本文關(guān)鍵詞：腸出血型大腸埃希菌O104∶H4轉(zhuǎn)運(yùn)蛋白AatA單克隆抗體制備及鑒定　出處：《中華疾病控制雜志》2017年06期 　論文類型：期刊論文

  更多相關(guān)文章： 腸出血型大腸桿菌 抗體 單克隆 抗體特異性

【摘要】：目的表達(dá)腸出血型大腸埃希菌O104∶H4的融合蛋白GST-Aat A,獲得單克隆抗體。方法人工合成腸出血型大腸埃希菌O104∶H4的轉(zhuǎn)運(yùn)蛋白基因aat A,連接至p MDl8-T載體,轉(zhuǎn)化E.coli DH5α,雙酶切回收,構(gòu)建原核表達(dá)質(zhì)粒PGEX-6P-1-GST-Aat A,測(cè)序鑒定后,轉(zhuǎn)化E.Coli BL21,IPTG誘導(dǎo)表達(dá),SDS-PAGE和Western Blot分析目的蛋白的表達(dá)并純化該重組蛋白。將純化后的融合蛋白GST-Aat A免疫Balb/c小鼠,制備相應(yīng)單克隆抗體;并對(duì)該單克隆抗體的純度、亞型、效價(jià)、特異性進(jìn)行鑒定和分析。結(jié)果構(gòu)建的原核表達(dá)載體PGEX-6P-1-GST-Aat A能表達(dá)融合蛋白GST-Aat A;親和層析純化后融合蛋白GST-Aat A純度達(dá)到95%,免疫小鼠后得到相應(yīng)特異性單克隆抗體。結(jié)論成功制備出一株抗轉(zhuǎn)運(yùn)蛋白Aat A的單克隆抗體,該抗體為Ig G1亞型,特異性好,效價(jià)高,純度可達(dá)97%。
[Abstract]:Objective to express the fusion protein GST-Aat A of Escherichia coli O104: H4. Methods the transporter gene aat A of Escherichia coli O104: H4 was synthesized and ligated to p MDl8-T vector. E. coli DH5 偽 was transformed into E. coli DH5 偽, and the prokaryotic expression plasmid PGEX-6P-1-GST-Aat A was constructed. After sequencing, E. Coli BL21 was transformed into E. coli BL21. IPTG induced expression. SDS-PAGE and Western Blot were used to analyze the expression of the target protein and purify the recombinant protein. The purified fusion protein GST-Aat A was immunized with Balb/c mice. To prepare the corresponding monoclonal antibody; The purity, subtype and titer of the monoclonal antibody were determined. Results the prokaryotic expression vector PGEX-6P-1-GST-Aat A could express the fusion protein GST-Aat A. The purity of fusion protein GST-Aat A was 95% after purification by affinity chromatography. Conclusion A monoclonal antibody against the transporter Aat A was successfully prepared, which is a subtype of Ig G1 with good specificity and high titer. The purity can be as high as 97.
【作者單位】： 南方醫(yī)科大學(xué)公共衛(wèi)生學(xué)院三級(jí)生物安全實(shí)驗(yàn)室;
【基金】：廣州市科技計(jì)劃項(xiàng)目(2014Y2-00090)
【分類號(hào)】：R392-33
【正文快照】： (Chin J Dis Control Prev 2017,21(6):572-576)2011年5月15日,德國(guó)暴發(fā)腸出血型大腸埃希菌(Enterohemorrhagic escherichia coli,EHEC)O104∶H4的感染疫情,在本次疫情中,德國(guó)感染病例累計(jì)達(dá)4 047例,48人死亡[1-3]。EHEC O104∶H4致病力強(qiáng)[4],傳播快且沒有有效的治療方法,引起，

本文編號(hào)：1383530