可定点整合的无筛选标记LacS基因表达载体的构建与鉴定

试验旨在构建一个能够定点整合且无筛选标记基因的半乳糖苷酶基因LacS表达载体.本研究以pEGFP-N1质粒为原始框架,通过PCR及限制性酶切位点在pEGFP-N1质粒的标记基因两端添加两个同向LoxP序列,在多克隆位点上游添加能够定点整合的attB序列,最后再将目的基因BC promoter-LacS-PolyA通过限制性酶切位点插入多克隆位点.每一步都进行PCR、酶切及测序鉴定.PCR产物及酶切片段大小符合预期结果,测序结果也与相应寡核苷酸序列一致,证明各个基因片段正确地连接到载体相应位置.本试验成功构建了可定点整合的无筛选标记半乳糖苷酶基因表达载体pEGFP-N1-LacS.

Construction and Identification of Site-specific Integrated and Marker-free LacS Gene Vector

The objective of this study was to solve the problem of construction a transgenic safety and site-specific integrated and marker-free LacS gene expression vector.Therefore,this experiment using plasmid pEGFP-N1 as the original framework,using PCR and restriction sites to add two with LoxP sequence at both ends of the marker genes in the pEGFP-N1 plasmid,attB sequence was added at the upstream of the multiple cloning sites for site-specific integration,then add the gene BC promoter-LacS-PolyA into the multiple cloning site.By restricted endonucleases digestion,these 3 fragments,Loxp,attB and LacS,were detected in the vector pEGFP-N1-LacS,PCR products and fragment size consistent with the expected results,the results of sequencing and oligonucleotide sequences also corresponding favorable,proved that each gene fragment was correctly connected to the corresponding position of the plasmid.In conclusion,the site-specific integrated and marker-free LacS gene vector was constructed successfully.