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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 investigate the role of cloned Taq RecA as an additive in single nucleotide polymorphism (SNP) genotyping. Methods The recombinant plasmid pET-28a-Taq RecA was constructed and expressed in BL21 (DE3) strain. Some of the extracellular proteins were removed by thermal denaturation and ammonium sulfate precipitation. The recombinant Taq RecA protein was purified by Ni-NTA affinity chromatography ; PCR and allele-specific PCR (AS-PCR) method to study its enhanced specificity and SNP genotyping improve the accuracy of typing. Results The expression plasmid was successfully constructed and expressed and purified to obtain higher purity protein, which was used to reduce the non-specific products in SNP genotyping and improve the typing accuracy. Conclusion As a PCR additive, Taq RecA protein can increase the accuracy of AS-PCR-mediated SNP genotyping with the help of ATP.