CAJ | 학술논문

目的：探讨人多巴胺D2受体(DRD2)基因第3内含子区51103 bp位点单核苷酸多态性(rs2075652)对DRD2基因转录活性的影响,进一步揭示DRD2基因第3内含子区的单核苷酸多态性影响创伤后应激障碍发病风险的分子机制。方法以人全血基因组DNA为模板,扩增DRD2基因第3内含子区序列(399 bp),51103 bp处分别为T或C,与报告基因载体pmirGlo相连接,构建重组荧光素酶表达载体,经限制性内切酶酶切及测序得以鉴定,然后分别与pRL-CMV共转染人胚肾癌细胞株HEK293,检测细胞中荧光素酶的相对表达量。结果双酶切及测序结果证实成功构建重组质粒pmirGlo-T/pmirGlo-C,荧光素酶检测结果显示,当第3内含子区51103 bp位点为C时荧光素酶活性显著高于为T时(P<0．01)。结论成功构建了pmirGlo-T/pmirGlo-C报告基因重组质粒,DRD2基因第3内含子区rs2075652位点由T突变为C后,可能增强基因转录活性,该结果为进一步研究DRD2基因内含子区的功能奠定了基础。
목적：탐토인다파알D2수체(DRD2)기인제3내함자구51103 bp위점단핵감산다태성(rs2075652)대DRD2기인전록활성적영향,진일보게시DRD2기인제3내함자구적단핵감산다태성영향창상후응격장애발병풍험적분자궤제。방법이인전혈기인조DNA위모판,확증DRD2기인제3내함자구서렬(399 bp),51103 bp처분별위T혹C,여보고기인재체pmirGlo상련접,구건중조형광소매표체재체,경한제성내절매매절급측서득이감정,연후분별여pRL-CMV공전염인배신암세포주HEK293,검측세포중형광소매적상대표체량。결과쌍매절급측서결과증실성공구건중조질립pmirGlo-T/pmirGlo-C,형광소매검측결과현시,당제3내함자구51103 bp위점위C시형광소매활성현저고우위T시(P<0．01)。결론성공구건료pmirGlo-T/pmirGlo-C보고기인중조질립,DRD2기인제3내함자구rs2075652위점유T돌변위C후,가능증강기인전록활성,해결과위진일보연구DRD2기인내함자구적공능전정료기출。
Objective To investigate the effect of single nucleotide polymorphism ( SNP ) in 51 103 bp (rs2075652) of Dopamine receptor D2 (DRD2) gene of the third intron region on transcription activity and to further study the effect of gene mechanism on onset risk of post-traumatic stress disorder ( PTSD) in human. Methods The third intron region sequences of DRD2 gene T or C at site 51 103 bp were amplified using whole blood genomic DNA as template, the obtained fragments of 399 bp (the transcription start site is marked as +1) were inserted into pmirGlo re-spectively. The recombinant vector or pmirGlo-Basic vector was co-transfected together with pRL-CMV vector containing renal luciferase reporter gene into primarily cultured HEK293 after identifying with restriction enzyme double digestion and sequencing, and the corresponding expression value of firefly luciferase was detected. Results The correct construc-tion of recombinant expression vector pmirGlo-DRD2-T/ pmirGlo-DRD2-C was confirmed by restriction enzyme double di-gestion and sequencing analysis. The luciferase detection showed that expression of firefly luciferase, when the site of 51 103 bp was C, was significantly higher than that when the site of 51 103 was T (P<0. 01). Conclusion Luciferase re-porter vectors pmirGlo-T /pmirGlo-C can be successfully constructed. The transcription activity of DRD2 gene may be en-hanced when the DRD2 gene of the third intron region 51 103 bp site changes from C to T, and these results may be the basis for further study of the function of DRD2 gene intron region.