[1]吴利,黎礼达,胡艺,等.类鼻疽杆菌Ⅲ型分泌系统BPSS1617蛋白的重组表达及其抗体制备[J].第三军医大学学报,2018,40(18):1663-1667.
 WU Li,LI Lida,HU Yi,et al.Recombinant expression and antibody preparation of BPSS1617 protein in type Ⅲ secretion system of Burkhlderia pseudomallei[J].J Third Mil Med Univ,2018,40(18):1663-1667.
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类鼻疽杆菌Ⅲ型分泌系统BPSS1617蛋白的重组表达及其抗体制备(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
40卷
期数:
2018年第18期
页码:
1663-1667
栏目:
基础医学
出版日期:
2018-09-30

文章信息/Info

Title:
Recombinant expression and antibody preparation of BPSS1617 protein in type Ⅲ secretion system of Burkhlderia pseudomallei
作者:
吴利黎礼达胡艺黎元莉陈海朱雄胡治强马腾飞韩丹李倩曹刘素尹小毛毛旭虎
海南省三亚市人民医院检验科;陆军军医大学(第三军医大学)药学与检验医学系临床微生物及免疫学教研室
 
Author(s):
WU Li LI Lida HU Yi LI Yuanli CHEN Hai ZHU Xiong HU Zhiqiang MA Tengfei HAN Dan LI Qian CAO Liusu YIN Xiaomao MAO Xuhu  

Department of Clinical Laboratory, Sanya People’s Hospital, Sanya, Hainan Province, 572000; Department of Clinical Microbiology and Immunology, Faculty of Pharmacy and Medical Laboratory Science, Army Medical University (Third Military Medical University), Chongqing, 400038, China

关键词:
类鼻疽杆菌蛋白表达抗体制备与鉴定
Keywords:
Burkhlderia pseudomallei protein expression polyclonal antibodies
分类号:
R341;R378.99;R392.7
文献标志码:
A
摘要:

目的    构建重组表达类鼻疽杆菌Ⅲ型分泌系统蛋白BPSS1617,并制备其抗体。方法    采用PCR扩增BPSS1617全长序列,克隆至原核表达载体pET-22b,将重组表达质粒转化至大肠杆菌BL21(DE3)中进行诱导表达,免疫新西兰兔,制备其多克隆抗体,并通过Western blot、免疫荧光和ELISA检测抗体的特异性,并分析该蛋白在类鼻疽杆菌中的亚细胞定位。结果    成功扩增类鼻疽杆菌BPC006株基因组中的BPSS1617基因;酶切和测序验证重组表达质粒pET-22b-BPSS1617构建成功;经IPTG诱导表达、包涵体洗涤、纯化以及变性复性,成功获得纯度>95%,分子量为25×103的目的蛋白;将纯化的BPSS1617重组蛋白免疫新西兰兔,获得抗BPSS1617蛋白多克隆抗体,经实验验证其特异性良好;同时BPSS1617蛋白主要定位于类鼻疽杆菌的细胞质中。结论    成功构建、表达、纯化获得BPSS1617蛋白,并成功制备了其特异性多克隆抗体,并且该蛋白定位于类鼻疽杆菌的细胞质中。

Abstract:

Objective     To construct a recombinant expression system for BPSS1617, a component of the type Ⅲ protein secretion system of Burkhlderia pseudomallei, and prepare polyclonal antibodies for this protein. Methods    The full-length coding sequence of BPSS1617 was amplified with PCR and cloned into the prokaryotic expression plasmid pET-22b. The recombinant plasmid was transformed into E.coli BL21 (DE3) strain for expression of the target protein, which, after purification, was used to immunize New Zealand white rabbits to obtain the polyclonal antibodies of BPSS1617. The specificity of the antibodies was evaluated by Western blotting, immunofluorescence assay and enzyme-linked immunosorbent assay (ELISA). The subcellular localization of BPSS1617 in Burkhlderia pseudomallei cells was investigated using the obtained antibodies. Results      The results of enzyme digestion and sequencing analysis verified the successful construction of the recombinant pET-22b-BPSS1617 expression plasmid. After IPTG induction of E.coli, inclusion body cleansing, purification, solubilization and renaturation, we obtained the target protein (relative molecular mass of 25×103) with a purity over 95%. From the white rabbits immunized with this protein, we obtained highly specific polyclonal antibodies against BPSS1617. Using these antibodies, we identified that BPSS1617 protein was mainly localized in the cytoplasm of Burkhlderia pseudomallei cells. Conclusion        We successfully obtain the recombinant BPSS1617 protein, and its specific polyclonal antibodies and identify that BPSS1617 is localized mainly in the cytoplasm of Burkhlderia pseudomallei cells.

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更新日期/Last Update: 2018-09-29