摘要
目的选用含高效价碱性成纤维细胞生长因子(bFGF)抗体的自身免疫病患者外周血淋巴细胞为材料,构建人噬菌体抗体库,并从中筛选抗bFGF抗体克隆。方法收集自身免疫病患者外周血,提取淋巴细胞总RNA后逆转录得到cDNA第一链,经PCR扩增重链Fd段和轻链基因,分别构建到噬菌粒载体pComb3中,将重组噬菌粒载体电转化大肠杆菌XL1Blue,以辅助噬菌体VCSM13超感染,构建人噬菌体抗体库。经过3轮固相筛选,获得能结合bFGF的抗体克隆,并用ELISA进行进一步鉴定及筛选,获得具有高亲和力的抗bFGF噬菌体抗体克隆。结果构建的人源噬菌体抗体库库容为2.5×107,经3轮筛选获得具有bFGF特异性的Fab噬菌体抗体克隆。结论构建了人噬菌体Fab抗体库,并能够从中筛选出与bFGF特异结合的抗体克隆,为bFGF抗体药物研究奠定基础。
Objective To construct human phage antibody library from autoimmunity patients＇ blood containing high titer of anti-bFGF antibody and select anti-bFGF phage antibody from the library. Methods The heavy chain and light chain of the Fab fragments of human IgG were separately amplified by RT-PCR from peripheral lymphocytes of autoimmunity patients＇ blood. The amplified Fd chain and light chain were inserted into vector pComb3 and electro-transferred into E. coli XL1-Blue cells. The recombinant phage was rescued by coculture with helper phage VCSM13 for constructing human phage antibody library. After three rounds of panning, the specific phage antibodies against bFGF were highly enriched. The high affinity phage antibodies against bFGF were identified by indirect ELISA. Results The results shown that the human phage antibody library was constructed （library content was 2.5× 10^7） and specific phage antibodies were obtained from the antibody library. The construction of the human phage antibody library and the preparation of the anti-bFGF phage antibodies provide a basis for the research of anti-bFGF drugs.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2006年第4期451-454,共4页
Immunological Journal
基金
国家自然科学基金(30471607)
教育部科学技术项目(105175)资助
关键词
bFGF抗体
噬菌体展示
抗体库
Anti-bFGF antibody
Phage display
Antibody library
