Expression And Functional Analysis Of ADAMs Related Genes In Hepatoma Cell

BackgroundThe members of ADAMs family belong to type I transmembrane protein,which share similar basic structure including of disintegrin and metalloprotease functional domain.ADAM functions are implicated in ECM degradation,cell-cell and cell-matrix interactions,signal transduction,and so on.Although many functions of ADAMs are already known,futher researchs are still required because of the complicated structure,numerous members and specificity of tissue distribution of this protein family.ObjectiveTo construct the cDNA library of the Changliver cells(human hepatoma cell)treated by DM(dedifferentiation medium),and to study their function,the differentiation-related gene of ADAMs was immunoscreened and cloned with the primary antibody of ADAMs.Methods1.Changliver cells treated by DM was checked by Western blot.2.The cDNA library of the Changliver cells treated by DM for 30 min and recovered for 24 h was constructed.3.The library was immunoscreened with the primary antibody of ADAMs and full-length cDNA was cloned,and then sequenced and registered.4.The prokaryotic expression vector of ARP1 was constructed,and the preliminary function was analyzed using SDS-G-PAGE and Western blot.5.The distribution and protein map of ADAMs and their protease activity in different tissues and organs including liver of rat were analyzed with SDS-G-PAGE and SDS-PAGE.Results1.30kD?50kD and 105 kD ADAMs were showed in the tested sample,and the 30 kD and 50 kD ADAMs were induced by DM.2.The cDNA library of the Changliver cells treated was constructed successfully.3.The library was immunoscreened with the primary antibody of ADAMs and full-length cDNA was cloned.Four Species-specific genes associated with ADAMs were sequenced and registered.4.The ARP1 was analyzed using SDS-G-PAGE and Western blot,and found it has alkaline protease activity,and could degrade gelatin,what`s more,could bind the primary antibody of ADAMS.5.The results show that the distribution of ADAMs and enzyme activity were very different in 17 tissues and organs of rat.ConclutionIn this study,the c DNA library of Changliver cells treated by DM was constructed and immunoscreened.It was preliminarily verified that 30 kD and 50 kD ADAMs were induced by DM.It was confirmed that the constructed cDNA library had standard quality and strong usability.Four Species-specific genes associated with ADAMs were sequenced and registered in Gencank.The prokaryotic expression vector of ARP1 was constructed and its expression product had alkali protease activity and able to bind the primary antibody of ADAMs.The distribution of ADAMs and enzyme activity was significantly different in 17 tissues and organs of rat.This study provides important information to further incestigate the function of ADAMs and its related genes.