CAJ | 학술논문

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saxiphilin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。
목적：마비성패류독소수체단백 Saxiphilin가특이성결합마비성패류독소,본문채용간상병독표체재체계통대Saxiphilin진행체외표체연구。방법종우와간장극륭득도saxiphilin기인(N단함유자신분비신호태),이용특이인물사기C단대상조안산표첨。결과사용간상병독표체재체계통구건대유목적기인적중조병독,용병독감염초지탐야아(Spodoptera frugiperda)세포 Sf9이표체 Saxiphilin,통과우화세포배양기중태우혈청함량급감염시간,학정사용무혈청배양기배양,중조병독감염Sf9세포72 h시,배양기중가용성목적단백표체량최대。결론통과얼주순화득도Saxiphilin단백,이기장차단백진일보용우마비성패류독소적검측。
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.