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

作　　者：胡艺[1] 胡志强[1] 马腾飞[1] 韩丹[1] 李倩[1] 方瑶[1] 毛旭虎[1] 

机构地区：[1]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,重庆400038

出　　处：《第三军医大学学报》2017年第10期941-945,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(30973402)~~

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32×10~3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。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 × 103. 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：类鼻疽杆菌 Ⅲ型分泌系统蛋白 抗体制备与鉴定 

分 类 号：R341[医药卫生—基础医学] R378.99