牛γ干扰素和白细胞介素4基因的原核表达及其产物的单克隆抗体研制
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摘要
牛结核病主要由牛型结核分枝杆菌(Mycobacterium bovis, M. bovis)引起的一种重要人兽共患慢性传染病。牛群感染结核分枝杆菌后,主要产生Th1型的细胞免疫应答,以IFN-γ的产生为特征,通过激活巨噬细胞的杀菌活性以抑制或清除体内的细菌,通常情况下病原菌不能被清除,而是被控制在一定区域,疾病进入潜伏期。研究发现随着结核病病情的发展,机体免疫应答逐渐由Th1型转向Th2型,伴随着细胞免疫应答的减弱和体液免疫应答的增强,后者是以IL-4的产生为主要特征。因此IFN-γ和IL-4是牛结核免疫应答中两个重要的细胞因子。为此本研究构建了原核表达牛IFN-γ和IL-4蛋白的重组大肠杆菌,获得相应的融合蛋白,并制备其特异性单克隆抗体。为研究分枝杆菌等牛传染病的致病机理、免疫机制和建立快速检测技术提供了重要条件。同时,牛IFN-γ和IL-4单克隆抗体的获得为研究牛的免疫生物学、免疫应答和免疫调控提供了有力的工具。
一、牛γ干扰素和白细胞介素4基因的原核表达及其产物特性
在实验室前期构建的重组菌BL21(pGEX-6p-1-BoIFN-γ)和BL21(DE3)(pET- BoIFN-γ)基础上,对两种重组菌进行了诱导表达,两种融合蛋白均主要以可溶性形式表达。经亲和层析对重组蛋白纯化后,SDS-PAGE鉴定结果表明表达蛋白rGST-BoIFN-γ、rHis-BoIFN-γ与预期的大小一致。
通过PCR将编码不含信号肽的牛白细胞介素4蛋白的基因从重组质粒pSP73-BoIL-4中克隆出来,分别克隆入表达载体pGEX-6p-1和pET-30a (+),将重组质粒转入DH5α,测序鉴定。测序鉴定正确后将重组质粒转入表达菌,构建出重组菌BL21(pGEX-6p-1-BoIL-4)和BL21(DE3)(pET-BoIL-4)。对重组菌的诱导表达发现两种重组蛋白均以包涵体形式表达。通过对包涵体的变性与复性得到了相应的纯化蛋白,SDS-PAGE鉴定表明得到的蛋白与预期的大小一致。
二、牛γ干扰素和白细胞介素4单克隆抗体的制备与鉴定
应用淋巴细胞杂交瘤技术,用纯化的重组蛋白rHis-BoIFN-γ、rHis-BoIL-4分别免疫7周龄BALB/c小鼠,腹部皮下和腹腔免疫,100μg/只。首免时,抗原与等量弗氏完全佐剂混合乳化经腹部皮下免疫;二免时抗原与等量不完全弗氏佐剂混合乳化经腹部皮下免疫;三免时用抗原通过腹腔直接免疫;间隔为两周;尾静脉加强免疫不加佐剂的抗原,100μg/只,3d后,取免疫鼠脾细胞和骨髓瘤SP2/0-Ag-14细胞进行融合。纯化的rGST-BoIFN-γ和rGST-BoIL-4分别作为检测抗原,用间接ELISA方法筛选阳性克隆。获得13株稳定分泌BoIFN-γMcAb的细胞株,命名为1C12、1F7、1G5、3E6、4D5、5E11、5G4、6F8、6G6、7E9、8D3、8F8、9G11,腹水的效价依次为640000、160000、640000、320000、160000、320000、2560000、2560000、320000、640000、80000、2560000、10000,除5G4 McAb亚类为IgG2b外,其余均为IgG1。获得7株稳定分泌BoIL-4 McAb的细胞株,命名为2B8、4A10、5D6、5D8、7G10、8B7、10F8,腹水的效价依次为5000、160000、10000、640000、5000、40000、5000、40000,McAb亚类均为IgG1。Dot-ELISA试验表明,所得单抗只与相应的融合蛋白反应,与其它融合蛋白和对照细菌不反应,显示良好的特异性。Western-blot试验显示所得单抗均能与相应的融合蛋白发生反应,出现特异性条带。单抗与牛IFN-γ和牛IL-4标准品的ELISA反应显示所获单抗均能与标准品反应,显示了良好的应用前景。
Bovine TB is mainly caused by Mycobacterium bovis which pose an infectious risk to cattle and humans. After infected with Mycobacterium bovis, cattle can elicite a Th1 cellular immune response charactered by IFN-γsecretion. IFN-γcan activate the microbicidal activity of macrophages to eliminate the pathogen. But pathogens usually can not be eliminated and be restricted in a certain region, then the pathogen lies latent in the host. Studies of tuberculosis have suggested a shift in dominance from a Th1 towards Th2 immune response that is associated with suppressed cell mediated immune responses and increased humoral responses as the disease progresses. IL-4 is the major cytokine of Th2 immune response. Therefore IFN-γand IL-4 is important in TB research. In this study, the recombinant bacteria were constructed to express IFN-γand IL-4, the recombinant proteins were used to develop monoclonal antibody. All these results could be useful for studying the pathogenicity and the immune mechanism of Mycobacterium bovis, and for developing the method and technique in the detection of Mycobacterium bovis, and furthermore for studying bovine immunological biology immune response and immune regulation.
1. Prokaryotic expression of bovine IFN-γand IL-4 genes in recombinant E.coli
Recombinant bacteria BL21(pGEX-6p-1-BoIFN-γ) and BL21(DE3)(pET-BoIFN-γ) were constructed previously. The recombinant proteins, named rGST-BoIFN-γand rHis-BoIFN-γ, are expressed in soluble form. After induced by IPTG, the two recombinant proteins were purified, and their sizes were consistent with the prediction.
The IL-4 gene without coding signal peptides was amplified from pSP73-BoIL-4 by PCR, then inserted into prokaryotic expression vector pGEX-6p-1 and pET-30a(+). The recombinant plasmids pGEX-6p-1-BoIL-4 and pET-BoIL-4 were transformed into DH5αfor sequencing. After sequencing confirmation, the two recombinant plasmids were transformed into expression bacteria BL21 and Bl21(DE3) respectively, and then recombinant bacteria BL21(pGEX-6p-1-BoIL-4) and BL21(DE3)(pET-BoIL-4) were developed. The recombinant proteins, named rGST-BoIL-4 and rHis-BoIL-4, are expressed in inclusion body form. After denaturation and renaturation of inclusion body, the two recombinant proteins were obtained, and their sizes were consistent with the prediction.
2. Production and characterization of monoclonal antibodies against bovine IFN-γand bovine IL-4
To prepare monoclonal antibodies against bovine IFN-γand bovine IL-4, purified protein rHis-BoIFN-γand rHis-BoIL-4 were respectively used as immunogen to immunize subcutaneously 7-week-old BALB/c mice. The immune dose was 100μg recombinant protein emulsified in complete Freund’s adjuvant for the first immunization and in incomplete Freund’s adjuvant for the second injections, the recombinant proteins without emulsified by adjuvant were immunized intraperitoneally for the third time, the interval of two immunizations is 2 weeks. Then an intravenous dose of purified protein was administrated. After 3 days, splenocytes from immunized mice were fused with Sp2/0-Ag-14 myeloma cells. Purified rGST-BoIFN-γand rGST-BoIL-4 were used as detecting antigen respectively, and the supernatant of hybridoma clones was screened by indirect ELISA. Thirteen hybridoma cell lines secreting McAbs against BoIFN-γ, named 1C12, 1F7, 1G5, 3E6, 4D5, 5E11, 5G4, 6F8, 6G6, 7E9, 8D3, 8F8, 9G11 were obtained. The immunoglobulin subclass of McAb 5G4 was IgG2b, the others were IgG1. The ascitic titers of these McAbs were 640000, 160000, 640000, 320000, 160000, 320000, 2560000, 2560000, 320000, 640000, 80000, 2560000, 10000 respectively. Seven hybridoma cell lines secreting McAbs against BoIL-4, named 2B8, 4A10, 5D6, 5D8, 7G10, 8B7, 10F8 were obtained. The immunoglobulin subclass were IgG1. The ascitic titers of these McAbs were 5000, 160000, 10000, 640000, 5000, 40000, 5000, 40000 respectively. In Dot-ELISA, all McAbs could only react with the immunogen and the detecting antigen. Western-blot analysis confirmed that all McAbs could only react with the corresponding recombinant proteins. The McAbs also reacted with the standard recombinant bovine IFN-γor IL-4 with biological activity. All these results suggested that the specific McAbs against bovine IFN-γand IL-4 were developed, which are very useful in both fundamental and practical studies.
一、牛γ干扰素和白细胞介素4基因的原核表达及其产物特性
在实验室前期构建的重组菌BL21(pGEX-6p-1-BoIFN-γ)和BL21(DE3)(pET- BoIFN-γ)基础上,对两种重组菌进行了诱导表达,两种融合蛋白均主要以可溶性形式表达。经亲和层析对重组蛋白纯化后,SDS-PAGE鉴定结果表明表达蛋白rGST-BoIFN-γ、rHis-BoIFN-γ与预期的大小一致。
通过PCR将编码不含信号肽的牛白细胞介素4蛋白的基因从重组质粒pSP73-BoIL-4中克隆出来,分别克隆入表达载体pGEX-6p-1和pET-30a (+),将重组质粒转入DH5α,测序鉴定。测序鉴定正确后将重组质粒转入表达菌,构建出重组菌BL21(pGEX-6p-1-BoIL-4)和BL21(DE3)(pET-BoIL-4)。对重组菌的诱导表达发现两种重组蛋白均以包涵体形式表达。通过对包涵体的变性与复性得到了相应的纯化蛋白,SDS-PAGE鉴定表明得到的蛋白与预期的大小一致。
二、牛γ干扰素和白细胞介素4单克隆抗体的制备与鉴定
应用淋巴细胞杂交瘤技术,用纯化的重组蛋白rHis-BoIFN-γ、rHis-BoIL-4分别免疫7周龄BALB/c小鼠,腹部皮下和腹腔免疫,100μg/只。首免时,抗原与等量弗氏完全佐剂混合乳化经腹部皮下免疫;二免时抗原与等量不完全弗氏佐剂混合乳化经腹部皮下免疫;三免时用抗原通过腹腔直接免疫;间隔为两周;尾静脉加强免疫不加佐剂的抗原,100μg/只,3d后,取免疫鼠脾细胞和骨髓瘤SP2/0-Ag-14细胞进行融合。纯化的rGST-BoIFN-γ和rGST-BoIL-4分别作为检测抗原,用间接ELISA方法筛选阳性克隆。获得13株稳定分泌BoIFN-γMcAb的细胞株,命名为1C12、1F7、1G5、3E6、4D5、5E11、5G4、6F8、6G6、7E9、8D3、8F8、9G11,腹水的效价依次为640000、160000、640000、320000、160000、320000、2560000、2560000、320000、640000、80000、2560000、10000,除5G4 McAb亚类为IgG2b外,其余均为IgG1。获得7株稳定分泌BoIL-4 McAb的细胞株,命名为2B8、4A10、5D6、5D8、7G10、8B7、10F8,腹水的效价依次为5000、160000、10000、640000、5000、40000、5000、40000,McAb亚类均为IgG1。Dot-ELISA试验表明,所得单抗只与相应的融合蛋白反应,与其它融合蛋白和对照细菌不反应,显示良好的特异性。Western-blot试验显示所得单抗均能与相应的融合蛋白发生反应,出现特异性条带。单抗与牛IFN-γ和牛IL-4标准品的ELISA反应显示所获单抗均能与标准品反应,显示了良好的应用前景。
Bovine TB is mainly caused by Mycobacterium bovis which pose an infectious risk to cattle and humans. After infected with Mycobacterium bovis, cattle can elicite a Th1 cellular immune response charactered by IFN-γsecretion. IFN-γcan activate the microbicidal activity of macrophages to eliminate the pathogen. But pathogens usually can not be eliminated and be restricted in a certain region, then the pathogen lies latent in the host. Studies of tuberculosis have suggested a shift in dominance from a Th1 towards Th2 immune response that is associated with suppressed cell mediated immune responses and increased humoral responses as the disease progresses. IL-4 is the major cytokine of Th2 immune response. Therefore IFN-γand IL-4 is important in TB research. In this study, the recombinant bacteria were constructed to express IFN-γand IL-4, the recombinant proteins were used to develop monoclonal antibody. All these results could be useful for studying the pathogenicity and the immune mechanism of Mycobacterium bovis, and for developing the method and technique in the detection of Mycobacterium bovis, and furthermore for studying bovine immunological biology immune response and immune regulation.
1. Prokaryotic expression of bovine IFN-γand IL-4 genes in recombinant E.coli
Recombinant bacteria BL21(pGEX-6p-1-BoIFN-γ) and BL21(DE3)(pET-BoIFN-γ) were constructed previously. The recombinant proteins, named rGST-BoIFN-γand rHis-BoIFN-γ, are expressed in soluble form. After induced by IPTG, the two recombinant proteins were purified, and their sizes were consistent with the prediction.
The IL-4 gene without coding signal peptides was amplified from pSP73-BoIL-4 by PCR, then inserted into prokaryotic expression vector pGEX-6p-1 and pET-30a(+). The recombinant plasmids pGEX-6p-1-BoIL-4 and pET-BoIL-4 were transformed into DH5αfor sequencing. After sequencing confirmation, the two recombinant plasmids were transformed into expression bacteria BL21 and Bl21(DE3) respectively, and then recombinant bacteria BL21(pGEX-6p-1-BoIL-4) and BL21(DE3)(pET-BoIL-4) were developed. The recombinant proteins, named rGST-BoIL-4 and rHis-BoIL-4, are expressed in inclusion body form. After denaturation and renaturation of inclusion body, the two recombinant proteins were obtained, and their sizes were consistent with the prediction.
2. Production and characterization of monoclonal antibodies against bovine IFN-γand bovine IL-4
To prepare monoclonal antibodies against bovine IFN-γand bovine IL-4, purified protein rHis-BoIFN-γand rHis-BoIL-4 were respectively used as immunogen to immunize subcutaneously 7-week-old BALB/c mice. The immune dose was 100μg recombinant protein emulsified in complete Freund’s adjuvant for the first immunization and in incomplete Freund’s adjuvant for the second injections, the recombinant proteins without emulsified by adjuvant were immunized intraperitoneally for the third time, the interval of two immunizations is 2 weeks. Then an intravenous dose of purified protein was administrated. After 3 days, splenocytes from immunized mice were fused with Sp2/0-Ag-14 myeloma cells. Purified rGST-BoIFN-γand rGST-BoIL-4 were used as detecting antigen respectively, and the supernatant of hybridoma clones was screened by indirect ELISA. Thirteen hybridoma cell lines secreting McAbs against BoIFN-γ, named 1C12, 1F7, 1G5, 3E6, 4D5, 5E11, 5G4, 6F8, 6G6, 7E9, 8D3, 8F8, 9G11 were obtained. The immunoglobulin subclass of McAb 5G4 was IgG2b, the others were IgG1. The ascitic titers of these McAbs were 640000, 160000, 640000, 320000, 160000, 320000, 2560000, 2560000, 320000, 640000, 80000, 2560000, 10000 respectively. Seven hybridoma cell lines secreting McAbs against BoIL-4, named 2B8, 4A10, 5D6, 5D8, 7G10, 8B7, 10F8 were obtained. The immunoglobulin subclass were IgG1. The ascitic titers of these McAbs were 5000, 160000, 10000, 640000, 5000, 40000, 5000, 40000 respectively. In Dot-ELISA, all McAbs could only react with the immunogen and the detecting antigen. Western-blot analysis confirmed that all McAbs could only react with the corresponding recombinant proteins. The McAbs also reacted with the standard recombinant bovine IFN-γor IL-4 with biological activity. All these results suggested that the specific McAbs against bovine IFN-γand IL-4 were developed, which are very useful in both fundamental and practical studies.
引文
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