STAT3基因shRNA真核表达载体的构建与鉴定

Construction of STAT3 shRNA eukaryotic expression vector

目的构建携带信号传导和转录激活子3(STAT3)基因短发夹状干扰RNA(shRNA)的真核表达载体。方法从GenBank STAT3mRNA上寻找到3条符合特征的靶序列,合成靶序列的DNA寡核苷酸链,合成双链DNA,并和BamHⅠ和HindⅢ酶切后的pRNAT-U6.1/Neo载体质粒连接产生重组质粒,PCR筛选阳性克隆,并行测序鉴定。重组质粒转染大肠癌LoVo细胞,RT-PCR检测STAT3的表达。结果PCR和测序证实重组质粒构建正确。3种重组质粒对大肠癌LoVo细胞STAT3的表达有较强的抑制作用。结论成功构建了携带有STAT3基因的短发夹状干扰RNA的重组质粒。

Objective To construct eukaryotic expression vectors of RNA interference of STAT3 gene. Methods Three sequences of siRNA targeting STAT3 gene were found from GenBank. The complementary DNA containing both sene and antisene Oligo DNA of the targeting sequence was designed,synthesized and cloned into the pRNAT-U6.1/Neo vector. The recombinant plasmids were confirmed by PCR and sequenced. LoVo cells were transfected with the recombinant plasmids and RT-PCR was used to certify the expression of STAT3 mRNA. Results PCR and DNA sequencing demonstrated that the eukaryotic expression vectors of RNA interference of STAT3 mRNA were constructed successfully. STAT3 shRNA could inhibit the expression of STAT3 gene in LoVo cells, which was transfected with the recombinant plasmids effectively. Conclusion The eukaryotic expression vectors of RNA interference of STAT3 gene are constructed successfully.