Construction of single-chain Fv library against hepatocellular carcinoma.
OBJECTIVE: To construct a phage display single-chain Fv (ScFv) library against hepatocellular carcinoma (HCC). METHODS: The mRNA extracted from the spleen cells of immunized BALB/C mice was purified and reverse transcriptase-polymerase chain reaction (RT-PCR) was employed for the seperate amplification of the gene fragments encoding the heavy- and light-chain variable regions respectively, which were subsequently assembled into ScFv gene. Amplification of the resultant ScFv gene was performed before it was introduced in to E.coli TG1 via a phagemid vector pCANTAB5E. A proportion of the transformed cells was loaded onto SOBAG plates and incubated and the number of bacterial colonies counted. PCR, nucleotides sequence analysis and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the constructed ScFV library. RESULT: The phage display ScFv library had a capacity of approximately 2.14x106, with 90% of the phagemids containing ScFv gene insertion as demonstrated by PCR. The nucleotides sequence of ScFv gene was approved, and ELISA showed that the ScFv library could specifically conjugate with HCC antigen. CONCLUSION: The phage display ScFv library against HCC is successfully constructed, which may facilitate the acquisition of special antibodies and may be instrumental in the research and clinical management of HCC.
Yang
Dong-Hua
DH
Department of Gastroentology, The First Affiliated Hospital, Medical College of Jinan University, Guangzhou 510630, China.
Bi
Xiang-Jun
XJ
Nong
Yu-Xin
YX