人降钙素原的原核表达、纯化和鉴定

发布时间：2018-06-16 23:41

  本文选题：降钙素原 + 原核表达　； 参考：《临床检验杂志》2017年03期

【摘要】：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。
[Abstract]:Objective to construct a prokaryotic expression vector of procalcitonin PCT (PCT) and obtain a high purity PCT recombinant protein. Methods according to the sequence of PCT gene on NCBI, the PCT gene was amplified by PCR. The recombinant expression vector PCT / p ET-22b () was constructed and transformed into E. coli BL21 to induce expression, and the recombinant protein was purified by nickel column affinity chromatography. It was identified by western blot and colloidal gold method. Results agarose gel electrophoresis showed that the PCR amplification product was about 350 BP. The results of homology analysis showed that pTG19-T vector was successfully inserted into pTG19-T vector without base mutation. The recombinant expression vector was digested with Bam H 鈪，

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