Construction of human phage display scFv library against mastocarcinoma

Objective:To construct a human anti-mastocarcinoma cells phage single-chain Fv antibody library by phage display technology.Methods:About 30 peripheral blood samples of breast cancer patients without chemotherapy were got from hospital.Then RNA was isolated from the peripheral blood mononuclear cells(PBMC)and reverse transcribed to cDNA by RT-PCR.VH and VL were amplified and linked together with a linker segment by overlapping PCR(SOE-PCR)to form a human scFv gene.Cut the scFv gene and phagmid vector pCantab5E by SfiⅠand Not,ligated them and electrically transformed to TG1.Consequently,a primary scFv phage display library was constructed.Results:The fragment of VH and VL were about 360 and 340 bp.They were linked in vitro to form scFv of about 750 bp.The insertion efficiency was about 80% tested by PCR method.Randomized clones from unselected library digested with BstNⅠshowed different patterns.Sequencing indicated that VH and VL genes were homologous with the variable region of human antibody.A recombinant phage display library with total of 2.4×106pfu/ml was established.Conclusion:We constructed a primary human anti-mastocarcinoma cells phage scFv antibody library by phage display technology.It can be used for the next selection of specific anti-mastocarcinoma cells phage antibody.