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目的:从宫颈癌组织中扩增与上皮细胞分化和人乳头瘤病毒(HPV)基因表达相关的转录因子Skn-1acDNA,进而在真核细胞中表达该转录因子以了解其功能。方法:从宫颈癌组织中提取总RNA,以此为模板,RT-PCR扩增Skn-1acDNA片段,将该cDNA片段克隆至pMD18-T载体并进行序列分析,构建Skn-1a真核表达载体,并转染Hela细胞,检测其表达情况。结果:RT-PCR扩增得到约1 300 bp的cDNA片段,将其克隆至pMD18-T载体,DNA测序表明,得到片段长度为1 308 bp,为转录因子Skn-1a基因编码的cDNA,获得的Skn-1acDNA序列与已公布的Skn-1acDNA序列具有99.85%同源性,有1个碱基的差异,但并未导致编码氨基酸的改变,继而构建了Skn-1a真核表达载体pcDNA3/Skn-1a,其转染Hela细胞后可正确表达。结论:转录因子Skn-1a在宫颈癌组织中有一定量的表达,并在体外培养的Hela细胞中获得表达。
OBJECTIVE: To amplify Skn-1acDNA, a transcription factor related to epithelial cell differentiation and human papillomavirus (HPV) gene expression, from cervical cancer tissues and further to express its function in eukaryotic cells. Methods: The total RNA was extracted from cervical cancer tissues and used as a template to amplify Skn-1acDNA fragment by RT-PCR. The cDNA fragment was cloned into pMD18-T vector and sequenced to construct Skn-1a eukaryotic expression vector. Hela cells were transfected to detect the expression of Hela cells. Results: The cDNA fragment of about 1 300 bp was amplified by RT-PCR and cloned into pMD18-T vector. DNA sequencing showed that the cDNA fragment was 1 308 bp in length and encoded by Skn-1a gene. The Skn-1acDNA sequence has 99.85% homology with the published Skn-1acDNA sequence with 1 base difference, but does not lead to the change of the encoded amino acid. The Skn-1a eukaryotic expression vector pcDNA3 / Skn- 1a, Hela cells transfected correctly expressed. Conclusion: The transcription factor Skn-1a is expressed in cervical cancer tissues and expressed in Hela cells cultured in vitro.