Recombinant expression and antibody preparation of BPSS1617 protein in type Ⅲ secretion system of <italic style="font-style: italic">Burkhlderia pseudomallei</italic>

Li WU; Lida LI; Yi HU; Yuanli LI; Hai CHEN; Xiong ZHU; Zhiqiang HU; Tengfei MA; Dan HAN; Qian LI; Liusu CAO; Xiaomao YIN; Xuhu MAO

doi:10.16016/j.1000-5404.201803142

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Recombinant expression and antibody preparation of BPSS1617 protein in type Ⅲ secretion system of Burkhlderia pseudomallei

Military Medicine | 更新时间：2026-01-09

- Recombinant expression and antibody preparation of BPSS1617 protein in type Ⅲ secretion system of Burkhlderia pseudomallei
- Journal of Third Military Medical University Vol. 40, Issue 18, Pages: 1663-1667(2018)
- 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：
- 基金信息：
  
  the Natural Science Foundation of Hainan Province(817398)
- DOI：10.16016/j.1000-5404.201803142
  CLC： R341;R378.99;R392.7
- Received：20 March 2018，
  
  Revised：2018-05-30，
  
  Published：30 September 2018
- 稿件说明：
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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：

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

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25×10

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-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

) 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.

关键词

Keywords

references

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Expression and identification of recombinant Burkholderia pseudomallei type Ⅲ secretion system BPSS1395 protein and preparation of its polyclonal antibodies

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Recombinant expression and antibody preparation of BPSS1617 protein in type Ⅲ secretion system of Burkhlderia pseudomallei

DOI：10.16016/j.1000-5404.201803142

摘要

Abstract

关键词

Keywords

references