鸡斑联蛋白基因的慢病毒干扰载体的构建及筛选  

Construction and Selection of Chicken Zyxin Recombinant Lenti-virus Interference Vectors

作　　者：戴国俊[1,2] 孙大辉[1,2] 林雨鑫[1,2] 孙明明[1,2] 王翔[1,2] 王金玉[1,2] 张跟喜[1,2] 谢恺舟[1,2] 施会强[3] 侍苗苗[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009 [2]江苏省动物遗传繁育与分子设计重点实验室,扬州225009 [3]江苏京海禽业集团有限公司,南通226103

出　　处：《畜牧兽医学报》2013年第8期1330-1336,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家肉鸡产业技术体系(nyctx-42-G1-05);江苏省高校自然科学研究重大项目(11KJA230001);江苏高校优势学科建设工程资助项目(PAPD)

摘　　要：本试验旨在构建4条鸡斑联蛋白(zyxin)基因的RNAi慢病毒载体,对其进行滴度测定,并进行有效干扰靶点的筛选,为以后开展鸡zyxin基因抗球虫机理的研究奠定基础。提取鸡胸腺组织的RNA,反转录,扩增zyxin基因的编码区,并将其克隆到真核表达载体Pex-6上。设计针对zyxin的shRNA序列,应用基因重组技术插入到Lv3慢病毒表达载体中,将lv3-shRNA载体与包装质粒pGag/Pol、pRev、Pvsv-G共转染293T细胞,进行病毒包装。将得到的病毒原液10倍稀释6个梯度,分别转染293T细胞,进行滴度的测定。将构建好的4条慢病毒载体与Pex-6-zyxin真核表达载体共转染293T细胞,运用RT-PCR和Western-blot方法进行有效干扰靶点的筛选。构建4条针对目的基因zyxin的RNAi慢病毒穿梭质粒表达载体CO1、CO2、CO3、CO4和1个阴性对照质粒,经测序鉴定正确;共转染293T细胞包装病毒并浓缩后滴度达1×108 TU·mL-1,适合感染目的细胞,经过筛选,CO3对zyxin在293T细胞中的表达干扰效果最佳。成功构建了zyxin基因RNA干扰慢病毒表达载体,并进行了有效干扰靶点的筛选,为进一步研究zyxin基因的功能奠定了基础。The aim of this study was to construct 4 Lenti-virus Interference Vectors for chicken zyxin gene,determine their titers,and filter the effective interference target,and lay the foundation for studying of anti-coccidial mechanism of chicken zyxin gene.The experiment was performed by extracting RNA from chicken thymus,then synthesizing the cDNA with reverse transcription,amplifying coding regions of zyxin,and cloning it into eukaryotic expression vector Pex-6 at last.The shRNA sequences for zyxin were designed,and were inserted into Lv3 lenti-virus vectors.Lv3-shRNA vectors and packaging plasmids which include pGag/Pol,pRev and Pvsv-G were cotransfected into 293T cells to package virus.Dilute virus liquid 10 times the 6 gradients,and transfect them into 293T cells respectively to determine the titer.Transfect 4 recombinant lenti-virus interference vectors and eukaryotic expression vectors Pex-6 into 293T cells,and filter the effective interference target by RT-RCR and Western-blot.The sequence identification results showed that the 4 constructed zyxin recombinant lenti-virus interference vectors including CO1,CO2,CO3,CO4 and a negative control plasmid were correctly constructed.By way of transfecting them into 293T cells and concentration,the titer reached 1×108 TU·mL-1 which showed that they were suit for being transfected into the target cells.The filter result showed that CO3 had the most significant interference effect on zyxin expression in 293T cells.Analysis of the data suggests that the zyxin recombinant lenti-virus interference vectors were constructed and filtered the effective interference target successfully,which laid the foundation for further research on zyxin gene function.

关 键 词：zyxin基因 RNAI 慢病毒干扰载体 真核表达载体 鸡 

分 类 号：S813.3[农业科学—畜牧学]