Construction of phage random eight-peptide library / 中国病理生理杂志

ABSTRACT

AIM: To construct random eight-peptide library for the study on atherosclerosis and restenosis. METHODS and RESULTS: Random oligodeoxynucleotides encoded eight peptides were synthesized and amplified by polymerase chain reaction(PCR). The product was cloned into phage surface display vector fUSE5 in Sfi I site and electroporated into competent MC1061. The library was identified through PCR, hybridization, DNA sequencing and affinity biopanning of streptavidin. Because the upstream primer is complementary to part vector clone site sequences and part exogenous gene sequences, and the other one complementary to pIII gene of vector, thus only clones inserted exogenous gene could be amplified easily. Additionally we used the probe oligodeoxynucleotide complementary to vector clone site sequences to identify clones which were not inserted exogeneous genes. Furthermore, two hybridizing positive clones were sequenced. Their sequences are consistent with two oligodeoxynucleotide probe sequences. As a result, 2.1?108 special clones were obtained. Affinity biopanning proved that the libraries could be amplified steadily. CONCLUSION: The eight-peptide library is reliable.