中文摘要：

目的：将PCR扩增产物人内皮型一氧化氮合酶（heNOS）cDNA定向克隆到载体pShuttle—IRES—hrGFP--2以构建重组质粒pShuttle—heNOS—EGFP。方法：在扩增目的基因的PCR引物5′端加上与线性化载体同源的15个碱基，使PCR反应产物两端分别带上15个和线性化载体两端同源的碱基序列。EcoR I酶切质粒pHCM．VsplA—heNOS，回收heNOS cDNA作为模板，进行PCR扩增。将纯化的PCR产物heNOS cDNA与EcoR V酶切后的线性化载体pShutde—IRES—hrGFP--2以摩尔数为8：1比例混和，在重组酶作用下，25℃反应30min，将PCR产物定向克隆入目标载体pShuttle—IRES—hrGFP--2上，获得阳性重组质粒pShuttle—heNOS—EGFP。重组质粒经PCR、酶切和测序鉴定。结果：经鉴定，成功构建出重组质粒pShutde—heNOS—EGFP。结论：无需传统的酶切、连接、载体去磷酸化等手段，利用PCR产物靶向克隆法，可快速、高效完成PCR产物的定向克隆。

英文摘要：

Objective To clone a PCR fragment of human endothelial nitric oxide synthase （heNOS） cDNA into vecto pShuttle - IRES - hrGFP - 2 to generate recombinant plasmid pShuttle - heNOS - EGFP. Methods PCR primers whicl have 15 bases of homology with sequences flanking the desired site of insertion in the cloning vector were designed, The plasmid pHCM VsplA- heNOS was digested with EcoR I and the recovered heNOS cDNA was used as the template fo PCR amplification of the fragment for PCR cloning. The vector pShuttle - IRES - hrGFP - 2 was linearized with EcoR V. The amplified heNOS cDNA for PCR cloning and hnearized vector pShutde - IRES - hrGFP - 2 were purified with low melt ing agarose gel. The PCR fragment heNOS cDNA and the linearized pShutde - IRES - hrGFP - 2 were mixed together at 8： 1 molar ratio within the tube containing recombinant enzyme in total volume 20 μL and incubated at 25 ℃ for 30 minutes, then transformed it to competent cells DH 5a. The positive clone pShutde - heNOS - EGFP was identified by PCR enzyme digestion, and DNA sequencing. Results heNOS cDNA was successfully inserted into the shuttle vector. Homolo gous recombination occurred between pShuttle - IRES - hrGFP - 2 and heNOS cDNA to generate pShutde - heNOS - EGFP. The recombinant vector was confirmed to be successfully constructed with PCR, digestion and DNA sequencing. Con closion PCR cloning technique is a simple, highly efficient, and convenient way to clone PCR products into the vector interest, without restriction enzyme digestion, blunt- end polishing, ligase, or phosphatase.