[转化生长因子-β1短发夹RNA真核表达载体的构建]

[Construction of eukaryotic expression vector of short hairpin RNA for transforming growth factor-beta1].

作者信息

Wang Jing, Wu Jun-Zheng, Guo Fu-Ping, Zhu Xiu-Li, Wen De-Sheng

机构信息

Dept. of Biology, College of Stomatology, The Fourth Military Medical University, Xi'an 710032, China.

出版信息

Hua Xi Kou Qiang Yi Xue Za Zhi. 2006 Apr;24(2):113-6.

PMID:16704085

Abstract

OBJECTIVE

To construct the plasmid containing short hairpin RNA (shRNA) of TGF-beta1 expression vector.

METHODS

Short chain oligonucleotide was designed according to the TGF-beta1 mRNA sequence provided by Genebank, then DNA segment was gained through annealing after chemosynthesis, and then was cloned to pWH1 vector. The recombinant TGF-beta1 shRNA expression vector was evaluated by using enzyme cutting. At last, the constructed TGF-beta1 expression vector was transfected into salivary gland mucoepidermoid carcinoma (Ms) cells by Lipofectomine TM 2000, and its effect on TGF-beta1 expression was observed by RT-PCR and immunohistochemistry.

RESULTS

Successful construction was identified by enzyme cutting and the constructed plasmid was called pWH1-TGF-beta1. The shRNA and it inhibited the TGF-beta1 mRNA and protein expression effectively.

CONCLUSION

The constructed TGF-beta1 shRNA expression vector can block the TGF-beta1 expression in salivary gland mucoepidermoid carcinoma cells.

摘要

目的

构建含转化生长因子-β1（TGF-β1）表达载体短发夹RNA（shRNA）的质粒。

方法

根据Genebank提供的TGF-β1 mRNA序列设计短链寡核苷酸，化学合成后经退火获得DNA片段，再克隆至pWH1载体。采用酶切法对重组TGF-β1 shRNA表达载体进行鉴定。最后，用脂质体TM 2000将构建的TGF-β1表达载体转染至涎腺黏液表皮样癌（Ms）细胞，通过逆转录聚合酶链反应（RT-PCR）和免疫组织化学观察其对TGF-β1表达的影响。