鴨新城疫病毒HN蛋白單克隆抗體的制備及間接ELISA方法的建立
發(fā)布時間：2018-11-26 17:50
【摘要】：鴨新城疫,又稱為鴨副黏病毒病(Duck paramyxovirus disease,DPMV)是由禽副黏病毒I型(APMV-I)引起的一種急性、高度接觸性傳染病,各日齡鴨均易感,給養(yǎng)鴨業(yè)帶來嚴重經濟損失。新城疫病毒粒子囊膜表面包含兩種糖蛋白,分別是血凝素-神經氨酸酶蛋白(hemagglutinin-neuraminidase protein,HN)和融合蛋白(fusion protein,F)。其中HN蛋白與病毒的毒力及致病性密切相關,是主要的保護性抗原,在新城疫病毒(NDV)的研究中具有十分重要的意義。本研究以Gen Bank已公布的鴨源NDV SDWF02株HN基因序列(Gen Bank登錄號:HM188399)為模板,在其抗原位點分布較為豐富區(qū)域,設計并合成一對特異性引物,運用RT-PCR的方法擴增出鴨源NDV的HN基因片段,經測序分析后,將陽性重組質粒進行原核表達,制備出針對HN蛋白的單克隆抗體,為鴨源NDV的深入研究及臨床檢測提供了新途徑。一、鴨源新城疫病毒HN基因的克隆及分析取實驗室保存的鴨源NDV SDWF02株病毒,傳胚收集尿囊液,提取RNA,運用設計的特異性引物,經RT-PCR的方法成功擴增出了NDV SDWF02株的HN基因片段,并將擴增片段克隆至p MD18-T Vector載體中。經測序分析,目的基因的核苷酸序列長度為822 bp,與鴨源NDV SDWF02株堿基序列一致。二、NDV HN蛋白的表達及間接ELISA(HN-ELISA)的建立將含有的HN基因片段的陽性重組載體p MD18-T-HN經Sac I和Hind III雙酶切,并將產物連接至p ET-28a表達載體。再將含有HN基因片段的陽性重組質粒轉化到E.coli Rosetta感受態(tài)細胞中,篩選含重組質粒p ET-28a-HN的重組菌,經IPTG誘導表達出融合蛋白,SDS-PAGE分析表明,獲得的融合蛋白主要以包涵體的形式出現,分子量約為30.5 k Da,與預期大小相一致,選用Ni-NTA Resin對融合蛋白進行純化,Western blot結果顯示,該融合蛋白能夠特異性識別抗鴨NDV血清,具有良好的免疫原性。并利用純化的HN蛋白作為包被抗原,對抗原包被濃度、血清稀釋度及包被條件等進行探索,建立出檢測鴨源NDV抗體的間接ELISA方法。三、抗NDV HN蛋白單克隆抗體的制備及鑒定以NDV HN重組蛋白作為免疫原免疫Balb/c小鼠后,三次免疫后,經加強疫取免疫小鼠的脾淋巴細胞與SP2/0骨髓瘤細胞融合,經間接ELISA方法檢測篩選出陽性雜交瘤細胞株,并采用有限稀釋法對其進行亞克隆,獲得1株能穩(wěn)定分泌抗NDV HN蛋白單克隆抗體的雜交瘤細胞株,命名為B1F12。單克隆抗體亞類的鑒定結果顯示,該株雜交瘤細胞分泌的單克隆抗體為Ig G2b型。用動物(Balb/c小鼠)體內生產系統(tǒng),制備大量腹水,并運用辛酸-硫酸銨法純化收集的腹水,最終獲得抗鴨源NDV HN蛋白的單克隆抗體。
[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.
【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.4
本文編號：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.
【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.4
【引證文獻】
相關會議論文 前2條
1 紀巍;刁有祥;王明亮;馬艷芳;孫寧;孫法良;吳煥榮;;一株經鴨胚傳遞的鴨副粘病毒的分離鑒定及生物學特性研究[A];山東畜牧獸醫(yī)學會禽病學專業(yè)委員會第一次學術研討會論文集[C];2009年
2 刁有祥;;鴨副粘病毒病流行情況與防治措施[A];禽類新發(fā)傳染病高層論壇論文集[C];2009年
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