Preparation of polyclonal antibodies and subcellular localization analysis of chicken apolipoprotein A-Ⅰ
Apolipoprotein A-Ⅰ(Apo A-Ⅰ)plays important roles in the regulation of atherosclerosis,viral infections,lipid metabolism and other aspects.However,there are few studies on chicken Apo A-Ⅰ(chApo A-Ⅰ),and its biological function is not well understood.Based on the bioinformatics analysis of chApo A-Ⅰ,this study further performed the expression and purification of the recombinant protein of chApo A-Ⅰ via the pET-28a prokaryotic expression system.Mouse polyclonal antiserum was prepared by immunizing mice with purified recombinant protein.Enzyme-linked immunosorbent assay(ELISA)was used to determine the titer of the polyclonal antiserum,and Western blot(WB)and indirect immunofluorescence assay(IFA)were used to determine its reactivity.Then,the polyclonal antiserum was further applied for subcellular localization analysis of chApo A-Ⅰ.Bioinformatic analysis revealed that the chApo A-Ⅰ protein contains signal peptide at 1-18 amino acids,which is composed of continuous alpha helix at the N-terminal.Homology analysis of amino acid sequences revealed that the chApo A-Ⅰ protein had the highest homology with turkey and the lowest homology with fish.The polyclonal antibody prepared using successfully expressed and purified recombinant protein His-chApo A-Ⅰhad an ELISA titer above 1×105 and specifically reacted with the eukaryotic expressed chApo A-Ⅰ protein in WB and IFA.Particularly,the antibody can recognize the Apo A-Ⅰ protein in chicken serum,but cannot cross-react with Apo A-Ⅰ proteins in the serums of mice,rabbits,cattle or pigs.This polyclonal antibody was further applied for subcellular localization analysis of full-length chApo A-Ⅰ(chApo A-Ⅰ-FL)and chApo A-Ⅰ without signal peptide(chApo A-Ⅰ-NS).Observed by confocal microscope,it was found that chApo A-Ⅰ-FL protein was mainly localized near the cell membrane,but chApo A-Ⅰ-NS protein was localized in the cytoplasm,and most of them were diffusely distributed.The specific polyclonal antibody and the results of subcellular localization of chApo A-Ⅰ provide a basis for further research on the biological function of Apo A-Ⅰ.
NETL
NSTL
万方数据
维普
