线虫Fat-1基因密码子的优化及其真核表达载体构建

Codon Optimization and Eukaryotic Expression Vector Construction of Fat-1 Gene Coming From Caenorhabditis elegans

目的:为使线虫Fat-1基因能够在牛中高效表达,通过密码子优化及全基因合成方法制备Fat-1基因,并构建其转基因表达载体。方法:通过生物信息学手段,将来源于线虫的Fat-1基因以牛为表达宿主进行密码子优化,将优化后的Fat-1基因命名为bFat-1;通过全基因合成方法获得bFat-1基因,构建pcDNA3.1-bFat-1真核表达载体。结果:通过密码子优化,影响Fat-1基因在牛中表达的密码子适应指数、优势密码子频率及GC含量等各项指标均有显著改善;全基因合成、克隆及测序结果显示已获得bFat-1基因;构建并鉴定了pcDNA3.1-bFat-1载体。结论:获得了适合牛体表达的Fat-1基因并构建了其转基因载体,为获得高效表达Fat-1基因的转基因牛细胞系奠定了基础。

Objective： In order to make Fat-1 gene from Caenorhabditis elegans be highly expressed in cattle, the Fat-1 gene was optimized and obtained by nucleotide synthesis, the transgenic vector was constructed in the end. Methods： The codons of Fat-1 gene from C.elegans were optimized by bio-information software according cattle as the expressional host. The optimized Fat-1 gene was named as bFat-1. The bFat-1 was obtained by nucleotide synthesis, and the transgenic vector pcDNA3.1-bFat-1 was consturcted in the end. Results： The results showed that some indexes which control the expression level in cattle were significantly improved, including codon adaptation index（CAI）, frequency of optimal codons（FOP） and GC content. The results of nucleotide synthesis, gene clone and sequencing aproved the bFat-1 gene was obtained successfully; the results of vector construction and identification showed that the construction of pcDNA3.1-bFat-1 was successful. Conclusion： The transgenic vector pcDNA3.1-bFat-1, which expresses optimized Fat-1 gene from C.elegans was successfully established and would be used to construct the Fat-1 transgenic cell lines in follows study.