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

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。

Construction of eukaryotic expression vector of short hairpin RNA targeting human xylosyltransferase-I gene

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 (PC). 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 Lipofectomine 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- I in transfected ACC-M cells and the protein expression of XT- I was detected by Western blot. RESULTS: The plasmids expressing shRNA targeting XT-I gene are called WJ1, WJ2, WJ3, WJ4, WJ5 and WJ6. Successful constructions were identified by digestion and sequencing. The mean rate of transfection was 50.26%. ACC-M cells transfected with WJ1-WJ6 expressed 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 mRNA level and 70.18% of protein level respectively. CONCLUSION: The XT-I shRNA expression vectors were constructed successfully which lays the foundation for RNAi study on the biosynthesis of PG in salivary gland tumors.