人DcR3 cDNA克隆及其在COS-7细胞中的表达

目的 克隆人DcR3分子的cDNA,将其重组入真核表达载体,并表达于COS-7细胞.方法 以PCR方法克隆编码人DcR3分子的cDNA,对其序列进行测定.将测序正确的DcR3 cDNA克隆入真核表达载体pAAV-IRES-hrGFP,构建重组表达载体.采用脂质体法转染COS-7细胞,用Western blot检测和激光共聚焦检测重组DcR3分子在COS-7细胞中的表达.结果 PCR方法扩增出一1 000 bp左右的基因片段,插入pAAV-IRES-hrGFP构建重组表达载体后,经序列测定证实扩增的片段为人DcR3分子cDNA.将重组子转染COS-7细胞,Western blotting和激光共聚焦检测到DcR3分子的表达.结论 成功地克隆并在COS-7细胞中表达了DcR3分子.

Cloning and expression of human DcR3 molecule in COS-7 cells

Objective To clone ORF of DcR3 gene and insert it into eukaryotic expression vector and express it in COS-7 cells. Methods Encoding sequence of human DcR3 gene was cloned by PCR and sequenced. The sequenced ORF was cloned into eukaryotic expression vector pAAV-IRES-hrGFP to construct recombinant plasmid. COS-7 cells were transfected with recombinant plasmid by lipofectamine2000. Expression of recombinant DcR3 gene was verified by Western blotting and confocal microscopy. Results A 1 000-bp gene segment was obtained by PCR and inserted into pAAV-IRES-hrGFP to construct recombinant plasmid. The gene segment was proved to be encoding sequence of human DcR3 gene by sequencing. DcR3 expression in COS-7 cells was verified by Western blotting and confocal microscopy. Conclusion DcR3 gene was successfully cloned and expressed in COS-7 cells.