Expression and identification of recombinant <italic style="font-style: italic">Burkholderia pseudomallei</italic> type Ⅲ secretion system BPSS1395 protein and preparation of its polyclonal antibodies

Yi Hu; Zhiqiang Hu; Tengfei Ma; Dan Hang; Qian Li; Yao Fang; Xuhu Mao

doi:10.16016/j.1000-5404.201611152

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

更新时间：2026-02-09

- Expression and identification of recombinant Burkholderia pseudomallei type Ⅲ secretion system BPSS1395 protein and preparation of its polyclonal antibodies
- Journal of Third Military Medical University Vol. 39, Issue 10, Pages: 941-945(2017)
- 作者机构：
  
  400038 重庆，第三军医大学西南医院医学检验系临床微生物及免疫学教研室
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(30973402)
- DOI：10.16016/j.1000-5404.201611152
  CLC： R341;R378.99;R392-33
- Received：17 November 2016，
  
  Revised：2016-12-26，
  
  Published：30 May 2017
- 稿件说明：
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Yi Hu, Zhiqiang Hu, Tengfei Ma, et al. Expression and identification of recombinant Burkholderia pseudomallei type Ⅲ secretion system BPSS1395 protein and preparation of its polyclonal antibodies[J]. Journal of Third Military Medical University, 2017, 39(10): 941-945.
DOI：

Yi Hu, Zhiqiang Hu, Tengfei Ma, et al. Expression and identification of recombinant Burkholderia pseudomallei type Ⅲ secretion system BPSS1395 protein and preparation of its polyclonal antibodies[J]. Journal of Third Military Medical University, 2017, 39(10): 941-945. DOI： 10.16016/j.1000-5404.201611152.

摘要

目的

重组表达类鼻疽杆菌Ⅲ型分泌系统蛋白BPSS1395，利用其抗体检测该蛋白在细菌中的分布。

方法

以pET-22b为表达系统，采用DNA重组技术在大肠杆菌中表达BPSS1395蛋白；用纯化重组蛋白免疫新西兰大白兔，Western blot及ELISA法鉴定其免疫学性质；以制备的抗血清Western blot分析该蛋白的亚细胞定位。

结果

从类鼻疽杆菌BPC006株基因组DNA中PCR得到了BPSS1395目的基因，并成功构建pET-22b-BPSS1395重组质粒，转化到

E. coli

宿主菌BL21（DE3）经IPTG诱导表达、纯化得到了相对分子质量为32 × 10

的高纯度BPSS1395蛋白，制备的兔抗血清ELISA效价均高达1: 1 280 000，并能与目的蛋白发生特异性抗原抗体反

应。亚细胞分离后BPSS1395在类鼻疽杆菌中主要定位于细胞质。

结论

重组表达了具有生物活性的BPSS1395蛋白，制备了其特异性多克隆抗体，证明了该蛋白定位于细胞质。

Abstract

Objective

To obtain recombinant protein of type Ⅲ secretion system protein BPSS1395 of

Burkholderia pseudomallei

(

B. pseudomallei

) and prepare its polyclonal antibodies.

Methods

The pET-22b expression system was used to express the recombinant protein BPSS1395. The recombinant protein obtained was used to immunize New Zealand white rabbits

and its immunogenicity was identified using enzyme-linked immunosorbent assay (ELISA) and Western blotting. The subcellular localization of BPSS1395 protein was analyzed with Western blotting using the antiserum from the immunized rabbits.

Results

The BPSS1395 gene was amplified by PCR from the genomic DNA of

B. pseudomallei

and the recombinant plasmid pET-22b-BPSS1395 was successfully constructed. The recombinant protein was obtained after transformation of

E. coli

BL21 (DE3) with the constructed plasmid

and the purified protein showed a relative molecular mass of 32 × 10

. In the rabbits immunized with the recombinant protein

the serum titer of the polyclonal antibodies reached 1: 1 280 000 as detected using ELISA. Western blotting showed that the subcellular fractionation of BPSS1395 was localized mainly in the cytoplasm.

Conclusion

We successfully obtained the recombinant protein BPSS1395 with biological activities and prepared its polyclonal antibodies from rabbits for further investigation of the role of type Ⅲ secretion system in the pathogenesis of

B. pseudomallei

infection.

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

DOI：10.16016/j.1000-5404.201611152

摘要

Abstract

关键词

Keywords

references