首页> 中文期刊> 《实验动物与比较医学》 >广西巴马小型猪α-1,3半乳糖转移酶基因真核表达载体的构建及鉴定

广西巴马小型猪α-1,3半乳糖转移酶基因真核表达载体的构建及鉴定

         

摘要

目的 通过RT-PCR扩增巴马小型猪GGTA1基因,分析其序列的结构特点并构建真核表达载体,为生产GGTA1基因沉默的广西巴马小型猪奠定基础.方法 根据NCBI上发布的猪α-1,3半乳糖转移酶基因cDNA序列(AF221508),设计合成一对含有 HindⅢ和BamHI酶切位点的特异性引物,以广西巴马小型猪肝脏组织总 RNA为模板进行RT-PCR,所得目的片段经纯化、克隆、鉴定和测序;将目的基因与pEGFP-N 1进行双酶切后连接构建真核表达载体pEGFP-GGTA1,通过测序,双酶切鉴定.结果 所克隆的广西巴马小型猪GGTA1基因存在缺失第五外显子(81-116) 的GGTA1序列,序列全长1 080 bp和未缺失第五外显子的GGTA1序列,全序列为1 116 bp.构建两个表达载体(缺失第五外显子 pEGFP-GGTA1-1、未缺失第五外显子pEGFP-GGTA1-2).结论 广西巴马小型猪GGTA1基因存在第五外显子缺失的现象,通过对GGTA1基因克隆序列进行分析以及构建两个真核表达载体,为后面在猪源PK-15细胞中对其进行表达鉴定以及巴马小型猪 α-1,3半乳糖转移酶基因的沉默实验奠定基础.%Objective For the purpose of producing GGTA1 gene silencing bama-mini pig, cloned and analyzed the function of a-1,3 galactosyltransferase gene of bama-mini pig then constructed two expression vectors. Methods The GGTA1 gene was amplified from liver tissue by RT-PCR using a specific pair of primers which were designed and synthesized according to the GGTAl gene sequence released in GenBank, the amplified cDNA fragment was ligated into pMD18-T vector after purification. The recombinant was verified by the method of PCR and sequencing, then the enzyme digested products was ligated into pEGFP-Ni to construct the expression vectors, identified by PCR and digesting reaction. Results The a-1,3 galactosyltransferase gene of bama-mini pig exist the absence of 5-exon(81-116) which containing 1080 bp, and existence of 5-exon which containing 1116 bp. Conclusions There exit the absence of 5-exon in the bama-mini pig, Construction of the both expression vectors (pEGFP-GGTA1-1 and pEGFP-GGTAl-2) can be transfected into PK-15 cells to identify their functional expression, also can be used in the experiment of gene silencing pig a-1,3 galactosyltransferase gene.

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