Zhao Xiao-ling, Yin Jun, Wang Hui, Jiang Ming, Hou Xiao-jun
Institute of Microbiology and Epidemiology, the Academy of Military Medical Sciences, Beijing 100071, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 Mar;20(2):243-7.
To construct a human phage displayed single chain Fv antibody (scFv) library and screen specific scFv against rabies virus.
Using a phage-display technique, IgVH and IgVL genes were cloned from peripheral blood lymphocytes from three donors immunized with the WISTAR PM strain vaccine. Genes encoding scFv fragments were constructed by random linkage of VH gene and VL gene by SOE PCR, and then the constructed scFv genes were cloned into phagemid vector and transfected into E. coli TG1. The recombinant phages were screened by four rounds of panning with rabies virus vaccine. The positive recombinant phages were sequenced and antigen-binding activity of recombinant scFv was detected by competition ELISA.
A human phage-displayed scFv library with about 7 x 10(8) sink size was constructed. A new human scFv was selected, which bound specifically to rabies virus and had higher affinity.
The results lay a solid foundation for preparation of human engineering antibody to rabies virus reported herein with higher affinity.
构建人噬菌体展示单链Fv抗体（scFv）文库，并筛选针对狂犬病病毒的特异性scFv。
采用噬菌体展示技术，从3名接种WISTAR PM株疫苗的供体的外周血淋巴细胞中克隆IgVH和IgVL基因。通过SOE PCR将VH基因和VL基因随机连接构建编码scFv片段的基因，然后将构建好的scFv基因克隆到噬菌粒载体中并转染到大肠杆菌TG1中。用狂犬病病毒疫苗进行四轮淘选筛选重组噬菌体。对阳性重组噬菌体进行测序，并通过竞争ELISA检测重组scFv的抗原结合活性。
构建了库容约为7×10(8)的人噬菌体展示scFv文库。筛选到一种新的人scFv，它能特异性结合狂犬病病毒且具有较高亲和力。
本研究结果为制备具有更高亲和力的狂犬病病毒人源工程抗体奠定了坚实基础。
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