棉铃虫围食膜肠粘蛋白HaIIM72真核表达及几丁质结合活性检测

Eukaryotic expression and analysis of chitin-binding of intestinal mucin HaIIM72 from Helicoverpa armigera(Hübner)(Lepidoptera: Noctuidae)

在前期工作中,本实验室已成功克隆得到棉铃虫粘蛋白基因haiim72。为了研究粘蛋白HaIIM72的生化特性,本试验将haiim72基因克隆到质粒pFastBacTM,转化大肠杆菌DH10Bac,构建重组杆状病毒bacmid-hai-im72,转染昆虫细胞系BTI-TN-5B1-4(HighFive)从而在昆虫细胞系中进行表达。结果表明:成功表达了分子量约260kDa的棉铃虫围食膜肠粘蛋白HaIIM72。几丁质结合活性试验表明,肠粘蛋白HaIIM72具有几丁质结合活性,能够被较强的变性剂洗脱,属于第三类围食膜蛋白。

Insect Intestinal Mucin（IIM）is an important protein constituent of the peritrophic membrane. In previous work, we cloned the intestinal mucin gene haiim72 of Helicoverpa ar- migera. Here the gene haiim72 was cloned into the vector pFastBacTM, then the recombinant pFastBac-haiim72 was transformed to Escherichia coli DH10Bac for getting bacmid-haiim72,and the recombinant baculovirus Bac-haiim72 was transfered into HighFive cell line to express the HaIIM72. The results showed that the HaIIM72 protein was expressed successfully in in- sect cell line BTI-TN-5B1-4 （HighFive） as a molecular weight about 260 kDa. The chitin-bind- ing assay exhibited that HaIIM72 could be combined with the regenerated chitin and be released from the complex by strong denaturant.