抗三唑磷基因工程抗体的研制及同源建模
发布时间：2018-12-11 17:12
【摘要】： 为了研制基因工程抗体,本文首先通过杂交瘤技术筛选能够稳定分泌高亲和力、高特异性抗体的杂交瘤细胞,以从中克隆编码抗体可变区的基因。利用杂交瘤细胞制备的单克隆抗体建立了三唑磷残留ELISA检测方法,并对方法进行了系统优化。优化后的ELISA应用于食品和环境样品中三唑磷残留检测,其良好的回收率和较小的变异系数一方面证明了ELISA在三唑磷等农药的残留检测中具有良好的应用前景,另一方面也说明筛选的杂交瘤细胞是成功的,可以用于基因工程抗体的研制。 然后从杂交瘤细胞株出发,通过RT-PCR技术,利用小鼠重链和轻链可变区通用引物扩增得到了抗三唑磷单克隆抗体轻、重链可变区基因序列。再通过重叠延伸PCR(SOE-PCR)方法,将两个可变区基因连接起来,构建了单链抗体(ScFv)核苷酸序列。然后将ScFv序列插入到pET-29a(+)载体,得到了重组表达质粒,转化大肠杆菌BL21(DE3)后,阳性菌株经IPTG诱导培养后,表达得到了ScFv蛋白。蛋白经过变性、纯化和复性处理后,通过竟争抑制ELISA进行了初步鉴定,结果表明,ScFv具有免疫亲和活性。 在此基础上,利用Accelrys公司的Discovery Studio~(TM)(DS)软件系统通过同源建模法模拟了抗三唑磷单链抗体的三级结构及ScFv与三唑磷农药小分子间的对接构象。首先通过模板分子搜索、序列比对、构建模型及模型合理性评价等过程分别构建了轻、重链可变区三维结构;然后再通过模板分子搜索、序列比对及叠合等过程,初步构建了ScFv的三维结构模型。模型经过动力学和能量最小化优化后,最终得到了评价合理的ScFv的三维结构。分子对接模拟结果显示,单链抗体与三唑磷主要以范德华力和氢键发生相互作用,抗体有13个氨基酸残基可以与农药分子发生作用。这些氨基酸作用位点和作用方式的确定为进一步进行抗体分子的改造和研究抗体与农药小分子间的作用机理打下了坚实的基础。 最后在二硫键稳定抗体(dsFv)的研制、dsFv同源建模及分子对接方面开展了部分工作。利用PCR定点突变法,通过设计突变引物,成功地将半胱氨酸突变位点引入了轻、重链可变区的相应位点,从而为dsFv小分子抗体的研制打下了基础。DS软件系统对dsFv三级结构及dsFv与三唑磷的对接构象的模拟结果表明,dsFv与ScFv具有相似的活性口袋,在与三唑磷发生对接过程中,也是以氢键和范德华力作为主要作用方式,参与作用的氨基酸残基大部分相同,但dsFv可参与反应的氨基酸数目更多,这意味着dsFv和ScFv对三唑磷的亲和力可能会有差异。
[Abstract]:In order to develop genetic engineering antibodies, hybridoma cells secreting high affinity and specific antibodies were screened by hybridoma technique to clone genes encoding variable regions of antibodies. A method for the detection of triazophos residue ELISA was established using monoclonal antibody prepared by hybridoma cells and the method was systematically optimized. The optimized ELISA was applied to the determination of triazophos residues in food and environmental samples. The good recovery rate and small coefficient of variation proved that ELISA had a good application prospect in the detection of triazophos pesticide residues. On the other hand, the selection of hybridoma cells is successful and can be used in the development of genetic engineering antibodies. Then the light and heavy chain variable region gene sequences of anti-triazophos monoclonal antibody were obtained from hybridoma cell lines by RT-PCR amplification using mouse heavy chain and light chain variable region universal primers. Then by overlapping extension PCR (SOE-PCR) method, two variable region genes were linked together to construct the single chain antibody (ScFv) nucleotide sequence. Then the ScFv sequence was inserted into the pET-29a () vector and the recombinant expression plasmid was obtained. After transformed into Escherichia coli BL21 (DE3), the positive strain was induced by IPTG to express ScFv protein. After denaturation, purification and renaturation, the protein was preliminarily identified by competitive inhibition of ELISA. The results showed that ScFv had immune affinity activity. On this basis, the third-order structure of anti-triazophos single-chain antibody and the conformation of docking between ScFv and triazophos pesticide small molecules were simulated by using the Discovery Studio~ (TM) (DS) software system of Accelrys Company by homologous modeling method. Firstly, through template molecular search, sequence alignment, model construction and model rationality evaluation, three dimensional structures of light and heavy chain variable region were constructed respectively. Then the 3D structure model of ScFv was constructed by template search sequence alignment and superposition. After the optimization of dynamics and energy minimization, the 3D structure of ScFv is obtained. The results of molecular docking simulation showed that the single chain antibody interacted with triazophos mainly by van der Waals force and hydrogen bond, and the antibody had 13 amino acid residues which could interact with pesticide molecules. The determination of the interaction sites and action modes of these amino acids laid a solid foundation for the further modification of antibody molecules and the study of the interaction mechanism between antibodies and pesticide small molecules. Finally, some work has been done on the preparation of disulfide stable antibody (dsFv), dsFv homology modeling and molecular docking. By using PCR site-directed mutation method and by designing mutated primers, cysteine mutation sites were successfully introduced into the corresponding sites with light and heavy chain variable regions. The simulation results of dsFv tertiary structure and the conformation of dsFv and triazophos show that dsFv and ScFv have similar active pockets, and in the process of docking with triazophos, dsFv and ScFv have similar active pockets. The hydrogen bond and van der Waals force were also used as the main action modes. The amino acid residues involved in the reaction were mostly the same, but the number of amino acids that dsFv could participate in the reaction was more, which meant that the affinity of dsFv and ScFv to triazophos might be different.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392
本文编号：2372924
[Abstract]:In order to develop genetic engineering antibodies, hybridoma cells secreting high affinity and specific antibodies were screened by hybridoma technique to clone genes encoding variable regions of antibodies. A method for the detection of triazophos residue ELISA was established using monoclonal antibody prepared by hybridoma cells and the method was systematically optimized. The optimized ELISA was applied to the determination of triazophos residues in food and environmental samples. The good recovery rate and small coefficient of variation proved that ELISA had a good application prospect in the detection of triazophos pesticide residues. On the other hand, the selection of hybridoma cells is successful and can be used in the development of genetic engineering antibodies. Then the light and heavy chain variable region gene sequences of anti-triazophos monoclonal antibody were obtained from hybridoma cell lines by RT-PCR amplification using mouse heavy chain and light chain variable region universal primers. Then by overlapping extension PCR (SOE-PCR) method, two variable region genes were linked together to construct the single chain antibody (ScFv) nucleotide sequence. Then the ScFv sequence was inserted into the pET-29a () vector and the recombinant expression plasmid was obtained. After transformed into Escherichia coli BL21 (DE3), the positive strain was induced by IPTG to express ScFv protein. After denaturation, purification and renaturation, the protein was preliminarily identified by competitive inhibition of ELISA. The results showed that ScFv had immune affinity activity. On this basis, the third-order structure of anti-triazophos single-chain antibody and the conformation of docking between ScFv and triazophos pesticide small molecules were simulated by using the Discovery Studio~ (TM) (DS) software system of Accelrys Company by homologous modeling method. Firstly, through template molecular search, sequence alignment, model construction and model rationality evaluation, three dimensional structures of light and heavy chain variable region were constructed respectively. Then the 3D structure model of ScFv was constructed by template search sequence alignment and superposition. After the optimization of dynamics and energy minimization, the 3D structure of ScFv is obtained. The results of molecular docking simulation showed that the single chain antibody interacted with triazophos mainly by van der Waals force and hydrogen bond, and the antibody had 13 amino acid residues which could interact with pesticide molecules. The determination of the interaction sites and action modes of these amino acids laid a solid foundation for the further modification of antibody molecules and the study of the interaction mechanism between antibodies and pesticide small molecules. Finally, some work has been done on the preparation of disulfide stable antibody (dsFv), dsFv homology modeling and molecular docking. By using PCR site-directed mutation method and by designing mutated primers, cysteine mutation sites were successfully introduced into the corresponding sites with light and heavy chain variable regions. The simulation results of dsFv tertiary structure and the conformation of dsFv and triazophos show that dsFv and ScFv have similar active pockets, and in the process of docking with triazophos, dsFv and ScFv have similar active pockets. The hydrogen bond and van der Waals force were also used as the main action modes. The amino acid residues involved in the reaction were mostly the same, but the number of amino acids that dsFv could participate in the reaction was more, which meant that the affinity of dsFv and ScFv to triazophos might be different.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 李鹏;抗重金属汞、铜、锌单抗可变区序列的克隆鉴定、真核表达及三维模拟[D];南京农业大学;2011年
，本文编号：2372924
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