RESUMEN
OBJECTIVE: To construct a mouse recombinant enamelin eukaryocyte expression system, and establish the stable cell line which can produce the protein continuously. METHODS: The mRNA transcript from the 3-day mouse jaw was extracted. and the enamelin gene fragment amplified with RT-PCR techniques. Then the PCR product was cat with two restriction enzymes, and subcloned into the eukaryotic gene expression vector pcDNA3.1TM/mycj His(-)B.The recombinant plasmid was transformed into E.coli DH5alpha bacterial cells, and harvested with plasmid midi kit. The recombinant expression plasmid was transferred to the HEK 293A eukaryocyte cells, cultured selectively with 800 mg/L G418, and examined with SDS-PAGE and Western Blot at the protein level. RESULTS: The mouse enamelin gene was cloned to the eukaryotic expression plasmid successfully by sequence measuring. After the recombinant plasmid was transferred into the HEK 293A cells, about 32,000 enamelin protein was checked out by SDS-PAGE and Western Blot. CONCLUSION: The recombinant eukaryocyte expression plasmid and the stable cell line were established. This is a basic research to obtain high-yeild biologically active enamelin protein, which may facilitate further investigation of its function.
Asunto(s)
Proteínas del Esmalte Dental/biosíntesis , Proteínas Recombinantes/biosíntesis , Transfección , Animales , Animales Recién Nacidos , Línea Celular , Clonación Molecular , Proteínas del Esmalte Dental/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos/genética , Ratones , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes/genéticaRESUMEN
A patient with severe hypertriglyceridemia and recurrent pancreatitis was found to have significantly decreased lipoprotein lipase (LPL) activity and normal apolipoprotein C-II concentration in post-heparin plasma. DNA analysis of the LPL gene revealed two mutations, one of which was a novel homozygous G-->C substitution, resulting in the conversion of a translation initiation codon methionine to isoleucine (LPL-1). The second was the previously reported heterozygous substitution of glutamic acid at residue 242 with lysine (LPL-242). In vitro expression of both mutations separately or in combination demonstrated that LPL-1 had approximately 3% protein mass and 2% activity, whereas LPL-242 had undetectable activity but normal mass. The combined mutation LPL-1-242 exhibited similar changes as for LPL-1, with markedly reduced mass, and for LPL-242, with undetectable activity. These results suggest that the homozygous initiator codon mutation rather than the heterozygous LPL-242 alteration was mainly responsible for the patient phenotypes.
Asunto(s)
Codón Iniciador/genética , Hiperlipoproteinemia Tipo IV/genética , Hiperlipoproteinemia Tipo I/genética , Lipoproteína Lipasa/genética , Pancreatitis/genética , Adolescente , Análisis Mutacional de ADN , Humanos , Hiperlipoproteinemia Tipo I/enzimología , Hiperlipoproteinemia Tipo IV/enzimología , Lipoproteína Lipasa/deficiencia , Mutación , Pancreatitis/enzimología , Biosíntesis de Proteínas/genética , RecurrenciaRESUMEN
Lecithin cholesterol acyltransferase (LCAT) is a key enzyme in the metabolism of high density lipoprotein (HDL), which has been found inversely correlated with atherosclerosis. Adenovirus mediated overexpression of human LCAT (hLCAT) in hamsters resulted in increased levels of plasma total cholesterol, HDL cholesterol, phospholipids and enlarged particle size of HDL. It also increased cholesterol and total bile acid concentrations in bile. Hepatic mRNA level of cholesterol 7alpha-hydroxylase increased 2.7-fold in hamsters. However, such effects were not observed in mice in a parallel experiment. This study suggests that overexpression of hLCAT in hamsters facilitated reverse cholesterol transport. Similar metabolic changes in humans might modify atherogenic risk.