| Found in 1994, human laminin α4 chain is 122kp long with 39 exons and located on chromosome 6q21. The ORF encoding 1851 amino acids is 5448 long, Laminin α4 chain mainly distributes in tissues originating from mesoderm, such as basement membrane of endothelium, adipocytes, skeletal muscle cells, myocardial cells and smooth muscle cells. It also appears in basement membranes of such epithelium as epiderm, salivary glands, pancreas, esophagus, intestinal lacuna, tubules in renal cortex and gastric glands, α4 chain is one of the subunits of laminin-8 and laminin-9. In capillaries of skeletal muscles, α4 chain appears at stage Ell and present until adulthood. The biological functions of α4 chain are still unknown. No relationship has been determined between α4 chain and human diseases and abnormal response. The extensive and specific expression of α4 chain in developing microvessel suggests that it may be involved in angiogenesis. Laminin α4-null mice experience widespread bleeding in subcutaneous tissues and muscles leading to anemia. Extensive bleeding in microvessel, movement defect, and damages in microvessel structures under electron microscope . laminin α4-null mice indicate that α4 chain is essential for the formation and maintenance of integrity of neovascular growth. Overexpression of α4 chain in brain tumours such as glioblastoma multiforme, astrocytoma, and meningioma might become one of the markers for the diagnosis of neuroglioma.Studies have indicated that the interaction between G domain of laminin and cell receptors as integrin and a-dystroglycan and other extracellular ligands is important for the integrity of basement membrane. This interaction may mediate the adhesiveness of integrin-dependant cells and is significant in metastasis and infiltration of such brain tumors as glioblastoma multiforme, astrocytoma, and meningioma. Following studies have been performed in order to investigate crystal structures and biological functions of LG1-3 modules.Prokaryotic expression of LG2 module gene in laminin α4 chain RT-PCR was employed to amplify the cDNA of LG2 and then was recombinated into cloning vector and prokaryotic expression vector. Cloning plasmid pMD-LG2 and prokaryotic expression plasmid pET-LG2 were constructed . E. Coli BL21(DE3) were used as host bacteria and induced with IPTG, the 26kDa LG2 recombinant protein were expressed. The recombinant protein LG2 were purified with SDS-PAGE. A single band was shown in SDS-PAGE and then it was used as the antigen for the screening for monoclonal antibody.The preparation of monoclonal antibody against LG2-3 module gene in laminin α4chain RT-PCR was used to amplify LG 2-3 cDNA in laminin α4 chain so as to detect the recombinant LG. Cloning plasmid pMD-LG2-3 and eukaryotic expression plasmid pVAX-LG2-3 were constructed. BALB/c mice were immunized with eukaryotic expression plasmid pVAX-LG2-3 by means of genetic immunity. 1 cell strain of hybridoma CF12 was established after conventional cell fusion, indirect ELISA screeeing and limiting dilution cloning. Monoclonal antibody CF12 has specific response to recombinantly expressed LG2 recombinant protein. Then immunohistochemical assay was performed to detect the reaction of monoclonal antibody CF12 to fetal kidney, small intestine and large artery. The results showed that the positive reaction occurred in basilar membrane of renal tubules, cell membrane of Bowman's capsules, serum membrane of small intestine, smooth muscle of small intestine and capillary endothelium, epithelial cell membrane of intestinal villi and endothelial membrane of large artery. The results were coincidence with the available reports, thus demonstrating the specificity of the monoclonal antibody prepared with gene immunization.The expression of LG1-3 module gene in laminin α4 chain in Pichia pastrois expression system The cDNA of LG1-3 in laminin 4 chain was obtained by RT-PCR. Cloning plasmid pMD-LGl-3 was constructed. By in vitro site mutation, Sac I endonuclease site in 960bp of LG 1-3 was mutated...
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