论文部分内容阅读
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 an eukaryotic expression vector carrying short hairpin RNA (shRNA) of signal transducer and activator of transcription 3 (STAT3) gene. Methods Three target sequences were found from GenBank STAT3 mRNA. The DNA oligonucleotide of target sequence was synthesized and double-stranded DNA was synthesized. The recombinant plasmid was ligated with plasmid pRNAT-U6.1 / Neo vector digested with BamHⅠ and HindⅢ Produce recombinant plasmid, PCR screening of positive clones, parallel sequencing identification. The recombinant plasmids were transfected into LoVo cells, and the expression of STAT3 was detected by RT-PCR. Results PCR and sequencing confirmed that the recombinant plasmid was constructed correctly. The three recombinant plasmids strongly inhibited the STAT3 expression in colorectal cancer LoVo cells. Conclusion The recombinant plasmid carrying short hairpin RNA carrying STAT3 gene was successfully constructed.