Cloning And Expression Of Apolipoprotein B MRNA Editing Enzyme 3A(APOBEC3A) And Its Biological Function

Introduction:Human cellular immunoglobulin APOBEC3A(A3A),also known as human apolipoprotein B mRNA editing enzyme-catalyzed polypeptide 3A(APOBEC3A,A3A),is amember of human apolipoprotein B mRNA editing enzyme-catalytic peptide-like protein3 family(A3A,A3B,A3C,A3D,A3F,A3G and A3H).A3A is a congenital host restriction factor and plays an important role in the innate immune response in human cells.It can inhibit retrovirus,endogenous reverse transcription factor and foreign DNA,and also can inhibit the replication of DNA viruses,such as HBV and HPV.A3A is mainly expressed in peripheral blood mononuclear cells,such as CD14~+monocytes and macrophages.The expression of A3A in hepatocytes is very low,but IFN-?can induce the expression of A3A.In recent years,it has been found that IFN-?-induced A3A can change C base into U base in the HBV cccDNA,which can cause HBV cccDNA specific degradation.This finding leads to the hope of removing antiviral studies of HBV cccDNA in the nucleus.However,the mechanism which A3A causes cccDNA degradation and whether or not to cause damage to genomic DNA requires further study.So we study A3A in vitro to prepare for the subsequent study of the mechanism by which A3A induces cccDNA degradation and the effect on genomic DNA.Objective:In order to assist in studying the mechanism by which A3A causes cccDNA degradation,we first investigated the in vitro enzymatic activity of A3A and its effect on cellular gene level and protein level.Methods:1.We construct A3A eukaryotic expression vector pcDNA3.0-A3A and transfect HEK239T and HepG2 cells with it.We identify of A3A protein by Western blotting and identify the localization of A3A in HEK239T and HepG2 cells by immunofluorescence.Then we enrich A3A protein by His tag protein purification method and detect A3A deaminase activity by fluorescence polarization technique.2.A3A was overexpressed in HEK293T cells to detect the effect of A3A on cell proliferation,cell cycle and apoptosis.The mutations induced by A3A were identified by high-throughput sequencing in the target region.3.The expression profile of A3A-related proteome was identified and quantified by TMT and LC-MS/MS.Results:1.DNA sequencing confirmed that a long 600bp A3A gene sequence was inserted in pcDNA3.0-A3A.The plasmid was able to express A3A in HEK239T and HepG2 cells.A3A is mainly located in cytoplasm in HEK239T cells and in cytoplasm and nuclear in HepG2 cells.The purified A3A has cytosine deamination activity in the TTCA sequence of single-stranded DNA.2.Overexpression of A3A inhibited the proliferation of HEK293T cells;overexpression of A3A led to cell cycle arrest in S phase;overexpression of A3A promoted apoptosis.3.Overexpression of A3A result in a significant difference in the expression of 259proteins in the cells,of which 134 proteins were up-regulated and 125 proteins down-regulated.Conclusions:1.A3A is mainly distributed in the cytoplasm in HEK239T and in the cytoplasm and nucleus in HepG2 cells,indicating that the intracellular localization of A3A is tissue-specific.2.In vitro expression of A3A has TTCA fragment-specific cytosine deaminase activity.3.A3A can inhibit the proliferation of HEK293T cells,lead to cell cycle S phase arrest and promote cell apoptosis;4.A3A can affect the genomic DNA in cells5.A3A can change the expression of related protein.