重链可变区基因随机CDR3 ScFv噬菌体抗体库的构建及筛选

Construction and screening of ScFv phage repertoire with random CDR3 of VH gene

目的构建VH 基因随机CDR3ScFv噬菌体抗体库 ,并筛选抗HFRS病毒NP抗原的噬菌体抗体。方法采用随机引物PCR法扩增抗HFRS病毒mAb1A8的VH 基因 ,并与1A8VL 基因共同克隆入噬菌粒表达载体 pHEN1后 ,构建VH 基因随机CDR3ScFv基因库。继用辅噬菌体VCSM13超感染后得到噬菌体抗体库 ,然后用HFRS病毒抗原进行筛选。随机挑取筛选的菌落超感染 ,用夹心ELISA检测超感染上清。结果获得库容量约为107 噬菌体抗体库。夹心ELISA的结果表明 ,筛选出的噬菌体抗体可与HFRS病毒NP抗原特异性结合。结论成功地构建了库容量较高的ScFv噬菌体抗体库 ,并获得可与HFRS病毒NP抗原结合的噬菌体抗体。

Aim To construct ScFv phage repertoire with random CDR3 of VH gene and to screen phage antibodies against NP antigen of HFRS virus. Methods The VH gene of mAb 1A8 against HFRS virus was amplified with random oligonucleotide primers. The ScFv gene repertoire was constructed by cloning the amplified VH gene into phagemid expression vector pHEN1 containing VL gene of mAb 1A8. The phage repertoire was obtained by superinfection with helper phage VCSM13 and then screened using NP antigen of HFRS virus. Subsequent clones were selected randomly and the phagemid were rescued with VCSM13 helper phage. The supernatants harvested were detected by indirect sandwich ELISA. Results The ScFv phage repertoire with capacity of 107 was obtained. The results of indirect sandwich ELISA indicated that screened phage antibodies could react specifically with the NP antigens. Conclusion ScFv phage repertoire with higher capacity is constructed successfully and phage antibodies with binding activities to NP antigen are obtained.