猪细小病毒VP2蛋白单克隆抗体的制备及鉴定
发布时间：2018-11-11 19:18
【摘要】：猪细小病毒(Porcine Parvovirus,PPV)是引起猪繁殖障碍的主要病原之一。PPV的流行范围非常广泛,在世界各地均有感染,给养猪业和畜牧业造成了巨大的经济损失。VP2蛋白是PPV的结构蛋白之一,也是构成该病毒的主要衣壳蛋白,其携带了主要的抗原决定簇,决定着病毒的组织嗜性、抗原性、致病性等多种主要的生物学特性。本研究将实验室保存的重组杆状病毒Ac NPV-VP2同步接种于昆虫细胞Sf9,36 h后收集病毒,并进行SDS-PAGE电泳,随后利用Western blot对其结果进行分析,结果显示蛋白成功的获得了高效表达。通过Ni柱树脂纯化,以及切胶纯化等方法,将表达的VP2蛋白进行纯化,并利用抗His标签蛋白单抗作为一抗,对纯化后的蛋白进行Western blot分析,结果表明,纯化效果良好,蛋白可用于后续的研究。将纯化后得到的VP2蛋白作为免疫原,采用皮下注射的方式免疫6周龄的BALB/c小鼠,经过三次基础免疫和一次加强免疫后,利用细胞融合技术,将脾细胞与杂交瘤细胞进行融合。之后用间接ELISA方法进行筛选,经过3次以上细胞亚克隆,最终成功获得了1B3、2E5、5F8三株能够稳定分泌的抗VP2蛋白抗体的杂交瘤细胞。经鉴定,3株杂交瘤细胞的亚型均为IgG1型;杂交瘤细胞培养上清效价为1:800-1 000,将杂交瘤细胞按适当的量腹腔注射已经注射石蜡一周的BALB/c小鼠,检测获得的腹水效价为1:32 000-64 000。为进一步鉴定3株单克隆抗体的免疫反应性,收集同步接种猪细小病毒后的ST细胞,并以此为检测用免疫原,检测单克隆抗体的免疫原性。对其及进行了Western blot检测,结果表明3株单抗均可以对PPV全病毒以及重组VP2蛋白特异性识别;间接免疫荧光结果表明,3株单抗均产生荧光,可以与PPV全病毒发生反应;特异性试验表明,3株单抗均不与TEGV和PEDV等病毒发生反应,仅特异性与PPV发生反应;将在体外连续传代3个月和冻存后再复苏的3株杂交瘤细胞做了稳定性分析,结果表明,3株单抗的稳定性较好。本研究成功筛选了3株具有较高的反应原性和特异性的猪细小病毒结构蛋白VP2的单克隆抗体,为后期建立有效的PPV特异性血清学检测方法奠定了理论依据,同时也为PPV受体及感染机制的研究提供了物质基础。
[Abstract]:Porcine parvovirus (Porcine Parvovirus,PPV) is one of the major pathogens causing reproductive disorders in pigs. PPV is widely prevalent and has been infected all over the world. VP2 protein is one of the structural proteins of PPV, and is also the main capsid protein of the virus. It carries the main antigenic determinant and determines the tissue and antigenicity of the virus. Pathogenicity and other major biological characteristics. In this study, recombinant baculovirus (Ac NPV-VP2) stored in laboratory was inoculated into insect cell Sf9,36 h and then collected by SDS-PAGE electrophoresis. The results were analyzed by Western blot. The results showed that the protein was highly expressed successfully. The expressed VP2 protein was purified by Ni column resin purification, and the monoclonal antibody against His label protein was used as the first antibody, and the purified protein was analyzed by Western blot. The results showed that the purification effect was good. Proteins can be used in subsequent studies. The purified VP2 protein was used as immunogen to immunize 6-week-old BALB/c mice by subcutaneous injection. After three basic immunization and one enhanced immunization, cell fusion technique was used. Spleen cells were fused with hybridoma cells. After being screened by indirect ELISA method, three hybridoma cells, 1B3O2E5O5F8, which could stably secrete antibodies against VP2 protein, were successfully obtained after more than 3 cell subclones. The subtypes of three hybridoma cells were identified as IgG1 type. The supernatant titer of hybridoma cell culture was 1: 800-1 000. The ascites titer of BALB/c mice which had been injected paraffin for one week was intraperitoneally injected with the appropriate amount of hybridoma cells. The ascites titer was between 1:32 and 64 000. In order to further identify the immunoreactivity of three strains of monoclonal antibodies, the ST cells inoculated with porcine parvovirus were collected and used as immunogen to detect the immunogenicity of monoclonal antibodies. The results of Western blot detection showed that all the three McAbs could specifically recognize the whole PPV virus and the recombinant VP2 protein, and the indirect immunofluorescence showed that all the McAbs produced fluorescence and reacted with the whole PPV virus. The specific test showed that the three McAbs did not react with TEGV and PEDV, but only with PPV. The stability of the three hybridoma cells which were subcultured in vitro for 3 months and resuscitated after cryopreservation were analyzed. The results showed that the stability of the three McAbs was better. In this study, three monoclonal antibodies against porcine parvovirus structural protein (VP2) with high reactivity and specificity were successfully screened, which laid a theoretical foundation for the establishment of an effective method for the detection of PPV specific serology. It also provides a material basis for the study of PPV receptor and infection mechanism.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651
本文编号：2325864
[Abstract]:Porcine parvovirus (Porcine Parvovirus,PPV) is one of the major pathogens causing reproductive disorders in pigs. PPV is widely prevalent and has been infected all over the world. VP2 protein is one of the structural proteins of PPV, and is also the main capsid protein of the virus. It carries the main antigenic determinant and determines the tissue and antigenicity of the virus. Pathogenicity and other major biological characteristics. In this study, recombinant baculovirus (Ac NPV-VP2) stored in laboratory was inoculated into insect cell Sf9,36 h and then collected by SDS-PAGE electrophoresis. The results were analyzed by Western blot. The results showed that the protein was highly expressed successfully. The expressed VP2 protein was purified by Ni column resin purification, and the monoclonal antibody against His label protein was used as the first antibody, and the purified protein was analyzed by Western blot. The results showed that the purification effect was good. Proteins can be used in subsequent studies. The purified VP2 protein was used as immunogen to immunize 6-week-old BALB/c mice by subcutaneous injection. After three basic immunization and one enhanced immunization, cell fusion technique was used. Spleen cells were fused with hybridoma cells. After being screened by indirect ELISA method, three hybridoma cells, 1B3O2E5O5F8, which could stably secrete antibodies against VP2 protein, were successfully obtained after more than 3 cell subclones. The subtypes of three hybridoma cells were identified as IgG1 type. The supernatant titer of hybridoma cell culture was 1: 800-1 000. The ascites titer of BALB/c mice which had been injected paraffin for one week was intraperitoneally injected with the appropriate amount of hybridoma cells. The ascites titer was between 1:32 and 64 000. In order to further identify the immunoreactivity of three strains of monoclonal antibodies, the ST cells inoculated with porcine parvovirus were collected and used as immunogen to detect the immunogenicity of monoclonal antibodies. The results of Western blot detection showed that all the three McAbs could specifically recognize the whole PPV virus and the recombinant VP2 protein, and the indirect immunofluorescence showed that all the McAbs produced fluorescence and reacted with the whole PPV virus. The specific test showed that the three McAbs did not react with TEGV and PEDV, but only with PPV. The stability of the three hybridoma cells which were subcultured in vitro for 3 months and resuscitated after cryopreservation were analyzed. The results showed that the stability of the three McAbs was better. In this study, three monoclonal antibodies against porcine parvovirus structural protein (VP2) with high reactivity and specificity were successfully screened, which laid a theoretical foundation for the establishment of an effective method for the detection of PPV specific serology. It also provides a material basis for the study of PPV receptor and infection mechanism.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651
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