腸出血性大腸桿菌O157：H7志賀樣毒素2B亞基的表達(dá)
發(fā)布時(shí)間：2019-05-16 21:35
【摘要】： 目的克隆表達(dá)腸出血性大腸桿菌(Enterohemorrhagic Escherichia coli,EHEC)0157:H7志賀樣毒素2B亞單位(Shiga-like toxin,Stx2B),并對(duì)其初步純化。 方法設(shè)計(jì)寡核苷酸引物,利用PCR法自EHEC 0157:H7基因組擴(kuò)增Stx2B的編碼基因stx2b,TA克隆后連接到表達(dá)載體pET-28a以構(gòu)建重組質(zhì)粒,經(jīng)測(cè)序鑒定后轉(zhuǎn)入表達(dá)宿主菌E.coli BL-21(DE3)感受態(tài)細(xì)胞,IPTG誘導(dǎo)表達(dá),以SDS-PAGE和Western-blot分析重組表達(dá)產(chǎn)物,固相親和層析純化重組蛋白。 結(jié)果PCR擴(kuò)增stx2b基因約210bp,成功構(gòu)建重組質(zhì)粒pET28a-stx2b,并在E.coli BL21(DE3)中得到高效表達(dá),目的蛋白的相對(duì)分子質(zhì)量約為7.5kD,約占總蛋白量的40%;免疫印跡顯示目的蛋白可與特異性抗體發(fā)生為特異性陽(yáng)性反應(yīng)。 結(jié)論成功克隆EHEC 0157:H7 Stx2b基因,在原核系統(tǒng)獲得目的蛋白的高效表達(dá),為0157:H7感染機(jī)制以及疾病的診斷和疫苗研究奠定基礎(chǔ)。
[Abstract]:Objective to clone and express Shiga-like toxin 2B subunit (Shiga-like toxin,Stx2B) of enterohemorrhagic Escherichia coli (Enterohemorrhagic Escherichia coli,EHEC) 0157:H7 and purify it. Methods oligodeoxynucleotides primers were designed and cloned from EHEC 0157:H7 genome to amplify Stx2B coding gene stx2b,TA by PCR and ligated to expression vector pET-28a to construct recombinant plasmid. After sequencing, the host strain E.coli BL-21 (DE3) receptive cells were transferred to express them. The expression was induced by IPTG. The recombinant expression products were analyzed by SDS-PAGE and Western-blot, and the recombinant proteins were purified by solid phase affinity chromatography. Results the stx2b gene was about 210 BP amplified by PCR, and the recombinant plasmid pET28a-stx2b, was successfully constructed and highly expressed in E.coli BL21 (DE3). The relative molecular weight of the target protein was about 7.5 KD, accounting for 40% of the total protein. Western imprinting showed that the target protein could react specifically with the specific antibody. Conclusion EHEC 0157:H7 Stx2b gene was successfully cloned and the high expression of target protein was obtained in prokaryotic system, which laid a foundation for the mechanism of 0157:H7 infection, disease diagnosis and vaccine research.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R378
本文編號(hào)：2478571
[Abstract]:Objective to clone and express Shiga-like toxin 2B subunit (Shiga-like toxin,Stx2B) of enterohemorrhagic Escherichia coli (Enterohemorrhagic Escherichia coli,EHEC) 0157:H7 and purify it. Methods oligodeoxynucleotides primers were designed and cloned from EHEC 0157:H7 genome to amplify Stx2B coding gene stx2b,TA by PCR and ligated to expression vector pET-28a to construct recombinant plasmid. After sequencing, the host strain E.coli BL-21 (DE3) receptive cells were transferred to express them. The expression was induced by IPTG. The recombinant expression products were analyzed by SDS-PAGE and Western-blot, and the recombinant proteins were purified by solid phase affinity chromatography. Results the stx2b gene was about 210 BP amplified by PCR, and the recombinant plasmid pET28a-stx2b, was successfully constructed and highly expressed in E.coli BL21 (DE3). The relative molecular weight of the target protein was about 7.5 KD, accounting for 40% of the total protein. Western imprinting showed that the target protein could react specifically with the specific antibody. Conclusion EHEC 0157:H7 Stx2b gene was successfully cloned and the high expression of target protein was obtained in prokaryotic system, which laid a foundation for the mechanism of 0157:H7 infection, disease diagnosis and vaccine research.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R378
【參考文獻(xiàn)】
中國(guó)期刊全文數(shù)據(jù)庫(kù) 前1條
1 汪華,史智揚(yáng);O157:H7大腸桿菌流行病學(xué)研究概況[J];江蘇衛(wèi)生保健;2001年01期
，本文編號(hào)：2478571
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