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。
To prepare the engineering antibody,hybridoma cells secreting antibody with high affinity and specificity were screened by hybridoma technology,from which variation region genes of the antibody would be cloned.Based on the anti-triazophos antibody produced by the selected cells,a direct competitive enzyme-linked immunosorbent assay(ELISA) for triazophos was developed.The influence of several physicochemical factors on the immunoassay was studied.The optimized ELISA has been used to quantify triazophos in foods and environmental samples spiked at different amounts.The excellent recoveries achieved not only confirmed the potential of the immunoassay for monitoring of triazophos in food and environmental samples, but also proved the hybridoma cell was good enough to prepare the engineering antibodies.
From the hybidoma cell lines,which can secrete anti-triazophos McAb with high affinity and specificity,the variable region genes of the McAb were amplified with universal primers by RT-PCR.To construct single-chain Fv antibody(ScFv) gene, methods splicing by overlap extension(SOE) was used for stringing variable region genes of heavy chain and light chain with a(G_4S)_3 linker.After sequencing identification,the ScFv gene was cloned into the expression vector PET-29a(+) with the direction of V_H-linker-V_L,which was constructed by endonuclease digestion and ligation.Then the recombinant vector was transformed into E.coli BL21(DE3) strain. The identified positive strain can express the anti-pesticide ScFv protein under the induction of IPTG.The collected ScFv protein was denaturalized with urea,purified with immunoaffinity column and renaturalized by dialysis with renaturalizing solution.The ScFv' immuno-activities were confirmed by competitive inhibition ELISA.
On the basis of the variable region genes of the McAb above,the amino acid sequences of the variable region were obtained.Then the three dimensional structure of the ScFv was constructed through homologous modeling by series of software of Discovery Studio~(TM)(Accelrys).The heavy variable region(V_H) and light variable region(V_L) domains were respectively modeled through recognition of target model, alignment between query sequence and target sequence,modeling,evaluation of structure rationality.Then the V_H and V_L model were superimposed upon the target model of antibody variable region by sequence alignment and the linker was added between V_H and V_L,thus the three-dimensional structure of ScFv was constructed.It was proved the modeling structure was reasonable after structure optimization by the methods of dynamics and energy minimization.Also the molecular modeling study of the interaction between ScFv and triazophos was performed by software.The docking showed that triazophos was deeply located within the binding pocket of ScFv.The result of the docking analysis indicated that triazophos mainly bear hydrogen-bond and Van(?)er Waals interaction with ScFv whose 13 residues can involve in the stable binding interactions.The results from the binding mode analysis may be useful in rational lapten design,developing engineering antibody with high affinity and specificitr,and studying the theory of interaction between antibody and pesticide.
Finally,we carried out the initial research for construction the dsFv(disulfide stabilized Fv fragments).Site-specific mutagenesis in the light and heavy chain DNA fragment had been created by PCR using mutagenic primers.Thus a pair of inter-chain disulfide bond would be introduced between the light and heavy chain variable region at the 44~(th) site of the heavy chain and the 100~(th) site of the light chain. The binding mode between dsFv and triazophos was virtually the same as that of ScFv and triazophos.Hydrogen-bond and Van der Waals were the main interactive force.Residues involved in the binding interaction were at similar position.However, dsFv had more residues involved in the docking.There seemed to be affinity differencr between dsFv and ScFv.
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