单链二硫键稳定抗体靶向超抗原SEA（D227A）分子的构建与活性研究
发布时间：2019-05-29 03:43
【摘要】：肝细胞肝癌(HCC),简称肝癌,是恶性程度极高的恶性肿瘤,素有“癌王”之称,目前临床对肝癌的诊治仍然十分困难,仍以手术治疗和放化疗为主。近年来,以抗体为基础的导向治疗方法的研究为肿瘤的诊治带来了生机,先后出现了以单链抗体、二硫键抗体、Fab抗体等抗体形式为导向载体,以细胞因子、毒素分子、超抗原、放射性核素为“弹头”的导向治疗制剂。 在单链抗体、二硫键抗体、Fab抗体这几种导向载体中,二硫键抗体、Fab抗体因重链和轻链之间有二硫键相连,稳定性较高,但表达困难或较难获得较高产量;单链抗体有较成熟的原核表达系统,产量较高,但稳定性较差。为了解决这一问题,我们构建了抗肝癌单链二硫键稳定抗体(这一新型抗体形式)靶向超抗原SEA(D227A)分子,以提高抗肝癌抗体靶向超抗原SEA(D227A)分子的产量和稳定性。单链二硫键稳定抗体(single-chain disulfide-stabilized Fv,scdsFv)是在单链抗体的基础上,在抗体的重链可变区和轻链可变区的框架区分别突变出一个半胱氨酸,以完整的一条链形式表达,这样可在轻、重链可变区之间形成二硫键,以达到提高稳定性和产量的目的。 本研究通过连接肽将抗肝癌二硫键稳定抗体的VH、VL连接起来,构建了单链二硫键稳定抗体,这一最具应用前景的Fv抗体形式,并将其同超抗原SEA(D227A)融合,构建了抗肝癌scdsFv-SEA(D227A)重组表达质粒,以期提高产量和稳定性。 ScdsFv-SEA(D227A)重组表达质粒经IPTG诱导后,在大肠杆菌BL21plusS中以包涵体形式表达,表达量占菌体蛋白的30%左右。包涵体经充分洗涤和变性、复性后,用Q Sepharose HP和Hiprep 26/60 Sephacryl S-200 HR进行纯化,纯度达90%以上,BCA蛋白定量显示每升诱导菌液的产量高达60mg,提高了产量。
[Abstract]:Hepatocellular carcinoma (HCC),) is a malignant tumor with a high degree of malignancy. It is known as the "king of cancer". At present, the diagnosis and treatment of HCC is still very difficult, and it is still dominated by surgical treatment and radiotherapy and chemotherapy. In recent years, the study of antibody-based directed therapy has brought vitality to the diagnosis and treatment of tumors. Single chain antibody, disulfide bond antibody, Fab antibody and other antibodies have been used as guiding vectors, with cytokines and toxin molecules. Superantigens, radionuclides are guided therapeutic agents for warheads. In the vector of single chain antibody, disulfide bond antibody and Fab antibody are more stable because of the connection of disulfide bond between heavy chain and light chain, but the expression is difficult or difficult to obtain higher yield. Single chain antibody has a mature prokaryotic expression system, the yield is high, but the stability is poor. In order to solve this problem, we constructed a single chain disulfide stable antibody (D227A) targeting superantigen SEA (D227A) molecule to improve the yield and stability of anti-HCC antibody targeting superantigen SEA (D227A) molecule. Single chain disulfide stable antibody (single-chain disulfide-stabilized Fv,scdsFv) is based on single chain antibody, which mutates a cysteine in the frame region of the heavy chain variable region and the light chain variable region of the antibody, respectively, and expresses it in the form of a complete chain. In this way, disulfide bonds can be formed between the variable regions of light and heavy chains in order to improve the stability and yield. In this study, the single chain disulfide stable antibody, the most promising form of Fv antibody, was constructed by ligating peptide to ligate the VH,VL of anti-HCC disulfide stable antibody, and it was fused with superantigen SEA (D227A). The recombinant expression plasmid of anti-HCC scdsFv-SEA (D227A) was constructed in order to improve the yield and stability. After the recombinant expression plasmid ScdsFv-SEA (D227A) was induced by IPTG, it was expressed in the form of inclusion body in E. coli BL21plusS, which accounted for about 30% of the bacterial protein. After full washing and denaturation, the inclusion body was purified with Q Sepharose HP and Hiprep 26 鈮，
本文编号：2487613
[Abstract]:Hepatocellular carcinoma (HCC),) is a malignant tumor with a high degree of malignancy. It is known as the "king of cancer". At present, the diagnosis and treatment of HCC is still very difficult, and it is still dominated by surgical treatment and radiotherapy and chemotherapy. In recent years, the study of antibody-based directed therapy has brought vitality to the diagnosis and treatment of tumors. Single chain antibody, disulfide bond antibody, Fab antibody and other antibodies have been used as guiding vectors, with cytokines and toxin molecules. Superantigens, radionuclides are guided therapeutic agents for warheads. In the vector of single chain antibody, disulfide bond antibody and Fab antibody are more stable because of the connection of disulfide bond between heavy chain and light chain, but the expression is difficult or difficult to obtain higher yield. Single chain antibody has a mature prokaryotic expression system, the yield is high, but the stability is poor. In order to solve this problem, we constructed a single chain disulfide stable antibody (D227A) targeting superantigen SEA (D227A) molecule to improve the yield and stability of anti-HCC antibody targeting superantigen SEA (D227A) molecule. Single chain disulfide stable antibody (single-chain disulfide-stabilized Fv,scdsFv) is based on single chain antibody, which mutates a cysteine in the frame region of the heavy chain variable region and the light chain variable region of the antibody, respectively, and expresses it in the form of a complete chain. In this way, disulfide bonds can be formed between the variable regions of light and heavy chains in order to improve the stability and yield. In this study, the single chain disulfide stable antibody, the most promising form of Fv antibody, was constructed by ligating peptide to ligate the VH,VL of anti-HCC disulfide stable antibody, and it was fused with superantigen SEA (D227A). The recombinant expression plasmid of anti-HCC scdsFv-SEA (D227A) was constructed in order to improve the yield and stability. After the recombinant expression plasmid ScdsFv-SEA (D227A) was induced by IPTG, it was expressed in the form of inclusion body in E. coli BL21plusS, which accounted for about 30% of the bacterial protein. After full washing and denaturation, the inclusion body was purified with Q Sepharose HP and Hiprep 26 鈮，
本文编号：2487613
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