| BackgroundFamilial hypercholesterolaemia(FH) is one of the autosomal dominantdisorder. It lead to the atherosclerosis easily and greatly increased therisk of the cardiovascular disease. Therefore, FH is a serious threat tohuman health. The studies have shown that hyperlipidemia mainlyelevated plasma low-density lipoprotein cholesterol (LDL-C) levels, and85%of LDL-C increasing cases are due to the mutations in the low-densitylipoprotein-receptor (LDL-R) encoding gene. The LDL-R is used forclearing LDL-C from the blood by means of endocytosis andintracellular degradation. In case the receptor is defective or lacking,this will lead to the rise of cholesterol. So Controlling plasma levels ofLDLR is the key to reduce LDL—C. Recently, reashers have foundHuman proprotein convertase subtilisin9(pcsk9) can increase the proteinof LDLR in the nucleus or lysosomal degradation, increasing theconcentration of LDL-C; also can increased LDL-C by reducing the LDLRinto the cytosol. Thus, there was a positive correlation between the proprotein convertase subtilisin/kexin type9(pcsk9) and the LDL-C. In addition,the polymorphism of pcsk9gene was related to the occurrence,developmentand prognosis of cardiovascular diseases. And different gene polymorphismwill cause different levels of LDL-C. Gain of function mutation causeshyperlipidemia, loss of function mutation reduces LDL-C, while alsoreducing the risk of cardiovascular disease in80%of patients.Therefore, realizing the characteristics of the human pcsk9genes,monitoring human pcsk9protein, can provide the basis for the clinicaldiagnosis, monitoring, prevention and treatment of hyperlipidemia. Which theInternational clinical use to treat the hyperlipidemia are antibodybiologics of pcsk9protein, mostly still in the clinical validation phase.The present results had shown that human pcsk9antibodies caneffectively control the level of LDL-C, reduce cardiovascular disease risk..At present, the manufacturers introduced the biologics of pcsk9aremainly imported products, which are espensive and primarily ELISAkit using to monitor the level of the pcsk9protein in the blood.which greatly limits the widespread promotion and use of the pcsk9gene in the clinic.According to above, this project will engage in a series of basicresearch on human pcsk9gene, building its prokaryotic expressionvector, induce it to express protein, prepare polyclonal and monoclonalantibodies against human pcsk9protein,which will make acontribution to clinic.As human pcsk9protein expressed mainly in human liver, andobtained liver tissue from the human body is relatively difficult.Traditional methods of separation and purification of pcsk9proteinsfrom human liver tissue are not only tedious and low yield, proteinpurity poor, it can not meet the clinical needs. Therefore, this studyused genetic engineering methods, and considering the E. coli’s geneticbackground is low, the rate of protein synthesis is aging, yield and other characteristics, compound the coding region sequence of pcsk9gene, build their prokaryotic expression vector, and induced theexpression vectors to express protein largely. Used the purifiedrecombinant protein immunogens animals, prepare the polyclonal andmonoclonal antibodies which had good specificity and potency. Laythe foundation for further development of human-related biologics pcsk9kit, also make preparations for pcsk9using in human clinicalmonitoring, prevention and treatment of hyperlipidemia andcardiovascular disease..Objective1. To construct the expression vector of Proprotein convertasesubtilisin/kexin type9;2. Induce the PET-28b-pcsk9to express and find the optimumconditions for the target gene expression., purified the expressedprotein;3. Immunize the New Zealand Rabbits with the purified protein,prepare the polyclonal antibody of human pcsk9and identify thepolyclonal antibodies;4. Immunize the BALB/c mice with the purified protein, preparethe monoclonal antibody of human pcsk9and identify the monoclonalantibodies.Methods1. Construct the expression vector of Proprotein convertase subtilisin/kexintype9(pcsk9)The sequence of pcsk9cDNA was synthesized by the biologycompany and amplied by PCR., the amplified products by electrophoresis, EB staining and observe the results. After determining the presence ofamplified products, the target gene was cloned into TA cloning vectorPMD-19T. By means of digestion and subcloning, the sequence wascloned into the expression vector PET-28b. The recombinant plasmid ofpET-28b-pcsk9was transformed into the competent cell of E.coli.BL21（DE3）.After sequencing, the gene was not mutated.2. Induced expression and purification of human pcsk9proteinUnder different conditions, induce the PET-28b-pcsk9to expressand compare the results to find the best expression conditions. Theninduce the recombinant plasmid of pET-28b-pcsk9to express largely.Finally receive the protein by nickel column for affinity chromatographypurification, the purified protein with western blot and BCA methodrespectively to detect the specificity and protein concentration.3. Prepare the polyclonal antibody of human pcsk9Every rabbit takes200ug immune protein for the first timewith the same amount of freund’s complete immune adjuvantemulsion blending by subcutaneous injection. Then after every2weekswith the purpose of100ug protein with the same amount ofincomplete freund’s immune adjuvant to immune every rabbit bysubcutaneous immunization. Moreover, immune to5times. Finally collectedserum, collected for polyclonal antibody, and purification of theantibody. The antiserum of purified were detected specificity by westernblot and titer determination.4. Prepare the monoclonal antibody of human pcsk9Every8weeks of SPF BALB/c mouse takes200ug immuneprotein for the first time with the same amount of freund’scomplete immune adjuvant emulsion blending by abdominal cavityinjection. Then after every3weeks with the purpose of100ugprotein with the same amount of incomplete freund’s immune adjuvantto immune every mouse by abdominal cavity injection.Moreover, immune to5times totally. Let the immunized mouse spleen cellsto fuse with mouse myeloma cells as hybridoma. Select the positive cellsto enlarged culture, then injected the cells into healthy BALB/c miceenterocoelia. Obtain the ascites,purify and identify the monoclonalantibodies.Results1. Construct the expression vector of Proprotein convertasesubtilisin/kexin type9(pcsk9)。The gene of pcsk9was amplified successfully. By electrophoresisobservation, there’s a target band in2000bp. After TA cloning, thetarget gene successfully constructed into the PMD–19T carrier. Then putthe PMD–19T-pcsk9vector plasmid and PET-28b expression vectorplasmid to double enzyme digest, connect. Morever, picked out positive coloniesby the colony PCR. Finally, after gene sequencing,the recombinant plasmid ofpET-28b-pcsk9was without the mutation.2. Induced expression and purification of human pcsk9proteinThe objective sequence was cloned and expression in E.coli.BL21（DE3）.The best expression conditions of PET-28b-pcsk9were atOD=0.6, induced by1.0mmol/L IPTG for8h at temperature of30℃. Theexpression of the recombinant expression vector protein is givenpriority to with inclusions. Compared to normal bacteria induced crackingliquid precipitation, found the target protein in100,500, tendency of imidazole/Lphosphate buffer washing column chromatography, and with a tendencyfor500mmol/L imidazole phosphate buffer when catharsis, the effectis much better. And purification of the target protein concentrationdetermination is about333ug/ml.3. Prepare the polyclonal antibody of human pcsk9The polyclonal antibody of pcsk9protein can identify the purification of purpose protein, but it can’t identify PET-28b protein. Homemadeantiserum the absorbance of A value falled with the rise of the antiserumdilution degrees. Regard the antiserum A value for the negative control formore than2times as the biggest dilution degrees and the antibodytiter, it is concluded that the homemade antiserum titer was morethan1:13200.4. Prepare the monoclonal antibody of human pcsk9One hybridmas producing antibodies against pcsk9protein wasobtained and named after H1. The monoclonal antibody of Pcsk9proteincan identify the purification of purpose protein, but it can’t identifyPET-28b protein. The absorbance,A value of the homemade monoclonalantibody falled with the rise of the monoclonal antibody dilution degrees.Regard the A value for the negative control for more than2times as the biggest dilution degrees and the antibody titer, it isconcluded that the homemade monoclonal antibody’s titer was morethan1:12800. The home-made monoclonal antibody subtypes was IgG2b,light chain for kappa chain predominate. The affinity of the antibody Kvalue is2.08145x10-7.Conclusion1. The gene of pcsk9was amplified successfully. the expressionvector was constructed and expressed well.2. The expression of the recombinant plasmid of PET-28b-pcsk9weresensitive to the induced temperature and time. The purification of thetarget protein concentration determination is about333ug/ml and they hadspecility.3. Get the polyclonal antibody of human pcsk9successfully, and thehomemade antiserum titer was more than1:13200.4. One hybridmas producing antibodies against pcsk9protein was obtained and named after H1. The homemade monoclonal antibody’stiter was more than1:12800and its subtypes was IgG2b, lightchain for kappa chain predominate. The affinity of the antibody Kvalue is2.08145x10-7. |
PDF Full Text Request