摘要
为克隆尖吻蝮蛇C型凝集素类蛋白基因至表达载体pBV2 2 0 ,在该载体的多克隆位点上选择适当的酶切位点 ,设计PCR引物 :在目的基因上、下游引物的 5′ 端分别加上对应于载体的粘性末端序列 ,建立两个单独反应体系 ,每个反应体系中只加入一种引物 ,用PfuDNA聚合酶催化模板DNA单链扩增 ;将所得的两种单链产物在适当条件下进行复性后直接与经过酶切的载体DNA连接 ,并转化大肠杆菌DH5α感受态细胞 ,获得足够数量的转化子 .经PCR鉴定单菌落中有目标DNA片段插入 ,测序表明克隆连接正确 .该方法以其简捷性和有效性 。
To clone Akitonin βcDNA into the expression vector p BV220, appropriate restriction endonuclease recognition sites were chosen at the multiple cloning sites (MCS) of the vector. PCR primers were designed with refe rence to the sites chosen: cohesive end sequences were appended onto the 5′-en d of the upstream and downstream primers of the target gene. Two independent PCR reaction systems were established, each using only one primer, to amplify sing le-stranded DNA catalyzed by Pfu DNA polymerase. The amplified single segments were annealed under proper conditions and ligated with the vector pBV220 heretof ore double-digested with the selected restriction enzymes, and transformed into DH5α competent cells. Sufficient transformants were obtained and the target DN A insertion was identified by PCR and confirmed by sequencing. This strategy pro vides a reliable approach to directional cloning characterized by the directness and efficiency in introducing cohesive ends, as well as its simplicity and effi ciency in manipulation.
