抗三唑磷基因工程抗體的研制及同源建模
發(fā)布時(shí)間：2018-12-11 17:12
【摘要】： 為了研制基因工程抗體,本文首先通過雜交瘤技術(shù)篩選能夠穩(wěn)定分泌高親和力、高特異性抗體的雜交瘤細(xì)胞,以從中克隆編碼抗體可變區(qū)的基因。利用雜交瘤細(xì)胞制備的單克隆抗體建立了三唑磷殘留ELISA檢測(cè)方法,并對(duì)方法進(jìn)行了系統(tǒng)優(yōu)化。優(yōu)化后的ELISA應(yīng)用于食品和環(huán)境樣品中三唑磷殘留檢測(cè),其良好的回收率和較小的變異系數(shù)一方面證明了ELISA在三唑磷等農(nóng)藥的殘留檢測(cè)中具有良好的應(yīng)用前景,另一方面也說明篩選的雜交瘤細(xì)胞是成功的,可以用于基因工程抗體的研制。 然后從雜交瘤細(xì)胞株出發(fā),通過RT-PCR技術(shù),利用小鼠重鏈和輕鏈可變區(qū)通用引物擴(kuò)增得到了抗三唑磷單克隆抗體輕、重鏈可變區(qū)基因序列。再通過重疊延伸PCR(SOE-PCR)方法,將兩個(gè)可變區(qū)基因連接起來,構(gòu)建了單鏈抗體(ScFv)核苷酸序列。然后將ScFv序列插入到pET-29a(+)載體,得到了重組表達(dá)質(zhì)粒,轉(zhuǎn)化大腸桿菌BL21(DE3)后,陽性菌株經(jīng)IPTG誘導(dǎo)培養(yǎng)后,表達(dá)得到了ScFv蛋白。蛋白經(jīng)過變性、純化和復(fù)性處理后,通過竟?fàn)幰种艵LISA進(jìn)行了初步鑒定,結(jié)果表明,ScFv具有免疫親和活性。 在此基礎(chǔ)上,利用Accelrys公司的Discovery Studio~(TM)(DS)軟件系統(tǒng)通過同源建模法模擬了抗三唑磷單鏈抗體的三級(jí)結(jié)構(gòu)及ScFv與三唑磷農(nóng)藥小分子間的對(duì)接構(gòu)象。首先通過模板分子搜索、序列比對(duì)、構(gòu)建模型及模型合理性評(píng)價(jià)等過程分別構(gòu)建了輕、重鏈可變區(qū)三維結(jié)構(gòu);然后再通過模板分子搜索、序列比對(duì)及疊合等過程,初步構(gòu)建了ScFv的三維結(jié)構(gòu)模型。模型經(jīng)過動(dòng)力學(xué)和能量最小化優(yōu)化后,最終得到了評(píng)價(jià)合理的ScFv的三維結(jié)構(gòu)。分子對(duì)接模擬結(jié)果顯示,單鏈抗體與三唑磷主要以范德華力和氫鍵發(fā)生相互作用,抗體有13個(gè)氨基酸殘基可以與農(nóng)藥分子發(fā)生作用。這些氨基酸作用位點(diǎn)和作用方式的確定為進(jìn)一步進(jìn)行抗體分子的改造和研究抗體與農(nóng)藥小分子間的作用機(jī)理打下了堅(jiān)實(shí)的基礎(chǔ)。 最后在二硫鍵穩(wěn)定抗體(dsFv)的研制、dsFv同源建模及分子對(duì)接方面開展了部分工作。利用PCR定點(diǎn)突變法,通過設(shè)計(jì)突變引物,成功地將半胱氨酸突變位點(diǎn)引入了輕、重鏈可變區(qū)的相應(yīng)位點(diǎn),從而為dsFv小分子抗體的研制打下了基礎(chǔ)。DS軟件系統(tǒng)對(duì)dsFv三級(jí)結(jié)構(gòu)及dsFv與三唑磷的對(duì)接構(gòu)象的模擬結(jié)果表明,dsFv與ScFv具有相似的活性口袋,在與三唑磷發(fā)生對(duì)接過程中,也是以氫鍵和范德華力作為主要作用方式,參與作用的氨基酸殘基大部分相同,但dsFv可參與反應(yīng)的氨基酸數(shù)目更多,這意味著dsFv和ScFv對(duì)三唑磷的親和力可能會(huì)有差異。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 李鵬;抗重金屬汞、銅、鋅單抗可變區(qū)序列的克隆鑒定、真核表達(dá)及三維模擬[D];南京農(nóng)業(yè)大學(xué);2011年
，本文編號(hào)：2372924
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