T4多聚核苷酸激酶的原核表达、纯化及初步应用

Prokaryotic Expression, Purification and Preliminary Application of T4 Poynucleotide Kinase

原核表达、纯化T4多聚核苷酸激酶,并尝试将纯化的T4 PNK用于短探针序列的连接。本研究以合成的pseT基因为模板,PCR扩增出带有NdeⅠ和Bam HⅠ位点的目的片段,构建pseT-pET-15b原核表达载体,并转入E. coli ER2566中诱导表达。Ni-Agarose亲和层析柱纯化重组蛋白后,再进行Western blot鉴定。用纯化后再浓缩的T4 PNK参与探针连接反应,并设置商品T4 PNK和阴性对照。PCR扩增成功获得大于900 bp的目的基因片段,原核表达载体pseT-pET-15b构建成功,经诱导表达的重组蛋白分子量大小约为35 kD,Western blotting确认蛋白表达正确,浓缩后的蛋白浓度达到826μg/m L。电泳结果显示,重组T4 PNK在探针连接中效果较好。本研究成功表达并纯化了可溶性的T4多聚核苷酸激酶,且具有较好的活性,该蛋白可进一步用于后续大批量探针连接反应或其他相关研究,具有一定实际应用价值。

The prokaryotic expression and purification of T4 polynucleotide kinase, was carried out and the purified T4 PNK was attempted to link short probe sequence. In this study, the synthetic pseT gene was used as a template,and the target fragments with Nde Ⅰ and Bam H Ⅰ were amplified by PCR. The prokaryotic expression vector of pseT-pET-15 b was constructed and transferred into E.coli ER2566 to induce expression. The recombinant protein was purified with Ni-Agarose affinity chromatography column and then identified with Western boltting. The purified and concentrated T4 PNK was involved in probe ligation reaction, and the commercial T4 PNK and negative control were also set. A target gene fragment of more than 900 bp was successfully obtained by PCR amplification, the prokaryotic expression vector pseT-pET-15 b was successfully constructed and the molecular weight of the induced recombinant protein was about 35 kD. The expression protein was also confirmed by Western blot. The concentration of the concentrated protein reached 826 μg/m L. The results of electrophoresis showed that the recombinant T4 PNK had good effect in the probe connection. In this study, soluble T4 polynucleotide kinase with good activity was successfully expressed and purified. The protein could be further used in subsequent probe ligation reactions in large quantities or other related studies, with certiain practical application value.