鸡传染性支气管炎病毒M蛋白单克隆抗体的制备及其抗原表位的鉴定
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摘要
鸡传染性支气管炎(Avian Infectious Bronchitis,IB)是由鸡传染性支气管炎病毒(Avian Infectious Bronchitis Virus,IBV)引起的一种急性、高度接触传染性、病毒性呼吸道疾病。由于IBV易变异,不同血清型毒株之间具有较小的交叉保护性甚至无交叉保护性,因此,IB仍是危害世界养禽业的重要疫病之一。通过单克隆抗体技术对IBV抗原表位的研究,不仅有助于了解IBV抗原结构与功能的关系,而且对该病的诊断以及设计安全有效的基因工程表位疫苗等具有重要的意义。
本研究首先通过RT-PCR克隆了IBV CK/CH/LHLJ/04Ⅴ毒株681bp的M蛋白基因,并将其克隆到pMD18-T载体进行测序,通过双酶切将羧基端387bp的M基因片段亚克隆到原核表达载体pET-30c(+)上,构建重组质粒pET-30c-M。然后将重组阳性质粒转化大肠杆菌BL21(DE3)并用IPTG于37℃诱导表达。SDS-PAGE分析显示,表达的鸡传染性支气管炎病毒重组M蛋白分子量约为20kDa。通过ProBondTM蛋白纯化试剂盒纯化此蛋白,逐步透析法对其进行复性。Western Blot检测表明重组M蛋白能与鸡抗IBV的抗血清发生特异性反应,证明重组M蛋白具有良好的抗原性。
以表达的IBV重组M蛋白为免疫原,免疫BALB/c小鼠并通过常规杂交瘤技术制备抗IBV M蛋白的单克隆抗体。经过间接ELISA和Western Blot筛选和克隆后融合后的杂交瘤细胞,最终获得了两株能稳定分泌抗IBV M蛋白单克隆抗体的杂交瘤细胞株,分别命名为13A3和15E2。其单抗亚类均为IgG1,轻链均为κ链。这两株单克隆抗体具有良好的特异性,均能与IBV CK/CH/LHLJ/04Ⅴ毒株的M蛋白发生特异性的反应,为M蛋白功能研究及抗原表位研究奠定了基础。为了详细而精确的研究IBV M蛋白的表位结构,本研究设计表达了一组总数为13个部分重
叠且覆盖羧基端387bp M基因的短肽融合蛋白,利用肽扫描技术对这两株M蛋白的单克隆抗体所针对的抗原表位进行了研究,鉴定出了一个新的M蛋白抗原表位:199FATFVYAK206。Western blot和同源性分析结果显示,此表位是IBV M蛋白的一个高度保守的线性B细胞抗原表位。
本研究结果对进一步阐明了IBV M蛋白抗原结构,探讨其表位生物学功能,设计科学合理的疫苗和诊断试剂具有重要的意义。
Avian infectious bronchitis (IB) , caused by infectious bronchitis virus(IBV), is an acute and highly contagious disease in chichens. Because of the high mutation frequency of IBV and little cross-protection between serologically distinct viruses , IB is still a major health problem affecting the chicken industry in most countries of the world. Antigenic epitope information of avian infectious bronchitis virus (IBV) is not only useful for investigating the relationship between antigenic structure and function of the IBV, but also significant for diagnosis of IBV infection and developing safe and effective multi-epitope vaccines against the disease.
To begin with, reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the 681bp membrane (M) glycoprotein gene of avian infectious bronchitis virus and the gene was cloned into the pMD18-T vector for sequencing. By digestion of restriction enzymes BamHⅠand HindⅢ, the C-terminal 387bp fragments of M gene were subcloned into pET-30c to construct recombinant plasmid pET-30c-M . Then the recombinant plasmid was transformed into E.coli BL21(DE3) and induced with IPTG. It was demonstrated by SDS-PAGE that a protein of 20 kDa (M) was expressed in E.coli. The recombinant M protein was subsequently purified by affinity chromatography. Western blotting assay indicated that the chicken antiserum against IBV could recognize this protein. The results showed that the recombinant M protein possessed a good antigenicity and laid foundation for the preparation of monoclonal antibody (McAb) agaist IBV M protein and other functional research of M protein.
Subsequently, SPF BALB/c mice were immunized subcutaneously(S/C)and intraperitoneally(I/P) with the recombinant M protein as antigen for the preparation of McAbs against M protein.Two McAbs against IBV M protein ,designated as 13A3 and 15E2, were finally identified by the indirect ELISA and Western blot assays using recombinant M protein and IBV as antigens. The results of subtype analysis showed that McAbs 13A3 and 15E2 all belonged to IgG1 and the light chains of all McAbs were theκchain. The two McAbs had good specificity and could specifically recognize both the recombinant M protein and IBV M protein,which were significant for the studies of both the function of M protein and epitope identification.
In order to accurately study epitope structures of IBV M protein, 13 partially overlapping or consecutive peptides (MP3-1—MP12) spanning 387bp C-terminus of M gene were expressed for epitopes screening by Pepscan technology. One linear epitope of IBV M protein,199FATFVYAK206, was finally identified. The results of Western blot and homologous analysis indicated that the epitope was a highly conserved linear B-cell epitope of IBV M protein.
To conclude, this study is not only significant for further understanding the epitope structure and immunological function of IBV M protein, but also useful for designing the diagnostic reagent and epitope vaccines.In addition, this study will lay foundation for the genetic and variant studies of the avian infectious bronchitis virus.
本研究首先通过RT-PCR克隆了IBV CK/CH/LHLJ/04Ⅴ毒株681bp的M蛋白基因,并将其克隆到pMD18-T载体进行测序,通过双酶切将羧基端387bp的M基因片段亚克隆到原核表达载体pET-30c(+)上,构建重组质粒pET-30c-M。然后将重组阳性质粒转化大肠杆菌BL21(DE3)并用IPTG于37℃诱导表达。SDS-PAGE分析显示,表达的鸡传染性支气管炎病毒重组M蛋白分子量约为20kDa。通过ProBondTM蛋白纯化试剂盒纯化此蛋白,逐步透析法对其进行复性。Western Blot检测表明重组M蛋白能与鸡抗IBV的抗血清发生特异性反应,证明重组M蛋白具有良好的抗原性。
以表达的IBV重组M蛋白为免疫原,免疫BALB/c小鼠并通过常规杂交瘤技术制备抗IBV M蛋白的单克隆抗体。经过间接ELISA和Western Blot筛选和克隆后融合后的杂交瘤细胞,最终获得了两株能稳定分泌抗IBV M蛋白单克隆抗体的杂交瘤细胞株,分别命名为13A3和15E2。其单抗亚类均为IgG1,轻链均为κ链。这两株单克隆抗体具有良好的特异性,均能与IBV CK/CH/LHLJ/04Ⅴ毒株的M蛋白发生特异性的反应,为M蛋白功能研究及抗原表位研究奠定了基础。为了详细而精确的研究IBV M蛋白的表位结构,本研究设计表达了一组总数为13个部分重
叠且覆盖羧基端387bp M基因的短肽融合蛋白,利用肽扫描技术对这两株M蛋白的单克隆抗体所针对的抗原表位进行了研究,鉴定出了一个新的M蛋白抗原表位:199FATFVYAK206。Western blot和同源性分析结果显示,此表位是IBV M蛋白的一个高度保守的线性B细胞抗原表位。
本研究结果对进一步阐明了IBV M蛋白抗原结构,探讨其表位生物学功能,设计科学合理的疫苗和诊断试剂具有重要的意义。
Avian infectious bronchitis (IB) , caused by infectious bronchitis virus(IBV), is an acute and highly contagious disease in chichens. Because of the high mutation frequency of IBV and little cross-protection between serologically distinct viruses , IB is still a major health problem affecting the chicken industry in most countries of the world. Antigenic epitope information of avian infectious bronchitis virus (IBV) is not only useful for investigating the relationship between antigenic structure and function of the IBV, but also significant for diagnosis of IBV infection and developing safe and effective multi-epitope vaccines against the disease.
To begin with, reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the 681bp membrane (M) glycoprotein gene of avian infectious bronchitis virus and the gene was cloned into the pMD18-T vector for sequencing. By digestion of restriction enzymes BamHⅠand HindⅢ, the C-terminal 387bp fragments of M gene were subcloned into pET-30c to construct recombinant plasmid pET-30c-M . Then the recombinant plasmid was transformed into E.coli BL21(DE3) and induced with IPTG. It was demonstrated by SDS-PAGE that a protein of 20 kDa (M) was expressed in E.coli. The recombinant M protein was subsequently purified by affinity chromatography. Western blotting assay indicated that the chicken antiserum against IBV could recognize this protein. The results showed that the recombinant M protein possessed a good antigenicity and laid foundation for the preparation of monoclonal antibody (McAb) agaist IBV M protein and other functional research of M protein.
Subsequently, SPF BALB/c mice were immunized subcutaneously(S/C)and intraperitoneally(I/P) with the recombinant M protein as antigen for the preparation of McAbs against M protein.Two McAbs against IBV M protein ,designated as 13A3 and 15E2, were finally identified by the indirect ELISA and Western blot assays using recombinant M protein and IBV as antigens. The results of subtype analysis showed that McAbs 13A3 and 15E2 all belonged to IgG1 and the light chains of all McAbs were theκchain. The two McAbs had good specificity and could specifically recognize both the recombinant M protein and IBV M protein,which were significant for the studies of both the function of M protein and epitope identification.
In order to accurately study epitope structures of IBV M protein, 13 partially overlapping or consecutive peptides (MP3-1—MP12) spanning 387bp C-terminus of M gene were expressed for epitopes screening by Pepscan technology. One linear epitope of IBV M protein,199FATFVYAK206, was finally identified. The results of Western blot and homologous analysis indicated that the epitope was a highly conserved linear B-cell epitope of IBV M protein.
To conclude, this study is not only significant for further understanding the epitope structure and immunological function of IBV M protein, but also useful for designing the diagnostic reagent and epitope vaccines.In addition, this study will lay foundation for the genetic and variant studies of the avian infectious bronchitis virus.
引文
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