猪β-防御素2多克隆和单克隆抗体的制备及初步应用
发布时间：2018-10-12 18:52
【摘要】： 抗菌肽是由动物机体自身分泌的抵抗外界病原微生物的一类微量的重要蛋白质,它在动物的先天免疫中发挥十分重要的作用。因其独特的抗菌机制、广泛的抗菌谱以及不易产生耐药性等特点,近年来成为研究的热门。防御素是其中一类重要的小分子阳离子抗菌肽。目前有不少研究者试图通过转基因动物大量表达防御素,以增强转基因动物的抗病能力。为了建立检测猪β-防御素2(PBD2)在猪和转基因动物中的表达,本研究制备了PBD2的多克隆抗体和单克隆抗体,建立了间接免疫荧光和免疫组织化学检测方法。 本研究根据PBD2的氨基酸序列,人工合成了PBD2的11肽,与钥匙孔血蓝蛋白KLH偶联成全抗原PBD2-KLH,免疫Balb/C小鼠,三次免疫后,以合成的37肽作为PBD2抗体的检测抗原,检测小鼠血清效价,均达到1：12800以上,取其血清制备小鼠抗PBD2的多克隆抗体,并利用该鼠源PBD2多抗建立了PBD2的间接免疫荧光检测方法。 通过间接ELISA方法筛选出抗体水平高的小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,用间接ELISA方法筛选出阳性克隆,用有限稀释法进行两次亚克隆,最后获得5株杂交瘤细胞株1C2、1F4、2H2、3A2和4B1,均能够稳定分泌特异性好、效价高的PBD2单克隆抗体,其细胞培养上清的抗体ELISA效价达到1：210以上,腹水的抗体的ELISA效价达到1：106以上。 分别取临床健康猪和胸膜肺炎放线杆菌血清1型4074株感染猪的肝脏、脾脏、肠系膜淋巴结、小肠等组织,用制备的单克隆抗体对其组织切片进行SABC免疫组化染色,分析PBD2在不同组织中的表达分布及表达差异。结果表明,猪PBD2在肝脏中表达量最高,PBD2主要分布于肝脏的血管内皮细胞,肝细胞中也有广泛分布,在细胞质和细胞核中均有分布。不同动物和组织中PBD2的表达差异需要进一步结合荧光定量PCR和双抗夹心ELISA进行验证。
[Abstract]:Antimicrobial peptide is a kind of important protein secreted by animal body to resist external pathogenic microorganisms. It plays a very important role in animal innate immunity. Due to its unique antibacterial mechanism, extensive antibacterial spectrum and resistance to drug resistance, it has become a hot topic in recent years. Defensins are one of the most important cationic antimicrobial peptides. At present, many researchers try to express defensins in large quantities through transgenic animals, in order to enhance the disease resistance of transgenic animals. In order to detect the expression of porcine 尾 -defensin 2 (PBD2) in pigs and transgenic animals, the polyclonal antibody and monoclonal antibody of PBD2 were prepared, and indirect immunofluorescence and immunohistochemical methods were established. According to the amino acid sequence of PBD2, the 11-peptide of PBD2 was synthesized and conjugated with keyhole hemocyanin KLH to complete antigen PBD2-KLH, to immunize Balb/C mice. After three times of immunization, the synthesized 37 peptide was used as the detection antigen of PBD2 antibody to detect the titer of mouse serum. All of them were above 1: 12800. The polyclonal antibody against PBD2 in mice was prepared from its serum, and the indirect immunofluorescence detection method for PBD2 was established by using the polyantibody of mouse PBD2. Mice with high antibody level were screened by indirect ELISA method, spleen cells were fused with SP2/0 myeloma cells, positive clones were screened by indirect ELISA method, and subclones were subcloned by limited dilution method. Finally, five hybridoma cell lines 1C2F4F4H2H2O3A2 and 4B1 were obtained. The monoclonal antibodies of PBD2 with good specificity and high titer could be secreted stably. The antibody ELISA titer of supernatant of cell culture was more than 1: 210, and the ELISA titer of ascites antibody was more than 1: 106. The liver, spleen, mesenteric lymph nodes and small intestine of 4074 strains of Actinobacillus pleuropneumoniae serotype 1 were collected from healthy pigs and Actinobacillus pleuropneumoniae respectively. The sections were stained by SABC immunohistochemical staining with monoclonal antibodies. To analyze the distribution and difference of PBD2 expression in different tissues. The results showed that the expression of porcine PBD2 was the highest in the liver. PBD2 was mainly distributed in the vascular endothelial cells of the liver, and was also widely distributed in the liver cells, and also in the cytoplasm and nucleus. The differences of PBD2 expression in different animals and tissues need to be further verified by fluorescence quantitative PCR and double antibody sandwich ELISA.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2267228
[Abstract]:Antimicrobial peptide is a kind of important protein secreted by animal body to resist external pathogenic microorganisms. It plays a very important role in animal innate immunity. Due to its unique antibacterial mechanism, extensive antibacterial spectrum and resistance to drug resistance, it has become a hot topic in recent years. Defensins are one of the most important cationic antimicrobial peptides. At present, many researchers try to express defensins in large quantities through transgenic animals, in order to enhance the disease resistance of transgenic animals. In order to detect the expression of porcine 尾 -defensin 2 (PBD2) in pigs and transgenic animals, the polyclonal antibody and monoclonal antibody of PBD2 were prepared, and indirect immunofluorescence and immunohistochemical methods were established. According to the amino acid sequence of PBD2, the 11-peptide of PBD2 was synthesized and conjugated with keyhole hemocyanin KLH to complete antigen PBD2-KLH, to immunize Balb/C mice. After three times of immunization, the synthesized 37 peptide was used as the detection antigen of PBD2 antibody to detect the titer of mouse serum. All of them were above 1: 12800. The polyclonal antibody against PBD2 in mice was prepared from its serum, and the indirect immunofluorescence detection method for PBD2 was established by using the polyantibody of mouse PBD2. Mice with high antibody level were screened by indirect ELISA method, spleen cells were fused with SP2/0 myeloma cells, positive clones were screened by indirect ELISA method, and subclones were subcloned by limited dilution method. Finally, five hybridoma cell lines 1C2F4F4H2H2O3A2 and 4B1 were obtained. The monoclonal antibodies of PBD2 with good specificity and high titer could be secreted stably. The antibody ELISA titer of supernatant of cell culture was more than 1: 210, and the ELISA titer of ascites antibody was more than 1: 106. The liver, spleen, mesenteric lymph nodes and small intestine of 4074 strains of Actinobacillus pleuropneumoniae serotype 1 were collected from healthy pigs and Actinobacillus pleuropneumoniae respectively. The sections were stained by SABC immunohistochemical staining with monoclonal antibodies. To analyze the distribution and difference of PBD2 expression in different tissues. The results showed that the expression of porcine PBD2 was the highest in the liver. PBD2 was mainly distributed in the vascular endothelial cells of the liver, and was also widely distributed in the liver cells, and also in the cytoplasm and nucleus. The differences of PBD2 expression in different animals and tissues need to be further verified by fluorescence quantitative PCR and double antibody sandwich ELISA.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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相关博士学位论文 前1条
1 马卫明;猪小肠抗菌肽分离鉴定及其生物活性研究[D];中国农业大学;2004年
相关硕士学位论文 前2条
1 刘红珍;兔肠源抗菌蛋白的分离纯化及其生物活性研究[D];山东农业大学;2007年
2 郑杰;分泌抗猪流感病毒（河南株）单克隆抗体杂交瘤细胞株的制备、鉴定和初步应用[D];河南农业大学;2004年
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