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

作　　者：曹玲[1,2] 吕敬章[1,2] 丁晶[1,2] 葛丽雅[1,2] 刘慧玲[1,2] 张恒[1,2] 杨凯[3] 汤慕瑾[1,2] 岳振峰[1,2] 

机构地区：[1]深圳出入境检验检疫局,深圳市食品安全检测技术研发重点实验室,深圳518045 [2]深圳市检验检疫科学研究院,深圳518010 [3]中山大学生命科学学院,深圳510275

出　　处：《食品安全质量检测学报》2014年第6期1739-1745,共7页Journal of Food Safety and Quality

基　　金：国家重大基础研究项目(973)(2009CB118903);国家质检总局科技计划项目(2010IK159);深圳市知识创新计划项目(JCYJ20120618172144503)~~

摘　　要：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.

关 键 词：麻痹性贝类毒素受体蛋白 杆状病毒表达载体系统 镍柱亲和纯化 

分 类 号：Q78[生物学—分子生物学]