Recombinant expression and immunological characterization of <italic style="font-style: italic">Burkholderia pseudomallei</italic> type Ⅲ secretion system protein BipD

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Recombinant expression and immunological characterization of Burkholderia pseudomallei type Ⅲ secretion system protein BipD

Basic Medicine | 更新时间：2026-01-09

- Recombinant expression and immunological characterization of Burkholderia pseudomallei type Ⅲ secretion system protein BipD
- Journal of Army Medical University Vol. 46, Issue 15, Pages: 1713-1720(2024)
- 作者机构：
  
  1.400038 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与免疫学教研室
  2.400038 重庆，陆军军医大学(第三军医大学)创伤与化学中毒全国重点实验室
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(32270190)
- DOI：10.16016/j.2097-0927.202311041
  CLC： R341;R378.99;R392.11
- Received：08 November 2023，
  
  Revised：2024-01-17，
  
  Published：15 August 2024
- 稿件说明：
移动端阅览
Dongqi NAN, Yuan WEN, Jiangao CHEN, et al. Recombinant expression and immunological characterization of Burkholderia pseudomallei type Ⅲ secretion system protein BipD[J]. Journal of Army Medical University, 2024, 46(15): 1713-1720.
DOI：

Dongqi NAN, Yuan WEN, Jiangao CHEN, et al. Recombinant expression and immunological characterization of Burkholderia pseudomallei type Ⅲ secretion system protein BipD[J]. Journal of Army Medical University, 2024, 46(15): 1713-1720. DOI： 10.16016/j.2097-0927.202311041.

摘要

目的

2

重组表达类鼻疽菌Ⅲ型分泌系统BipD蛋白，制备多克隆抗体并鉴定其免疫学性质。

方法

2

利用pET-28a表达系统在

Eschericahia coli

(

E.coli

) BL21(DE3)中重组表达BipD蛋白，通过His Trap亲和层析纯化获得rBipD蛋白，免疫BALB/c小鼠获得rBipD多克隆抗体；分别用兔抗类鼻疽菌血清和类鼻疽菌感染阳性患者血清进行Western blot实验检测rBipD的免疫反应性；通过Western blot及免疫荧光染色检测鉴定rBipD的免疫原性；以rBipD建立间接ELISA检测临床类鼻疽患者血清抗体。

结果

2

成功构建pET-28a-BipD重组质粒并转化至

E.coli

BL21(DE3)中诱导表达、纯化获得了相对分子质量约为36×10

3

的rBipD蛋白，纯度约为95.4%。rBipD蛋白具有良好的免疫原性和免疫反应性。免疫小鼠可产生特异性抗体，制备鼠抗rBipD多克隆抗体，效价达1∶512 000。5.0 μg/mL rBipD蛋白可与类鼻疽患者血清发生免疫反应，而不与结核患者血清发生免疫反应，差异有统计学意义(

P

<

0.01)。

结论

2

成功制备了具有免疫学活性的rBipD蛋白及其多克隆抗体，为其用于临床免疫学诊断和类鼻疽菌感染免疫机制研究提供了良好的工具。

Abstract

Objective

2

To express recombinant

Burkholderia pseudomallei

(

B. pseudomallei

) type Ⅲ secretion system BipD protein

prepare its polyclonal antibodies and verify their immunological traits.

Methods

2

The recombinant pET-28a-BipD plasmid was generated

and the pET-28a-BipD-carried

E.coli

BL21 (DE3) bacteria were induced with isopropyl-β-d-thiogalactoside (IPTG) to express recombinant BipD (rBipD) protein. The rBipD was obtained by affinity chromatography using His Trap column

then mixed with Fredrick's adjuvant to immunize BALB/c mice by intraperitoneal injection in order to obtain anti-rBipD polyclonal antibodies. The immunoreactivity of rBipD was detected by Western blot assay using rabbit anti-melioidosis serum and the serum from melioidosis patients. The immunogenicity of rBipD was evaluated using Western blotting and immunofluorescence staining. Finally

rBipD was used to establish an indirect ELISA to detect serum antibodies of clinical melioidosis patients.

Results

2

The recombinant plasmid pET-28a-BipD was successfully constructed and transformed into

E.coli

BL21 (DE3) to induce rBipD expression with IPTG treatment. The obtained rBipD had a relative molecular weight of 36×10

3

and a purity of 95.4%

and had good immunogenicity and immunoreactivity. It could induce the production of specific antibodies after immunizing mice

and mouse polyclonal antibodies against rBipD were prepared with the titer of 1∶512 000. rBipD of 5.0 μg/mL produced specific immune response with the serum of melioidosis patients

but had no specific reaction with the serum of tuberculosis patients

with statistical difference (

P

<

0.01).

Conclusion

2

rBipD with immunological

activity is successfully prepared and purified

and its polyclonal antibodies are also developed

which provide a good tool for clinical immunological diagnosis and study of immune mechanism of

B. pseudomallei

infection.

关键词

Keywords

references

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