Cloning And Prokaryotic Expression Of The Domain Ⅳ Of The Laminin5 γ₂ Chain

Objective: Laminin 5 is a hetertrimeric glycoprotein with a strong adhesive function consisting of three polypeptide chains (03, f^ Y 2 ) that is secreted by keratinocytes. It has been demonstrated that Laminin 5 is close related to skin tissue engineering, genesis and metastasis of rumour ,and inherited junctional epidermolysis bullosa(JEB). The domain IV of the Y 2 chain in Laminin 5 plays an important role in cell adhesion. The recombinant plasmids including domain IV of the Y 2 chain were constructed by applying modern molecular biological technology and expressed in E.coli. DH5 a . Methods: The target gene of the domain was obtained by RT-PCR from cultured human keratinocytes coming from isolation and culture of human skin. The target gene fragment with 647 bp was loaded into cloning vector pGEM-T and transforming E.coli. DH5 a . Then the target gene fragment was cloned into the expression vector pBV220 for transforming E.coli. DH5 a and going on prokaryotic expression after the gene was appraised and validated as target fragment by measuring DNA sequences. The product was determined by SDS-PAGE for its biological activity . Results: . The molecular weight of target protein is about 26kD that was in accordance with the expected size and the expressed protein quantity acccounted for 60% of total proteins of bacterium. Conclusion: The target gene of the domain IV of thechain V 2 could be gained from keratinocytes and expressed in prokaryotic cells in high efficiency.