reached 69%.This study successfully screened out the efficient siRNA vector which could exclusively interfere porcine BPI gene expression,which provided experimental basis for further studying the BPIgene function and mechanism for the re-sistance to gram-negative bacteria infection in porcine intestinal tract at the cellular level.

关 键 词：猪 BPI基因 siRNA载体 RNA干扰 

分 类 号：S828[农业科学—畜牧学]