人降钙素原的原核表达、纯化和鉴定

Prokaryotic expression,purification and identification of human procalcitonin

目的构建人降钙素原(procalcitonin,PCT)原核表达载体,获得高纯度PCT重组蛋白。方法根据NCBI上PCT基因序列设计引物,利用PCR技术扩增PCT基因,构建PCT/p ET-22b(+)重组表达载体,转化至大肠埃希菌(E.coli)BL21中诱导表达;采用镍柱亲和层析法纯化重组蛋白,用western blot和胶体金法对其进行鉴定。结果琼脂糖凝胶电泳结果显示PCR扩增产物约为350 bp。同源性比对分析结果表明,PCT基因片段(348 bp)成功插入p TG19-T载体,未出现碱基突变。用Bam HⅠ和HindⅢ对重组表达载体双酶切,得到约为350 bp和5 500 bp片段。SDS-PAGE电泳显示PCT重组蛋白以可溶性形式存在,Mr约14 000,经镍柱亲和层析法纯化后即可获得。western blot和PCT胶体金法结果呈阳性,显示融合蛋白含组氨酸标签(His-tag),成功表达出PCT重组蛋白。结论应用重组DNA技术成功构建PCT基因融合重组表达载体,通过蛋白质表达纯化技术获得PCT融合蛋白。

Objective To construct the prokaryotic expression vector of human procalcitonin（PCT） and obtain the highly purified recombinant PCT protein. Methods PCT primers were designed based on the sequence of PCT gene from NCBI, and PCT gene was amplified by PCR. Then, the recombinant vector PCT/pET-22b（ ＋ ） was constructed and transferred into E. coli BI21 to induce the expression of recombinant PCT protein. Last, the recombinant PCT protein was purified by the nickel column affinity chromatography and identified by Western blot and the colloidal gold method, respectively. Results The results of agarose gel electrophoresis showed that the product of PCR amplification was about 350 bp. The homology comparison analysis revealed that the PCT gene fragment（348 bp） was successfully inserted into pTG19-T vector, and that no any base mutation was found. When the recombinant plasmid of PCT/pET-22b（ ＋ ） was diges- ted with BamH I and Hindlll, two pieces of about 350 bp and 5 500 bp were obtained. SDS-PAGE showed that the recombinant PCT pro- tein with about 14 000 of Mr was existed in soluble form, and was easily obtained by the nickel column affinity chromatography. Moreover, western blot and the colloidal gold method demonstrated that the recombinant PCT protein was successfully expressed and contained histidine label（His-tag）. Conclusion The recombinant vector PCT/pET-22b（ ＋ ） is successfully constructed by the recombinant DNA technology, and the recombinant PCT fusion protein is successfully obtained by the nickel column affinity chromatography.