广西巴马小型猪α-1,3-半乳糖转移酶基因RNAi载体构建与检测  

Construction and Identification Guangxi Bama-mini Pig α-1,3-Galactose Transferase Gene RNAi Vector

作　　者：郭晓萍[1] 陈时锦[1] 张笠[1] 蒋钦杨[1] 兰干球[1] 郭亚芬[1] 

机构地区：[1]广西大学动物科学技术学院,南宁530004

出　　处：《基因组学与应用生物学》2015年第6期1165-1171,共7页Genomics and Applied Biology

基　　金：广西科技基础条件平台建设项目(10-108-23)资助

摘　　要：α-1,3-半乳糖转移酶基因(α-1,3 GT)在异种器官移植中的免疫排斥反应有重要的作用,本研究旨在构建α-1,3-半乳糖转移酶基因的干扰载体,在m RNA和蛋白质水平鉴定并筛选出干扰效果最佳的序列。根据巴马小型猪GGTA1基因CDS序列,设计合成3对特异性单链si RNA序列以及一对阴性序列,构建GGTA1 RNA干扰载体分别命名为p LLU2G-sh GGTA1-1、p LLU2G-sh GGTA1-2、p LLU2G-sh GGTA1-3和p LLU2G-sh GGTA1-NC。将构建好的载体分别转染转染PK15细胞,通过q RT-PCR和Western blot检测GGTA1 m RNA及其蛋白的相对表达量。结果显示本研究成功构建了3个重组RNAi表达载体,与转染p LLU2G-NC和空白组相比,p LLU2G-sh GGTA1-1、p LLU2G-sh GGTA1-2和p LLU2G-sh GGTA1-3转染组的GGTA1 m RNA和蛋白表达量均明显下降,对m RNA表达的抑制效率分别为85.02%、86.05%和83.85%。本研究成功获得有效抑制广西巴马小型猪GGTA1基因表达的RNAi载体,为下一步生产沉默GGTA1基因广西巴马小型猪奠定基础。α-1,3 galactose transferase gene play an important role in hyperacute immune rejection of clinical Xenotransplantation. The objective of this study was to construct the RNAi vectors of porcine α-1,3-galactose transferase gene and screen the best RNAi vector on cell m RNA and protein level. According to CDS sequence of Guangxi Bama mini-pig's α-1,3-galactose transferase gene cloned in our lab, three si RNA sequences and a negative sequence were designed and synthesized, directly cloned into p LLU2 G vector to produce p LLU2G-sh GGTA1-1,p LLU2G-sh GGTA1-2, p LLU2G-sh GGTA1-3 and p LLU2G-sh GGTA1-NC vectors. The constructed vectors were transfected into PK-15 cells, relative transcription level of GGTA1 gene m RNA and protein expression were determined by Real-time PCR and Western blot, respectively. The results showed that 3 GGTA1 gene RNAi vectors were constructed successfully. Compared with p LLU2G-sh GGTA1-NC, the relative level of GGTA1 gene m RNA and protein expression decreased in PK-15 cells which transfected with p LLU2G-sh GGTA1-1,p LLU2G-sh GGTA1-2 and p LLU2G-sh GGTA1-3. The m RNA inhibition ratios were 85.02%, 86.05% and 83.85,respectively. In conclusion, effective RNAi vectors for α-1,3-galactose transferase gene have been successfully constructed, which would lay a foundation for further producing and breeding GGTA1 gene silencing Guangxi Bama mini-pig.

关 键 词：巴马小型猪 α-1 3半乳糖转移酶基因 RNAI 

分 类 号：S828[农业科学—畜牧学]