摘要
为了克隆广西巴马小型猪硫氧还蛋白相互作用蛋白基因(TXNIP)序列并构建真核表达载体,为生产转TXNIP基因的广西巴马小型猪奠定基础,试验采用RT-PCR技术从广西巴马小型猪胰腺组织中扩增出TXNIP基因编码序列,连接至p MD18-T载体,测序鉴定后提取质粒,用SalⅠ和EcoRⅠ限制性内切酶进行双酶切,回收的TXNIP片段连接至p EGFP-C1载体上,构建含TXNIP的真核表达载体p EGFP-C1-TXNIP;利用双酶切和测序对重组质粒p EGFP-C1-TXNIP进行鉴定,并将重组质粒转染β-TC6细胞24 h后观察细胞荧光表达情况。结果表明:广西巴马小型猪TXNIP基因编码区序列长1 176 bp,编码391个氨基酸,与Gen Bank已公布的猪TXNIP基因c DNA序列(序列号为DQ278874)对应区段同源性为99.7%;重组表达载体p EGFP-C1-TXNIP质粒转染β-TC6细胞能表现出绿色荧光。
To clone the thioredoxin - interacting protein （TXNIP） gene sequence and construct the eukaryotic expression vector of Guangxi Bama miniature pig, and to lay the foundation for producing TXNIP gene - transfer Guangxi Bama miniature pig. The coding - sequence （CDS） of TXNIP gene was amplified from the pancreatic tissue of Guangxi Bama miniature pig using RT - PCR technique, and connected the pMD18 -T vector. The positive plasmids were extracted after sequencing and identification, and used for the double digestion by Sal I and EcoR I , and then the recovered TXNIP gene fragments were connected into pEGFP - C1 vector to construct the expression vector pEGFP - C1 -TXNIP. The recombinant plasmid pEGFP--C1 -TXNIP was identified using double digestion and sequencing, and the recombinant plasmid was transfected into the 13 - TC6 ceils, and then the fluorescence expressions of the cells were observed under the inverted fluorescence microscope after 24 hours. The results showed that the length of TXNIP gene CDS of Guangxi Bama miniature pig was 1 176 bp, encoding for a protein of 391 amino acids, and the sequence shared 99.7% homology with the cDNA sequence of pig TXNIP gene published in GenBank（ accession number DQ278874）. The results indicate that the 13 -TC6 ceils transfected with the plasmid of recombinant expression vector pEGFP - C1 -TXNIP after 24h, can present green fluorescence.
出处
《黑龙江畜牧兽医》
CAS
北大核心
2015年第5期6-9,266,共5页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金项目(81360135)
广西科学研究与技术开发计划项目"应用转基因技术构建广西巴马小型猪2型糖尿病模型"(桂科攻1347003-2)
广西自然科学基金项目(2013GXNSFAA019187)
