甜菜夜蛾围食膜几丁质结合蛋白SeCBP66在不同体系中的表达

Expression of peritrophic membrane chitin binding protein SeCBP66 of Spodoptera exigua in different systems

分析甜菜夜蛾(Spodoptera exigua)围食膜几丁质结合蛋白SeCBP66氨基酸序列,利用PCR技术扩增获得缺失信号肽编码区围食膜几丁质结合蛋白的基因△(s)-secbp66。将△(s)-secbp66克隆到表达载体pET28a和pPIC9K,分别转化大肠杆菌BL21和毕赤酵母GS115进行原核及真核表达;将△(s)-secbp66克隆到质粒pFastBacTM,转化大肠杆菌DH10Bac构建重组Bacmid,转染昆虫细胞系Sf9及BTI-TN-5B1-4(HighFive)从而在昆虫细胞系中进行表达。结果表明:SeCBP66在原核细胞中未见表达,在酵母细胞中表达产物呈弥散状,而在昆虫细胞系中则能稳定的表达116kDa的特异蛋白。

Amino acid sequence of chitin-binding protein SeCBP66 of Spodoptera exigua peritrophic membrane was analyzed and a △（s）-secbp66 encoding SeCBP66 without signal peptide was get by PCR method from the midgut cDNA library of Spodoptera exigua. The △（s）- secbp66 gene was cloned into pET28a and pPIC9K and transformed into E. coli BL21 ,P. [asto- ris GSllS,resulting recombinant E. coli and P. pastoris,respectively. Using Bac to Bac system, the △（s）-secbp66 gene was transfected into insect cell lines Sf9 and BTI-TN-5B1-4（High- Five）. The results showed SeCBP66 was expressed in P. pastoris and insect cell lines, but not in E. coli. However, SeCBP66 appeared as a dispersion of unequal size in P. pastoris while a stable expression was showed as a molecular weight 116 kDa in insect cell lines.