苦荞过敏原TBa和TBb基因的共表达及其包涵体复性的研究

基因TBa和TBb分别编码苦荞过敏蛋白的两个亚基，构建这两个基因的表达质粒pET-28a-TBa和pET-32m-TBb，利用双抗生素筛选法，获得稳定遗传的双质粒转化子，经IPTG诱导，两个基因在同一宿主菌中共表达，表达蛋白以包涵体的形式存在。在共表达产物复性过程中，两个亚基互为分子伴侣，相互促进了蛋白质的重新正确折叠，ELISA检测表明：复性后的蛋白免疫学活性得到了提高。由此获得了有活性的蛋白质，并且建立了不相容双质粒共表达外源基因和包涵体复性的方法。

Coexpression of Tartary Buckwheat Allergen TBa and TBb

Both TBa and TBb genes encoded tartary buckwheat allergenic protein’s two sununits, respectively. The genes were ligated into expression vector pET-28a and pET-32m for construct plasmids pET-28a-TBa and pET-32m-TBb. The two plasmids could be stably transformed and maintained in E.coli BL21(DE3) when both ampicilln and kanamycin were presented in the selective medium. After induction with IPTG, both TBa and TBb genes were coexpressed in E.coli, with their production as inclusion body. In the process of renaturation of coexpressed product, two subunits acted as molecular chaperones with each other, which promoted the refolding of protein TBa and TBb. ELISA indicated that the renatured protein had the improvement on the immunological activity and gained the activated protein. A new method for coexpression of proteins in E.coli containing two incompatible plasmids in which two different antibiotic resistant markers were included and inclusion body renaturation were also established.