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

Cloning and Identifying Expression Vectors of α-1,3 Galactosyltransferase Gene from Bama Minipig in Guangxi

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-N1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1080bp和未缺失第五外显子的GGTA1序列，全序列为1116bp。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 α- 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 GGTAlgene sequence released in GenBank, the amplified cDNA fragment was ligated into pMD 18-T vector after purification. The recombinant was verified by the method of PCR and sequencing, then the enzyme digested products was ligated into pEGFP-N1 to construct the expression vectors, identified by PCR and digesting reaction. Results The α-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-GGTA1-2） can be transfected into PK-15 cells to identify their functional expression, also can be used in the experiment of gene silencing pig α-1,3 galactosyltransferase gene.