Screening Of The Proteins Which Can Interact With Nef By Using Yeast Two Hybrid System

The human immunodeficiency virus1(HIV-1) is a kind of single strand RNA retrovirus which cause the acquired immunodeficiency syndrome (AIDS). It has high copying rate, high recombination rate and mutation probability and it can escape from its host’s immune system because of its changeable genome. HIV has been epidemic in the whole world for tens of years, which make it very significant to find out useful method to avoid or treat this dangerous virus. Negative factor (NEF) is one of HIV’s important accessory proteins, which has a very conservative gene sequence in HIV-1, HIV-2and Simian immunodeficiency virus (SIV). Many researches related to this protein haven been done home and abroad. As a whole this interesting protein mainly has several important functions as follows:(1) down regulates CD4,(2) down regulates CD28,(3) down regulates MHCⅡ,(4) interfere the viron’s replication and infection,(5) control the cells’apoptosis. However, the researches about this protein is still relatively limited, thus we use the yeast two-hybrid screening system to search for potential proteins which can interact with Nef, in order to know better about this protein’s function and the pathogenesis of HIV-1.The yeast two-hybrid screening system is a kind of proteins’interaction detection method and has a very high efficiency.Our research use the plasmid pNL4-3to clone the Nef gene and then insert it to the plasmid pGBKT7making a recombinational plasmid.After that we use the yeast two-hybrid screening system and B lymphocyte to search for potential proteins after the self-activation and toxicity detection. Particularly, we first activate the yeast stain AH109, transfect the plasmid pGBKT7-Nef into AH109, transfect the library gene to the yeast with pGBKT7-Nef, and test the positive result using X-agal. After achieving the positive yeast strain, we extract the plasmid from monoclonal strain and transfect it to Escherichia Coli DH5a. Then we extract the genes from transfected DH5a, sequence them, and do homology analysis using the NCBI’s ’Blastx’function to find out the proteins which can interact with Nef. As a result, we find two potential ones, one is ficolin and the other is yementin.Through yeast two-hybrid screening system we find out some proteins which can interact with Nef, however, this interaction is found in yeast cells which do not definitely mean they can happen in animal cells for the difference between these two kinds of cells. Thus, related researches in animal cells still need to be done based on animal cells like co-ip, GST pull-down, in order to further understand the interaction. If the interactions of ficolin, yementin and Nef of HIV-1are determined, it will be very important for understanding the regulation mechanism of Nef and the pathogenesis of HIV-1.