二甲氧基硫代磷酸酯类农药单克隆抗体研制及单链抗体三维结构模拟
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摘要
依据二甲氧基硫代磷酸酯类有机磷农药的共有结构,设计了含有二甲氧基硫代磷酸酯结构和苯环结构的通用半抗原,即3-[4-(O,O-二甲基硫代磷酰氧基)苯基]-1-丙酸(Hapten1, H1)。采用活泼酯法分别将H1与牛血清白蛋白(BSA)、卵清白蛋白(OVA)偶联合成免疫原和包被原。将制备好的免疫原H1-BSA,免疫6-8周龄雌性BALB/c鼠,每次每只小鼠的免疫原用量100μg。以HI-OVA作为包被原,用非竞争间接酶联免疫分析法(ELISA)检测多次免疫后的小鼠血清效价并选择效价较高的小鼠用于细胞融合。采用PEG介导的细胞融合技术将小鼠脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,建立可稳定分泌抗二甲氧基硫代磷酸酯类有机磷农药抗体的杂交瘤细胞株。经ELISA法筛选及有限稀释法亚克隆,成功建立了两株对甲基对硫磷、甲基毒死蜱、倍硫磷、甲基立枯磷、杀螟硫磷和马拉硫磷六种二甲氧基硫代磷酸酯类有机磷农药具有明显的识别作用的杂交瘤细胞株(D12-B5、E5-H2)。通过体内与体外培养大量制备单克隆抗体,并经盐析、蛋白亲和层析获得纯度较高的抗体。用非竞争间接ELISA法检测效价,非竞争酶免疫实验测定亲和力,竞争间接ELISA法测定抗体的特异性。选效价较高的杂交瘤细胞株D12-B5为研究对象,用小鼠亚型分析试剂盒测定出所分泌的抗体为IgG1、κ型,亲和常数达1.709×109L/mol。以H1-OVA作为包被原,采用竞争间接ELISA法测定出抗体对甲基对硫磷、甲基毒死蜱、倍硫磷、甲基立枯磷、杀螟硫磷和马拉硫磷六种农药的150分别为42.1μg/mL、60.2μg/mL、75μg/mL、13.58μg/mL、117μg/mL和172.9μg/mL。
为了分析不同结构的异源包被原对抗体检测灵敏度的影响,将与甲基对硫磷、甲基毒死蜱、倍硫磷和杀螟硫磷结构相似的半抗原用活泼酯法与OVA偶联形成不同化学结构的异源包被原。实验结果表明与分析物(农药)的结构相似的异源半抗原并不能提高单克隆抗体对该农药的检测灵敏度,但宽谱性抗体与H7-OVA的组合可使甲基对硫磷和甲基毒死蜱竞争间接ELISA的150值分别可达到0.58μg/mL、0.81μg/mL。
根据以上异源包被原的分析结果选择H7-OVA作为包被原,采用矩阵滴定法确定抗原抗体的最佳组合,通过对离子强度、pH值等影响因子的研究,确定ELISA的最佳工作参数,建立定量测定的间接竞争ELISA方法。通过ELISA条件优化,确定了甲基对硫磷和甲基立枯磷的ELISA的最佳工作条件,建立了定量测定甲基对硫磷和甲基立枯磷的间接竞争ELISA方法,甲基对硫磷的150和检测限(120)分别为0.165μg/mL、0.0144μg/mL,甲基立枯磷的150和检测限(120)分别为0.95μg/mL、0.033μg/mL。
在此基础上,选择效价较高的小鼠脾细胞和较高亲和力的杂交瘤细胞株,提取总RNA,用鼠类重链、轻链可变区通用引物,通过反转录PCR,扩增两种细胞可变区基因。通过重叠延伸PCR (SOE-PCR)将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T载体中,通过PCR和测序进行鉴定。其结果显示从小鼠脾细胞和杂交瘤细胞中成功地扩增到了重链和轻链可变区基因,大小在300 bp左右；采用SOE-PCR获得约750 bp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符合小鼠可变区基因的特征,轻重链之间是由45 bp连接肽基因连接。
在Discovery Studio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三维结构模型,并验证了三维结构的合理性。从构建的单链抗体(ScFv)三维结构模型中看出,单链抗体的轻、重链的超可变区(CDR)均暴露在抗体结构的表面,并形成桶状结构,这种结构非常有利于抗体与抗原的结合。单链抗体三维模型的模建为进一步分析轻、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奠定了基础。
According to the common structure of O,O-dimethyl organophosphorus pesticides, the generic hapten (H1,3-(4-Dimethoxyphosphorothioyloxy phenyl)propanoic acid) was designed with roxyphenylpropionic acid and 0,0-dimethyl phosphorochloridothioate. H1 was covalently attached to BSA as immunogen and to OVA as homologous coating antigen with the active ester method. BALB/c female mice (8 weeks old) were immunized with the H1-BSA conjugate. The dose consisted of 100μg of conjugate intraperitoneally injected. One week after the third booster injection, mice were tail-bled and antisera were tested for anti-hapten antibody titer by a noncompetitive indirect enzyme-linked immunosorbent assay (ELISA) using a homologous coating antigen (H1-OVA). The mice showing higher serum reactivity were selected as the donors of spleen cells for hybridoma production. Two hybridoma cell lines that can secrete broad-specific McAbs were established by cell fusion technique. Purified McAbs were prepared by salt precipitation and protein chromatography. The titer and affinity of McAbs were determined by noncompetitive indirect ELISA, the class and subclass of McAbs were identified by Monoclonal Antibody Isotyping Kit, the specificity of McAbs was determined by competitive indirect ELISA. The McAb affinity of D12-B5 is 1.709×109L/mol. The class and subclass of D12-B5 were IgGl andκ. In the competitive indirect ELISA, the I50 of Parathion-methyl、Chlorpyrifos-methyl、Fenthion、Tolclofos-methyl、Fenitrothion and Malathion was respectively 42.1μg/mL,60.2μg/mL, 75μg/mL,13.58μg/mL,117μg/mL and 172.9μg/mL
To study the effect of heterologous coating antigens on immunoassay sensitivity, eight haptens designed were four pesticides' derivatives. The result showed that the haptens whose structure were similar to analytes could not improve the sensitivity, exactly the sensitivity of generic-antibody was not associated with the homology of heterologous coating antigen and analyte. Whereas the combination of mAb with coating antigen (H7-OVA) gave the lowest I50 values.
According to the analysis of heterologous coating antigens, we choosed the H7-OVA as the coating antigen. Under the optimization of the ionic strength and pH, the standard curves were estblished by competitive indirect ELISA. The I50 values of specific to parathion-methyl was 0.165μg/mL, showing the lowest detection limit of 0.0144μg/mL, the I50 value achieved for tolclofos-methyl was 0.95μg/mL, with the lowest detection limit of 0.033μg/mL.
The total RNA of spleen cells and hybridoma cell was isolated with trizol reagent, and the variable region genes were amplified with variable region primers by RT-PCR (reverse transcriptase-polymerase chain reaction). The variable region genes of heavy chain and light chain were strung together by SOE-PCR (splicing by overlap extension PCR) and the ScFv gene was constructed. After cloned into T vector, the recombinant ScFv gene was identified by PCR and sequencing. The sequencing result shows that the ScFv gene consists of about 750 bp, with more than 300 bp of heavy chain variable region gene and light chain variable region gene, which were linked by a 45 bp peptide.
On Discovery Studio, with homologous protein-structure-prediction, a three-dimensional model of ScFv was mimiced according to variable region gene. The three-dimension of ScFv antibody provided a method for improving antibody affinity, investigating antibody structure and analyzing function of light, as well as heavy chain in antibody activity.
为了分析不同结构的异源包被原对抗体检测灵敏度的影响,将与甲基对硫磷、甲基毒死蜱、倍硫磷和杀螟硫磷结构相似的半抗原用活泼酯法与OVA偶联形成不同化学结构的异源包被原。实验结果表明与分析物(农药)的结构相似的异源半抗原并不能提高单克隆抗体对该农药的检测灵敏度,但宽谱性抗体与H7-OVA的组合可使甲基对硫磷和甲基毒死蜱竞争间接ELISA的150值分别可达到0.58μg/mL、0.81μg/mL。
根据以上异源包被原的分析结果选择H7-OVA作为包被原,采用矩阵滴定法确定抗原抗体的最佳组合,通过对离子强度、pH值等影响因子的研究,确定ELISA的最佳工作参数,建立定量测定的间接竞争ELISA方法。通过ELISA条件优化,确定了甲基对硫磷和甲基立枯磷的ELISA的最佳工作条件,建立了定量测定甲基对硫磷和甲基立枯磷的间接竞争ELISA方法,甲基对硫磷的150和检测限(120)分别为0.165μg/mL、0.0144μg/mL,甲基立枯磷的150和检测限(120)分别为0.95μg/mL、0.033μg/mL。
在此基础上,选择效价较高的小鼠脾细胞和较高亲和力的杂交瘤细胞株,提取总RNA,用鼠类重链、轻链可变区通用引物,通过反转录PCR,扩增两种细胞可变区基因。通过重叠延伸PCR (SOE-PCR)将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T载体中,通过PCR和测序进行鉴定。其结果显示从小鼠脾细胞和杂交瘤细胞中成功地扩增到了重链和轻链可变区基因,大小在300 bp左右；采用SOE-PCR获得约750 bp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符合小鼠可变区基因的特征,轻重链之间是由45 bp连接肽基因连接。
在Discovery Studio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三维结构模型,并验证了三维结构的合理性。从构建的单链抗体(ScFv)三维结构模型中看出,单链抗体的轻、重链的超可变区(CDR)均暴露在抗体结构的表面,并形成桶状结构,这种结构非常有利于抗体与抗原的结合。单链抗体三维模型的模建为进一步分析轻、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奠定了基础。
According to the common structure of O,O-dimethyl organophosphorus pesticides, the generic hapten (H1,3-(4-Dimethoxyphosphorothioyloxy phenyl)propanoic acid) was designed with roxyphenylpropionic acid and 0,0-dimethyl phosphorochloridothioate. H1 was covalently attached to BSA as immunogen and to OVA as homologous coating antigen with the active ester method. BALB/c female mice (8 weeks old) were immunized with the H1-BSA conjugate. The dose consisted of 100μg of conjugate intraperitoneally injected. One week after the third booster injection, mice were tail-bled and antisera were tested for anti-hapten antibody titer by a noncompetitive indirect enzyme-linked immunosorbent assay (ELISA) using a homologous coating antigen (H1-OVA). The mice showing higher serum reactivity were selected as the donors of spleen cells for hybridoma production. Two hybridoma cell lines that can secrete broad-specific McAbs were established by cell fusion technique. Purified McAbs were prepared by salt precipitation and protein chromatography. The titer and affinity of McAbs were determined by noncompetitive indirect ELISA, the class and subclass of McAbs were identified by Monoclonal Antibody Isotyping Kit, the specificity of McAbs was determined by competitive indirect ELISA. The McAb affinity of D12-B5 is 1.709×109L/mol. The class and subclass of D12-B5 were IgGl andκ. In the competitive indirect ELISA, the I50 of Parathion-methyl、Chlorpyrifos-methyl、Fenthion、Tolclofos-methyl、Fenitrothion and Malathion was respectively 42.1μg/mL,60.2μg/mL, 75μg/mL,13.58μg/mL,117μg/mL and 172.9μg/mL
To study the effect of heterologous coating antigens on immunoassay sensitivity, eight haptens designed were four pesticides' derivatives. The result showed that the haptens whose structure were similar to analytes could not improve the sensitivity, exactly the sensitivity of generic-antibody was not associated with the homology of heterologous coating antigen and analyte. Whereas the combination of mAb with coating antigen (H7-OVA) gave the lowest I50 values.
According to the analysis of heterologous coating antigens, we choosed the H7-OVA as the coating antigen. Under the optimization of the ionic strength and pH, the standard curves were estblished by competitive indirect ELISA. The I50 values of specific to parathion-methyl was 0.165μg/mL, showing the lowest detection limit of 0.0144μg/mL, the I50 value achieved for tolclofos-methyl was 0.95μg/mL, with the lowest detection limit of 0.033μg/mL.
The total RNA of spleen cells and hybridoma cell was isolated with trizol reagent, and the variable region genes were amplified with variable region primers by RT-PCR (reverse transcriptase-polymerase chain reaction). The variable region genes of heavy chain and light chain were strung together by SOE-PCR (splicing by overlap extension PCR) and the ScFv gene was constructed. After cloned into T vector, the recombinant ScFv gene was identified by PCR and sequencing. The sequencing result shows that the ScFv gene consists of about 750 bp, with more than 300 bp of heavy chain variable region gene and light chain variable region gene, which were linked by a 45 bp peptide.
On Discovery Studio, with homologous protein-structure-prediction, a three-dimensional model of ScFv was mimiced according to variable region gene. The three-dimension of ScFv antibody provided a method for improving antibody affinity, investigating antibody structure and analyzing function of light, as well as heavy chain in antibody activity.
引文
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