抗重金属镉、铅单克隆抗体、单链抗体的研制及单链抗体三维结构的模建
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摘要
重金属是一类很危险的污染物，在生物体内长期积累不可降解，在极其微量的情况下也会产生不良后果。各种生态系统都不同程度受到重金属的影响。重金属通过食物链沉积到人体中，可引起各种疾病甚至癌症，危害还能遗传到下一代。因此重金属在环境、农产品中残留监测都是非常重要的。
本文旨在建立能稳定分泌高亲和力、高特异性抗重金属镉和铅单克隆抗体的杂交瘤细胞株，在对其亲和力、特异性进行鉴定的基础上，建立其免疫分析方法并得到初步应用，克隆抗重金属镉、铅单克隆抗体的重链和轻链可变区基因，构建抗重金属单链抗体基因，在大肠杆菌中诱导表达，并对表达产物进行初步鉴定，利用计算机平台和软件进行模拟分析抗镉的单链抗体(ScFv)三维结构。重金属离子通过双功能螯合剂与血蓝蛋白的偶联物作为免疫原，免疫BALB／c鼠。采用杂交瘤技术建立能稳定分泌高亲和力抗重金属镉、铅单克隆抗体的杂交瘤细胞株，大量制备并经盐析、蛋白亲和层析获得纯化的单克隆抗体，间接ELISA法检测效价，非竞争酶免疫实验测定亲和力，竞争ELISA法测定抗体的特异性。采用矩阵实验确定抗原抗体的最佳组合，通过对离子强度、pH值、封闭物等影响因子的研究，确定酶联免疫吸附分析(ELISA)的最佳工作参数，建立定量测定金属离子的间接竞争ELISA方法。在此基础上，选择杂交瘤细胞株，用Trizol试剂提取总RNA，通过RT-PCR，采用小鼠重链可变区和轻链可变区通用引物，扩增抗体可变区基因。通过重叠延伸PCR(SOE-PCR)将两个可变区基因通过连接肽基因连接起来，构建单链抗体基因，克隆到pGEM-T Easy载体中，通过酶切、PCR和测序进行鉴定。然后用限制性内切酶双酶切pGEM-metal-ScFv质粒和pET-30a(+)质粒，构建pET-metal-ScFv重组表达质粒。转化大肠杆菌BL21(DE3)后，筛选阳性菌株，通过酶切、PCR进行鉴定。阳性菌株经IPTG诱导培养后，分离包涵体，进行变性和复性处理，通过SDS-PAGE和竞争抑制ELISA对表达蛋白的分子量和活性进行初步鉴定。在SWISS-MODEL软件服务器上，利用同源蛋白结构预测的方法模建单链抗体分子结构，建立其三维结构模型，运用分子生物学、分子动力学，验证三维结构的合理性。成功建立了七株能稳定分泌抗镉和铅单克隆抗体的杂交瘤细胞株，获得了高纯度的单克隆抗体，蛋白浓度达1.58-5.0mg／mL，Aa6为IgG1亚类，其余的均为IgM亚类，亲和力达10~8-10~(10)L／mol，选亲和力高的抗体建立标准曲线，抗体Aa6和4／7的IC_(50)分别为4.19μg／L和2.72μM，镉和铅的最低检出浓度分别为0.313μg／L和0.056μM，自来水、超纯水等水样中重金属标准液的添加回收率为80-114％。从杂交瘤细胞中成功地扩增出重链和轻链可变区基因，采用重叠延伸PCR获得约750bp大小的单链抗体基因，并克隆到T载体。经测序证实，轻重链可变区基因均在300bp左右，轻重链之间是由45bp连接肽基因连接。然后，成功地将抗金属ScFv基因按设计的VH-linker-VL方向插入表达载体pET-30a(+)，转化大肠杆菌，阳性菌落经ITPG诱导后，表达出目的蛋白，目的蛋白主要以包涵体形式存在，经变性和复性处理，可获得分子量大约29KD的蛋白，但亲和力不是很高，抑制率不超过10.6％。获得抗体的分子结构模型，单链抗体的轻、重链联系精密且位置得当，并可分析H链CDR3区主要的抗原结合位点。重金属镉、铅单克隆抗体Aa6和4／7能有效地用于实验室水样的重金属镉和铅残留检测。从杂交瘤细胞株扩增抗体可变区，成功构建了抗金属镉和铅单链抗体基因，在大肠杆菌获得了功能性表达，为进一步开展以抗重金属ScFv为基础的研究奠定了基础，抗体三维模型的建立为进一步分析轻、重链在抗体功能中的作用，为改造抗体、提高抗体活性等方面提供了一种途径。
Heavy metals are a kind of contaminants that can lead to side effects even if they arepresent in extremely minute quantities. They are the most insidious pollutants because oftheir nonbiodegradable nature and ability to persist for long periods. All forms ofecological systems are effected to various extends by heavy ,metals. Through the foodchain, heavy metals may deposit into the humane body, cause serious health problems,specifically cancer, and even transfer to the next generation on the genetic level. Therefore,the quantitative analysis of trace heavy metals is extremely important in environmental andagricultural food monitoring,
To establish hybridoma cell lines that can secrete anti-Cd/Pb monoclonal antibody(McAb) with high affinity and specificity, and develop an effective enzyme-linkedimmunosorbent assay(ELISA) for Cd/Pb. Based on the identification of their affinity,specificity and function, clone the variable region genes of the McAb and constructrecombinant anti-metal single chain Fv (ScFv) gene, then transform and express the ScFvgene in E. coli, and identify the resulting protein. According to variable region gene athree-dimensional model of ScFv of anti-Cadmium single chain antibody was mimiced.Heavy metal was coupled to protein carrier (keyhole limpet hemocyanin, KLH; bovineserum albumin, BSA) via bifuntional chelate reagents (1-(4-isothiocyanaobenzyl)-ethylene-diamine N, N, N', N'-tetraacetic acid; ITCBE; S-2(4-aminobenzyl)-diethylenetriaminepentaccetic acid; 9-NH2-Bn-DTPA; S-2-(4-isothiocyanaobenzyl)-diethylenetriaminepentaccetic acid, SCN-Bn-DTPA). The hybridoma cell lines that can secrete anti-metal (Cd,Pb) McAbs with high affinity were established by hybridoma technique. Purified McAbs were prepared by salt precipitation and protein chromatography. The titer and affinity ofMcAbs were determined by indirect ELISA, the class and subclass of McAbs wereidentified by Monoclonal Antibody Isotyping Kit, the specificity of McAbs was determinedby competitive inhibition ELISA. Under the optimized blocking reagents, ionic strengthand pH value, the standard curves were established by indirect competitive ELISA. Thetotal RNA of hybridoma cell lines was isolated with Trizol reagent, and the variable regiongenes were amplified with variable region primers by RT-PCR. The variable region genesof heavy chain and light chain were strung together by splicing by overlap extension(SOE-PCR) and the ScFv gene was constructed. After cloned into pGEM-T Easy vector,the recombinant ScFv gene was identified by endonuclease digestion, PCR and sequencing.The recombinant expression vector pET-heavy metal-ScFv was constructed byendonuclease digestion and ligation, and then transformed into E. coli BL21 (DE3) strain.After screened on LB plate with Kanamycin and identified by endonuclease digestion andPCR, the positive strain was cultured under IPTG. The inclusion body of the inducedbacteria was collected, denaturalized with urea and renaturalized by dialysis withrenaturalizing solution. The molecular weight and immuno-activities of expressed proteinwere identified by SDS-PAGE and competitive inhibition ELISA respectively OnSWISS-MODEL, with homologous protein-structure-prediction, we constructed andanalyzed molecular models of single chain antibody(Aa). Seven hybridoma strains that cansecrete anti-heavy metal McAbs were established designed Aa4, Aa6, Ac4, Ba2, 4/7, 7/4and 6E11. The concentration of the purified antibodies ranges from 1.58 mg/mL to 5.0mg/mL, with titer of 1:200-1:500 and affinity conatants of 10~8-10~(10)L/mol. Thecross-reactivities in this ELISA were less than 3％for other metal ions. The standard curveswere obtained by the antibodies with higher affinity. The IC_(50) values of Aa6 specific tocadmium was 4.19μg/mL, showing the lowest detection limit of 0.313μg/mL, the IC_(50)value of 4/7 achieved for lead was 2.72μM, showing the detection range of with the lowestdetection limit of 0.056μM. Recoveries from the analyte-fortified into tap water andultrapure water were in the range of 80-114％. The variable region genes were amplifiedfrom hybridoma cell lines by RT-PCR, and recombinant ScFv gene was constructed. Thesequencing result shows that the ScFv gene consists of about 750bp, with more than 300bpof heavy chain variable region gene and light chain variable region gene, which werelinked by a 45bp peptide. Then the ScFv gene was cloned into expression vector pET-30a(+) by the direction of VH-linker-VL. The recombinant ScFv gene was expressed successfully in E.coli under the induction of IPTG. The anti-heavy metal ScFv protein wasexpressed mainly in the form of inclusion body and with a molecular weight of about29KD. After denaturalization and renaturalization treatment, the immuno-activity ofanti-heavy metal ScFv protein was verified as it can not competitively inhibit the bindingbetween McAband ScFv with inhibitory rate of about 10.6％. The structure model ofantibody is constructed successfully.Seven of hybridoma cell lines that can secreteanti-heavy metal McAb with high affinity and specificity were established. The variableregion genes of the McAbs were amplified and ScFv gene was constructed. Therecombinant anti-heavy metal ScFv gene was then transformed and successfully expressedin E. coli as a fusion protein witn immuno-activity. The three-dimension of anti-cadmiumsingle chain antibody was provided a method for improving antibody affinity, investigatingantibody structure and analyzing function of light, as well as heavy chain in antibodyactivity.
本文旨在建立能稳定分泌高亲和力、高特异性抗重金属镉和铅单克隆抗体的杂交瘤细胞株，在对其亲和力、特异性进行鉴定的基础上，建立其免疫分析方法并得到初步应用，克隆抗重金属镉、铅单克隆抗体的重链和轻链可变区基因，构建抗重金属单链抗体基因，在大肠杆菌中诱导表达，并对表达产物进行初步鉴定，利用计算机平台和软件进行模拟分析抗镉的单链抗体(ScFv)三维结构。重金属离子通过双功能螯合剂与血蓝蛋白的偶联物作为免疫原，免疫BALB／c鼠。采用杂交瘤技术建立能稳定分泌高亲和力抗重金属镉、铅单克隆抗体的杂交瘤细胞株，大量制备并经盐析、蛋白亲和层析获得纯化的单克隆抗体，间接ELISA法检测效价，非竞争酶免疫实验测定亲和力，竞争ELISA法测定抗体的特异性。采用矩阵实验确定抗原抗体的最佳组合，通过对离子强度、pH值、封闭物等影响因子的研究，确定酶联免疫吸附分析(ELISA)的最佳工作参数，建立定量测定金属离子的间接竞争ELISA方法。在此基础上，选择杂交瘤细胞株，用Trizol试剂提取总RNA，通过RT-PCR，采用小鼠重链可变区和轻链可变区通用引物，扩增抗体可变区基因。通过重叠延伸PCR(SOE-PCR)将两个可变区基因通过连接肽基因连接起来，构建单链抗体基因，克隆到pGEM-T Easy载体中，通过酶切、PCR和测序进行鉴定。然后用限制性内切酶双酶切pGEM-metal-ScFv质粒和pET-30a(+)质粒，构建pET-metal-ScFv重组表达质粒。转化大肠杆菌BL21(DE3)后，筛选阳性菌株，通过酶切、PCR进行鉴定。阳性菌株经IPTG诱导培养后，分离包涵体，进行变性和复性处理，通过SDS-PAGE和竞争抑制ELISA对表达蛋白的分子量和活性进行初步鉴定。在SWISS-MODEL软件服务器上，利用同源蛋白结构预测的方法模建单链抗体分子结构，建立其三维结构模型，运用分子生物学、分子动力学，验证三维结构的合理性。成功建立了七株能稳定分泌抗镉和铅单克隆抗体的杂交瘤细胞株，获得了高纯度的单克隆抗体，蛋白浓度达1.58-5.0mg／mL，Aa6为IgG1亚类，其余的均为IgM亚类，亲和力达10~8-10~(10)L／mol，选亲和力高的抗体建立标准曲线，抗体Aa6和4／7的IC_(50)分别为4.19μg／L和2.72μM，镉和铅的最低检出浓度分别为0.313μg／L和0.056μM，自来水、超纯水等水样中重金属标准液的添加回收率为80-114％。从杂交瘤细胞中成功地扩增出重链和轻链可变区基因，采用重叠延伸PCR获得约750bp大小的单链抗体基因，并克隆到T载体。经测序证实，轻重链可变区基因均在300bp左右，轻重链之间是由45bp连接肽基因连接。然后，成功地将抗金属ScFv基因按设计的VH-linker-VL方向插入表达载体pET-30a(+)，转化大肠杆菌，阳性菌落经ITPG诱导后，表达出目的蛋白，目的蛋白主要以包涵体形式存在，经变性和复性处理，可获得分子量大约29KD的蛋白，但亲和力不是很高，抑制率不超过10.6％。获得抗体的分子结构模型，单链抗体的轻、重链联系精密且位置得当，并可分析H链CDR3区主要的抗原结合位点。重金属镉、铅单克隆抗体Aa6和4／7能有效地用于实验室水样的重金属镉和铅残留检测。从杂交瘤细胞株扩增抗体可变区，成功构建了抗金属镉和铅单链抗体基因，在大肠杆菌获得了功能性表达，为进一步开展以抗重金属ScFv为基础的研究奠定了基础，抗体三维模型的建立为进一步分析轻、重链在抗体功能中的作用，为改造抗体、提高抗体活性等方面提供了一种途径。
Heavy metals are a kind of contaminants that can lead to side effects even if they arepresent in extremely minute quantities. They are the most insidious pollutants because oftheir nonbiodegradable nature and ability to persist for long periods. All forms ofecological systems are effected to various extends by heavy ,metals. Through the foodchain, heavy metals may deposit into the humane body, cause serious health problems,specifically cancer, and even transfer to the next generation on the genetic level. Therefore,the quantitative analysis of trace heavy metals is extremely important in environmental andagricultural food monitoring,
To establish hybridoma cell lines that can secrete anti-Cd/Pb monoclonal antibody(McAb) with high affinity and specificity, and develop an effective enzyme-linkedimmunosorbent assay(ELISA) for Cd/Pb. Based on the identification of their affinity,specificity and function, clone the variable region genes of the McAb and constructrecombinant anti-metal single chain Fv (ScFv) gene, then transform and express the ScFvgene in E. coli, and identify the resulting protein. According to variable region gene athree-dimensional model of ScFv of anti-Cadmium single chain antibody was mimiced.Heavy metal was coupled to protein carrier (keyhole limpet hemocyanin, KLH; bovineserum albumin, BSA) via bifuntional chelate reagents (1-(4-isothiocyanaobenzyl)-ethylene-diamine N, N, N', N'-tetraacetic acid; ITCBE; S-2(4-aminobenzyl)-diethylenetriaminepentaccetic acid; 9-NH2-Bn-DTPA; S-2-(4-isothiocyanaobenzyl)-diethylenetriaminepentaccetic acid, SCN-Bn-DTPA). The hybridoma cell lines that can secrete anti-metal (Cd,Pb) McAbs with high affinity were established by hybridoma technique. Purified McAbs were prepared by salt precipitation and protein chromatography. The titer and affinity ofMcAbs were determined by indirect ELISA, the class and subclass of McAbs wereidentified by Monoclonal Antibody Isotyping Kit, the specificity of McAbs was determinedby competitive inhibition ELISA. Under the optimized blocking reagents, ionic strengthand pH value, the standard curves were established by indirect competitive ELISA. Thetotal RNA of hybridoma cell lines was isolated with Trizol reagent, and the variable regiongenes were amplified with variable region primers by RT-PCR. The variable region genesof heavy chain and light chain were strung together by splicing by overlap extension(SOE-PCR) and the ScFv gene was constructed. After cloned into pGEM-T Easy vector,the recombinant ScFv gene was identified by endonuclease digestion, PCR and sequencing.The recombinant expression vector pET-heavy metal-ScFv was constructed byendonuclease digestion and ligation, and then transformed into E. coli BL21 (DE3) strain.After screened on LB plate with Kanamycin and identified by endonuclease digestion andPCR, the positive strain was cultured under IPTG. The inclusion body of the inducedbacteria was collected, denaturalized with urea and renaturalized by dialysis withrenaturalizing solution. The molecular weight and immuno-activities of expressed proteinwere identified by SDS-PAGE and competitive inhibition ELISA respectively OnSWISS-MODEL, with homologous protein-structure-prediction, we constructed andanalyzed molecular models of single chain antibody(Aa). Seven hybridoma strains that cansecrete anti-heavy metal McAbs were established designed Aa4, Aa6, Ac4, Ba2, 4/7, 7/4and 6E11. The concentration of the purified antibodies ranges from 1.58 mg/mL to 5.0mg/mL, with titer of 1:200-1:500 and affinity conatants of 10~8-10~(10)L/mol. Thecross-reactivities in this ELISA were less than 3％for other metal ions. The standard curveswere obtained by the antibodies with higher affinity. The IC_(50) values of Aa6 specific tocadmium was 4.19μg/mL, showing the lowest detection limit of 0.313μg/mL, the IC_(50)value of 4/7 achieved for lead was 2.72μM, showing the detection range of with the lowestdetection limit of 0.056μM. Recoveries from the analyte-fortified into tap water andultrapure water were in the range of 80-114％. The variable region genes were amplifiedfrom hybridoma cell lines by RT-PCR, and recombinant ScFv gene was constructed. Thesequencing result shows that the ScFv gene consists of about 750bp, with more than 300bpof heavy chain variable region gene and light chain variable region gene, which werelinked by a 45bp peptide. Then the ScFv gene was cloned into expression vector pET-30a(+) by the direction of VH-linker-VL. The recombinant ScFv gene was expressed successfully in E.coli under the induction of IPTG. The anti-heavy metal ScFv protein wasexpressed mainly in the form of inclusion body and with a molecular weight of about29KD. After denaturalization and renaturalization treatment, the immuno-activity ofanti-heavy metal ScFv protein was verified as it can not competitively inhibit the bindingbetween McAband ScFv with inhibitory rate of about 10.6％. The structure model ofantibody is constructed successfully.Seven of hybridoma cell lines that can secreteanti-heavy metal McAb with high affinity and specificity were established. The variableregion genes of the McAbs were amplified and ScFv gene was constructed. Therecombinant anti-heavy metal ScFv gene was then transformed and successfully expressedin E. coli as a fusion protein witn immuno-activity. The three-dimension of anti-cadmiumsingle chain antibody was provided a method for improving antibody affinity, investigatingantibody structure and analyzing function of light, as well as heavy chain in antibodyactivity.
引文
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