猪β_2-AR基因重组质粒的快速筛选

Rapid screen for recombinant clones of porcine β_2-AR gene

为建立一种高效快速鉴定重组质粒的实验方法。将猪β2 -肾上腺素能受体 (β2 -AR)基因与pET - 32C重组 ,构建 β2 -AR基因表达载体pET32CAR。以T7启动子引物和猪 β2 -肾上腺素能受体基因特异性引物为PCR引物 ,挑取重组质粒转化单菌落直接进行PCR。产物经电泳发现 ,5个候选克隆中有 3个克隆扩增出了一条约 2kb的特异性条带 ,并且插入方向正确。随机选取其中 1个克隆用限制性酶切鉴定以及DNA测序验证PCR筛选的正确性。研究结果表明 :在采用PCR方法对重组克隆进行鉴定时 ,可以直接使用细菌菌落参与反应 ,而无须提取克隆的DNA。与传统的实验方法相比 ,筛选的时间缩短至 3～ 4h ,大大提高了重组DNA的筛选效率。

This article reports a new method for rapid screening recombinant bacteria.The gene encoding for porcine β 2-adrenergic receptor (β 2-AR ) was subcloned into expression vector pET32C. Primers for PCR screen were designed on the basis of T7 promoter in pET32C vector and porcine β 2-AR cDNA. The recombinant bacteria were transferred into PCR reaction mixture directly. After PCR and agrose gel electrophoresis, three positive clones of five candidates with expected DNA size and direction were detected. One positive clone was further confirmed by restriction enzyme characterization and DNA sequencing. The results show that recombinant plasmid in bacterial cells can be directly applied in PCR detection. This convenient and timesaving procedure eliminates the need for preparation and purification of the plasmid DNA.