Si B, Yang P, Qin E
Institute of Microbiology and Epidemiology, Academy of Military Medical Scienes, Beijing 100850.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 1998 Sep;12(3):229-32.
Hybridoma 3D3 which secreted monoclonal antibody against Dengue type 3 virus was used to extract mRNA and then was reverse-transcripted into cDNA for amplifying heavy and light chain variable domain genes. The amplified light and heavy chain variable domain genes were connected by flexible linker to form a single chain antibody fragment (ScFv) gene of 750 bp, which was cloned into phage pCANTAB5 vector using the recombinant phage antibody system. The ScFv gene was expressed as fusion protein with phage g3p coat protein and displayed on the phage surface. The phagemid was used to transform competent E. coli TG1 cells and then infected with M13K07 helper phage to rescue the phagemid and antibody ScFv gene. 12 of the 20 randomized clones were shown to react with dengue type 3 virus by immunofluorescence.
分泌抗3型登革病毒单克隆抗体的杂交瘤3D3用于提取mRNA，然后反转录成cDNA以扩增重链和轻链可变区基因。扩增的轻链和重链可变区基因通过柔性接头连接，形成一个750 bp的单链抗体片段（ScFv）基因，使用重组噬菌体抗体系统将其克隆到噬菌体pCANTAB5载体中。ScFv基因与噬菌体g3p外壳蛋白表达为融合蛋白，并展示在噬菌体表面。该噬菌粒用于转化感受态大肠杆菌TG1细胞，然后用M13K07辅助噬菌体感染以拯救噬菌粒和抗体ScFv基因。20个随机克隆中有12个通过免疫荧光显示与3型登革病毒发生反应。
