摘要
目的 构建人源性胶质瘤噬菌体抗体库 .方法 从胶质瘤患者外周血淋巴细胞中提取细胞总 RNA,经逆转录后用套式 PCR分别扩增抗体轻链 Vκ 和重链 VH 基因 .分别构建 Vκ5 .5× 10 6,VH3.5× 10 6基因库 ,连接 Vκ和 VH构建单链抗体 (Sc Fv)基因库 ,并克隆入表达载体 p DNA5内进行重组 ,得到 Sc Fv噬菌体抗体库 .结果 表明经 Cre- L oxp5 11系统重组后 ,噬菌体抗体库库容量达到 6 .0× 10 8,测序证实抗体基因与人源性抗体基因数据库同源 .结论 利用 Cre- L oxp5 11胞内重组系统成功地构建了较大容量的人源性脑胶质瘤噬菌体抗体库 ,为进一步筛选特异性人源性胶质瘤 Sc Fv奠定了基础 .
AIM To construct recombination library of human glioma ScFv antibody by phage display technology. METHODS Total RNA was extracted from peripheral blood lymphocytes of patients with glioma and reversely transcripted into cDNA. Variable region genes (V κ and V H gene) were amplified from the cDNA using nested polymerase chain reaction. V κ (3.5×10 6) and V H (1.5×10 6) gene library was firstly constructed and V κ gene with V H gene were connected, then ScFv gene was cloned into pDNA5. Positive colonies were randomly selected and analyzed by sequencing. RESULTS The phage antibody library was successfully constructed by Cre Loxp recombination system. The library capacity was 6.0×10 8. The sequencing results indicated that the cloned genes encoded variable regions of human antibodies. CONCLUSION The Cre Loxp511 vivo recombination system could enlarge library capacity effectively. The study may be used as a foundation for next screening of specific antibody against human glioma.
出处
《第四军医大学学报》
北大核心
2001年第23期2153-2157,共5页
Journal of the Fourth Military Medical University
基金
国家自然科学基金资助项目 ( 39970 85 2 )
