In-fusion 连接技术优化及其在大片段重组载体构建中的应用

摘要

With the completion of large-scale genome sequencing and the establishment of expression se-quence tag databases,the genome research had a great conversion from structural genomics to functional genomics and the key way to study gene function was gene fusion technology.To construct large recombi-nant vectors efficiently and precisely,we constructed large recombinant vectors using In-fusion technology and optimized the In-fusion technology system by changing the time of gene fusion.The quality of these vectors were tested by cell transfection and immunoblotting.The efficiency of clones could be improved by optimization of reaction time.Large recombinant vectors could be successfully achieved with the combina-tion of In-fusion technology and Fusion reagent.We constructed several eukaryotic expression vectors with the inserted DNA fragments of 10 ku,8 ku and 4 ku after optimizing connecting time during reaction and they were translated successfully.The optimized In-fusion technology make it more efficient and easier to fuse seamlessly and clone large PCR products into a vector of interest.This lays foundation for further re-search about the function of large sequence.%随着大规模基因组测序的完成和表达序列标签数据库的建立，基因组的研究已由结构基因组向功能基因组转化，而基因功能研究的关键是基因融合技术。为高效率高保真地构建融合大片段的重组载体，本试验利用 In-fusion 技术进行大片段载体构建，以不同的融合时间进行比较，通过细胞转染以及免疫印迹等方法对载体质量进行验证。通过对体系中连接时间的优化，构建出了含插入片段10、8、4 ku 的真核表达载体，并可正确表达。In-fusion 技术的优化，提高了大片段真核表达载体构建的效率，为研究大片段基因的生物学功能奠定了基础。