鸭新城疫病毒HN蛋白单克隆抗体的制备及间接ELISA方法的建立
发布时间：2018-11-26 17:50
【摘要】：鸭新城疫,又称为鸭副黏病毒病(Duck paramyxovirus disease,DPMV)是由禽副黏病毒I型(APMV-I)引起的一种急性、高度接触性传染病,各日龄鸭均易感,给养鸭业带来严重经济损失。新城疫病毒粒子囊膜表面包含两种糖蛋白,分别是血凝素-神经氨酸酶蛋白(hemagglutinin-neuraminidase protein,HN)和融合蛋白(fusion protein,F)。其中HN蛋白与病毒的毒力及致病性密切相关,是主要的保护性抗原,在新城疫病毒(NDV)的研究中具有十分重要的意义。本研究以Gen Bank已公布的鸭源NDV SDWF02株HN基因序列(Gen Bank登录号:HM188399)为模板,在其抗原位点分布较为丰富区域,设计并合成一对特异性引物,运用RT-PCR的方法扩增出鸭源NDV的HN基因片段,经测序分析后,将阳性重组质粒进行原核表达,制备出针对HN蛋白的单克隆抗体,为鸭源NDV的深入研究及临床检测提供了新途径。一、鸭源新城疫病毒HN基因的克隆及分析取实验室保存的鸭源NDV SDWF02株病毒,传胚收集尿囊液,提取RNA,运用设计的特异性引物,经RT-PCR的方法成功扩增出了NDV SDWF02株的HN基因片段,并将扩增片段克隆至p MD18-T Vector载体中。经测序分析,目的基因的核苷酸序列长度为822 bp,与鸭源NDV SDWF02株碱基序列一致。二、NDV HN蛋白的表达及间接ELISA(HN-ELISA)的建立将含有的HN基因片段的阳性重组载体p MD18-T-HN经Sac I和Hind III双酶切,并将产物连接至p ET-28a表达载体。再将含有HN基因片段的阳性重组质粒转化到E.coli Rosetta感受态细胞中,筛选含重组质粒p ET-28a-HN的重组菌,经IPTG诱导表达出融合蛋白,SDS-PAGE分析表明,获得的融合蛋白主要以包涵体的形式出现,分子量约为30.5 k Da,与预期大小相一致,选用Ni-NTA Resin对融合蛋白进行纯化,Western blot结果显示,该融合蛋白能够特异性识别抗鸭NDV血清,具有良好的免疫原性。并利用纯化的HN蛋白作为包被抗原,对抗原包被浓度、血清稀释度及包被条件等进行探索,建立出检测鸭源NDV抗体的间接ELISA方法。三、抗NDV HN蛋白单克隆抗体的制备及鉴定以NDV HN重组蛋白作为免疫原免疫Balb/c小鼠后,三次免疫后,经加强疫取免疫小鼠的脾淋巴细胞与SP2/0骨髓瘤细胞融合,经间接ELISA方法检测筛选出阳性杂交瘤细胞株,并采用有限稀释法对其进行亚克隆,获得1株能稳定分泌抗NDV HN蛋白单克隆抗体的杂交瘤细胞株,命名为B1F12。单克隆抗体亚类的鉴定结果显示,该株杂交瘤细胞分泌的单克隆抗体为Ig G2b型。用动物(Balb/c小鼠)体内生产系统,制备大量腹水,并运用辛酸-硫酸铵法纯化收集的腹水,最终获得抗鸭源NDV HN蛋白的单克隆抗体。
[Abstract]:Duck Newcastle disease (Duck paramyxovirus disease,DPMV) is an acute and highly contagious disease caused by avian paramyxovirus type I (APMV-I). The surface of Newcastle disease virus (NDV) particle capsule contains two kinds of glycoproteins: hemagglutinin-neuraminidase protein (hemagglutinin-neuraminidase protein,HN) and fusion protein (fusion protein,F). The HN protein is the main protective antigen which is closely related to the virulence and pathogenicity of the virus. It is of great significance in the study of Newcastle disease virus (NDV) (NDV). In this study, a pair of specific primers were designed and synthesized using (Gen Bank accession number: HM188399 of HN gene sequence of duck NDV SDWF02 strain published by Gen Bank as template. The HN gene fragment of duck NDV was amplified by RT-PCR. After sequencing, the positive recombinant plasmid was expressed in prokaryotic cells and monoclonal antibody against HN protein was prepared. It provides a new way for further study and clinical detection of duck NDV. 1. Cloning and analysis of the HN gene of duck Newcastle disease virus (NDV). The specific primers designed for the use of RNA, were extracted by collecting allantoic fluid and collecting allantoic fluid from duck NDV SDWF02 strain preserved in laboratory. The HN gene fragment of NDV SDWF02 strain was successfully amplified by RT-PCR and cloned into p MD18-T Vector vector. The nucleotide sequence length of the target gene was 822 bp, which was consistent with that of duck NDV SDWF02 strain. The expression of two, NDV HN protein and the establishment of indirect ELISA (HN-ELISA). The recombinant vector p MD18-T-HN containing the HN gene fragment was digested by Sac I and Hind III, and the product was ligated to the expression vector of p ET-28a. Then the positive recombinant plasmid containing HN gene fragment was transformed into E.coli Rosetta receptive cells. The recombinant bacteria containing recombinant plasmid p ET-28a-HN were screened, and the fusion protein was induced by IPTG. SDS-PAGE analysis showed that, The fusion protein was obtained in the form of inclusion body, and the molecular weight of the fusion protein was about 30.5 k Da, which was consistent with the expected size. Ni-NTA Resin was used to purify the fusion protein. The fusion protein can specifically recognize anti-duck NDV serum and has good immunogenicity. Using purified HN protein as coating antigen, the indirect ELISA method for detection of duck NDV antibody was established by exploring the concentration of antigen coating, the dilution of serum and the conditions of encapsulation. 3. Preparation and Identification of Monoclonal Antibody against NDV HN protein. After immunizing Balb/c mice with NDV HN recombinant protein, after three times immunization, spleen lymphocytes of immunized mice were fused with SP2/0 myeloma cells. The positive hybridoma cell line was screened by indirect ELISA method and subcloned by limited dilution method. A hybridoma cell line which can secrete monoclonal antibody against NDV HN protein stably was obtained and named as B1F12. The identification of monoclonal antibody subclass showed that the monoclonal antibody secreted by the hybridoma cell was Ig G 2b type. A large amount of ascites were prepared by using animal (Balb/c) production system in vivo, and the collected ascites were purified by octanoic acid-ammonium sulfate method. Finally, monoclonal antibodies against duck NDV HN protein were obtained.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4
本文编号：2359221
[Abstract]:Duck Newcastle disease (Duck paramyxovirus disease,DPMV) is an acute and highly contagious disease caused by avian paramyxovirus type I (APMV-I). The surface of Newcastle disease virus (NDV) particle capsule contains two kinds of glycoproteins: hemagglutinin-neuraminidase protein (hemagglutinin-neuraminidase protein,HN) and fusion protein (fusion protein,F). The HN protein is the main protective antigen which is closely related to the virulence and pathogenicity of the virus. It is of great significance in the study of Newcastle disease virus (NDV) (NDV). In this study, a pair of specific primers were designed and synthesized using (Gen Bank accession number: HM188399 of HN gene sequence of duck NDV SDWF02 strain published by Gen Bank as template. The HN gene fragment of duck NDV was amplified by RT-PCR. After sequencing, the positive recombinant plasmid was expressed in prokaryotic cells and monoclonal antibody against HN protein was prepared. It provides a new way for further study and clinical detection of duck NDV. 1. Cloning and analysis of the HN gene of duck Newcastle disease virus (NDV). The specific primers designed for the use of RNA, were extracted by collecting allantoic fluid and collecting allantoic fluid from duck NDV SDWF02 strain preserved in laboratory. The HN gene fragment of NDV SDWF02 strain was successfully amplified by RT-PCR and cloned into p MD18-T Vector vector. The nucleotide sequence length of the target gene was 822 bp, which was consistent with that of duck NDV SDWF02 strain. The expression of two, NDV HN protein and the establishment of indirect ELISA (HN-ELISA). The recombinant vector p MD18-T-HN containing the HN gene fragment was digested by Sac I and Hind III, and the product was ligated to the expression vector of p ET-28a. Then the positive recombinant plasmid containing HN gene fragment was transformed into E.coli Rosetta receptive cells. The recombinant bacteria containing recombinant plasmid p ET-28a-HN were screened, and the fusion protein was induced by IPTG. SDS-PAGE analysis showed that, The fusion protein was obtained in the form of inclusion body, and the molecular weight of the fusion protein was about 30.5 k Da, which was consistent with the expected size. Ni-NTA Resin was used to purify the fusion protein. The fusion protein can specifically recognize anti-duck NDV serum and has good immunogenicity. Using purified HN protein as coating antigen, the indirect ELISA method for detection of duck NDV antibody was established by exploring the concentration of antigen coating, the dilution of serum and the conditions of encapsulation. 3. Preparation and Identification of Monoclonal Antibody against NDV HN protein. After immunizing Balb/c mice with NDV HN recombinant protein, after three times immunization, spleen lymphocytes of immunized mice were fused with SP2/0 myeloma cells. The positive hybridoma cell line was screened by indirect ELISA method and subcloned by limited dilution method. A hybridoma cell line which can secrete monoclonal antibody against NDV HN protein stably was obtained and named as B1F12. The identification of monoclonal antibody subclass showed that the monoclonal antibody secreted by the hybridoma cell was Ig G 2b type. A large amount of ascites were prepared by using animal (Balb/c) production system in vivo, and the collected ascites were purified by octanoic acid-ammonium sulfate method. Finally, monoclonal antibodies against duck NDV HN protein were obtained.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4
【引证文献】
相关会议论文 前2条
1 纪巍;刁有祥;王明亮;马艳芳;孙宁;孙法良;吴焕荣;;一株经鸭胚传递的鸭副粘病毒的分离鉴定及生物学特性研究[A];山东畜牧兽医学会禽病学专业委员会第一次学术研讨会论文集[C];2009年
2 刁有祥;;鸭副粘病毒病流行情况与防治措施[A];禽类新发传染病高层论坛论文集[C];2009年
，本文编号：2359221
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2359221.html
