中文摘要：

目的分析人MUC16基因的转录起始位点和重要的调控区域，确定人MUC16基因的核心启动子可能存在区域。方法用生物信息学分析人MUC16基因启动子所在区域。培养SKOV3细胞株并提取细胞基因组DNA，用PCR技术扩增启动子区域的594（-1844／-1250／）、548（-1798／-1250）、326（-1576／-1250）和170bp（-1420／-1250）的启动子片段，分别构建重组PGEM—T质粒，再克隆入荧光素酶报告基因载体PGL3载体，构建PGL3—170、PGL3—326、PGL3—548和PGL3—594重组表达质粒。用脂质体介导的方法瞬时转染SKOV3细胞，测定荧光素酶的表达活性。结果成功构建4种人MUC16基因启动子片段的荧光素酶报告基因表达体系，分别为PGL3—170（-1420／-1250）、PGL3—326（-1576／-1250）、PGL3—548（-1798／-1250）和PGL3—594（-1844／-1250），各缺失片段在SK—OV3细胞中均有活性。MUC16的核心启动子5’端可能为PGL3—326的3’端，3’端固定在-1250处，5’端由-1844向-1420移动时启动子活性逐渐增强，且变化幅度较大，提示这一区间内可能存在重要的顺式作用元件。结论成功构建了人MUC16基因启动子片段的荧光素酶报告基因载体，确定人MUC16基因的核心启动子区域。

英文摘要：

Objective To analyze the transcription start point and important regulatory regions of human MUC16 gene and determine possible core promoter of human MUC16 gene. Methods The promoter region of MUC16 gene was analyzed by bioinformatics. The cultured cell lines of SKOV3 was cultured and its genomic DNA was extracted. The 594 bp （ - 1 844/- 1 250/） ,548 bp （ - 1 798/- 1 250） ,326（ - 1 576/- 1 250） and 170 bp （ - 1 420/ - 1 250） promoter fragments were produced and amplified by PCR, then PGEM-T-promoter recombinant plasmid were constructed and transformed into JM109 bacteria being competent cell, respectively. Then these promoter fragments were cloned into luciferase reporter gene （ pGL3 ） vector, so PGL3-170,PGL3-326,PGL3-548 and PGL3- 594 recombinant expression plasmid were constructed and transformed into JM109 bacteria being competent cell. With liposome-mediated transient transfection way, PGL3-basie-promoter expression plasmids were transient transfected into SKOV3 cells, and luciferase activities were measured in cells. Results The four expression systems carrying human MUC16 gene promoter fragment and luciferase reporter gene were successfully constructed, they were PGL3-170 （ - 1 420/- 1 250 ）, PGL3-326 （ - 1 576/- 1 250 ）, PGL3-548 （ - 1 844/- 1 750 ） and PGL3-594 （ -1798/- 1750）,respectively. These promoter fragments all had activities in SKOV3 cells. The 5 end of human MUC16 core promoter was 5 end of PGL3-326, and its 3 end was determined in the position of - 1 750 sequences. When 5 end moved from - 1 844 to - 1 420, promoter activity gradually increased, and the changes were high in magnitude. This suggests that there were possible essential cis-acting elements in this sequence. Conclusion The four expression voctors carrying human MUC16 gene promoter fragment and luciferase reporter gene are successfully constructed. The possible core promoter of human MUC16 gene is determined.