【摘要】 目的:构建人Prp8基因全长真核表达质粒。方法:从HeLa细胞中提取总体RNA,两步法合成cDNA,利用逆转录聚合酶链反应(RT-PCR)法,扩增出人Prp8基因的四段序列,首先克隆至pTZ57R/T载体,再定向克隆至真核表达载体pcDNA3.1(+),构建pcDNA3.1(+)-Prp8重组质粒。结果:RT-PCR法获得Prp8基因的4段序列,长度分别为2025、1090、1966、1963bp,分别与pTZ57R/T载体连接后,选择合适的酶切位点再连接成全长序列,然后将全长序列和pcDNA3.1(+)真核表达载体连接、转化、酶切鉴定及序列分析后,证实pcDNA3.1(+)-Prp8重组质粒构建成功。结论:成功克隆了人Prp8的编码基因,并构建了表达载体pcDNA3.1(+)-Prp8。更多还原

【Abstract】 Objective: To construct the eukaryotic expression plasmid about the whole Prp8 sequence. Methods: The total RNA was extracted from Hela cell and the cDNA was synthesized by two steps,and the four fragments of human Prp8 were amplified by RT-PCR. The four fragments were cloned to the pTZ57R/T vector,subcloned to eukaryotic expression vector pcDNA3.1(+)and constructed the pcDNA3.1(+)-Prp8 constitutive plasmid. Results: The four fragments of Prp8 produced by RT-PCR were 2 025 bp,1 090 bp,1 966 bp and 1 963 bp in size respectively,they were ligated with the pTZ57R/T vector,and formed the whole sequence by selecting suitable enzyme-digested sites and subcloned into the pcDNA3.1(+). The constitutive plasmid was successful confirmed by sequencing. Conclusion: The cloning of Prp8 gene and the construction of the eukaryotic expression vector pcDNA3.1(+)-Prp8 are achieved.更多还原