摘要
目的构建人的生物钟基因Period2（hPer2）真核表达载体pEGFP-N1-hPer2，观察其在骨肉瘤细胞MG63中的表达。方法应用实时定量荧光反转录聚合酶链反应（FQ—PCR）技术从MG63细胞中扩增hPer2，并将PCR产物经双酶切后定向克隆入pEGFP—N1的多克隆位点，采用脂质体介导转染骨肉瘤细胞MC63，采用FQ-PCR和Westernblot分别检测hPer2的表达。结果重组质粒经PstI、KpnI双酶切和测序与hPer2基因序列一致，利用脂质体介导将pEGFP—N1-hPer2重组质粒成功转染到骨肉瘤细胞MG63中并获得了hPer2基因的过表达。FQ-PCR显示实验组（pEGFP—N1-hPer2组）的mRNA水平（7．84±1．17）明显高于空载体组（pEGFP—N1组，1．42±2．11）和空白对照组（1．21±1．61）；Westemblot显示实验组hPer2蛋白（0．382±1．131）的表达明显高于空载体组（0．178±2．110）和空白对照组（0．166±1．951），差异均有统计学意义（P〈0．05）。结论成功构建pEGFP—N1-hPer2真核表达载体，并能够在骨肉瘤细胞MG63中过表达。
Objective To construct the eukaryotic expression vector of pEGFP - N1 - hPer2 and its expression in human osteosarcoma cell line MG63. Methods Total mRNA was extracted from human osteosarcoma MG63 cells, hPer2 gene was obtained by fluorescent quantitative polymerase chain reaction （FQ -PCR） and cloned into pEGFP- N1 vector, then the recombinant pEGFP- N1 -hPer2 plasmid was constructed and transfected into MG63 cells by lipofectamine 2000, the expression of hPer2 in MG63 cells was detected by FQ -PCR and Western blotting, respectively. Results Corrected construction of pEGFP - N1 -hPer2 was identified by double enzyme digestion and DNA sequencing, hPer2 gene expressed by the transfected cells was testified by FQ - PCR and Western blotting, hPer2 mRNA expression was significantly higher in the pEGFP - N1 - hPer2 group （7.84 ± 1.17） as compared to the pEGFP - N1 （ 1.42 ± 2. 11 ） or control groups （ 1. 21 ± 1.61, P 〈 0. 05）. Meanwhile, bPer2 protein was also markedly higher in the pEGFP - N1 - hPer2 group （ 0. 382 ± 1.131 ） as compared to the pEGFP - N1 （0. 178 ± 2. 110 ） or control groups （0. 166 ± 1. 951, P 〈 0. 05 ）. Conclusion The recombinant pEGFP - N1 - hPer2 plasmid has been constructed successfully, which is expressed effectively in MG63 cells.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第3期737-740,共4页
Chinese Journal of Experimental Surgery
