| Transporting the therapeutic proteins and genes to the target cells efficiently is the reseach field of malignant tumor therapy. But because of the poor permeability of cell membrane, it's very difficult to do so. Nowadays some proteins or some domains of the protein were found to have the ability to translocate across membranes and lead therapeutic molecule into cytoplasma. They were named translocating prteins or cell penetrtrating peptides.Pseudomonas exotoxin A is a single-chain cytotoxin composed of three structural domains. Domain I a (l-252aa) is responsible for sell binding, domain II (253-364aa) for membrane translocation enabling access to the cytosol, domain 111(400-613aa) for the catalytic inactivation of protein synthesis, and domain I b(265-399aa) which has no clear function. PE enters cells by receptor-mediated endocytosis and in acidified endosome Furin proteinase cleaves substrates marked with paries of basic amino acids in PE translocation domain, with the cleavage ofpeptide bond between Arg279 and Gly280, sequently release its catalytic domain to the cytosol.Using the translocation domain of PE as a vehicle to transport effectors into cells has evaluated a novel application in biological therapy. But there are many reports to illustrate the length of PE functional translocation domain. Shorten translocation domain and find the smallest but still have translocating function domain can reduce it's space obstruction to the C-terminal molecule and immunogenicity in human.PCR ampliation was used to obtain three recombinant DNAs encoding antibody/granzymeB containing different truncated PE translocation domain (e23sFv-PE II -GrB), which were constructed by fusing single-chain antibody against HER-2 gene and GrB gene to 5'-end and 3'-end of PE translocation domain sequence receptively, or directly fuse antibody against HER-2 gene to 5'-end of GrB. We compared these three recombinant genes with ImmunoGrB(PE253-358). The resulte indicated that ImmunoGrB containing PE translocation domain whether shor or long can enter the HER-2 positive SKBR-3 cells specificly, in the low pH enviroment of endosome, products PE-GrB after Furin cleavage was translocated into cytosol, and induce apoptotic cells death. But the time of cell death and the degree of cell growth inhibition varied, the time of cell death induced by ImmunoGrB(PE275-280) was postponed, and cell growth inhibition was weaker than ImmunoGrB(PE253-358) and ImraunoGrB(PE275-358). However, ImmunoGrB(PE-null) displayed no cytotoxity.We next tested antitumor activity of ImmunoGrB in nude mouse models. The ImmunoGrB fusion genes were packaged by Lipofectemin and intramuscle administrated into the nude mice with HER-2 SKBR-3 xenograft. The groupstreated with ImmunoGrB(PE253-358) , ImmunoGrB(PE275-358) andlmmunoGrB(PE275-280) showed high antitumor activity and survival time of nude mice were prolonged, which is somewhat different with the results of cell in vitro. ImmunoGrB was nightly located selectively in the HER-2 positive tumor tissue by immunohistochemistry staining, while in other tissues, such as heart, liver, lung, spleen, kidney, the staining of ImmunoGrB were negative or low positive. Although ImmunoGrB(PE-null) fusion protein can also selectively locate in tumor tissue, the tumor volumn and nude mice survival days after treatment with ImmunoGrB(PE-null) were similar with control group as a result of it's cell killing inability.In this paper, we construted ImmunoGrB(PE253-358) into retrovirous vector to obtained pLNCX-ImmunoGrB(PE253-358)(PE253-358) and packaged, we obtained monoclone cell lines secreting high titer virus, used media with virus paticles to infect T cells in vitro which later were administrated to nude mice bearing HER-2 positive tumor. As a result, the speed of tumor growth became slow and life span of nude mice were prolonged. The above suggest that it is feasibile to administrate immunoGrB-secreting autologous lymphocytes to suppress tumor in vivo.In summary, when PE translocation do...
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