宫颈癌组织中转录因子Skn-1 a cDNA的克隆及表达

cDNA cloning and expressing of human Skn-1a cDNA from cervical carcinoma tissue

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 clone cDNA of human transcription factor .Skn-1a （related to epidermal differentiation and HPV gene expression） gene from cervical carcinoma tissue and construct the eukaryotic expression vector of the Skn-1a cDNA. Methods： The Skn-1a cDNA fragment was amplified by RT-PCR from total RNA that was extracted from cervical carcinoma tissue,the PCR product was cloned into pMD18-T vector and the nucleotide sequence was analyzed, subsequently, the eukaryotic expression vector of the Skn-1a cDNA was constructed, and transfected Hela cells, the expression of Skn -1a was examined. Results： A cDNA fragment about 1300 bp was amplified by RT-PCR,the result of sequencing showed the fragment was 1 308 bp in length, containing a cDNA coding human Skn-1a . The nuclcotide sequence homology to the previously published sequence of human .Skn-1a was 99.85%, there was one conversion in nucleic acid sequences, but the conversion didn＇t induce the change of coding amino acid; the eukaryotic expression vector of Skn-1a can express correctly after transfecting Hela cells. Conclusions： The results suggested that Skn- 1a gene expresses at some level in the cervical carcinoma tissue, and Skn-1a cDNA can express in vitro culture Hela cells. The research provide basis for further exploring the relationship between Skn-1a and cervical carcinoma.