The Protein Expression,Purification And Antibody Preparation Of Low-Density Lipoprotein Receptor

Low density lipoprotein receptor(LDLR)is a kind of transmembrane glycoprotein,which encoded by low density lipoprotein receptor gene and belongs to the family of low density lipoprotein receptor.Cells can obtain exogenous cholesterol through LDL endocytosis mediated by LDLR,therefore,Low density lipoprotein(LDL)plays an important role in blood cholesterol level regulation.LDLR mutation or afunction is the crucial of blood cholesterol content increase in healthy body.Metabolic disorders of cholesterol can result in a series of serious diseases.Such as cardiovascular disease,atherosclerosis,and gallstones result from cholesterol metabolism disorder.At the same time,there are evidences indicate that obesity,diabetes,Alzheimer's disease are also associated with the disturbance of cholesterol metabolism.Meaning the relative LDLR content variation is usually required in the studies of detailed pathogenesis of related diseases.Hence,the preparation of an effective LDLR antibody becomes very meaningful.In this study,a LDLR fragment amino acids813?862 was amplified from pmLDLR-5×Myc plamid by PCR and the PCR product was cloned,then recombinant pGEX-6P-1-LDLR-C plamid was transformed into Escherichia coli BL21,the target protein of 32 KD was produced by induction of IPTG.Then the protein was injected into New Zealand rabbits after it was purified and analyesed quantitatively.Afterwards,we test the titer of LDLR protein polyclonal antibody through Western Blot assay,the results reveal that the prepared LDLR antibody shows high titer and specificity.Finally,after further affinity purification,the prepared LDLR polyclonal antibody can be applied in immunofluorescence assay,which illustrates that the preparation of LDLR antibody has been successfully completed.As a result,the prepared LDLR antibody can be used in Western Blot assay,Immunofluorescence assay and so on.The successful preparation of LDLR polyclonal antibody can be applied for the subsequent studies,such as LDLR distribution detection,localization in cell and interaction with other proteins.