本文選題：類鼻疽伯克霍爾德菌 + BPSS蛋白　； 參考：《免疫學雜志》2017年09期

【摘要】：目的重組表達類鼻疽伯克霍爾德菌(Burkholderia pseudomallei)BPSS1622蛋白并鑒定其免疫原性及亞細胞定位。方法以pET-22b為表達載體,在大腸埃希菌BL21中表達BPSS1622蛋白;親和層析純化目的蛋白;用純化后蛋白免疫新西蘭兔獲得抗血清;ELISA及Western blot鑒定純化蛋白的免疫原性,并對類鼻疽桿菌BPSS1622蛋白進行亞細胞定位,探討其生物學功能。結果以菌株BPC006基因組DNA為模板,通過PCR獲得目的基因,構建pET-22b/BPSS1622重組質粒;轉化至E.coli BL21獲得工程菌株,經IPTG誘導表達、純化得到了相對分子質量為22 000的目的蛋白;制備的抗血清ELISA效價高達1∶1 200 000,并能與目的蛋白發(fā)生特異性抗原抗體反應;亞細胞定位發(fā)現BPSS1622蛋白定位于類鼻疽桿菌的胞壁及胞膜中。結論成功克隆和表達了類鼻疽桿菌的BPSS1622蛋白,制備了免疫血清,該蛋白定位于細菌的細胞膜和細胞壁中,預測該蛋白具有ATP依賴的RNA解旋酶活性,為疾病診斷和預防奠定了基礎。
[Abstract]:Objective to express BPSS1622 protein of Burkholderia pseudomallei and identify its immunogenicity and subcellular localization. Methods using pET-22b as expression vector, BPSS1622 protein was expressed in Escherichia coli BL21, the target protein was purified by affinity chromatography, and the immunogenicity of purified protein was identified by Elisa and Western blot. The subcellular localization of BPSS 1622 protein from Bacillus mallei was carried out to investigate its biological function. Results using the genomic DNA of strain BPC006 as template, the target gene was obtained by PCR, and the recombinant plasmid pET-22b / BPSS1622 was constructed. The recombinant plasmid was transformed into E. coli BL21 and expressed by IPTG, and the target protein with relative molecular weight of 22 000 was purified. The Elisa titer of the prepared antiserum was as high as 1:1 200000, and BPSS1622 protein was found to be located in the cell wall and membrane of Bacillus mallei by subcellular localization. Conclusion the BPSS1622 protein of Bacillus mallei was cloned and expressed successfully, and the immune serum was prepared. The protein was located in the cell membrane and cell wall of bacteria, and predicted that the protein had ATP-dependent RNA helicase activity. It lays a foundation for disease diagnosis and prevention.
【作者單位】： 第三軍醫(yī)大學第一附屬醫(yī)院醫(yī)學檢驗系臨床微生物及免疫學教研室;第三軍醫(yī)大學第二附屬醫(yī)院感染控制科;
【基金】：國家自然科學基金(30973402)
【分類號】：R378
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本文編號：2109506