Construction Of Truncated FAM96B Recombinants And Function Study On Its Interaction With Apoptin

FAM96B(family with sequence similarity96,member B),also named MIP18, CGI-128.This gene located on chromosome16q22.1-q22.3. Its protein contain163amino acids andweights18kd. The protein is highly conserved, its function has not been fully elucidated.Tanaka K reported that FAM96B together with MMS19,Ciao1,XPD form amacromolecular complex. It is able to adjust the activity of TFⅡH which plays a role in theprocess of mitosis and DNA repair; Silence the expression of FAM96B can result inabnormal mitosis. Weiwen Yang reported that FAM96B can regulate the expression ofE2-2protein, and play a role in the vascular endothelial cell’s proliferation, migration, andmicrotubule formation. From the above materials,FAM96B may play a key role in cellgrowth, mitosis, development of tumor and cell apoptosis.Apoptin is a small protein containing121amino acids encoded by Chicken AnemiaVirus. It only induces the tumorigenic phenotype of cells to apoptosis,but don’t induceapoptosis in normal human cells. One of the key factors of Apoptin specifically inducestumor cell to apoptosis is the nuclear localization. The preliminary laboratory studies byyeast two-hybrid screening has confirmed that FAM96B can interact withApoptin.Co-immunoprecipitation confirmed that this interaction exists in the tumor cellsand normal cells.Objective：1. Explore which peptides on FAM96B’s primary structure influences the interactionbetween FAM96B and Apoptin;2. Explore which peptides on FAM96B’s primary structure influences the subcellularlocalization of Apoptin.Through studying the relationship between the FAM96B’s structure and function,thesestudies will provide new information for FAM96B and Apoptin research. Methods：Construct eukaryotic expression vector of FAM96B and its recombinational segmentwith myc tag by molecular cloning techniques. They are pcDNA3.1-myc-FAM96B△1（deletionamino acid sequence from1-42）, pcDNA3.1-myc-FAM96B△2（deletionaminoacid sequence from43-121）, pcDNA3.1-myc-FAM96B△3（deletion amino acid sequencefrom122-163）.Transfect these vector respectively with flag-Apoptin plasmid into HEK293Tcells. After extracting total protein,detect whether FAM96B and its recombinant interact withApoptin in HEK293T cells by Co-immunoprecipitation,detect wether FAM96B and itsrecombinant affect the subcellular localization of Apoptin in HEK293T cells byImmunofluorescence technology.Results：We have successfully construct three truncated FAM96B recombinants with myctag.They can express successfully when transfected into HEK293T cells.We extract wholeprotein after transfecting these recombinants respectively with pcDNA3.1-flag-Apoptinplasmid into HEK293T cells. Results of Co-immunoprecipitation and immunoblottinexperiments show that the full-length FAM96B, FAM96B△1and FAM96B△3can interactwith Apoptin in HEK293T cell while this interaction does not exist between FAM96B△2and Apoptin. Results of immunofluorescence show that the full-length FAM96B,FAM96B△1can make Apoptin locate in the cytoplasm, and FAM96B△2, FAM96B△3don’t have the above effects.Conclusions：Full-length FAM96B, FAM96B△1and FAM96B△3can interact with Apoptin inHEK293T cell while this interaction does not exist between FAM96B△2andApoptin.Above result tell us that the missing area of FAM96B△2is the key region whereFAM96B directly interact with Apoptin, this region plays an important function inFAM96B’s interaction with Apoptin, deleting this region in FAM96B can make thedirectly interaction between FAM96B and Apoptin disappear.Full-length FAM96B, FAM96B△1can make Apoptin locate in the cytoplasm, andFAM96B△2,FAM96B△3can’t.Above results tell us that the missing areas of FAM96B△2,FAM96B△3are the key region where FAM96B directly affect Apoptin’ssubcellular localization.It is most likely that these two regions constitute a completefunctional domains together to make Apoptin locate in the cytoplasm.