Tree Shrew And Human Apolipoprotein A5 Gene Cloning And Expression And Apolipoprotein A5 And Apolipoprotein C3 Gene Polymorphism

The apolipoprotein A5 has repeatedly been shown to play an important role in determining plasma triglyceride concentrations in human and mice, which have been proved to code a new Apolipoprotein in human originally. To systemically study the mechanism of tree shrew's insusceptibility to atheroselerosis, we have successfully cloned several genes about metabolism of lipids and lipoprotein, for example APOA1, APOC1, APOE, APOC2, LPL, ABCA1, PLTP, LACT, and CETP, etc.To obtain the full-length cDNA and amino acid sequences of Apolipoprotein A5 from tree shrew-a good model for its insusceptibility to atheroselerosis, the cDNA sequence of tree shrew Apolipoprotein A5 was obtained by utilizing SMART-RACE method. Its amino acid sequence was deduced from cDNA sequence and primary and secondary structures were predicted. Two transcripts of the Apolipoprotein A5 from tree shrew were obtained from different clones. The longer transcript comprised 1948 base, the shorter one covered 1937 base. The full-length sequence of tree shrew cDNA indicated that the two transcripts in tree shrew were likely the results of alternative polyadenylation, so the predicted open reading frame (ORF) were completely the same and the two transcripts of tree shrew also encoded 369 amino acids. It was worth noticing that the splicing patterns of Apolipoprotein A5 alternative polyadenylation in tree shrew, human and mouse are identical. After comparing this amino acid sequence with those of the other published animals' Apolipoprotein A5, it revealed that the identity among tree shrew, human, rat and mouse was 81.0%, 68.7%, 66.7% respectively. Protein structure analysis predicted N-terminal signal peptide including 23 amino acids, characteristic feature of lipid-binding Apolipoproteins. Through designing primer once again, the EcoRI site was designed in the forward primer and the Xhol site in the reverse primer. After the products of amplification were digested by double corresponding restriction endonucleases, the expression vector pET30a, including 343 amino acids of tree shrew mature Apolipoprotein A5, was successfully constructed. The protein was expressed in the E.coli BL21(DE3) in a fusion protein, consisted 34.6% of total celluar protein. The APOA5 protein was purified by...