Lipoprotein Metabolism And Activity Of Protein Structure And Function

ATP-binding cassette transporter Al (ABCA1) is one of the important members involved in lipid metabolism . It plays a key role during the course of reverse cholesterol transport (RCT) and the production of high density lipoprotein (HDL), thus, it is also called the "gatekeeper" of RCT.Tree shrew (TS) is considered to be a model for its insusceptibility to atherosclerosis, but the exact mechanism needs to be further investigated.To systematically study this mechanism, we have successfully cloned several genes related to lipid metabolism, such as APOAI, APOCI, APOE, APOCII, PLTP, LCAT, APOAV and CETP, and analyzed their structural feathers. In order to more comprehensively understand other related genes, the purpose of this work is to obtain the full length cDNA of ABCA1 as well as deduce the amino acid sequence and analyze the relative expression quantity in different tissues.The first strand cDNA was reversely transcribed from mRNA, which isolated from total RNA extracted from TS liver. Special primers were designed by consulting highly conserved sequences, which occur to identical cDNA sequences of human and other kinds of species. Several amplified fragments were observed by the technique of SMART(Switching Mechanism At 5'end of RNA Transcript) and RACE(Rapid Amplification of cDNA End) from the first strand of cDNA.The sequencing results were compared with data on GenBank .On the basis of the overlapping principle , the cDNA sequence of TS ABCA1 was acquired by connecting these fragments .Its amino acid sequence was deduced from corresponding cDNA . The primary and secondary structures of TS ABCA1 were predicted with software for molecular biology. In addition, we use the real-time PCR technology to analyze the relative ABCA1 mRNA expression in different tissues.The C-terminal of TS ABCA1 and plasmid vector of pET43.1a were digested with SmaI and XhoI enzymes, thus the recombinant expression vector was successfully constructed. After transforming into E.coli BL21(DE3) and IPTG induction, purification, we got the TS ABCA1 protein,...