Prokaryotic Expression, Perforation Of The Hormone N-terminal Peptide Antibody Preparation And Quantitative Detection Of Messenger Rna

Perform (cytolysin;pore-forrning protein), a 67 kD complement-like protein stored in cytoplasm of cytotoxic T lymphocytes (CTL)and natural killer (NK)cells, is thought to be a major mediator responsible for the cytolytic properties of these cells. Perform initiates T-cell cytolysis following aggregation and pore formation in target membranes. The resulting pores cause a breakdown of the transmembrane osmotic gradient and allow other cytolytic mediators to enter the target cell and initiate apoptosis. The recent generation of perform knock-out mice has demonstrated a crucial role for the pore-forming protein in both NK cells and CTL-mediated cytolysis. Perform plays an important immunoregulatory role in the prevention of humoral autoimmunity through the elimination of both autoreactive B cells and Ag-specific T cells. Perform has relative roles in the mechanisms of rejection following organ and tissue transplantation. Perform has the powerful role in clearance of viral infection and control of tumor growth.The studies on perform, although already extensive, have been hampered by the limited amount of material available from killer lymphocytes. To overcome the difficulty in obtaining sufficient amounts of perform, we have attempted in this study to produce recombinant human perform peptide. Because of the fact that the amino-terminal domain of perform account for most of the lysis activity, by a mechanism that is similar to that of holoperforin, the amino-terminal 118 amino acids polypeptide (22 1 39aa) of human perform (hPFP-N) was selectively expressed in E.coli as a fusion protein to get enhanced antigenity. The recombinant expressive vector was constructed by inserting DNA encoding hPFP-N into the plasmid pGEX-KG. The glycine-rich linker of this plasmid greatly increases the thrombin cleavage efficiency of fusion proteins. Upon the induction of IPTG, GST/hPFP-N fusion protein was expressed in E.coli BL21(DE3). The expressed fusion protein was localized in inclusion bodies, whichcould be solubilized by sonication after the detergent lauroylsarcosine was added. The fusion protein was purified by affinity chromatography with glutathione agarose. After being cleaved by thrombin, GST and uncleaved fusion protein were removed by glutathione agarose beads once more, then purified recombinant hPFP-N protein was obtained. Functional characterization showed the purified recombinant hPFP-N to be capable of lysing rabbit red cells. Using this protein as antigen, polyclonal antiserum specific to PFP was raised from New Zealand rabbits. Results of Western blot analysis demonstrate the specificity of the antibody. Immunohistochemical staining indicated that this antibody could identify natural PFP in human lymphocytes. It can be utilized in quantification or qualification tests of PFP. The recombinant hPFP-N protein will be a useful tool for immunological, pathological and structural studies.Quantification of the transcripts of perform by competitive RT-PCR method could represent a valuable tool for the investigation of surveillance function in patients with cancers. The quantitative RT- PCR assay was established and used to detecting the perform mRNA level in peripheral blood of cancer patients and healthy adults. Average perform mRNA level of six healthy adults is 54.6?2.2 fg/~.tgRNA. The perform mRNA level of patients with gastroenteric tumors is significantly lower than that of healthy adults (P