The Construction Of The Recombinant Plasmid And Gene Cloning Of Low Density Lipoprotein Receptor-related Protein Associated Protein 1

ObjectiveCardiovascular and cerebrovascular diseases have been plaguing the health of human beings,and atherosclerosis are the main foundation of the pathology of atherosclerosis,indepth study of the molecular mechanisms of cardiovascular and cerebrovascular disease have great significance for prevention and treatment of cardiovascular and cerebrovascular disease,in recent study to have found that oxidized LDL(oxLDL) involved in atherosclerosis formation,and the oxidation of LDL required low-density lipoprotein receptor-related protein(LRP) the participation,low-density lipoprotein receptor related protein(LRP) is an endocytic protein,belong to low-density lipoprotein receptor(LDLR) gene family. Receptor-related protein(RAP) is a permanent endoplasmic reticulum protein,is low-density lipoprotein receptor(LDLR) family of molecular chaperones,the family of receptor from the endoplasmic reticulum to the plasma membrane transport, sophisticated and mature folded right course of ligand binding from other attacks played a vital role.RAP exhibits high-affinity binding for LRP,when RAP is bound to LRP is the inhibition of binding and/or uptake of all known LRP ligands when the combination of RAP with LRP,the LRP and the other be able to prevent all known ligand binding / uptake.it has into a versatile tool that has greatly facilitated the study of the biological functions of the entire LDL receptor gene family.The object of this experiment is that to obtain low-density lipoprotein receptor related protein (LRPAP1) Cloning and expression vector of rat.For a better understanding the relations of LRP and the LDL oxidation process and further explore the relationship between Atherosclerosis and lipid metabolism,this experiment provides a favorable the instrument protein. MethodsThe use of rat adrenal cell line Pc1 2,using RT-PCR cloning of the full-length RAP cDNA gene fragments,gene fragments upstream with Kozak sequence and restriction sites HindⅢ,downstream add histidine tag protein sequence and restriction sites XhoⅠ. With restriction endonuclease HindⅢand XhoⅠto target gene and the expression plasmid pcDNA3.1 + into sticky end to connect to build ligase HisTag-LRPAP1 fusion protein vector,transformed into bacteria Feel state DH-5α,positive clones picked, plasmid extraction kit plasmid extraction,sequencing verification.ResultsProved to be a success by building a coding sequence HisTag-LRPAP1 fusion protein expression vector.DiscussionThis experiment accesses to the full-length gene fragment of RAP from the rat adrenal pheochromocytoma cells Pc12,and then full-length fragment is connected with the expression plasmid pcDNA3.1 +,and then amplification,extracted plasmid containing the target gene,In order to further pave the way to do a good job of protein expression.In this experiment,Primers deplete directly to 5 'signal peptide sequence of the target gene in the design,while upstream and downstream primers at both ends of the restriction sites add 5'HindⅢand 3'XhoⅠ,At both ends of the restriction sites add the three base to protection of the efficiency of the dual-enzyme digestion and inserted into plasmid pcDNA3.1 + at the time of the direction,for the next step the right expression of protein to lay a good base.At the same time,the design of primers added six histidine sequence for purification of proteins to do the next step.RAP is a wide range of molecular chaperones and ligand molecules for lipoprotein receptor,the study of structure and function of lipoprotein receptors plays an important role,while RAP is also related to the occurrence of certain diseases,it can be used such as membranous nephropathy,senile dementia research and other diseases.The study is successful construction of RAP,which can be the basis of RAP expression,further,can be used RAP to purify the receptor fragment of LDLR family,for the study provide a good tool. ConclusionSuccessful HisTag-LRPAP1 construct encoding the fusion protein of the plasmid vector,the future can be used for eukaryotic expression of this fusion protein.