The Preparation Of Human Recombinant Apolipoprotein M And Monoclonal-antibody And Preliminary Clinical Application

Chapter One Expression and Purification of apolipoprotein MBackground:apolipoprotein M (apoM) is a novel apolipoprotein which belongs to the lipocalin family and predominantly in HDL particles. Studies in vivo and in vitro shown that apoM can not only enhance reverse cholesterol transport by participating in the formation of preB-HDL, but also display anti-inflammatory and anti-oxidant. Therefore, apoM is regarded as a protective factor against atherogenesis, but the mechanism is still unknown. It is difficult to extract apoM from human plasma because it is a 24kD micromolecular apolipoprotein, anchored to phospholipid monolayer by a hydrophobic signal ligand. In order to investigate the structure and function of apoM, we cloned and expressed apoM in vector to generate its monoclonal-antibody (mAb) in mice.Purpose:To express full length recombinant human apoM for further purification, for gaining the target recombinant apoM with high concentration and purity.Methods:apoM DNA was amplified by PCR according to the published cDNA database from human liver. The confirmed sequence of target gene (apoM) was inserted into the vector pET according to the protocol, and amplified in E.coli JM109. The positive clones are confirmed by genetic sequencing. Protein was expressed by IPTG induced expression system after transfection with E.coli BL21(DE3). Immunoaffinity chromatography was used to purify the protein, which was consequently confirmed, measured and characterized by SDS-PAGE, Western blotting and Lowry methods.Results:1. apoM gene was amplified by PCR and a 560bp PCR product was showed on agarose eletrophoresis, which is consistent with the published human sequence in GenBank.2. We successfully established the pET-apoM vector. By BamH I and XhoI enzymes cutting and SDS-PAGE gel electrophoresis, it was showed two specific bands which are respectively consistent with the vector size before the insertion of the gene and target gene size.3. We expressed an IPTG-induced recombined protein after transfecting pET-apoM E.coli BL21 (DE3), and SDS-PAGE finding showed a 34kD new protein consistent with the target protein size.4. The band in SDS-PADE gel electrophoresis was clean after purifying the recombined Trx-apoM protein by immunoaffinity chromatography method.5. The protein was confirmed as Trx combined protein by anti-Trx antibody Western blotting.Conclusion:We have successfully cloned human apoM gene and expressed Trx-apoM recombinant protein. The cloned human apoM can be used as an antigen for apoM antibody to develop commercial kit for clinically measuring plasma concentration of apoM. It can also be used in animal or celluar models to further determine the role of apoM in lipid metabolism in vivo. Chapter two Preparation and identification of apolipoprotein M monoclonal-antibodyBackground:apoM has been suggested to play an important role of anti-atherosclerosis due to its genetic structure and its distribution among human tissue. Studying the structure and functions of apoM may help us to understand the pathogenesis of atherosclerosis and eventually preventing and treating this disease. Antibodies can act as a probe to study the relationship between structures and functions of antigens from three levels of molecule, cell and organ. In addition, antibodies can also be used as a ligand for the immunoaffinity chromatography, to couple to columns to separate and purify target proteins. Therefore, identification of antibodies is a key step of basic research methods to study proteins. One of common methods in biomedical field is to make antibody from hybridoma. The virtues of mAbs are including high homogenicity in physical, high specialty in chemical characteristics, strong binding to antigens, as well as easy to quality control, prepare and acquire, which make mAbs widely used. However, apoM in blood is a micromolecular protein and tightly combined with lipoprotein, which make this protein difficult to separate from plasma by physical methods. In our initial study we made highly purified recombined Trx-apoM protein, whereby we can make anti-apoM mAbs. Purpose:To prepare anti-apoM mAbs with high affinity and purity, and then analyse as well as identify their content, immunoglobulin subclass, affinity and specificity.Methods:Balb/c mouse was immunized with recombined Trx-apoM protein raised in the chapter one. After cytomixis, screening and cloning, we obtained sub-cloned hybridoma of well growth and steadily secreting, acquired ascites by injecting Balb/c mice intraperitoneally and gained anti-apoM mAbs by standard techniques. Purified immunoglobulin was, tested by 12% SDS-PAGE for purity clarify, and immunoglobulin sub-class was tested by immunoglobulin sub-class kit, and the antigenic determinants of immunoglobulin was judged by ELISA, and the relative affinity was measured by ELISA, and the specificity of monoclonal antibodies was tested by blocking test, and the expression of anti-apoM monoantibodies in human tissues, cells and plasma was examined by immunohistochemistry and Western blotting.Results:1. We gained three strains of hybridoma with well-grown and steadily secreting. After intraperitoneally injecting of mice with these hybridoma, the efficiencies of three groups of ascite were up to 1:106-1:107。 2. Three mAbs were IgG2aic by analysis of sub-type of IgG shown.3.1E2F12 and 8F12C6 had the different sites in apoM antigenic determinants, either were 1E2F12 and 14H1B7. But the capacity of 1E2F12/8F12C6 combining to apoM was stronger than 1E2F12 /14H1B7.4. The affinities of three mAbs were 1E2F12>8F12C6>14H1B7.5. Three mAbs had high specificity in blocking experiments.6. The reactivity of recombinant apoM for mAbs was examined by Western blotting, and specific bands were shown clearly at 34kD in each mAb without non-specific bands, which are consistent with predicted position.7. In Western blotting experiments with liver-matrix lysate or mixed human plasma and mAbs, the specific band was shown at 24kD, consistent with apoM size.8. The expression of apoM was detected in various cells by immunohistochemistry, and the positive expression were shown in normal human liver cells and monkey kidney cells.9. By means of the detection of three mAbs, the expression of apoM was positive in human liver cytolymph and pancreas vessel-epithelia membrane, but negative in heart, lung, spleen and intestine, which was 100% consistent with all samples. Conclusion:We have successfully made three strains of anti-apoM mAbs. The antibodies can react not only with recombined apoM, but also with apoM in human serum. It is the first report about the mice-anti-human apoM mAb in China. That will be useful in quantitative analyzing apoM concentration in human serum. chapter Three The concentration of apoM in human serum detected by ELISA and its relationship to lipidsBackground:apoM is mainly associated with HDL, expressed in liver and kidney. Studies suggested that apoM is involved in lipid metabolism and transportation in vivo. Recently, Xu et al reported that the plasma concentration of apoM is correlated with other lipids in human plasma. Their studies show that apoM is positively correlated with BMI and leptin, negatively correlated with cholesterol, but the mechanisms unknown. The apoM belongs to lipocalin superfamily, it may sever as a ligand binding site for hydrophobic molecules in plasma for lipoprotein transport and metabolism. The role of apoM remains further study. At present, the measurement of apoM concentration is only available through the Western blotting and Dot-blotting. It is essential to establish a standard, stable and easy method to measure the apoM concentration for clinic research.Purpose:To establish an ELISA to detecting the concentration of apoM in human plasma, and evaluate the sensitivity and stability of this method, as well as establish the normal range of apoM in human plasma for reference purpose. To observe the correlations between apoM and lipids in human plasma, and compare the apoM concentration in normal and different groups of coronary heart diseases.Method:We chose the optimal working concentration of both coating-antibody and HRP-marked antibody by titration ELISA and measured the standard curve of recombinant human apoM by protein titrated curve. The lowest detectable concentration, the day-to-day variation and between-plate-within-day variation, the interference of other non-specialized apolipoproteins were measured with ELISA. We established the reference range of our laboratory by detecting the plasma concentration of apoM in 124 healthy subjects.We also compared and analysed the trend of apoM in plasma from SA (n=32), ACS (n=34) and healthy subjects (n=35).Result:1. The linear range of the method presented 31.25-375 ng/ml with the lowest detectable concentration 11.8 ng/ml. The day-to-day variation and between-plate-within-day variation in the ELISA were 5.24-5.83% and 4.32-5.69%, respectively.2. The results of plasma dilution curve showed that good linearity was obtained when plasma dilution rate was from 1:50 to 1:400 and the best optimal dilute rate for the determination of apoM in plasma was 1:200.3. No significant interference was obtainted with different concentration of apoAI, apoB and Lp(a). 4. The reference ranges of healthy people were 13.15±2.68ug/ml5. apoM was significantly positive correlation with HDL-C and HDL-C/TC (r=0.336; r=0.345 respectively) and was negative with TG,TC and LDL-C.6.The concentration of apoM in SA group and ACS group were both significantly lower than health control. But there was no significant difference between SA and ACS group.Conclusion:We have developed the sandwich ELISA to detect the concentration of apoM in human serum. By examining its sensitivity, repetition and ability of resisting disturbance, it is show that ELISA is excellent in its stability, sensitivity and specificity. Compared with the reports from others, ELISA established here has the virtues of easily-operating, steady resource of standard and easy to largely examine for clinically application. It offers a convenient and reliable tool widely to use apoM for clinical study.