噬菌体PaP3推定基因tls核酸内切酶活性的初步验证

Experimental identification of endonuclease activity of the putative gene tls from phage PaP3

目的对BLAST推定的铜绿假单胞菌噬菌体PaP3的tls基因功能进行实验验证。方法采用PCR方法从噬菌体PaP3基因组扩增出tls基因,克隆至pMD18-T载体中,再经酶切纯化后将目的基因插入表达载体pQE31,转化入大肠杆菌JM109,IPTG诱导表达目的蛋白,超声裂菌后收集包涵体,并用裂解缓冲液溶解包涵体蛋白。然后利用Ni-NTA亲合层析纯化蛋白,通过透析使H6-TLS复性。最后构建含有末端酶大亚基酶切位点的底物质粒pMD-cos,检测复性蛋白活性。结果通过上述方法,成功构建了pQE-tls表达载体,融合蛋白H6-TLS表达量占菌体总量的30%。同时成功构建了目的蛋白的底物质粒pMD-cos。经由亲和层析初步纯化及透析复性后,H6-TLS可检测出核酸内切酶的活性,能将底物质粒部分线性化。结论成功地获得了具有内切酶活性的H6-TLS融合蛋白,为tls基因的认证提供了实验证据,为进一步鉴定和利用该基因奠定了基础。

Objective To experimentally verify the putative function of gene tls from Pseudomonas aeruginosa phage PAP3. Methods The gene tls was amplified from the genome of phage PaP3 by PCR and subcloned into pMD18-T vector. Then the gene tls cut down from the vector was inserted into the plasmid pQE31, which could give the 6-His tag at the N side of the expressed protein. The recombinant vector pQE-tls was transformed to E. coli JM109. After induction with IPTG, the expressed bacteria was resuspended and sonicated, then the inclusion body was obtained after centrifugalization. The inclusion body was then dissolved with lysis buffer. The inclusion body solution, which has the target protein, was then purified by affinity chromatography and renatured by dialysis. Finally the nuclease activity of the fusion protein H6-TLS was tested on the plasmid pMD-cos which contains the cutting site of terminase large subunit. Results The expression plasmid pQE31-tls was successfully constructed, and the target protein yield was up to 30% of the total bacterial proteins. Mean while the substrate vector pMD-cos was also successfully constructed. After purification and renaturation, the fusion protein H6-TLS could partially cut the substrate vector pMD-cos. Conclusion The fusion protein H6-TLS was successfully expressed, purified and renatured, and it was shown to possess nuclease function. The experiment lays the foundation for further research of the gene tls.