大鼠OPN真核表达载体的构建和体外表达

In Vitro Study on the Construction and Expression of Recombinant Plasmid pEGFP-OPN

目的:体外构建携带大鼠骨桥蛋白(osteopontin,OPN)基因的荧光真核表达载体,并观察其在人胚肾293T(humanembryo kidney 293T,HEK293T)细胞中的表达。方法:从SD大鼠骨组织标本提取RNA,应用逆转录PCR(reversaltranscript polymerase chain reaction,RT-PCR)方法扩增目的片断,双酶切克隆到增强型绿色荧光蛋白(enhanced greenfluorescent proteins,EGFP)报告基因的真核表达载体pEGFP-C1中,构成重组质粒pEGFP-OPN,经酶切鉴定和测序验证。通过脂质体介导重组质粒瞬时转染HEK293T细胞,并用RT-PCR检测OPN的表达。结果:成功构建重组质粒pEGFP-OPN,测序结果与GenBank中公布的序列(Genbank NM_012881)同源性为100%。将其转染HEK293T细胞后,观察到绿色荧光蛋白的表达,并通过RT-PCR检测到OPN的表达。结论:本实验成功构建了大鼠OPN荧光真核表达载体pEGFP-OPN,并能成功地在HEK293T细胞中表达,为OPN的进一步研究奠定了一定的实验基础。

Objective：To construct a recombinant plasmid of fluorescent eukaryote expression vector containing rat osteopontin（OPN）,and to study its expression in human embryonic kidney 293T（HEK293T） cells.Methods：The total RNA of OPN was abstracted from the SD rat bone tissue.The OPN cDNA was constructed by reversal transcript polymerase chain reaction（RT-PCR）,and cloned into a eukaryote expression vector pEGFP-C1 which is the report gene of enhanced green fluorescent proteins（EGFP） by directed cloning after restrictive endonucleases digestion.The pEGFP-OPN was checked and verified by digestion and DNA sequence analysis.HEK293T cells were transiently transfected with pEGFP-OPN by lipofectamine 2000 in vitro.RT-PCR was conducted to verify the expression of OPN.Results：The OPN was correctly inserted into pEGFP-C1,the sequencing results were 100% identical with those reported in GenBank.RT-PCR showed the expression of rat OPN after pEGFP-OPN transfection.Conclusion：Recombinant plasmid pEGFP-OPN was constructed successfully in vitro,which lays a foundation to the further study on the OPN gene.