Studies On Cloning And Expression Of Gene Of G Domain Of Laminin α5 Chain Of Mouse

Laminin is not only the mainly structural molecule of extracellular matrix ,but also the important signal molecule. Laminin and collagen IV compose the net-structure of the extracellular matrix,given the tissue of elastic, tenacity, water-holding power, amortization of mechanical power , ect. Laminin also has important factions in the control of cellular mutiplicatiom cellular migration, cellular differentiation, etc. Laminin is a heterotrimer assembled from α , β , γ chain subunits,twelve Laminins have been found in the mammal. Laminin a 5 chian is widly expressed in the adult tissue , blieved to be the mainly a chian of mammal. G domain is the most important domain of a chain .In this experiment, E3(LG4-5),LG4 and LG5 was cloned from the mouse kidney through RT-PCR. The product of PCR was digested with restriction endonuclease of BamH I and Xho I and ligated directly to the prokaryotic expression vector pET-28a got the three recombination vectors of pET-28a-E3, pET-28a-LG4, pET-28a-LG5. The recombination vectors were transformed into host strain of BL21 and induced with IPTG. All the recombinant protein was expressed into Inclusion Body . The recombinant protein was identified with SDS-PAGE and Westen-blot and purified through elution method .The muticlone antibody was got by immuning the rabbit with the purified protein.E3 and E8 fragments were got from the prokaryotic expression vector used the newly primer. The fragments were ligated directly to the pichia pastrois expression vector pPIC9 to got pPIC9-E3and pPIC9-E8.Vectors were amplificated in the E.coli DH5 a and were linearized with Bgl II .The linearizedvctors were transformed into host strain GS115.The recombinated strain was selected though phynotype and PCRThe positive strain was induced with methyl alcohol and was selected by Dot-ELISA.The recombinated protein was detected with SDS-PAGE and Western-blot as before .