BAFF-R胞内区与TRAF3相结合关键分子结合域的确定

Identification of the Key Molecular Binding Domains of the Cytoplasmic Domain of BAFF-R Binding with TRAF3

目的:研究BAFF-R胞内区与TRAF3相互作用的关键结合域。方法:分别钓取BAFF-R胞内区和TRAF3的c DNA片段,克隆到酵母双杂交系统中,验证两者之间的相互作用。利用缺失PCR和重叠PCR的方法获取11个TRAF3的突变体,验证TRAF3的11个突变体和BAFF-R胞内区的相互作用。结果:TRAF3与BAFF-R胞内区发生相互作用时,有三个关键的分子结合域,分别是N端的螺旋结构382-400位氨基酸、中间的428-463位氨基酸以及TRAF3 C端的543-560位氨基酸。结论:在体内得到了BAFF-R胞内区与TRAF3相互作用的三个关键分子结合域,有助于展开对BAFF-R胞内区与TRAF3相互作用的进一步研究。

Objective： To identify the key cytoplasmic molecular domain of BAFF-R binding with TNFR-associated factor 3（TRAF3）. Methods： At first, we gained the c DNA of cytoplasmic domain of BAFF-R and TRAF3 respectively by RT-PCR. Then, we used yeast two-hybrid assay to testify the interactions between the cytoplasmic domain of BAFF-R with TRAF3. In order to gain the key molecular binding domains of BAFF-R with TRAF3, the eleven deletion mutants were constructed by PCR and overlapped PCR. At last,we used yeast two-hybrid assay to testify the interaction between the eleven mutants with TRAF3. Results： 382-400 aa coil, 428-463 aa,543-560 aa of TRAF3 were three important domains which play critical part in binding to BAFF-R. Conclusion： The result will provide a molecular binding base for the association of cytoplasmic domain of BAFF-R with TRAF3.