We paid our main attention to cloning tb22kda gene from tartary buckwheat , expressing the structure gene and getting the purified recombinant protein , and these provide foundation for father study on the relationship between structure and function of the allergenic protein tb22kda , the orientation of the corresponding epitope and the exploration of allergenic reaction mechanism 本研究的目的在于分离克隆苦荞中的主要过敏蛋白( tb22kda )基因,并表达纯化出其结构基因编码的蛋白。从而为进一步研究过敏蛋白tb22kda的结构与功能,寻找其中的抗原决定簇,探讨过敏原与其相应抗体相互作用机理提供依据。
A new e . coli promoter - probe vector phn1005 was constructed by using a red - shift enhanced gfpmut3 as reporter gene and showed the following characters : 1 , bamhi at the 5 " end of gfpmut3 structure gene could be used to clone promoter - active fragment and the strength of promoter could be quantitatively assayed . 2 , a rrnbtlt2 terminator from rrna gene at the 3 " end of gfpmut3 could permit clone strong promoter . 3 , another rrnbt1t2 terminator was inserted upstream bamhi to prevent reading through of promoters from puc19 进一步以红移且荧光强度提高21倍的gfpmut3为报告基因,构建了大肠杆菌启动子探针载体phn1005 ,该载体上gfpmut3结构基因5 ’端的bamhi位点可用来克隆具有启动子活性的dna片段并定量分析插入的启动子强度;其3 ’端含rrnat1t2终止子,可允许克隆强启动子;在bamhi上游同样插入rrnat1t2终止子以防止载体puc19上的启动子的转录通读; gfpmut3结构基因上游还插入一段内含子序列使正反六种读码框的翻译均可被终止,可保证gfpmut3以正确的读码框翻译。