日本七鳃鳗pcna基因克隆、生物信息学分析及真核表达

增殖细胞核抗原(proliferating cell nuclear antigen,PCNA),也称周期蛋白或DNA聚合酶的辅助蛋白,是真核细胞合成所必需的核蛋白,在DNA复制中起重要作用。前期实验发现日本七鳃鳗肝脏cDNA文库的表达序列标签(Expressed Sequence Tag,EST)中存在与高等脊椎动物pcna基因同源的序列。提取日本七鳃鳗(Lampetra japonica)肝脏组织RNA,通过RT-PCR方法扩增七鳃鳗pcna基因,对其进行生物信息学分析,并将Lj-pcna基因成功构建到pGFP-N2真核表达载体上,重组质粒PGFP-N2-Lj-pcna转染人Hela细胞,荧光显微镜下观察有荧光蛋白的表达。日本七鳃鳗pcna基因的真核表达载体成功构建和转染,为探讨七鳃鳗pcna基因功能研究及其它七鳃鳗相关研究提供条件。

Cloning, Eukaryotic Expression and Bioinformatics Analysis of PCNA gene from Lampetra Japonica

Proliferating cell nuclear antigen （PCNA）, also known as the cycle of protein or DNA polymerase accessory protein, is necessary for synthesis of nuclear protein for eukaryotic cells and plays an important role in DNA replication. The previous research found that there is a homology EST sequence tags with PCNA of the higher vertebrates in liver cDNA library from Lampetra japonica. Total RNA was extracted from liver tissue of Japanese lamprey, then PCNA gene was amplified by RT-PCR and analysised by the bioinformatics methods. In addition, Lj-pcna gene was successfully constructed to pGFP-N2 eukaryotic expression vector, then the recombinant plasmid PGFP-N2-Lj-pcna w.as transfected to Hela cells, and green fluorescent protein was expressed under fluorescence microscopy. Eukaryotic expression vector of lamprey pcna gene was constructed and transfeced, which lays a foundation for the lamprey PCNA gene function research and other related studies.