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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
Recombinant expression and antibody preparation of BPSS1617 protein in type Ⅲ secretion system of
Burkhlderia pseudomallei - 第三军医大学学报 2018年40卷第18期 页码：1663-1667
- 作者机构：
1.572000 海南 三亚，海南省三亚市人民医院检验科
2.400038重庆，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
- 作者简介：
[ 毛旭虎，E-mail: mxh95xy@tom.com MAO Xuhu, E-mail: mxh95xy@tom.com
- 基金信息：2017年海南省自然科学基金(817398)
DOI：10.16016/j.1000-5404.201803142
中图分类号： R341;R378.99;R392.7收稿：2018-03-20，
修回：2018-05-30，
纸质出版：2018-09-30
- 稿件说明：
移动端阅览
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.
Li WU, Lida LI, Yi HU, et al. Recombinant expression and antibody preparation of BPSS1617 protein in type Ⅲ secretion system of
Burkhlderia pseudomallei [J]. Journal of Third Military Medical University, 2018, 40(18): 1663-1667.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. DOI： 10.16016/j.1000-5404.201803142.
Li WU, Lida LI, Yi HU, et al. Recombinant expression and antibody preparation of BPSS1617 protein in type Ⅲ secretion system of
Burkhlderia pseudomallei [J]. Journal of Third Military Medical University, 2018, 40(18): 1663-1667. DOI： 10.16016/j.1000-5404.201803142.
摘要
目的
2
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
2
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
2
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25×10
3
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
2
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
2
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
2
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
2
The results of enzyme digestion and sequencing analysis verified the successful construction of the recombinant pET-22b-BP
SS1617 expression plasmid. After IPTG induction of
E.coli
inclusion body cleansing
purification
solubilization and renaturation
we obtained the target protein (relative molecular mass of 25×10
3
) 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
2
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.
关键词
Keywords
references
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