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目的克隆大鼠galectin-9基因全长cDNA,构建含大鼠galectin-9基因的重组腺病毒载体,并予以鉴定。方法从大鼠的肝脏组织中用RT-PCR的方法克隆扩增大鼠galectin-9基因,再定向插入到带NotⅠ和HindⅢ酶切的pDC316-GFP穿梭质粒中,获得穿梭质粒pDC316-GFP-galectin-9。经PCR、NotⅠ和HindⅢ酶切及测序鉴定后,用脂质体将穿梭质粒pDC316-GFP-galectin-9与腺病毒骨架质粒pBHGlox△E1.3Cre共转染HEK-293细胞。经位点特异性重组获得含目的基因的重组腺病毒Ad5-galectin-9,行PCR鉴定,经HEK-293细胞扩增及纯化制备高滴度病毒液,用细胞培养方法测定病毒TCID50滴度。结果 PCR、酶切及测序证实穿梭质粒构建正确,PCR鉴定证实大鼠galectin-9基因重组腺病毒载体构建正确,病毒的感染滴度为1.4×109U/ml。结论成功构建了含大鼠galectin-9基因的重组腺病毒表达载体,为进一步研究大鼠galectin-9基因的功能奠定了基础。
Objective To clone the cDNA of rat galectin-9 gene and construct a recombinant adenovirus vector containing rat galectin-9 gene. Methods Rat galectin-9 gene was cloned and amplified by RT-PCR from rat liver tissue and inserted into pDC316-GFP shuttle plasmid digested with NotⅠand HindⅢ. The shuttle plasmid pDC316-GFP-galectin -9. The recombinant plasmid pDC316-GFP-galectin-9 and adenovirus backbone plasmid pBHGlox △ E1.3Cre were co-transfected into HEK-293 cells by lipofectamine after PCR, NotⅠ and HindⅢ digestion and sequencing. The recombinant adenovirus Ad5-galectin-9 containing the gene of interest was obtained by site-specific recombination. High-titer virus liquid was prepared by amplification and purification of HEK-293 cells. The virus TCID50 titer was determined by cell culture method. Results PCR, restriction enzyme digestion and sequencing confirmed that the shuttle plasmid was constructed correctly. PCR identification confirmed that the recombinant adenovirus vector of rat galectin-9 gene was constructed correctly. The virus titer was 1.4 × 109U / ml. Conclusion The recombinant adenovirus expression vector containing rat galectin-9 gene was successfully constructed, which laid the foundation for further study on the function of rat galectin-9 gene.