Interaction Mechanism Of LMP1 And TNFR-associated Protein And Assembly,Purification And Crystallization Of Complexs

Epstein-Barr virus(EBV),also known as Human Herpesvirus 4(HHV-4),belongs to the genus Herpesvirus,a linear double-stranded DNA virus.After infection with lymphocytes,they often exist in a latent state,and generally do not develop disease.When the human immunity is low or stimulated by the external environment,the state of the EB virus-infected cells will be converted from a latent state to an activated state,causing various malignant tumors,for example,nasopharyngeal cancer,breast cancer,lymphoma,gastric cancer et al.Latent membrane protein 1(LMP1)is a six-pass transmembrane protein encoded by Epstein-Barr virus,which is closely related to the malignant transformation and tumorigenesis of Epstein-Barr virus infection.Existing studies have shown that oligomerized LMP1 protein can hijack the tumor necrosis factor receptor CD40-mediated signaling pathway in normal cells,and interact with tumor necrosis factor receptor(TNFR)binding protein TRAF、RIP and lethal structure Domain proteins such as TRADD,thus inhibitting apoptosis and inducing cell proliferation and canceration.Blocking the interaction between LMP1 and TRAF by inhibiting LMP1 oligomerization is an important direction for the prevention and treatment of various related diseases.The analysis of the structure of LMP1 and protein complexes such as TRADD,RIP and TRAF will help to elucidate the precise molecular mechanism of LMP1 intervention in normal cell signaling pathways,laysing a structural foundation for drug and vaccine development of various related diseases.In this current study,we recombinantly expressed and purified the C-terminal intracellular domain protein of LMP1 and its interaction proteins TRAF3 and TRADD in E.coli cells to construct a stable protein complex.In addition,the LMP1-TRAF3 protein complex was successfully assembled.Preliminary analysis and research on their biochemical properties and functions were carried out with various methods.In vitro biochemical experiments showed that LMP1 protein existed in many different aggregation states.We have crystallized the LMP1-TRAF3 protein complex and obtained a diffractable crystal.After preliminary optimization,the highest resolution of the crystal diffraction can reach 3-5?,However,asignificant anisotropy existed in the diffraction.Further optimization of the crystal is still in progress in order to achieve complete diffraction data.