类鼻疽杆菌毒素分子BLF1的重组表达及生物学活性

Recombinant expression and biological characterization of Burkholderia pseudomallei toxin BLF1

目的重组、表达类鼻疽杆菌毒素BLF1并研究其生物学活性。方法运用DNA重组技术,以p GEX为表达系统在大肠杆菌中表达BLF1蛋白;以纯化重组蛋白r BLF1免疫新西兰大白兔,获得抗r BLF1血清,Western blot及ELISA鉴定抗血清;不同浓度r BLF1蛋白腹腔注射BALB/c小鼠,观察小鼠生存率;A549细胞模型中,通过CCK-8实验研究目的蛋白的细胞毒性效应及抗体阻断作用。结果从类鼻疽杆菌基因组DNA中PCR得到了BLF1目的基因,连接到p GEX-6p-2质粒,经双酶切及测序表明重组质粒构建成功。GST-BLF1蛋白经诱导表达、酶切纯化得到了相对分子质量为23×103的高纯度r BLF1,免疫家兔抗血清ELISA效价达1∶1 280 000,Western blot鉴定在目的蛋白位置处出现特异印记条带。重组r BLF1蛋白腹腔注射BALB/c小鼠,小鼠死亡率与蛋白剂量呈正相关。r BLF1蛋白能明显杀伤A549细胞,抗体可有效阻断其杀伤作用。结论成功重组表达了具有生物活性的r BLF1,该蛋白具有明显杀伤A549肿瘤细胞的作用,同时能够导致小鼠死亡但表现出一定耐受性。

Objective To express recombinant Burkholderia pseudomallei toxin BLF1 and observe its biological characterization. Methods The p GEX expression system was adopted to express recombinant BLF1 protein through recombinant DNA technology. The target protein was used to immunize New Zealand white rabbits,and the immunogenicity of the expressed protein was identified by ELISA and Western blotting.The survival rate of BALB / c mice was observed after intraperitoneal injection of different concentrations of r BLF1 protein. The purified protein acted on A549 cells to study its cytotoxicity and the blocking effect of antibody by detecting the number of living cells with CCK-8 kit. Results BLF1 gene was amplified by PCR from genomic DNA of Burkholderia pseudomallei and cloned into vector p GEX-6P-2. The recombinant plasmid highly expressing GST-BLF1 protein was identified by restricting enzyme digestion, PCR and DNA sequencing. The expressed protein was about 23 × 10^3 at molecular weight. After immunization,the titer of polyclonal antibody reached 1∶ 1 280 000 by ELISA. A specific positive band was found in the target protein position by Western blotting. After intraperitoneal injection of r BLF1 protein,the mortality of BALB / c mice was positively correlated with the dosage of r BLF1 protein. r BLF1 protein can obviously kill A549 cells and antibodies could inhibit its role effectively. Conclusion Recombinant r BLF1 protein with biological activity is successfully expressed,and can effectively kill A549 cells. The protein shows cytotoxicity to normal cells,but the normal cells can tolerate the toxin to some extent. All these results provide a basis for further studying pathogenesis of Burkholderia pseudomallei,seeking active prevention and treatment measures,and widening the application of BLF1 toxin in antitumor field.