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类鼻疽杆菌Ⅲ型分泌系统BPSS1395蛋白表达及其抗体制备与鉴定
Expression and identification of recombinant
Burkholderia pseudomallei type Ⅲ secretion system BPSS1395 protein and preparation of its polyclonal antibodies- 第三军医大学学报 2017年39卷第10期 页码：941-945
- 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：
[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(30973402)
DOI：10.16016/j.1000-5404.201611152
中图分类号： R341;R378.99;R392-33收稿：2016-11-17，
修回：2016-12-26，
纸质出版：2017-05-30
- 稿件说明：
移动端阅览
胡艺, 胡志强, 马腾飞, 等. 类鼻疽杆菌Ⅲ型分泌系统BPSS1395蛋白表达及其抗体制备与鉴定[J]. 第三军医大学学报, 2017,39(10):941-945.
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.胡艺, 胡志强, 马腾飞, 等. 类鼻疽杆菌Ⅲ型分泌系统BPSS1395蛋白表达及其抗体制备与鉴定[J]. 第三军医大学学报, 2017,39(10):941-945. DOI： 10.16016/j.1000-5404.201611152.
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
2
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
2
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
2
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
3
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
2
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
2
To obtain recombinant protein of type Ⅲ secretion system protein BPSS1395 of
Burkholderia pseudomallei
(
B. pseudomallei
) and prepare its polyclonal antibodies.
Methods
2
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
2
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
3
. 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
2
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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