CTX-M-3型超广谱β-内酰胺酶的原核表达和纯化
发布时间：2018-11-16 14:43
【摘要】：目的:对肺炎克雷伯菌所产CTX-M-3型超广谱β-内酰胺酶(extended-spectrumβ-lactamase ,ESBLs)进行基因重组表达及纯化。方法:对保存重组细菌DH5α/pGEM-T-CTX-M-3菌种鉴定和超广谱β-内酰胺 酶确证后,对其所产的CTX-M-3型酶基因进行序列分析,明确酶基因表型。构建原核表达载体pET-28a(+)与CTX-M-3编码基因的重组体,并在大肠杆菌BL21(DE3)中原核表达。IPTG诱导CTX-M-3融合蛋白表达,表达产物经过亲和层析纯化,用SDS-PAGE电泳和免疫印迹法(Western-blot)鉴定该纯化蛋白。 结果: (1)pGEM-T-CTX-M-3重组菌质粒经NdeI和EcoRI酶切后获得的片段大小约为876 bp和3000 bp。核苷酸序列分析结果显示,目的基因片段大小为876 bp,经GenBank同源性比较,与GenBank上登录的CTX-M-3 100%符合。 (2)成功构建pET-28a(+)-CTX-M-3重组质粒,经NdeI和EcoRI双酶切后,获得大小约为876 bp和5000 bp的片段,与预期大小相符,证明目的基因已经成功插入pET-28a(+)载体中。 (3)可溶性检测表明表达产物以可溶性形式(上清中)和包涵体形式(沉淀中)两种形式存在。通过蛋白表达条件的优化实验,SDS-PAGE电泳结果显示18℃、0.8mmol/L IPTG诱导24h的CTX-M-3重组蛋白的可溶性表达最佳。 (4)利用镍琼脂糖凝胶亲和柱层析对重组蛋白进行了初步的纯化研究。SDS-PAGE电泳和Western-blot检测表明我们获得纯化的重组32kD蛋白。 结论:本研究成功构建了pET-28a(+)- CTX-M-3基因的原核表达载体,并在大肠杆菌中BL21表达,用His亲和层析柱纯化表达产物,并获得高纯度可溶性的重组蛋白。纯化蛋白为进一步酶生物学特性及酶的抗体制备研究奠定了基础。
[Abstract]:Aim: to express and purify CTX-M-3 extended spectrum 尾 -lactamase (extended-spectrum 尾-lactamase, ESBLs) from Klebsiella pneumoniae. Methods: after the identification of DH5 伪 / pGEM-T-CTX-M-3 bacteria and the identification of extended-spectrum 尾 -lactam enzyme, the CTX-M-3 type gene was sequenced and the phenotype of the enzyme gene was determined. A prokaryotic expression vector pET-28a () and a recombinant encoding CTX-M-3 gene were constructed and expressed in E. coli BL21 (DE3). IPTG induced CTX-M-3 fusion protein expression, and the expression product was purified by affinity chromatography. The purified protein was identified by SDS-PAGE electrophoresis and Western blot (Western-blot). Results: (1) the fragment size of pGEM-T-CTX-M-3 recombinant plasmid digested by NdeI and EcoRI was about 876 bp and 3000 bp.. The result of nucleotide sequence analysis showed that the size of the target gene was 876 bp, by GenBank homology, which was consistent with 100% of the CTX-M-3 registered on GenBank. (2) the recombinant plasmid of pET-28a ()-CTX-M-3 was successfully constructed. The fragments of 876 bp and 5000 bp were obtained by NdeI and EcoRI digestion, which were in accordance with the expected size. It is proved that the target gene has been successfully inserted into pET-28a () vector. (3) Solubility detection showed that the expression product existed in two forms: soluble form (supernatant medium) and inclusion body form (precipitate form). The results of SDS-PAGE electrophoresis showed that the soluble expression of CTX-M-3 recombinant protein induced by 0.8mmol/L IPTG at 18 鈩，
本文编号：2335802
[Abstract]:Aim: to express and purify CTX-M-3 extended spectrum 尾 -lactamase (extended-spectrum 尾-lactamase, ESBLs) from Klebsiella pneumoniae. Methods: after the identification of DH5 伪 / pGEM-T-CTX-M-3 bacteria and the identification of extended-spectrum 尾 -lactam enzyme, the CTX-M-3 type gene was sequenced and the phenotype of the enzyme gene was determined. A prokaryotic expression vector pET-28a () and a recombinant encoding CTX-M-3 gene were constructed and expressed in E. coli BL21 (DE3). IPTG induced CTX-M-3 fusion protein expression, and the expression product was purified by affinity chromatography. The purified protein was identified by SDS-PAGE electrophoresis and Western blot (Western-blot). Results: (1) the fragment size of pGEM-T-CTX-M-3 recombinant plasmid digested by NdeI and EcoRI was about 876 bp and 3000 bp.. The result of nucleotide sequence analysis showed that the size of the target gene was 876 bp, by GenBank homology, which was consistent with 100% of the CTX-M-3 registered on GenBank. (2) the recombinant plasmid of pET-28a ()-CTX-M-3 was successfully constructed. The fragments of 876 bp and 5000 bp were obtained by NdeI and EcoRI digestion, which were in accordance with the expected size. It is proved that the target gene has been successfully inserted into pET-28a () vector. (3) Solubility detection showed that the expression product existed in two forms: soluble form (supernatant medium) and inclusion body form (precipitate form). The results of SDS-PAGE electrophoresis showed that the soluble expression of CTX-M-3 recombinant protein induced by 0.8mmol/L IPTG at 18 鈩，
本文编号：2335802
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