人穿孔素羧基端肽段的表达纯化与活性鉴定

Expression, Purification and Hemolytic Activity of Recombinant Human Perforin Carboxylterminal Peptide

穿孔素 ,即成孔蛋白 (poreformingprotein ,PFP) ,其溶细胞作用与免疫调节和自身免疫病以及其它多种疾病过程中的免疫性病理损伤相关 .为得到足够量的PFP建立与之相关的免疫学研究手段用于基础和临床研究 ,在已克隆人PFPcDNA的基础上 ,用基因工程方法表达了人PFPC端 12 4个氨基酸肽段 (hPFP C) ,并通过谷胱甘肽琼脂糖亲和层析获得纯化的GST/hPFP C融合蛋白 ,经凝血酶酶切和再次亲和层析去除GST部分 ,得到了纯化的hPFP C蛋白 .纯化的hPFP C蛋白与兔红细胞共育 ,呈现钙依赖的溶血活性 .

Perforin (also known as pore forming protein, PFP) is one of the main effector molecules which natural killer cells (NK) and cytotoxic T lymphocytes (CTL) utilize to kill their targets both in vivo and in vitro. It has been shown to be capable of undergoing polymerization to form pores in cell membranes and cause osmotic lysis and apoptosis of target cells, and its expression is now considered a specific marker of functionally active CTL and NK cells. Recent studies suggested that perforin have relative roles of immune regulation and immunopathological damage in allograft rejection,various viral and autoimmune diseases. The studies on perforin, although already extensive, have been hampered by the limited amount of material available from killer lymphocytes. To overcome this difficulty, we have attempted in this study to produce recombinant human perforin antigen. Because of the fact that the central one third of the perforin molecule is homologous, both structurally and functionally, to the putative membrane spanning domain of the complement components C6 through C9, only the N terminal portion (about 40aa residues) and C terminal portion (about 100 aa residues) are unique for the perforin molecule. The carboxylterminal 124 amino acids polypeptide (410 534aa) of human perforin (hPFP C) was selectively expressed in Escherichia coli to ensure that the recombinant protein has a antigenic specificity of perforin. Moreover, to get enhanced antigenity, the recombinant hPFP C protein is expressed as a fusion protein. The cDNA fragmants of hPFP C were obtained by PCR from the plasmid pCR2.1 (in which full length of hPFP cDNA had been cloned by us before), and then inserted into pGEX 2T to construct a recombinant expressive vector. Upon the induction of IPTG, GST/hPFP C fusion protein was expressed in E.coli BL21(DE3). The recombinant fusion protein was localized to include bodies which could be solubilized with urea. The denatured fusion protein was refolded by using dialysis which leads to purification of GST/hPFP C fusion protein by affinity chromatography with glutathione agarose. After thrombin cleavage, GST and uncleaved fusion protein were removed, thus recombinant hPFP C protein being purified by affinity chromatography on glutathione agarose columns once more. The purified recombinant hPFP C produced in E. coli showed a significant hemolytic activity when tested with rabbit red cells. These results suggest that the domain responsible for the lytic function lies not only in the N terminal portion but also in the C terminal portion of the perforin molecule. The recombinant hPFP C protein will be useful as a biological reagent for immunological, pathological and structural studies.