载脂蛋白M重组蛋白和单克隆抗体的制备及其初步临床应用
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摘要
第一部分人载脂蛋白M的表达与纯化
研究背景：
人载脂蛋白M (apolipoprotein M, apoM)是新发现的一种载脂蛋白,属于疏水性分子结合蛋白家族,主要存在于HDL中。动物和细胞试验的结果表明,apoM通过参与前β-HDL的形成,促进体内胆固醇逆转运的过程,同时还具抗炎和抗氧化的作用。因此,认为apoM具有抗动脉粥样硬化功效,但其抗动脉粥硬化作用机制目前还不十分清楚。由于apoM是一种小分子的载脂蛋白,分子量仅24kD,且成熟分子中的疏水信号肽介导该蛋白“锚着”在磷脂单层上,直接从血清中提取apoM蛋白较困难。本研究拟通过基因工程手段,体外表达apoM重组蛋白,而制备抗apoM的单克隆抗体,为apoM蛋白结构和功能的研究打下基础。
目的：
重组表达人全长apoM蛋白并将其纯化,得到浓度高、纯度好的重组apoM蛋白。
方法：
利用人类肝脏cDNA文库做模板,聚合酶链反应(PCR)法扩增apoMDNA,将扩增正确的目的基因片段插入载体pET相应位点,转化大肠杆菌E.coli JM109进行克隆,挑取阳性菌落进行基因测序；再用测序正确的质粒转染大肠杆菌E.coli BL21(DE3),IPTG诱导蛋白的表达；用亲和纯化的方法纯化大肠杆菌表达的目的蛋白,并用SDS-PAGE凝胶电泳、Westren印迹及Lowry蛋白定量的方法对蛋白质的含量、纯度及特性进行鉴定。
结果：
1、PCR扩增产物经琼脂糖凝胶电泳证实扩增出一条特异的560bp的基因片段,测序结果与GenBank公布的人apoM基因序列完全一致。
2、成功构建了pET-apoM质粒。经BamH I和XhoⅠ做双酶切,1%琼脂糖凝胶电泳,出现2条特异性的条带,其中1条与酶切前质粒位置相近,另1条与目的基因大小基本一致。
3、含重组质粒pET-apoM的大肠杆菌E. coli BL21 (DE3)经IPTG诱导后表达重组蛋白,SDS-PAGE电泳分析表明在相对分子量34kD左右出现新的蛋白条带,与目的蛋白分子量一致。
4、用亲和层析的方法纯化后的Trx-apoM重组蛋白,SDS-PAGE电泳表明纯度高,无明显杂带。
5、用抗Thioredoxin (Trx)抗体进行Western印迹检测证实,该重组蛋白为Thioredoxin (Trx)融合蛋白。
结论：
本研究成功克隆出人apoM基因,并表达出Trx-apoM重组蛋白。通过克隆表达人apoM蛋白,一方面可以将其作为抗原制备针对apoM的抗体,从而可以进一步开发人体血液中apoM含量的检测试剂盒,为临床研究和诊断提供工具；另一方面,可以对表达的apoM进行理化处理,在细胞和动物模型上对apoM的功能进行研究,从而进一步研究apoM在人体脂类代谢中的生理功能。
第二部分载脂蛋白M单克隆抗体的制备及其鉴定
研究背景：
载脂蛋白M (apoM)由于其基因结构和组织分布特点,被认为其可在抗动脉粥样硬化中发挥重要作用；对该蛋白结构及功能的认识,有助于揭示其抗动脉粥样硬化作用的机制,从而对疾病的预防及治疗起着重要的推动作用。抗体作为研究工作中的探针,可以从分子、细胞和器官等不同水平上,研究抗原物质的结构与功能的关系；还能作为亲合层析的配体,固定在层析柱上,通过亲合层析从复杂的混和物中分离、纯化某种蛋白,是蛋白质功能研究的基本手段。利用杂交瘤技术制备抗体,是生物学和医学研究领域常用的方法；单克隆抗体的理化性状高度均一,生物活性单一,与抗原结合的特异性强,便于人为处理和质量控制,来源容易等特点,使它的应用一直广泛地受到关注。血清中apoM蛋白分子量小,与脂蛋白结合紧密,物理方法不易分离,我们在前期研究中制备了高纯度的重组Trx-apoM蛋白,为单克隆抗体的制备提供了基础。
目的：
制备亲和力高、纯度好的apoM单克隆抗体,并对抗体的浓度、免疫球蛋白亚类、相对亲和力和特异性进行分析鉴定。
方法：
用重组Trx-apoM蛋白免疫Ba1b/c小鼠,经细胞融合、筛选及克隆化,得到三株生长良好,分泌稳定的杂交瘤细胞株；腹腔注射于Balb/c小鼠,收取腹水,纯化后得到三个抗apoM单克隆抗体。纯化后的抗体经12%SDS-PAGE电泳观察纯化效果；用抗体亚类测定试剂盒检测单抗免疫球蛋白亚类；通过直接法ELISA相加试验判断三株单克隆抗体的抗原识别位点；用直接ELISA法测定三株亚克隆抗体的相对亲和力；用间接法ELISA阻断试验判断单克隆抗体的特异性；用免疫荧光和免疫组织化学的方法判断单克隆抗体与细胞和组织的反应特性；用Westren印迹法比较单克隆抗体与重组Trx-apoM蛋白、人血浆蛋白的反应。
结果：
1、得到了三株分泌稳定、生长良好的亚克隆杂交瘤细胞株。用该三株杂交瘤细胞接种小鼠得到的腹水单抗效价达到了1：106-1：107。
2、经亚型鉴定结果显示三株mAb均为IgG2aκ。
3、1E2F12和8F12C6两株单抗识别抗原的位点不同,1E2F12特异性结合的抗原位点也不同于14H1B7；同时结合抗原的能力1E2F12/8F12C6强于1E2F12/14H1B7。
4、三株单克隆抗体亲和力由大到小次为1E2F12>8F12C6>14H1B7。
5、阻断试验显示三株抗体均具有较好的特异性。
6、Western印迹实验检测重组Trx-apoM蛋白与单克隆抗体的反应情况,在约34kD处三株单抗均有特异性条带,与预测的分子大小位置符合,背景清楚。
7、与正常人肝细胞裂解液和混合人血清的Western印迹实验中,在分子量24kD处有特异性条带出现。
8、用制备的单抗检测apoM在不同种属细胞中的表达,表明在人正常肝细胞、猴肾细胞中呈阳性表达。
9、用三株单克隆抗体检测人组织中apoM蛋白表达,发现在人肝组织细胞浆及人胰腺组织导管上皮细胞浆、膜中呈阳性表达,在心、肺、脾、大肠和小肠组织中未检测到该蛋白的表达,符合率达100%。
结论：
成功制备了三株抗apoM的单克隆抗体,不仅能与重组蛋白反应,还可与人体内的天然蛋白结合,是国内首次报道制备出了针对人血清天然apoM蛋白的鼠抗人单克隆抗体,为下一步定量检测人血清中apoM蛋白含量打下了基础。
第三部分EL ISA法测定血清中载脂蛋白M浓度的方法学评价及其与血脂相关性分析
研究背景：
apoM主要存在于HDL中,在肝脏和肾脏中表达,提示该蛋白与血脂的转运和代谢有关。最近Xu等发现血液中apoM浓度与体重指数(BMI)以及血液中瘦素(Leptin)含量呈正相关,而与血液中总胆固醇呈负相关,但相关机制还未明确。另外,apoM属于Lipocalin超家族,推测可能与血浆中疏水性生物活性分子的结合、转运和代谢有关联,其确切功能尚有待进一步研究。目前国内定量检测apoM浓度的方法仅限于半定量的Western印迹和Dot-Blotting法,需要建立标准统一、方法稳定、操作简单的定量检测方法,这对于临床研究的深入进行将有极大的帮助。
目的：
建立测定人血浆中apoM浓度的双抗体夹心法,并对该方法的敏感性、重复性、抗干扰能力等作一评价；并建立该方法的正常人参考范围；观察血浆中apoM与血脂之间的相关关系；初步比较正常人与冠心病各组人群之间血浆apoM浓度的变化趋势。
方法：
用棋盘滴定法选择包被抗体和酶标抗体的最佳工作浓度；用辣根过氧化物酶标记抗apoM 8F12C6抗体；用蛋白稀释度曲线确定蛋白标准曲线的范围；用血浆稀释度曲线确定实验中人血浆的稀释倍数；确定了该方法的最低检测限,测定批间、内变异及非特异性脂蛋白的干扰,以观察方法的敏感性和特异性。通过测定124例健康体检人群血浆apoM的浓度,建立本实验室的正常参考值范围。比较稳定型心绞痛组(SA)32例和急性冠脉综合征组(ACS)34例病人与35例健康人群的血浆蛋白浓度,分析apoM在不同人群中的变化趋势。
结果：
1、根据重组蛋白制备标准曲线,该方法的线性范围为31.25-375ng/ml,最低检测限为11.8 ng/ml。批内变异4.32-5.69%；批间变异5.24-5.83%。
2、血浆滴度曲线结果表明,人血浆在1：50到1：400范围内线性关系良好,取线性范围中间整数倍1：200稀释度,,作为实验中人血浆apoM浓度检测的最佳稀释倍数。
3、不同浓度的脂蛋白apoAI、apoB及Lp(a)对该法干扰小,其测定的吸光度值均低于0.33,在该方法的线性范围外。
4、该方法的正常参考值范围为13.15±2.68μg/ml。
5、apoM与TG、TC及LDL-C呈负相关趋势,但差异并无统计学意义；与HDL-C、HDL-C/TC呈正相关(r=0.336和r=0.345)。
6、两组冠心病患者血浆apoM浓度(SA组11.02±1.96μg/ml, ACS组10.76±1.32μg/ml)均低于正常对照组(12.83±2.28μg/ml),但两组冠心病组之间浓度差异不明显。
结论：
我们建立了测定人血浆中apoM的浓度的双抗体夹心法,通过检测该方法的敏感性、重复性、抗干扰能力等指标,认为该方法稳定性、敏感性和特异性均好,与国内外检测apoM浓度的方法相比,我们建立的ELISA法具有操作简单,标准蛋白来源恒定,易于大批检测和适应临床检测的特点,为apoM在临床研究中的广泛开展提供了方便、可靠的工具。
Chapter One Expression and Purification of apolipoprotein M
Background:
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-antibody
Background:
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 lipids
Background:
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/ml
5. 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.
研究背景：
人载脂蛋白M (apolipoprotein M, apoM)是新发现的一种载脂蛋白,属于疏水性分子结合蛋白家族,主要存在于HDL中。动物和细胞试验的结果表明,apoM通过参与前β-HDL的形成,促进体内胆固醇逆转运的过程,同时还具抗炎和抗氧化的作用。因此,认为apoM具有抗动脉粥样硬化功效,但其抗动脉粥硬化作用机制目前还不十分清楚。由于apoM是一种小分子的载脂蛋白,分子量仅24kD,且成熟分子中的疏水信号肽介导该蛋白“锚着”在磷脂单层上,直接从血清中提取apoM蛋白较困难。本研究拟通过基因工程手段,体外表达apoM重组蛋白,而制备抗apoM的单克隆抗体,为apoM蛋白结构和功能的研究打下基础。
目的：
重组表达人全长apoM蛋白并将其纯化,得到浓度高、纯度好的重组apoM蛋白。
方法：
利用人类肝脏cDNA文库做模板,聚合酶链反应(PCR)法扩增apoMDNA,将扩增正确的目的基因片段插入载体pET相应位点,转化大肠杆菌E.coli JM109进行克隆,挑取阳性菌落进行基因测序；再用测序正确的质粒转染大肠杆菌E.coli BL21(DE3),IPTG诱导蛋白的表达；用亲和纯化的方法纯化大肠杆菌表达的目的蛋白,并用SDS-PAGE凝胶电泳、Westren印迹及Lowry蛋白定量的方法对蛋白质的含量、纯度及特性进行鉴定。
结果：
1、PCR扩增产物经琼脂糖凝胶电泳证实扩增出一条特异的560bp的基因片段,测序结果与GenBank公布的人apoM基因序列完全一致。
2、成功构建了pET-apoM质粒。经BamH I和XhoⅠ做双酶切,1%琼脂糖凝胶电泳,出现2条特异性的条带,其中1条与酶切前质粒位置相近,另1条与目的基因大小基本一致。
3、含重组质粒pET-apoM的大肠杆菌E. coli BL21 (DE3)经IPTG诱导后表达重组蛋白,SDS-PAGE电泳分析表明在相对分子量34kD左右出现新的蛋白条带,与目的蛋白分子量一致。
4、用亲和层析的方法纯化后的Trx-apoM重组蛋白,SDS-PAGE电泳表明纯度高,无明显杂带。
5、用抗Thioredoxin (Trx)抗体进行Western印迹检测证实,该重组蛋白为Thioredoxin (Trx)融合蛋白。
结论：
本研究成功克隆出人apoM基因,并表达出Trx-apoM重组蛋白。通过克隆表达人apoM蛋白,一方面可以将其作为抗原制备针对apoM的抗体,从而可以进一步开发人体血液中apoM含量的检测试剂盒,为临床研究和诊断提供工具；另一方面,可以对表达的apoM进行理化处理,在细胞和动物模型上对apoM的功能进行研究,从而进一步研究apoM在人体脂类代谢中的生理功能。
第二部分载脂蛋白M单克隆抗体的制备及其鉴定
研究背景：
载脂蛋白M (apoM)由于其基因结构和组织分布特点,被认为其可在抗动脉粥样硬化中发挥重要作用；对该蛋白结构及功能的认识,有助于揭示其抗动脉粥样硬化作用的机制,从而对疾病的预防及治疗起着重要的推动作用。抗体作为研究工作中的探针,可以从分子、细胞和器官等不同水平上,研究抗原物质的结构与功能的关系；还能作为亲合层析的配体,固定在层析柱上,通过亲合层析从复杂的混和物中分离、纯化某种蛋白,是蛋白质功能研究的基本手段。利用杂交瘤技术制备抗体,是生物学和医学研究领域常用的方法；单克隆抗体的理化性状高度均一,生物活性单一,与抗原结合的特异性强,便于人为处理和质量控制,来源容易等特点,使它的应用一直广泛地受到关注。血清中apoM蛋白分子量小,与脂蛋白结合紧密,物理方法不易分离,我们在前期研究中制备了高纯度的重组Trx-apoM蛋白,为单克隆抗体的制备提供了基础。
目的：
制备亲和力高、纯度好的apoM单克隆抗体,并对抗体的浓度、免疫球蛋白亚类、相对亲和力和特异性进行分析鉴定。
方法：
用重组Trx-apoM蛋白免疫Ba1b/c小鼠,经细胞融合、筛选及克隆化,得到三株生长良好,分泌稳定的杂交瘤细胞株；腹腔注射于Balb/c小鼠,收取腹水,纯化后得到三个抗apoM单克隆抗体。纯化后的抗体经12%SDS-PAGE电泳观察纯化效果；用抗体亚类测定试剂盒检测单抗免疫球蛋白亚类；通过直接法ELISA相加试验判断三株单克隆抗体的抗原识别位点；用直接ELISA法测定三株亚克隆抗体的相对亲和力；用间接法ELISA阻断试验判断单克隆抗体的特异性；用免疫荧光和免疫组织化学的方法判断单克隆抗体与细胞和组织的反应特性；用Westren印迹法比较单克隆抗体与重组Trx-apoM蛋白、人血浆蛋白的反应。
结果：
1、得到了三株分泌稳定、生长良好的亚克隆杂交瘤细胞株。用该三株杂交瘤细胞接种小鼠得到的腹水单抗效价达到了1：106-1：107。
2、经亚型鉴定结果显示三株mAb均为IgG2aκ。
3、1E2F12和8F12C6两株单抗识别抗原的位点不同,1E2F12特异性结合的抗原位点也不同于14H1B7；同时结合抗原的能力1E2F12/8F12C6强于1E2F12/14H1B7。
4、三株单克隆抗体亲和力由大到小次为1E2F12>8F12C6>14H1B7。
5、阻断试验显示三株抗体均具有较好的特异性。
6、Western印迹实验检测重组Trx-apoM蛋白与单克隆抗体的反应情况,在约34kD处三株单抗均有特异性条带,与预测的分子大小位置符合,背景清楚。
7、与正常人肝细胞裂解液和混合人血清的Western印迹实验中,在分子量24kD处有特异性条带出现。
8、用制备的单抗检测apoM在不同种属细胞中的表达,表明在人正常肝细胞、猴肾细胞中呈阳性表达。
9、用三株单克隆抗体检测人组织中apoM蛋白表达,发现在人肝组织细胞浆及人胰腺组织导管上皮细胞浆、膜中呈阳性表达,在心、肺、脾、大肠和小肠组织中未检测到该蛋白的表达,符合率达100%。
结论：
成功制备了三株抗apoM的单克隆抗体,不仅能与重组蛋白反应,还可与人体内的天然蛋白结合,是国内首次报道制备出了针对人血清天然apoM蛋白的鼠抗人单克隆抗体,为下一步定量检测人血清中apoM蛋白含量打下了基础。
第三部分EL ISA法测定血清中载脂蛋白M浓度的方法学评价及其与血脂相关性分析
研究背景：
apoM主要存在于HDL中,在肝脏和肾脏中表达,提示该蛋白与血脂的转运和代谢有关。最近Xu等发现血液中apoM浓度与体重指数(BMI)以及血液中瘦素(Leptin)含量呈正相关,而与血液中总胆固醇呈负相关,但相关机制还未明确。另外,apoM属于Lipocalin超家族,推测可能与血浆中疏水性生物活性分子的结合、转运和代谢有关联,其确切功能尚有待进一步研究。目前国内定量检测apoM浓度的方法仅限于半定量的Western印迹和Dot-Blotting法,需要建立标准统一、方法稳定、操作简单的定量检测方法,这对于临床研究的深入进行将有极大的帮助。
目的：
建立测定人血浆中apoM浓度的双抗体夹心法,并对该方法的敏感性、重复性、抗干扰能力等作一评价；并建立该方法的正常人参考范围；观察血浆中apoM与血脂之间的相关关系；初步比较正常人与冠心病各组人群之间血浆apoM浓度的变化趋势。
方法：
用棋盘滴定法选择包被抗体和酶标抗体的最佳工作浓度；用辣根过氧化物酶标记抗apoM 8F12C6抗体；用蛋白稀释度曲线确定蛋白标准曲线的范围；用血浆稀释度曲线确定实验中人血浆的稀释倍数；确定了该方法的最低检测限,测定批间、内变异及非特异性脂蛋白的干扰,以观察方法的敏感性和特异性。通过测定124例健康体检人群血浆apoM的浓度,建立本实验室的正常参考值范围。比较稳定型心绞痛组(SA)32例和急性冠脉综合征组(ACS)34例病人与35例健康人群的血浆蛋白浓度,分析apoM在不同人群中的变化趋势。
结果：
1、根据重组蛋白制备标准曲线,该方法的线性范围为31.25-375ng/ml,最低检测限为11.8 ng/ml。批内变异4.32-5.69%；批间变异5.24-5.83%。
2、血浆滴度曲线结果表明,人血浆在1：50到1：400范围内线性关系良好,取线性范围中间整数倍1：200稀释度,,作为实验中人血浆apoM浓度检测的最佳稀释倍数。
3、不同浓度的脂蛋白apoAI、apoB及Lp(a)对该法干扰小,其测定的吸光度值均低于0.33,在该方法的线性范围外。
4、该方法的正常参考值范围为13.15±2.68μg/ml。
5、apoM与TG、TC及LDL-C呈负相关趋势,但差异并无统计学意义；与HDL-C、HDL-C/TC呈正相关(r=0.336和r=0.345)。
6、两组冠心病患者血浆apoM浓度(SA组11.02±1.96μg/ml, ACS组10.76±1.32μg/ml)均低于正常对照组(12.83±2.28μg/ml),但两组冠心病组之间浓度差异不明显。
结论：
我们建立了测定人血浆中apoM的浓度的双抗体夹心法,通过检测该方法的敏感性、重复性、抗干扰能力等指标,认为该方法稳定性、敏感性和特异性均好,与国内外检测apoM浓度的方法相比,我们建立的ELISA法具有操作简单,标准蛋白来源恒定,易于大批检测和适应临床检测的特点,为apoM在临床研究中的广泛开展提供了方便、可靠的工具。
Chapter One Expression and Purification of apolipoprotein M
Background:
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-antibody
Background:
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 lipids
Background:
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/ml
5. 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.
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[6]Faber K, Axler O, Dahlback B, Nielsen LB. Characterization of apoM in normal and genetically modified mice. J Lipid Res,2004; 45(7):1272-8
[7]Zhang XY, Dong X, Zheng L, et al. Specific tissue expression and cellular localization of human apolipoprotein M as determined by in situ hybridization. Acta Histochem,2003;105(1):67-72
[8]Faber K, Hvidberg V, Moestrup SK, Dahlback B, Nielsen LB. Megalin is a receptor for apolipoprotein M, and kidney-specific megalin-deficiency confers urinary excretion of apolipoprotein M. Mol Endocrinol,2006; 20(1):212-8
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[11]Xu N, Nilsson-Ehle P, Hurtig M, Ahren B. Both leptin and leptin-receptor are essential for apolipoprotein M expression in vivo. Biochem Biophys Res Commun,2004;321(4):916-21
[12]Xu N, Nilsson-Ehle P, Ahren B. Correlation of apolipoprotein M with leptin and cholesterol in normal and obese subjects. J Nutr Biochem 2004;15(10):579-82
[13]Xu N, Zhang XY, Dong X, Ekstrom U, Ye Q, Nilsson-Ehle P. Effects of platelet-activating factor, tumor necrosis factor, and interleukin-alpha on the expression of apolipoprotein M in HepG2 cells. Biochem Biophys Res Commun, 2002;292(4):944-50
[14]Richter S, Shih DQ, Pearson ER, Wolfram C, Fajans SS, Hattersley AT, Stoffel M. Regulation of apolipoprotein M gene expression by MODY3 gene hepatocyte nuclear factor-lalpha:haploinsufficiency is associated with reduced serum apolipoprotein M levels. Diabetes,2003;52(12):2989-95
[15]焦国庆,张晓膺.甲基泼尼松龙对肾癌细胞株786-0载脂蛋白M的影响.南京医科大学学报,2005；25(3)：166-168
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