类鼻疽伯克霍尔德菌BPSS1622蛋白免疫原性鉴定与亚细胞定位

发布时间：2018-07-09 13:26

  本文选题：类鼻疽伯克霍尔德菌 + BPSS蛋白　； 参考：《免疫学杂志》2017年09期

【摘要】：目的重组表达类鼻疽伯克霍尔德菌(Burkholderia pseudomallei)BPSS1622蛋白并鉴定其免疫原性及亚细胞定位。方法以pET-22b为表达载体,在大肠埃希菌BL21中表达BPSS1622蛋白;亲和层析纯化目的蛋白;用纯化后蛋白免疫新西兰兔获得抗血清;ELISA及Western blot鉴定纯化蛋白的免疫原性,并对类鼻疽杆菌BPSS1622蛋白进行亚细胞定位,探讨其生物学功能。结果以菌株BPC006基因组DNA为模板,通过PCR获得目的基因,构建pET-22b/BPSS1622重组质粒;转化至E.coli BL21获得工程菌株,经IPTG诱导表达、纯化得到了相对分子质量为22 000的目的蛋白;制备的抗血清ELISA效价高达1∶1 200 000,并能与目的蛋白发生特异性抗原抗体反应;亚细胞定位发现BPSS1622蛋白定位于类鼻疽杆菌的胞壁及胞膜中。结论成功克隆和表达了类鼻疽杆菌的BPSS1622蛋白,制备了免疫血清,该蛋白定位于细菌的细胞膜和细胞壁中,预测该蛋白具有ATP依赖的RNA解旋酶活性,为疾病诊断和预防奠定了基础。
[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.
【作者单位】： 第三军医大学第一附属医院医学检验系临床微生物及免疫学教研室;第三军医大学第二附属医院感染控制科;
【基金】：国家自然科学基金(30973402)
【分类号】：R378
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本文编号：2109506