CAJ | 학술논문

目的：通过克隆激活转录因子-2(ATF-2)基因以及真核表达载体的构建，初步研究ATF-2对小鼠成釉细胞基质金属蛋白酶-20(MMP-20)基因表达的影响。方法：根据引物设计原则设计ATF-2基因逆转录-聚合酶链反应(PCR)引物，从小鼠成釉细胞中提取总RNA逆转录所得cDNA为模板，进行PCR法扩增。得出含有Hind Ⅲ和xhol酶切位点的ATF-2目的基因连接到pcDNA 3.1/myc-HisA真核表达载体上；用重组质粒转染小鼠MMP-20基因，观察其对MMP-20基因转录活性的影响。结果：经过PCR引物扩增得到1463 bp基因片段，将获得的重组质粒pcDNA 3.1/myc-HisA-ATF-2双酶切分析鉴定，测序结果与Gen Bank登录基因序列完全一致；双荧光素酶结果显示ATF-2可以促进MMP-20启动子的表达(P<0.01)，且在-825~+23(848 bp)启动子区段促进作用最明显。结论：成功实现了ATF-2基因克隆及真核表达载体的构建，并发现ATF-2对MMP-20启动子区域活性表达起正向调节作用。
목적：통과극륭격활전록인자-2(ATF-2)기인이급진핵표체재체적구건，초보연구ATF-2대소서성유세포기질금속단백매-20(MMP-20)기인표체적영향。방법：근거인물설계원칙설계ATF-2기인역전록-취합매련반응(PCR)인물，종소서성유세포중제취총RNA역전록소득cDNA위모판，진행PCR법확증。득출함유Hind Ⅲ화xhol매절위점적ATF-2목적기인련접도pcDNA 3.1/myc-HisA진핵표체재체상；용중조질립전염소서MMP-20기인，관찰기대MMP-20기인전록활성적영향。결과：경과PCR인물확증득도1463 bp기인편단，장획득적중조질립pcDNA 3.1/myc-HisA-ATF-2쌍매절분석감정，측서결과여Gen Bank등록기인서렬완전일치；쌍형광소매결과현시ATF-2가이촉진MMP-20계동자적표체(P<0.01)，차재-825~+23(848 bp)계동자구단촉진작용최명현。결론：성공실현료ATF-2기인극륭급진핵표체재체적구건，병발현ATF-2대MMP-20계동자구역활성표체기정향조절작용。
Objective:To clone the activating transcription factor-2 ( ATF-2 ) gene, then construct the recombinant eukaryotic expression vector with ATF-2,and explore the effects of ATF-2 on the gene expression of matrix metalloproteinase-20(MMP-20) in mice ameloblasts. Methods:The reverse transcription PCR primers of ATF-2 were designed. The total RNA from the mice ameloblasts was extracted to obtain cDNA for PCR amplification. The ATF-2 gene containing HindⅢand xhol restriction sites was inserted into the eukaryotic expression vector(pcDNA 3. 1/myc-HisA). The effects of ATF-2 on the transcriptional activity of MMP-20 was observed after transfecting the recombinant plasmid. Results:One thousand four hundred and sixty-three bp gene fragment was amplified through PCR. The recombinant eukaryotic expression vector with ATF-2 ( pcDNA 3. 1/myc-HisA-ATF-2 ) was successful constructed by the verification of dual enzyme digestion and sequencing. The luciferase test showed that ATF-2 gene could promote the expression of MMP-20 promoter(P<0. 01). Conclusions:The recombinant pcDNA 3. 1/myc-HisA-ATF-2 expression vector is successful constructed. ATF-2 can positive regulate the promoter activity of MMP-20.