全人源肝癌噬菌体单链抗体的筛选及特异性鉴定

目的对全人源肝癌噬菌体单链抗体库进行鉴定,筛选肝癌抗体,同时对抗体的活性及特异性进行鉴定。方法PCR鉴定阳性重组菌TG1中人肝癌ScFv的插入率。先以人成纤维细胞吸附后再以体外培养的肝癌细胞SMMC7721为抗原对所建抗体库进行3轮“吸附洗脱扩增”的亲和筛选。将筛选后的ScFv进行PCR鉴定及双酶切鉴定;通过ELISA法及FCM鉴定其与人肝癌细胞及正常细胞的结合活性。结果ScFv基因插入率为70%。在亲和筛选过程中,肝癌噬菌体单链抗体得到富集,收获率逐轮得到提高,第3轮为第1轮的214倍。筛选后的ScFv进行PCR鉴定及双酶切鉴定,均可检测到目的基因。ELISA分析结果显示18个克隆与SMMC7721呈阳性反应,阳性率为90%,15个克隆与成纤维细胞有交叉反应。得到3株肝癌单链抗体。ScFv的FCM鉴定表明,以正常胎肝细胞L02为对照,ScFv与肝癌结合比率为41.3%。特异性鉴定表明,其与肝癌细胞结合活性明显高于正常细胞。结论利用噬菌体抗体库技术结合减数筛选得到了肝癌噬菌体单链抗体及其基因,且筛选后的抗体片段与人肝癌细胞有特异性的结合活性。

Screening of human phage antibodies with single-chain variable fragment and identiflication of its specificity to liver cancer cells

OBJECTIVE: To identify the human phage a ntibodies with single-chain variable fragment(ScFv), to select the ScFv specifi c to hepatocellular cancer and to detcct the avidity and specificity of the ScFv s. METHODS: The recombinant phages inserted with ScFv were determined by PCR.The recombinant phages were panned by human liver cancer ce ll line of SMMC-7721 after they were counter-selected with human fibroblast ce lls and human embrro liver cells L-02.After 3 rounds of biopanning,the ScFvs we re identified by PCR and enzymes digestion,the specificity of ScFv from the mono clonal bacteria was determined by ELISA and FCM.RESULTS:Th e rate of recombinant bacteria inserted with ScFv was 70%. From the 1st round to the 3th round of the panning,the number of the eluted phages increased 214 tim es.The inserted fragments were detected from the selected ScFvs by PCR and enzym es digestion. Positive reactions to SMMC-7721 were detected by ELISA in 18 clon es.The positive rate was 90%.Cross reactions to fibroblast cells were detected i n 15 clones. Three ScFvs to liver cancer cells were acquired. ScFv can combined 41.3% human liver cancer cell line of SMMC-7721 using normal liver cells as co ntrol by FCM. CONSLUSION:ScFvs to liver cancer cells and i ts genes were acquired by the phage antibody panning assisted by subtraction sel ection.The selected ScFvs has avidity specifically to human liver cancer cells. [