Luffin P1-KDEL的构建、表达、纯化及鉴定

Construction,expression,and identification of Luffin P1-KDEL

目的构建植物毒素Luffin P1的原核表达质粒PET32a(+)-Luffin P1,并对其进行诱导表达,以及表达蛋白的纯化及鉴定。方法采用基因工程技术将重组基因片段Luffin P1克隆到表达载体pET32a(+)中,经酶切及DNA序列分析正确后,转化至表达宿主菌BL21(DE3)中,经IPTG诱导表达Luffin P1融合蛋白,并用His标签抗体对该融合蛋白进行Western blot鉴定,对鉴定正确的融合蛋白进行纯化,肠激酶酶切及再纯化。结果成功的构建了原核表达载体pET32a(+)-Luffin P1,获得了纯度较高的Luffin P1蛋白。结论通过基因工程合成Luffin P1蛋白是成功的,并为进一步对Luffin P1功能研究及制备免疫毒素的弹头鉴定了实验基础。

Objective Luffin P1 as a new member of luffin family is an effective ribosome inactivating protein（RIP）.In this study,we aimed to construct prokaryotic expression plasmid PET32a（＋）-Luffin P1 for expressing,purifying,and identifying the expression protein Luffin P1.We subcloned a recombinant LuffinP1 DNA fragment into an expression plasmid pET32a（＋）,and identified the recombined plasmid by restriction enzyme digestion and DNA sequencing.After the recombinant plasmid was transformed into bacteria BL21（DE3）,the expression of fusion protein Luffin P1 was induced by IPTG and identified by Western blotting using anti-His Ab,then the fusion protein was purified,digested by enterokinase,and re-purified.The new recombinant plasmid pET32a（＋）-Luffin P1 was successfully constructed,and the correct fusion protein of Luffin P1 was highly purified.These results imply that Luffin P1 protein cand be acquired through genetic engineering, which provides a experimental basis for function research of Luffin P1 and production of immunotoxin warheads.