树鼩IgG分离纯化与抗体制备
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摘要
目前全球大约有1.7～2.0亿人感染丙型肝炎病毒(HCV),约占全球总人口的3%。感染HCV后,大部分感染者转变为慢性感染,还有较大比例的病人最终发展成肝硬化甚至肝癌,这表明丙型肝炎已成为威胁人类健康的一个主要公共卫生问题。由于缺少合适的动物模型,严重阻碍了人们对HCV发病机理、治疗药物及相关疫苗的研究。黑猩猩是目前已证实的唯一对HCV易感的动物模型,但是其价格昂贵,且濒临灭绝,不易推广使用。因此,寻找经济实用的动物模型就成为目前丙型肝炎研究的当务之急。国内外许多研究学者发现树鼩——一种类似松鼠的小型哺乳动物具有成为HCV动物模型的潜力,但树鼩作为动物模型当前最大的困难就是没有合适的检测工具,即各种商业化的检测抗体,而制备多克隆或单克隆抗体的关键就是分离纯化出纯度较高的免疫球蛋白。
本研究采用辛酸-硫酸铵沉淀并结合Sephacryl-S200凝胶过滤层析和DEAE-Sephadex A-50阴离子交换层析分离纯化树鼦血清免疫球蛋白G(IgG)。SDS-PAGE结果表明纯化的IgG纯度较高,同时测定了树鼦血清中IgG的分子量约为160kD,其重链分子量为53.5kD,轻链分子量为28.4kD。以提纯的免疫球蛋白为抗原免疫新西兰兔,制备了兔抗树鼦IgG多克隆抗体,免疫双扩散法检测多克隆抗体效价达到1∶8,酶联免疫吸附(ELISA)检测效价达1∶25600;同时建立了检测抗体的间接ELISA法,实验的最佳反应条件:抗原包被浓度为1.2μg/ml,待测血清的最佳稀释度为1∶1000,酶标抗体的最佳工作浓度为1∶8000。运用免疫印记法(Western-blotting)检测多克隆抗体的特异性,结果表明所制备的兔抗树鼩IgG抗体可以用于树鼩血清中抗原特异性IgG的检测,其能与树鼩血清中IgG的重链、轻链反应。以上结果表明我们获得了高效价、高特异性的兔抗树鼩IgG多克隆抗体。
以纯化的IgG作为抗原,与等量弗氏完全佐剂(第一次)、弗氏不完全佐剂(第二次)混合后皮下及腹腔注射免疫6周龄BALB/c小鼠(100μg/只),每次间隔2周。静脉加强免疫3天后,取免疫小鼠的脾细胞和对HAT敏感的骨髓瘤细胞SP2/0细胞进行融合。本研究采用化学法融合,融合剂为分子量1500的聚乙二醇(PEG-1500)。融合过程在50ml灭菌离心管中进行,按5∶1的比例将脾细胞和骨髓瘤细胞置于离心管内,使之充分接触,按严格的剂量逐滴加入融合剂使细胞发生融合,然后加入HAT培养基,滴加到铺满饲养细胞(小鼠腹腔巨噬细胞)的96孔培养板内培养,第二天检查是否污染。当细胞长满培养板时,取细胞上清液,用间接EUSA进行阳性孔的筛选。
我们对树鼩血清免疫球蛋白的纯化进行了探讨,得到了高纯度的树鼩IgG,并制备了兔抗树鼩多克隆抗体,同时对树鼩IgG单克隆抗体进行了初步制备,为树鼩在抗体方面的检测提供了有效的工具,并为HCV疫苗评价奠定了基础。
It is estimated that 170 to 200 million people are chronically infected with HCV worldwide, which up to 3% of the total population, approximately 3 to 4 million new infected cases occurring each year. The majority of HCV-infected individuals may developed into chronic hepatitis, which led to liver cirrhosis and hepatocellular carcinoma(HCC) after a long progress. Following with AIDS, hepatitis C is becoming a pathogen which threat human health seriously. So far, we understand HCV and hepatitis C poorly. Progress in the pathogenic mechanisms of HCV, the development of a vaccine against HCV and therapy of hepatitis C has been hampered by the lack of an appropriate small animal model. Although chimpanzees are easily infected with HCV, these animals are rare expensive and thus inappropriate for use in research. Therefore, the establish of a small animal model, which easy to breed and with low hold cost, would be of great importance in facilitating progress of HCV research. The HCV susceptibility on tree shrew has been proved by previous researches. However, there are still some impediments in the detection of antibody in tree shrew serum, which is important for evaluation on immune response. There are not commercial kits or methods yet. It is necessary to isolate and purify immunoglobulin from this animal, and then prepare polyclonal antibody and monoclonal antibody.
Here, tree shrew serum IgG was purified by caprylic acid-ammonium sulfate precipitation, and then purified by Sephacryl-S200 gel filtration and DEAE-Sephadex A-50 anion-exchange chromatogram. The result of SDS-PAGE revealed that the IgG had been further purified by each step of purification. The molecular weight of IgG is 160kD, and that of heavy chain is 53.5kD, while that of light chain is 28.4kD. The purified IgG was used as the antigen to immunize rabbit, then the anti-IgG polyclonal antibody was prepared. The determined titer of anti-serum with double immunodiffusion was 1:8, and the highest titer of the anti-serum determined by indirect ELISA procedure was 1:25600. An indirect ELISA for detecting serum antibody was established. The optimized reaction system is 1.25μg/ml of coated antigen in working concentration, and the optimum diluted concentration of the tested serum is 1:1000, and the optimum diluted concentration of the enzyme-linked antibody is 1:8000. The result of western blotting showed that the antibody can be used to detect antigen-specific IgG and had immunological activity by reaction with heavy chain and light chain of tree shrew serum IgG. An anti-IgG polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with tree shrew IgG.
The 6-week-old female BALB/c mice were immunized subcutaneously and intraperitoneally with 100μg purified IgG, which emulsified in Freund's complete adjuvant for the first primary injection and in Freund's incomplete adjuvant for the three enhanced immunization. Injections were given at 2-week intervals, followed by an intravenous dose of the antigen without adjuvant. On the third day after the final immunization, splenocytes were isolated and fused with a HAT-sensitive mouse myelomane SP2/0, by the polyethylene glycol (PEG) method. Highly purified Polyethylene 1500(PEG-1500) was used for cell fusion. Cell fusion was performed in a 50ml centrifuge tube. Mingle splenocytes and myeloma cells (5:1) into the centrifuge tube, and then instill PEG-1500 scrupulously at a certain dose. Put the mixed cells into the cell culture wells that had been paved with feeder cells, and selected-culture with HAT. The screening of positive cells with indirect ELISA is going on.
In summary, the methods of immunoglobulin purification from tree shrew serum, and preparation of the polyclonal antibody on rabbit have been established. Meanwhile, we have prepared purified IgG and high titer polyclonal antibody. Monoclonal antibody was also prepared preliminary. All of these provide a useful tool for tree shrew antibody detection, will be the basis for HCV vaccine evaluation.
本研究采用辛酸-硫酸铵沉淀并结合Sephacryl-S200凝胶过滤层析和DEAE-Sephadex A-50阴离子交换层析分离纯化树鼦血清免疫球蛋白G(IgG)。SDS-PAGE结果表明纯化的IgG纯度较高,同时测定了树鼦血清中IgG的分子量约为160kD,其重链分子量为53.5kD,轻链分子量为28.4kD。以提纯的免疫球蛋白为抗原免疫新西兰兔,制备了兔抗树鼦IgG多克隆抗体,免疫双扩散法检测多克隆抗体效价达到1∶8,酶联免疫吸附(ELISA)检测效价达1∶25600;同时建立了检测抗体的间接ELISA法,实验的最佳反应条件:抗原包被浓度为1.2μg/ml,待测血清的最佳稀释度为1∶1000,酶标抗体的最佳工作浓度为1∶8000。运用免疫印记法(Western-blotting)检测多克隆抗体的特异性,结果表明所制备的兔抗树鼩IgG抗体可以用于树鼩血清中抗原特异性IgG的检测,其能与树鼩血清中IgG的重链、轻链反应。以上结果表明我们获得了高效价、高特异性的兔抗树鼩IgG多克隆抗体。
以纯化的IgG作为抗原,与等量弗氏完全佐剂(第一次)、弗氏不完全佐剂(第二次)混合后皮下及腹腔注射免疫6周龄BALB/c小鼠(100μg/只),每次间隔2周。静脉加强免疫3天后,取免疫小鼠的脾细胞和对HAT敏感的骨髓瘤细胞SP2/0细胞进行融合。本研究采用化学法融合,融合剂为分子量1500的聚乙二醇(PEG-1500)。融合过程在50ml灭菌离心管中进行,按5∶1的比例将脾细胞和骨髓瘤细胞置于离心管内,使之充分接触,按严格的剂量逐滴加入融合剂使细胞发生融合,然后加入HAT培养基,滴加到铺满饲养细胞(小鼠腹腔巨噬细胞)的96孔培养板内培养,第二天检查是否污染。当细胞长满培养板时,取细胞上清液,用间接EUSA进行阳性孔的筛选。
我们对树鼩血清免疫球蛋白的纯化进行了探讨,得到了高纯度的树鼩IgG,并制备了兔抗树鼩多克隆抗体,同时对树鼩IgG单克隆抗体进行了初步制备,为树鼩在抗体方面的检测提供了有效的工具,并为HCV疫苗评价奠定了基础。
It is estimated that 170 to 200 million people are chronically infected with HCV worldwide, which up to 3% of the total population, approximately 3 to 4 million new infected cases occurring each year. The majority of HCV-infected individuals may developed into chronic hepatitis, which led to liver cirrhosis and hepatocellular carcinoma(HCC) after a long progress. Following with AIDS, hepatitis C is becoming a pathogen which threat human health seriously. So far, we understand HCV and hepatitis C poorly. Progress in the pathogenic mechanisms of HCV, the development of a vaccine against HCV and therapy of hepatitis C has been hampered by the lack of an appropriate small animal model. Although chimpanzees are easily infected with HCV, these animals are rare expensive and thus inappropriate for use in research. Therefore, the establish of a small animal model, which easy to breed and with low hold cost, would be of great importance in facilitating progress of HCV research. The HCV susceptibility on tree shrew has been proved by previous researches. However, there are still some impediments in the detection of antibody in tree shrew serum, which is important for evaluation on immune response. There are not commercial kits or methods yet. It is necessary to isolate and purify immunoglobulin from this animal, and then prepare polyclonal antibody and monoclonal antibody.
Here, tree shrew serum IgG was purified by caprylic acid-ammonium sulfate precipitation, and then purified by Sephacryl-S200 gel filtration and DEAE-Sephadex A-50 anion-exchange chromatogram. The result of SDS-PAGE revealed that the IgG had been further purified by each step of purification. The molecular weight of IgG is 160kD, and that of heavy chain is 53.5kD, while that of light chain is 28.4kD. The purified IgG was used as the antigen to immunize rabbit, then the anti-IgG polyclonal antibody was prepared. The determined titer of anti-serum with double immunodiffusion was 1:8, and the highest titer of the anti-serum determined by indirect ELISA procedure was 1:25600. An indirect ELISA for detecting serum antibody was established. The optimized reaction system is 1.25μg/ml of coated antigen in working concentration, and the optimum diluted concentration of the tested serum is 1:1000, and the optimum diluted concentration of the enzyme-linked antibody is 1:8000. The result of western blotting showed that the antibody can be used to detect antigen-specific IgG and had immunological activity by reaction with heavy chain and light chain of tree shrew serum IgG. An anti-IgG polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with tree shrew IgG.
The 6-week-old female BALB/c mice were immunized subcutaneously and intraperitoneally with 100μg purified IgG, which emulsified in Freund's complete adjuvant for the first primary injection and in Freund's incomplete adjuvant for the three enhanced immunization. Injections were given at 2-week intervals, followed by an intravenous dose of the antigen without adjuvant. On the third day after the final immunization, splenocytes were isolated and fused with a HAT-sensitive mouse myelomane SP2/0, by the polyethylene glycol (PEG) method. Highly purified Polyethylene 1500(PEG-1500) was used for cell fusion. Cell fusion was performed in a 50ml centrifuge tube. Mingle splenocytes and myeloma cells (5:1) into the centrifuge tube, and then instill PEG-1500 scrupulously at a certain dose. Put the mixed cells into the cell culture wells that had been paved with feeder cells, and selected-culture with HAT. The screening of positive cells with indirect ELISA is going on.
In summary, the methods of immunoglobulin purification from tree shrew serum, and preparation of the polyclonal antibody on rabbit have been established. Meanwhile, we have prepared purified IgG and high titer polyclonal antibody. Monoclonal antibody was also prepared preliminary. All of these provide a useful tool for tree shrew antibody detection, will be the basis for HCV vaccine evaluation.
引文
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