Taq RecA蛋白的克隆表达及其在SNP基因分型中的应用

Cloning and expression of Taq RecA protein and its application in single nucleotide polymorphisms genotype analysis

目的研究克隆表达的Taq RecA蛋白作为一种添加剂在单核苷酸多态性(SNP)基因型分析中的作用。方法构建pET-28a-Taq RecA表达质粒,在BL21(DE3)菌株中诱导表达,用热变性和饱和硫酸铵沉淀法去除部分杂蛋白,再经Ni-NTA亲和层析柱纯化分离Taq RecA蛋白;用PCR和等位基因特异性PCR(AS-PCR)方法研究其增强特异性和在SNP基因分型中提高分型准确性的作用。结果成功构建了表达质粒,表达纯化得到较高纯度蛋白,用于SNP基因分型中减少了非特异性产物,提高了分型准确性。结论 Taq RecA蛋白作为一种PCR中的添加剂,在ATP辅助作用下,适量添加能提高AS-PCR介导的SNP基因分型的准确度。

Objective To clone and express Thermus aquaticus ReeA protein in E. coli expression system and to research the effect of the bacterial-expressed Taq RecA protein on genotype analysis. Meth- ods To construct pET-28a-Taq RecA expression plasmid and overexpress the Taq RecA in BL21 （DE3）. The bacterial pellet was resuspended and sonicated. After heat treatment and （NH4 ）2 SO4 precipitation of the supernatant, the purified protein was obtained by the Ni-chelating resin affinity chromatography. For functional studies, various amount Taq RecA protein was added into PCR systems and the effect of Taq RecA protein was studied by comparing PCR results with or without Taq RecA protein. Results Taq Re- cA protein was successfully expressed in BI21 （ DE3 ） and purified. Results of PCR experiments showed that Taq RecA protein combining with its cofactor ATP effectively eliminates the nonspecific PCR product and can improve veracity of genotype analysis. Conclusion Taq RecA protein combining with its cofac- tor ATP in AS-PCR can significantly improve the accuracy of genotype analysis.