类鼻疽菌Ⅲ型分泌系统BipD蛋白的重组表达及其免疫学特性研究

南栋琪; 文远; 陈建高; 饶承龙; 吴潘; 张子元; 王施韦; 闫晶敏; 李倩; 毛旭虎

doi:10.16016/j.2097-0927.202311041

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类鼻疽菌Ⅲ型分泌系统BipD蛋白的重组表达及其免疫学特性研究

基础医学 | 更新时间：2026-01-09

- 类鼻疽菌Ⅲ型分泌系统BipD蛋白的重组表达及其免疫学特性研究
- Recombinant expression and immunological characterization of Burkholderia pseudomallei type Ⅲ secretion system protein BipD
- 陆军军医大学学报 2024年46卷第15期页码：1713-1720
- 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：
  
  [ 毛旭虎，E-mail：maoxh2012@hotmail.com MAO Xuhu, E-mail: maoxh2012@hotmail.com
- 基金信息：
  
  国家自然科学基金面上项目(32270190)
- DOI：10.16016/j.2097-0927.202311041
  中图分类号： R341;R378.99;R392.11
- 收稿：2023-11-08，
  
  修回：2024-01-17，
  
  纸质出版：2024-08-15
- 稿件说明：
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南栋琪, 文远, 陈建高, 等. 类鼻疽菌Ⅲ型分泌系统BipD蛋白的重组表达及其免疫学特性研究[J]. 陆军军医大学学报, 2024,46(15):1713-1720.

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.
南栋琪, 文远, 陈建高, 等. 类鼻疽菌Ⅲ型分泌系统BipD蛋白的重组表达及其免疫学特性研究[J]. 陆军军医大学学报, 2024,46(15):1713-1720. DOI： 10.16016/j.2097-0927.202311041.

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

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

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

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

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(

0.01)。

结论

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

To express recombinant

Burkholderia pseudomallei

(

B. pseudomallei

) type Ⅲ secretion system BipD protein

prepare its polyclonal antibodies and verify their immunological traits.

Methods

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

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

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 (

0.01).

Conclusion

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

LIMMATHUROTSAKUL D, WONGSUVAN G, AANENSEN D, et al . Melioidosis caused by Burkholderia pseudomallei in drinking water, Thailand, 2012[J ] . Emerg Infect Dis, 2014, 20(2): 265-268. DOI: 10.3201/eid2002.121891..

CHENG A C, JACUPS S P, GAL D, et al . Extreme weather events and environmental contamination are associated with case-clusters of melioidosis in the Northern Territory of Australia[J ] . Int J Epidemiol, 2006, 35(2): 323-329. DOI: 10.1093/ije/dyi271.

WIERSINGA W J, VIRK H S, TORRES A G, et al . Melioidosis[J ] . Nat Rev Dis Primers, 2018, 4: 17108. DOI: 10.1038/nrdp.2017.108.

SILVA E B, DOW S W. Development of Burkholderia mallei and pseudomallei vaccines[J ] . Front Cell Infect Microbiol, 2013, 3: 10. DOI: 10.3389/fcimb.2013.00010..

CHAPARTEGUI-GONZÁLEZ I, BOWSER S, TORRES A G, et al . Recent progress in Shigella and Burkholderia pseudomallei vaccines[J ] . Pathogens, 2021, 10(11): 1353. DOI: 10.3390/pathogens10111353..

FISHER D A, HARRIS P N A. Melioidosis: refining management of a tropical time bomb[J]. Lancet, 2014, 383(9919): 762-764. DOI: 10.1016/s0140-6736(13)62143-1.

GALÁN J E, LARA-TEJERO M, MARLOVITS T C, et al . Bacterial type Ⅲ secretion systems: specialized nanomachines for protein delivery into target cells[J ] . Annu Rev Microbiol, 2014, 68: 415-438. DOI: 10.1146/annurev-micro-092412-155725.

SUN G W, GAN Y H. Unraveling type Ⅲ secretion systems in the highly versatile Burkholderia pseudomallei [J ] . Trends Microbiol, 2010, 18(12): 561-568. DOI: 10.1016/j.tim.2010.09.002.

VANDER BROEK C W, STEVENS J M. Type Ⅲ secretion in the melioidosis pathogen Burkholderia pseudomallei [J ] . Front Cell Infect Microbiol, 2017, 7: 255. DOI: 10.3389/fcimb.2017.00255.

毛旭虎, 胡治强. 类鼻疽的实验室诊断方法及评价[J]. 第三军医大学学报, 2016, 38(11): 1200-1204. DOI: 10.16016/j.1000-5404.201602028.

MAO X H, HU Z Q. Progress of research on laboratory diagnosis of melioidosis[J]. J Third Mil Med Univ, 2016, 38(11): 1200-1204. DOI: 10.16016/j.1000-5404.201602028.

GASSIEP I, BAUER M J, PAGE M, et al . Comparative evaluation of Panther Fusion and real-time PCR for detection of Burkholderia pseudomallei in spiked human blood[J ] . Access Microbiol, 2022, 4(3). DOI: 10.1099/acmi.0.000333..

FAIRLEY L, SMITH S, MAISRIKROD S, et al . Systematic review and meta-analysis of diagnostic tests for diagnosis of melioidosis[J ] . Acta Trop, 2021, 214: 105784. DOI: 10.1016/j.actatropica.2020.105784.

ADLER N R L, STEVENS J M, STEVENS M P, et al . Autotransporters and their role in the virulence of Burkholderia pseudomallei and Burkholderia mallei [J ] . Front Microbio, 2011, 2: 151.DOI: 10.3389/fmicb.2011.00151..

PUAH S M, PUTHUCHEARY S D, WANG J T, et al . Molecular characterization of putative virulence determinants in Burkholderia pseudomallei [J ] . Sci World J, 2014, 2014: 590803. DOI: 10.1155/2014/590803..

ABBY S S, ROCHA E P C. The non-flagellar type Ⅲ secretion system evolved from the bacterial flagellum and diversified into host-cell adapted systems[J]. PLoS Genet, 2012, 8(9): e1002983. DOI: 10.1371/journal.pgen.1002983.

STEVENS M P, WOOD M W, TAYLOR L A, et al . An Inv/Mxi-Spa-like type Ⅲ protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen[J ] . Mol Microbiol, 2002, 46(3): 649-659. DOI: 10.1046/j.1365-2958.2002.03190.x..

STEVENS M P, FRIEBEL A, TAYLOR L A, et al . A Burkholderia pseudomallei Type Ⅲ secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity[J ] . J Bacteriol, 2003, 185(16): 4992-4996. DOI: 10.1128/jb.185.16.4992-4996.2003..

STEVENS M P, HAQUE A, ATKINS T, et al . Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa type Ⅲ secretion mutant in murine models of melioidosis[J ] . Microbiology, 2004, 150(8): 2669-2676. DOI: 10.1099/mic.0.27146-0..

GONG L, CULLINANE M, TREERAT P, et al . The Burkholderia pseudomallei type Ⅲ secretion system and BopA are required for evasion of LC3-associated phagocytosis[J ] . PLoS One, 2011, 6(3): e17852. DOI: 10.1371/journal.pone.0017852..

MACEK B, FORCHHAMMER K, HARDOUIN J, et al . Protein post-translational modifications in bacteria[J ] . Nat Rev Microbiol, 2019, 17(11): 651-664. DOI: 10.1038/s41579-019-0243-0.

WILLIAMSON E D. Plague[J]. Vaccine, 2009, 27: D56-D60. DOI: 10.1016/j.vaccine.2009.07.068.

TIPPAYAWAT P, PINSIRI M, RINCHAI D, et al . Burkholderia pseudomallei proteins presented by monocyte-derived dendritic cells stimulate human memory T cells in vitro [J ] . Infect Immun, 2011, 79(1): 305-313. DOI: 10.1128/iai.00803-10..

CURRIE B J, MAYO M, WARD L M, et al . The Darwin prospective melioidosis study: a 30-year prospective, observational investigation[J ] . Lancet Infect Dis, 2021, 21(12): 1737-1746. DOI: 10.1016/S1473-3099(21)00022-0.

CHANTRATITA N, PHUNPANG R, YARASAI A, et al . Characteristics and one year outcomes of melioidosis patients in Northeastern Thailand: a prospective, multicenter cohort study[J ] . Lancet Reg Health Southeast Asia, 2023, 9: 100118. DOI: 10.1016/j.lansea.2022.100118.

LIMMATHUROTSAKUL D, WONGRATANACHEEWIN S, DAY N P J, et al . Increasing incidence of human melioidosis in northeast Thailand[J ] . Am J Trop Med Hyg, 2010, 82(6): 1113-1117. DOI: 10.4269/ajtmh.2010.10-0038.

CURRIE B J. Melioidosis and Burkholderia pseudomallei : progress in epidemiology, diagnosis, treatment and vaccination[J ] . Curr Opin Infect Dis, 2022, 35(6): 517-523. DOI: 10.1097/qco.0000000000000869.

吴华, 黄东良, 王旭明. 如何避免类鼻疽的漏诊和误诊[J]. 现代预防医学, 2013, 40(19): 3681-3683.

WU H, HUANG D L, WANG X M. How to avoid the misdiagnosis and missed diagnosis of meliodosis[J]. Mod Prev Med, 2013, 40(19): 3681-3683.

毛旭虎. 加强类鼻疽的研究[J]. 第三军医大学学报, 2011, 33(13): 1315-1317. DOI: 10.16016/j.1000-5404.2011.13.013.

MAO X H. Progress on melioidosis[J]. J Third Mil Med Univ, 2011, 33(13): 1315-1317. DOI: 10.16016/j.1000-5404.2011.13.013.

杨小敏, 方瑶, 顾江, 等. 特异引物PCR鉴定类鼻疽伯克霍尔德菌方法的建立和优化[J]. 第三军医大学学报, 2011, 33(14): 1427-1431. DOI: 10.16016/j.1000-5404.2011.14.001.

YANG X M, FANG Y, GU J, et al . Establishment and optimization of a method to identify Burkholderia pseudomallei based on primer-specific polymerase chain reaction[J ] . J Third Mil Med Univ, 2011, 33(14): 1427-1431. DOI: 10.16016/j.1000-5404.2011.14.001..

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