人源性天然抗体库的构建及初步鉴定

Construction of a large naive human antibody library

目的 构建较大容量的人源性天然抗体库。方法 以未经铭疫的健康人外周血单个核细胞为基因来源，扩增多样性的Ｋ轻链基因和 ＩｇＧ／ＩｇＭ重链Ｆｄ段基因，分别构建铡库和重链库，然后组合为Ｆａｂ段面展示的噬菌体抗体库。通过多次重组积累达到理想的库容，对抗体库进行筛选和鉴定。结果 经过５次轻、重链基因的重组和转化，获得了库容为１×1０～９的人源性天然抗体库。该抗体库性能良好，用３种抗原进行“亲和吸附－洗脱－扩增”淘筛，获得针对其中两种抗原的７株特异性噬菌体抗体。结论天然抗体库为用于不经免疫制备人源性单克隆抗体创造了良好的条件。

Aim To construct a large naive human antibody library. Methods DNAs coding for K chain and Fd fragments of human IgG and IgM repertoires were amplified by PCR from the peripheral blood mononuclear cells of non-immununized healthy donors and cloned into phagemid vector to construct light and heavy chain variable region li- braries. And then a Fab fragment phage library was generated by com- bining the light chain and heavy chain genes. Three different antigens were used to select antibodies from the library. Results By combi- nation of light chain and heavy chain genes, an antibody library con- taining 1 × lO^9 different clones was obtained, in which 80 percent clones have K and Fd genes. 7 clones of specific phage antibody a- gainst 2 antigens (keratin and ferritin) were isolated after 3 to 4 rounds panning Conclusion The successful construction of naive human phage antibody library create favourable conditions for prepar- ing human monoclonal antibodies by-passing immunization.