【摘要】：目的:對肺炎克雷伯菌所產(chǎn)CTX-M-3型超廣譜β-內酰胺酶(extended-spectrumβ-lactamase ,ESBLs)進行基因重組表達及純化。方法:對保存重組細菌DH5α/pGEM-T-CTX-M-3菌種鑒定和超廣譜β-內酰胺 酶確證后,對其所產(chǎn)的CTX-M-3型酶基因進行序列分析,明確酶基因表型。構建原核表達載體pET-28a(+)與CTX-M-3編碼基因的重組體,并在大腸桿菌BL21(DE3)中原核表達。IPTG誘導CTX-M-3融合蛋白表達,表達產(chǎn)物經(jīng)過親和層析純化,用SDS-PAGE電泳和免疫印跡法(Western-blot)鑒定該純化蛋白。 結果: (1)pGEM-T-CTX-M-3重組菌質粒經(jīng)NdeI和EcoRI酶切后獲得的片段大小約為876 bp和3000 bp。核苷酸序列分析結果顯示,目的基因片段大小為876 bp,經(jīng)GenBank同源性比較,與GenBank上登錄的CTX-M-3 100%符合。 (2)成功構建pET-28a(+)-CTX-M-3重組質粒,經(jīng)NdeI和EcoRI雙酶切后,獲得大小約為876 bp和5000 bp的片段,與預期大小相符,證明目的基因已經(jīng)成功插入pET-28a(+)載體中。 (3)可溶性檢測表明表達產(chǎn)物以可溶性形式(上清中)和包涵體形式(沉淀中)兩種形式存在。通過蛋白表達條件的優(yōu)化實驗,SDS-PAGE電泳結果顯示18℃、0.8mmol/L IPTG誘導24h的CTX-M-3重組蛋白的可溶性表達最佳。 (4)利用鎳瓊脂糖凝膠親和柱層析對重組蛋白進行了初步的純化研究。SDS-PAGE電泳和Western-blot檢測表明我們獲得純化的重組32kD蛋白。 結論:本研究成功構建了pET-28a(+)- CTX-M-3基因的原核表達載體,并在大腸桿菌中BL21表達,用His親和層析柱純化表達產(chǎn)物,并獲得高純度可溶性的重組蛋白。純化蛋白為進一步酶生物學特性及酶的抗體制備研究奠定了基礎。
[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 偽 / 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，

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