麻痹性贝类毒素受体蛋白Saxiphilin的克隆与表达

Cloning and expression of the receptor protein Saxiphilin of paralytic shellfish poison

目的麻痹性贝类毒素受体蛋白Saxiphilin可特异性结合麻痹性贝类毒素,本文采用杆状病毒表达载体系统对Saxiphilin进行体外表达研究。方法从牛蛙肝脏克隆得到ssziphilin基因(N端含有自身分泌信号肽),利用特异引物使其C端带上组氨酸标签。结果使用杆状病毒表达载体系统构建带有目的基因的重组病毒,用病毒感染草地贪夜蛾(Spodoptera frugiperda)细胞Sf9以表达Saxiphilin,通过优化细胞培养基中胎牛血清含量及感染时间,确定使用无血清培养基培养,重组病毒感染Sf9细胞72 h时,培养基中可溶性目的蛋白表达量最大。结论通过镍柱纯化得到Saxiphilin蛋白,以期将此蛋白进一步用于麻痹性贝类毒素的检测。

ABSTRACT:Objective Saxiphilin, the receptor protein of paralytic shellfish poisons, could specifically bind to the paralytic shellfishtoxins.In this paper, the baculovious expression vector system was used in vitro for the study of Saiphilin expression. Methods Saxiphilin gene with its own secretory signal peptide in the N-terminus was obtained by reverse transcription from bullfrog liver, and a histidine tag was attached to the C-terminus of saxiphilin gene. Results Using the baculovirus expression vector system, the recombinant baculovirus (vAc-sax-e) was constructed to overexpress Saxiphilin protein. By optimizing the concentration of fetal bovine serum in the medium and the infection time, the maximum amount of the soluble Saxiphilin secreted into the supernatant was expressed after infected Sf9 cells with vAc-sax-e cultured in serum-free medium for 72 h. Conclusion Followed by ultrafiltration concentration, Saxiphilin was purified using the nickel column chromatograph. Purified Saxiphilin could be further used for the detection of paralytic shell-fish toxins.