摘要
本研究旨在克隆猪脂联素球状结构域gAd基因,构建重组质粒并将其转化至乳酸乳球菌NZ9000中进行表达,并在体外对重组猪脂联素球状结构域进行生物学活性分析。提取猪脂肪组织总RNA,扩增得到脂联素基因全长并测序,设计引物引入酶切位点和His-tag,把gAd亚克隆到表达载体pNZ8048,将重组质粒转化乳酸乳球菌NZ9000中诱导表达。通过Ni 2+亲和层析柱对重组蛋白纯化后再Western blotting检测。然后,将gAd蛋白注射到高糖饲喂的小鼠体内鉴定生物学活性。结果表明成功获得了gAd基因并在乳酸乳球菌NZ9000中表达,表达产物相对分子质量约17ku。经纯化后用Western blotting检验具有反应原性,动物试验证明制备的gAd蛋白能显著降低小鼠的血糖水平。本研究为利用外源重组脂联素球状结构域调节猪的脂肪代谢打下了良好的基础。
This experiment was conducted to clone, express porcine globular Adiponectin （gAd） gene in recombinant Lactococcus lactis NZ9000 and analyze the biological activity of expression product. The total RNA was extracted from porcine adipose tissue, and the Adiponectin gene was cloned into T plasmid for gene sequencing. Primers containing the corresponding restriction digestion sites and His-tag sequence were designed, and then gad gene was subcloned into expression vector pNZ8048 and expressed in recombinant L. lactis NZ9000. After purification, the immunological aeitivity of recombinant protein was determined by Western blotting. To eval- uate the biological activity of recombinant gAd, purified gad was injected into the mice fed high- glucose diet. The results showed that the gad gene was successfully obtained and expressed in recombinant L. lactis NZ9000. The molecular weight of gad protein is 17 ku. The recombinant gAd significantly decreased blood glucose of mice. This work established a good foundation for further study on the regulation of porcine lipometabolism by recombinant globular adiponeetin.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2012年第3期353-357,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
广东省东莞市科技局(2008D24)
广东省科技厅工业攻关重大项目(2008A010900003)
国家生猪产业技术体系(2009-2010)
关键词
猪
脂联素
球状结构域
乳酸乳球菌
porcine
adiponectin
globular domain
Lactococcus lactis
