Abstract:

Objective According to the need of large-scale gene cloning and the construction of protein interaction map in human liver proteome project (HLPP),to reconstruct the yeast two-hybrid plasmids pPC86 and pDBLeu,and then set up the Chinese liver cDNA library,and lay the foundation for further study of HLPP.Methods The specific oligonucleotides were designed and synthesized.They were treated to form double-strand DNA fragments with specifc sticky end,and inserted into pPC86 or pDBLeu vector at the appropriate restrictive enzyme sites to form reconstructed plasmids.The reconstructed vector,Chinese normal liver tissue mRNA and Clontech’s SMART cDNA library construction kit were used to construct NpDBLeu-cDNA library.Results Restriction enzyme digestion and DNA sequencing confirmed the insertion sites and correct sequences of reconstructed vectors.The NpPC86 and NpDBLeu were obtained successfully.Titer tests showed the content of constructed NpDBLeu-Chinese liver cDNA library reached 1.93×10⁶ cfu，in which the percentage of recombinant clones was 95.2%.The titer of amplified library reached 109cfu·mL^-1.Conclusion The Chinese normal liver cDNA library is successfully constructed using SMART technique.

Key words: SMART technique；liver tissue；reconstruct vector；cDNA library