Construction of a phage-displayed library with cDNA ofhuman hepatocarcinoma cell / 重庆医科大学学报

ABSTRACT

Objective:To identify the human hepatocarcinoma cell line with the highest reactivity to hALR and to construct the phage-displayed library with its cDNA as the basis of the selection for special protein binding hALR from this library.Methods:The reactivity to hALR of HepG2,7701 and QGY was compared by 3?H-TdR methods in vitro.The mRNA of QGY with the highest reactivity to hALR was isolated with PloyATtract System 1000 kit.The phage-displayed library with cDNA fragments of QGY was constructed withT7Select10-30rient Express cDNA Cloning System and Random Primer kit.The quality of this library was evaluated by phage titer assay and PCR.Results:hALR could stimulate the proliferation of QGY,HepG2,and 7701 in dose-dependent way in vitro,but QGY showed the highest reactivity to hALR.The phage-displayed library with cDNA of QGY was successfully constructed and contained approximate 2?107 primary recombinants.It was indicated by PCR that the size of the cDNA inserts was from 0.2 to 2kb,the average 0.6kb.Thorough information about mRNA of QGY cells could be soundly represented by this library.Conclusion:The results show that QGY had the highest reactivity to the hALR among three strains detected.The high quality phage-displayed library is successfully constructed with cDNA of QGY.