Granulysin真核表达载体的构建与鉴定

Construction of Eukaryotic Expression Vector of Granulysin and its DNA Sequencing

目的:构建颗粒溶解素(granulysin)cDNA真核表达载体。方法:采用逆转录聚合酶链反应(RT-PCR)技术从健康志愿者外周血单个核细胞(PBMC)中扩增出颗粒溶解素蛋白cDNA,经限制性核酸内切酶消化后,插入真核表达载体pcDNA3.1(-)的相应酶切位点,将此重组质粒进行序列测定,并将重组质粒转染COS7细胞,进行基因表达鉴定。结果:重组质粒插入基因序列测定证实为颗粒溶解素cDNA,并能在COS7细胞稳定表达。结论:成功构建颗粒溶解素真核表达载体,为进一步研究其抗结核作用奠定了基础。

Objective： To construct eukaryotic expression vector of Granulysin. Methods： Granulysin cDNA was amplified from PBMC of healthy volunteer by RT-PCR, and correctly inserted into corresponding sites of eukaryotic expression vector pcDNA3. 1 （-） after restriction endonuclease digestion, and the recombinant plasmid was transfected into COS7 cells, gene expression was determined by RT-PCR. Results： The gene fragment inserted into the vector pcDNA3.1（-） was confirmed by nucleotide sequencing, and the mRNA of the recombinant plasmid was successfully expressed in mammalian cells. Conclusion： The successful construction of recombinant eukaryotic expression vector based on the cDNA of granulysin, provides the experiment basis for further researching.