【摘要】 为了建立一种Klenow(exo-)蛋白的制备方法,并对其在RAA(recombinase-aid amplification)技术中的应用进行初步研究,通过Overlap点突变法构建获得目的基因片段,并构建Klenow(exo-)蛋白表达载体进而转化至大肠杆菌BL21(DE3)菌株进行诱导表达。表达产物通过Ni-NTA螯合层析进行初步纯化,然后用Heparine柱层析进一步纯化,通过SDS-PAGE鉴定纯化产物,获得了纯度较高的目的蛋白。用纯化后的目的蛋白配制RAA反应体系,针对肉源实际样本设计突变位点进行检测。实验结果显示该体系具有较强的特异性,在模板基因序列3′端出现双碱基差异时不会进行有效扩增,相较于常规的PCR检测方式该体系能够实现双碱基差异位点的有效区分。该技术未来有望大规模应用于SNPs的检测。更多还原

【Abstract】 A preparation method of Klenow(exo-) protein was established and its application in RAA technology was preliminary studied. The target gene fragment was constructed by the Overlap point mutation method, and the Klenow(exo-) protein expression vector was constructed and transformed into Escherichia coli BL21(DE3) strain to induce expression. The expressed product was subjected to preliminary purification by Ni-NTA chelate chromatography, followed by Heparine column chromatography for further purification, and the purified product was identified by SDS-PAGE. A protein of higher purity was obtained. The purified RAA reaction system was prepared using the purified target protein, and the mutation site was designed for the actual sample of the meat source. The experimental results showed that the system has strong specificity and don’t amplified when there is a double base difference at the 3′ end of the template gene sequence, compared with the conventional PCR detection method, the system could effectively distinguish double base mutation. The system was expected to applied in detection of SNPs in the future.更多还原