抗破伤风类毒素单抗可变区基因的克隆及在大肠杆菌中表达的三种工程抗体

The heavy and light chain variable region genes of anti-tetanus toxoid (TT) antibody and the heavy chain Fd genes were amplified and cloned through RT-PCR from mouse hybridoma cells. The sequences of VH and VK were determined. Fd gene fragments were expressed in E. coli. The ELISA results indicated that the expressed Fd showed antigen binding activity but was nonspecific. Furthermore, through SOE and PCR techniques, the VH and VK gene fragments together with ScFv linker were assembled into single chain antibody (ScFv) gene fragment. While together with human heavy chain CH 1 gene fragment and Fab linker, they were assembled into chimeric Fab gene fragment. The two assembled gene fragments were separately inserted into phagemid pHEN 1, which was a fd-based vector containing gene 3 encoding the minor coat protein. In presence of helper phage M 13-VCS the anti-TT phage-ScFv or phage-Fab were displayed on the surface of phage particles respectively. Results from phage-ELISA indicated that both phage antibodies were TT-specific.     

人-鼠嵌合Fab片段的温度诱导表达

构建了具有λＰＲＰＬ启动子高效表达人鼠嵌合Ｆａｂ片段的温度诱导表达型载体ｐＨＺ０１,并在大肠杆功中表达了三种嵌合Ｆａｂ片段：抗前列腺特异抗原（ＰＳＡ）的嵌合Ｆａｂ,抗溶菌酶（ＨＥＬ）的嵌合Ｆａｂ和抗破伤风类毒素（ＴＴ）的嵌合Ｆａｂ,三种表达的可溶性嵌合Ｆａｂ都具有特异结合抗原的能力,嵌合Ｆａｂ的ＣＨ１和ＣＫ区均为人源的,较之鼠源Ｆａｂ具有更好的应用前景。     

抗前列腺特异抗原的单链抗体的克隆和表达

本文克隆了抗前列腺特异抗原(PSA)单抗526的轻、重链可变区基因,构建了在大肠杆菌中表达单链抗体的具有强启动子PR和PL的温度诱导型表达载体,表达了抗前列腺特异抗原的单链抗体。表达产物经ELISA测定证明具有特异结合PSA的能力。     

抗破伤风类毒素单抗可变区基因的克隆及在大肠杆菌中表达的三种工程抗体

我们采用RT-PCR,从小鼠杂交瘤细胞中扩增并克隆了抗破伤风类毒素(TT)抗体轻、重链可变区,重链Fd区基因,测定了其VH、Vk序列。并在大肠杆菌中表达了Fd片段,ELISA分析的结果表明Fd片段具有抗原结合的能力,但特异性很差。进一步采用SOE,和PCR技术,将VH、VK基因与ScFv连接片段组装成单链抗体(ScFv)基因片段,以及将人重链CH1和Fab基因连接片段组装成Fab基因片段。将它们分别插入含噬菌体fd外壳蛋白3基因的phagem-id pHEN 1中,在辅助噬菌体M 13-VCS作用下,噬菌体表面表达了抗TT的噬菌体单链抗体(phage-ScFv)与噬菌体Fab(phage-Fab),经ELISA检测,表明它们都能与TT特异结合。     

人-鼠嵌合Fab片段的温度诱导表达

构建了具有λPRPL启动子高效表达人鼠嵌合Fab片段的温度诱导表达型载体pHZ01。并在大肠杆菌中表达了三种嵌合Fab片段：抗前列腺特异抗原(PSA)的嵌合Fab．抗溶菌酶(HEL)的嵌合Fab和抗破伤风类毒素(TT)的嵌合Fab．三种表达的可溶 性嵌合Fab都具有特异结合抗原的能力,嵌合Fab的CHI和CK区均为人源的。较之鼠源 Fab具有更好的应用前景。