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目的从人源噬菌体抗体库中筛选与肝癌细胞系HepG2特异性结合的单链抗体,为寻找肝癌细胞表面特异性标志及靶向研究奠定基础。方法以正常肝细胞系L02为阴性筛选细胞,以肝癌细胞系HepG2为阳性筛选靶细胞,从人源噬菌体抗体库(Griffin.1Library)经三轮筛选后,阳性菌质粒PCR确定其中含有单链抗体基因的克隆,通过细胞ELISA及FCM筛选特异性的单链抗体噬菌体,通过ABI3730全自动荧光测序仪测序,并通过GeneBank比对进行同源性分析,IPTG诱导可溶性单链抗体表达,并检测其与肝癌细胞株结合的特异性。结果获得了2个特异性较高的阳性噬菌体单个克隆。经DNA测序后,在Genebank中与人的免疫球蛋白库进行比对,并用IMGTVQuest软件进行分析,确定为2个插入序列不同的单链抗体片段。经细胞ELISA证明其阳性克隆菌培养上清可与肝癌细胞株特异性结合。获得的序列成功登陆Genebank,序列号为AY686498AY686499。结论从人源噬菌体抗体库中筛选到与肝癌细胞系HepG2特异性结合的具有功能活性的单链抗体,为进一步寻找肝癌细胞表面特异性抗原及靶向研究奠定了基础。
Objective To screen single-chain antibodies specific for HepG2 cells from human phage antibody library and lay a foundation for the study of specific surface markers of hepatocellular carcinoma cells and their targeting. Methods The normal liver cell line L02 was screened for negative cells and the hepatoma cell line HepG2 was selected as the positive cell line. After three rounds of screening from the human phage antibody library (Griffin.1 Library), the positive bacteria plasmid PCR confirmed that it contained single chain antibody The specific clones were screened by ELISA and FCM. The phage were screened by ABI3730 automatic fluorescence sequencer and analyzed by GeneBank. IPTG induced the expression of soluble single chain antibody, Specificity of hepatoma cell line binding. Results Two single positive clones with higher specificity were obtained. After DNA sequencing, human immunoglobulin pools were aligned in Genebank and analyzed with IMGTVQuest software to identify two single-chain antibody fragments with different insertions. The cell ELISA proved that the positive clone culture supernatant can be combined with the liver cancer cell lines. The obtained sequence successfully landed in Genebank with the sequence number of AY686498AY686499. Conclusion The scFv of human phage antibody was screened for specific binding to HepG2 cell line HepG2, which laid the foundation of further research on the surface antigen-specific targeting of HepG2 cells.