Studies On Cloning And Expression Of Recombinant Human Apolipoprotein A-Ⅳ In Pichia Pastoris

Human Apolipoprotein A4 which is one of the Human apolipoproteins was found in 1977. apoA-Ⅳis synthesized by small intestina and secreted with other triglyceride-rich lipoproteins together.Human apoA-Ⅳwhose molecular weight is 46000Da is one kind of glycoproteins.It contains 396 amino acids,with glycine as N-terminal and lysine as C-terminal.ApoA-Ⅳhas the lowest ability of affinity and surface tension of fat in all apolipoproteins because it is difficult to penetrate the lipid monolayer,apoA-Ⅳgene's CDS is 1191bp and that of mature peptide is 1128bp.In humans,this gene cluster locates on long arm of the chromosome 11.Large amount of ApoA-Ⅳcould be found in human peripheral blood,liver and small intestine,especially jejunum and ileum.After the intake of high fat foods,apoA-Ⅳis transfered into the blood circulation as chylomicrons through intestinal lymphatic system,and then departs from the lipoprotein through the metabolism of chylomicrons.One-fourth of apoA-Ⅳexists in HDL and the remaining exists in human plasma in the form of non-free lipoprotein.The two forms of apoA-Ⅳmake it of great importance in the metabolism of lipid.The main physiological functions of apoA-Ⅳare as follows:1.It causes a reduction of feeding and delays gastric emptying as a result of inhibiting gastric acid secretion.Therefore, apoA-Ⅳinvolves in lipid-induced inhibition of gastric function.2.ApoA-Ⅳcan prevent the occurrence of artery atherosclerosis by influencing plasma high-density cholesterol(HDL-C) levels.Since a high level of HDL-C can delay the formation of atherosclerotic plaques,and apoA-Ⅳis possible to play a role through the "endogenous" lipid's antioxidants.3.The occurrence of the typeⅡdiabetes mellitus(NIDDM) may be associated with the high level of apoA-Ⅳ.4.apoA-Ⅳcan inhibit appetite,It has been confirmed that the level of apoA-Ⅳin serum after intake of fat increases,which results in the sense of satiety,Therefore,low level of apoA-Ⅳin serum may cause the increase of appetite with the result of obesity.1 The Cloning of Human apoA-ⅣGeneIn this study,the sequence of apoA-Ⅳgene was obtained from small intestine of the people through RT-PCR and PCR.With the application of gene recombinate technology, apoA-Ⅳwas connected with PMD18-T and the cloning vector of pMD18-T-apoA-Ⅳwas established finally.Because PMD-18T can be amplified in E.coli,the pMD18-T-apoA-Ⅳwas transformed into E.coli and apoA-Ⅳgene was amplified at last.With the use of plasmid extraction kit,the plasmid extraction from Escherichia coli was finished.The apoA-Ⅳgene was identified by using PCR technology.The right cloning gene was used for the following experiment.2 The construction of pPICZα-apoA-Ⅳrecombinant plasmidThe sequence of the ApoA-Ⅳmature peptide was obtained with the specific expressive primers and the templates of the correct clone apoA-Ⅳgene.Separately with XhoI and XbaI enzyme,the sequence of the apoA-Ⅳmature peptide and the expressive vector of pPICZαwere digested,Then the apoA-Ⅳgene was connected to the expressive vector of pPICZα. with the use of DNA Connection technology.The apoA-Ⅳgene was cloned into the pPICZαC vector between the XhoI and XbaI restriction sites and the recombinant plasmid of the pPICZα-apoA-Ⅳwas established.3 The establishment of recombinant apoA-ⅣPichia pastoris eukaryotie expression systemThe linearized recombinant expression vectors were introduced into P.pastoris X-33 by electroporation using a Micropulser.With the application of specific expression primers to identify pPICZα-apoA-Ⅳthe result showed that the gene pPICZα-apoA-Ⅳhad stabilized into the Pichia pastoris genome.The Pichia pastoris started to express apoA-Ⅳwith the vector signal peptide and inductiong of methanol.The Pichia pastoris strains of the high expressive apoA-Ⅳat high level was obtained.Recombinant apoA-Ⅳwas cumulative.The output of apoA-Ⅳwould increase with the time of expression at a certain period of time,the result shows that the output get its maximum on the day 4.Optimize the best expressive environment and pH value is the main influence on the expression of recombinant apoA-Ⅳ.When the positive clone was incubated in BMMY media with different pH varing from 4.0 to 7.8,the secretion of recombinant protein had a highest yield at pH 6.5 Recombinant human apoA-Ⅳwas first obtained at a high level expression in Pichia pastoris and the fermentative condition of apoA-Ⅳwas studied.