Ma Xiao-bing, Chang Ji-wu, Wang Hai-tao, Wang Xin
Tianjin Institute of Urology,The Second Hospital of Tianjin Medical University, Tianjin 300211, China.
Wei Sheng Yan Jiu. 2006 Mar;35(2):206-8.
To construct a naive human Fab fragment phage display library, provide a platform for human antibody preparation and a new therapy for the malignant tumors.
Peripheral blood lymphocytes were isolated from 200 ml blood, which was obtained from a healthy blood donor. The heavy chain Fd fragments and light chain cDNA synthesized from the total RNA of lymphocytes were amplified by PCR and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E. coli XL1-Blue. The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody of Fabs. The phagemids abstracted from amplified E. coli were cut with endonucleases such as Sac I, Xba I, Spe I and Xho I and amplified by PCR to monitor the insertion of the light chain or heavy chain Fd genes. Human albumin and interleukin-2 were utilized as antigens to conduct respectively three rounds of panning to the original Fab antibody library.
By combination of light chain and heavy chain genes, an antibody library containing 1.2 x 10(6) clones was obtained, and both the cutting of enzymes and PCR showed that there were the light chain or heavy chain Fd genes of the phagemids. After the original antibody library having been panned respectively by two kinds of antigen proteins, it gained enrichment in different degree.
Utilizing the technology of phage surface display, special antibody can be gained from the human naive Fab phage display library, which can be used as a new therapy for tumors.
构建天然人Fab片段噬菌体展示文库，为制备人抗体提供平台，并为恶性肿瘤提供新的治疗方法。
从一名健康献血者提供的200ml血液中分离外周血淋巴细胞。从淋巴细胞总RNA合成的重链Fd片段和轻链cDNA经PCR扩增，扩增产物连接到噬菌粒载体pComb3中，然后将连接产物转化到感受态大肠杆菌XL1-Blue中。用VCSM13辅助噬菌体感染转化细胞，产生Fabs重组噬菌体抗体。从扩增的大肠杆菌中提取的噬菌粒用Sac I、Xba I、Spe I和Xho I等内切酶切割，并用PCR扩增以监测轻链或重链Fd基因的插入。用人白蛋白和白细胞介素-2作为抗原，分别对原始Fab抗体文库进行三轮淘选。
通过轻链和重链基因的组合，获得了一个包含1.2×10(6)个克隆的抗体文库，酶切和PCR结果均显示噬菌粒中有轻链或重链Fd基因。原始抗体文库经两种抗原蛋白分别淘选后，均有不同程度的富集。
利用噬菌体表面展示技术，可从人天然Fab噬菌体展示文库中获得特异性抗体，可作为肿瘤的新治疗方法。
