| Apolipoprotein E, a 34kDa protein composed of 299 amino acids, is a multifunctional molecular, which is an important functional component of chylomicrons and very low density lipoprotein, intermediate density lipoprotein and high density lipoproteins. As a ligand for the low density lipoprotein receptor (LDLR), it plays an important role in the metabolism of plasma lipoproteins. It is predominantly synthesized in liver and brain, but it is also synthesized by a wide variety of tissues and cell types. The expressed apoE protein in central neuronal system functions potentially in deposition and clearance of amyloidal peptide, sustaining the stabilization of microtubule proteins, cellular signal transduction, immune regulation, glucose metabolism and oxidation stress. ApoE plays a key biological active role in vivo.ApoE is a polymorphism protein, which exists as three common isoform, apoE2, apoE3 and apoE4. The most common isoform, apoE3, has cysteine and arginine at position 112 and 158, respectively, whereas apoE2 has cysteine and apoE4 has arginine at both positions. ApoE2 binds poorly to the LDLR, and homozygous inheritance of the apoE2 allele is strongly associated with type III dyslipoproteinemia.ApoE2 and apoE4 are associated with hypercholesterolemia and atherosclerosis(AS).The apoE4 is also associated with higher incidence and earlier age of onset of late onset familial Alzheimer's disease(AD), whereas apoE2 may have the opposite effect.Deficiency of apoE results in severe hypercholesterolemia and diffuse atherosclerotic disease in humans and gene-targeted mice. Deficiency of apoE may promote to produce and develop atherosclerotic lessions. The apoE gene-targeted mice will result in marked regression of both early and advanced atherosclerotic lesions by injected apoE recombinant protein, or by transfected adviral vector with apoE cDNA to express human apoE transgene in liver, or by transplantation of bone marrow with normal rat apoE gene .This demonstrates that apoE gene and its expressing product can inhibit progression of atherogenesis. ApoE3 hasa more effective prevention from AS than apoE2 and apoE4.Epidemiological studies are shown that apoE4 gene is a risk factor for Alzheimer's disease. Compared with apoEs, the subjects with apoE4 gene increase the risk for Alzheimer's disease and decrease the age with Alzheimer's disease. ApoE4 gene dosage effect is accompanied with the severe degree of Alzheimer's disease and earlier age of onset of Alzheimer's disease. Conversely, the subjects with apoEi gene have a long life span. It may have a preventional function from Alzheimer's disease. Molecular biological studies are shown that apoE isoform has a isoform-specific role to neurodegeneration. ApoE3 and apoEi aid to sustain the stabilization of cytoskeleton protein tau, while apoE4hyperphosphorylates tau to destabilize the microtubule protein, which will make neurocyte easy to form neurofibrillary tangles in AD. Clearance of amyloidal peptide by apoE2 and apoEa is more effective than by apoE4. ApoE2 has the strongest ability to clear the amyloidal peptide. It is shown that apoEi has a stronger prevention from Alzheimer's disease. As to the mechanism of apoE how to affect the neurodegenerative diseases is yet uncertain.The thesis is based on the above research advance. We have been trying to explore the prospection of apoE in prevention from atherosclerosis and Alzheimer's disease .The apoE gene was separated, cloned and expressed, as well as its function was studied in the thesis.The human total RNA was extracted from the foetal liver tissue sample of spontaneous abortion. mRNA was separated and purified from total RNA. ApoE3 cDNA was amplified by RT-PCR with a signal or without a signal sequence. Its sequence is consistent with the reported apoEs sequence in the Gene Bank. The apoE3 cDNA fragment was inserted into pQEso expressing vector at BamEl and Hindlll site. The recombinant plasmid was induced to express in BL21 host cell by ImM IPTG. The expressed protein is a 36kDa fusion protein...
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