豬β-防御素2多克隆和單克隆抗體的制備及初步應(yīng)用
發(fā)布時(shí)間：2018-10-12 18:52
【摘要】： 抗菌肽是由動(dòng)物機(jī)體自身分泌的抵抗外界病原微生物的一類微量的重要蛋白質(zhì),它在動(dòng)物的先天免疫中發(fā)揮十分重要的作用。因其獨(dú)特的抗菌機(jī)制、廣泛的抗菌譜以及不易產(chǎn)生耐藥性等特點(diǎn),近年來(lái)成為研究的熱門。防御素是其中一類重要的小分子陽(yáng)離子抗菌肽。目前有不少研究者試圖通過(guò)轉(zhuǎn)基因動(dòng)物大量表達(dá)防御素,以增強(qiáng)轉(zhuǎn)基因動(dòng)物的抗病能力。為了建立檢測(cè)豬β-防御素2(PBD2)在豬和轉(zhuǎn)基因動(dòng)物中的表達(dá),本研究制備了PBD2的多克隆抗體和單克隆抗體,建立了間接免疫熒光和免疫組織化學(xué)檢測(cè)方法。 本研究根據(jù)PBD2的氨基酸序列,人工合成了PBD2的11肽,與鑰匙孔血藍(lán)蛋白KLH偶聯(lián)成全抗原PBD2-KLH,免疫Balb/C小鼠,三次免疫后,以合成的37肽作為PBD2抗體的檢測(cè)抗原,檢測(cè)小鼠血清效價(jià),均達(dá)到1：12800以上,取其血清制備小鼠抗PBD2的多克隆抗體,并利用該鼠源PBD2多抗建立了PBD2的間接免疫熒光檢測(cè)方法。 通過(guò)間接ELISA方法篩選出抗體水平高的小鼠,取其脾細(xì)胞與SP2/0骨髓瘤細(xì)胞融合,用間接ELISA方法篩選出陽(yáng)性克隆,用有限稀釋法進(jìn)行兩次亞克隆,最后獲得5株雜交瘤細(xì)胞株1C2、1F4、2H2、3A2和4B1,均能夠穩(wěn)定分泌特異性好、效價(jià)高的PBD2單克隆抗體,其細(xì)胞培養(yǎng)上清的抗體ELISA效價(jià)達(dá)到1：210以上,腹水的抗體的ELISA效價(jià)達(dá)到1：106以上。 分別取臨床健康豬和胸膜肺炎放線桿菌血清1型4074株感染豬的肝臟、脾臟、腸系膜淋巴結(jié)、小腸等組織,用制備的單克隆抗體對(duì)其組織切片進(jìn)行SABC免疫組化染色,分析PBD2在不同組織中的表達(dá)分布及表達(dá)差異。結(jié)果表明,豬PBD2在肝臟中表達(dá)量最高,PBD2主要分布于肝臟的血管內(nèi)皮細(xì)胞,肝細(xì)胞中也有廣泛分布,在細(xì)胞質(zhì)和細(xì)胞核中均有分布。不同動(dòng)物和組織中PBD2的表達(dá)差異需要進(jìn)一步結(jié)合熒光定量PCR和雙抗夾心ELISA進(jìn)行驗(yàn)證。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
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1 馬衛(wèi)明;豬小腸抗菌肽分離鑒定及其生物活性研究[D];中國(guó)農(nóng)業(yè)大學(xué);2004年
相關(guān)碩士學(xué)位論文 前2條
1 劉紅珍;兔腸源抗菌蛋白的分離純化及其生物活性研究[D];山東農(nóng)業(yè)大學(xué);2007年
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，本文編號(hào)：2267228
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