靶向抑制人木糖基转移酶-I基因的shRNA真核表达载体的构建
Construction of eukaryotic expr ession vector of shor t hairpin RNA targeting human xylosyltr ansfer ase - I gene
作者：石宏 王洁 王旭 顾洪涛 侯亚丽 于利洁
Author：SHI Hong, WANG Jie, WANG Xu, GU Hong- tao, HOU Ya- li, YU Li- jie
收稿日期：2008-04-25 年卷（期）页码：2008,26(02):206-
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：人木糖基转移酶- Ⅰ,短发夹状shRNA,唾液腺肿瘤,肌上皮细胞,蛋白多糖,
Key words：xylosyltransferase- Ⅰ,short hairpin RNA,salivary gland tumor,myoepithelial cells,proteoglycans,
基金项目：
河北省自然科学基金资助项目（C2006000796）；河北省卫生厅医学科学研究重点课题指令性计划资助项目（07126）
中文摘要
目的构建靶向抑制人木糖基转移酶- Ⅰ（XT- Ⅰ）基因的短发夹状RNA（shRNA）真核表达载体，为探讨唾液腺肿瘤性肌上皮细胞合成及分泌蛋白多糖（PG）的研究奠定基础。方法根据GenBank提供的XT- Ⅰ基因序列，设计短链寡核苷酸，化学合成后经退火形成双链DNA片段，克隆到Pgenesil- 1载体中，构建shRNA真核表达载体WJ1-WJ6，行酶切及核酸测序鉴定；将构建的XT- Ⅰ特异性shRNA表达载体转染体外培养的人唾液腺腺样囊性癌细胞株ACC-M，在荧光倒置显微镜下观察绿色荧光蛋白的表达，流式细胞术检测转染效率，并采用半定量RT- PCR和Western blot分别检测转染后细胞XT- Ⅰ基因mRNA和蛋白表达水平的变化。结果经酶切、连接后构建的6个质粒命名为WJ1、WJ2、WJ3、WJ4、WJ5、WJ6。酶切及核酸测序鉴定证实，构建的shRNA表达载体WJ1-WJ6序列正确；转染WJ1-WJ6后ACC-M细胞均可表达绿色荧光；流式细胞术测定转染效率平均为50.26%；RT- PCR结果显示，WJ3显著抑制XT- Ⅰ mRNA的表达，抑制率为72.39%；Western blot结果显示，WJ3有效抑制XT- Ⅰ的蛋白表达，抑制率为70.18%。结论成功构建靶向抑制XT- Ⅰ的shRNA真核表达载体WJ1-WJ6，其中WJ3可高效抑制XT- Ⅰ基因mRNA及蛋白水平的表达，为唾液腺肿瘤中PG的RNAi研究奠定了基础。
英文摘要
Objective To design and construct the plasmids expressing short hairpin RNA（shRNA）targeting human xylosyltransferase- Ⅰ（XT- Ⅰ）which is the initiating enzyme in the biosynthesis of proteoglycans（PG）. Methods Short chain oligonucleotides were designed according to the sequence of XT- Ⅰ provided by GenBank. The DNA segments were gained through annealing after chemosynthesis, and were cloned into Pgenesil- 1 vector. The recombinant XT- Ⅰ shRNA expression vectors were identified by digestion and sequencing analysis. At last the constructed XT- Ⅰ expression vectors were transfected into salivary adenoid cystic carcinoma cell line（ACC-M） by LipofectomineTM 2000. The expression of green fluorescent protein（GFP） was detected by inverted fluorescent microscope and the rates of transfection were detected by flow cytometer. Semiquantitative RT- PCR was used to detect the expression of mRNA level of XT- Ⅰin transfected ACC-M cells and the rotein expression of XT- Ⅰ was detected by Western blot. Results The plasmids expressing shRNA targeting XT- Ⅰgene are called WJ1, WJ2, WJ3, WJ4, WJ5 and J6. Successful constructions were identified by digestion and sequencing. The mean rate of transfection was 50.26%. ACC-M cells transfected with WJ1 -WJ6 xpressed GFP successfully. And by RT- PCR and Western blot, WJ3 showed the most powerful RNAi gene silencing of inhibitory. The inhibition rate was 72.39% of RNA level and 70.18% of protein level respectively. Conclusion The XT- Ⅰ shRNA expression vectors were constructed successfully which lays the foundation for NAi study on the biosynthesis of PG in salivary gland tumors.
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