BpHi008A基因的体外表达分析

Analyze the expression of BpHi008A gene in vitro

BpHi008A是一种水稻诱导性防卫基因,以单拷贝序列存在,当秧苗受到褐飞虱咬食或机械损伤后,该基因便大量表达,编码一个含82个氨基酸的蛋白质.将此基因重组到表达型载体pET28a后,转化入能产生T7RNA聚合酶的大肠杆菌菌株中,以10%SDS—聚丙烯酰胺凝胶电泳来分离并鉴定未经IPTG诱导和经IPTG诱导后不同时刻的表达产物.结果表明利用T7 RNA聚合酶/启动子表达系统可使蛋白质在大肠杆菌中高水平表达,并且表达产物的分子量约为9.6 KDa.本实验表达的蛋白质可用为抗原刺激抗体的产生,为免疫定位和Western杂交做准备.

The BpHi008A, an inducible defense gene, present as a single -copy in the rice genome. Its transcript level was increased in rice seedling after being fed by brown platthopper nilapar vata lugens stal or damanged by mechanical wounding. The gene encodes a putative 82 amino acid protein. The gene was inserted into the expression vector of pET28a, and introduced into BL21 （ DE3 ） which can synthesize T7 RNA polymerase. Then we can isolate and characterize the expression product that was induced by IPTG by10% SDS -PAGE. The result indicates that overexpression of the proteins in E. coli can be achieved using T7 RNA polymerase/promoter system, and the molecular weight of the expression product was approximately 9.6 KDa. The protein synthesized in this experiment can be used as antigen to stimulate the synthesis of antibody, which can be applied in lmmunolocalization and western blotting analysis.