Preliminary Study Of PA/LFn Translocation System In Enhancing Immune Response

Anthrax toxin,a major virulence factor for Bacillus anthracis,produces a tripartite anthrax toxin composed of protective antigen(PA),lethal factor(LF)and edema factor(EF).PA can bind to anthrax toxin receptor on host cell surfaces and eventually translocate LF and EF into the cytosol of the host cells via a series of interaction so that LF and EF independently exert their toxic effects respectively.LFn is a 254aa N-terminal domain of LF,turning out the carboxy-terminal toxic domain of LF and binding to PA with highαffinity.On the basis of anthrax toxin structures and the intoxication mechanism,a PA/LFn translocation system has been developed to deliver exogenous protein into cytosol for inducing specific CTL response.CTL plays an important role in the elimination of pathogen(virus and some intracellular bacteria) and the repression of the growth of tumor cells.Prostate stem cell antigen(PSCA)encodes a 123aa glycoprotein,a member of the Thy-1/Ly-6 family of GPI-anchored cell surface antigen.PSCA is prostate-specific and significantly increased in prostate tumor cells,So it can be used as a target for prostate cancer diagnosis and therapy.This research foucs on the possibility of using of PA/LFn translocation system in cancer immunotherapy.We succeeded in construction of the pET21-LFn-PSCA expressed plasmid,then the recombinant plasmid was transformed to E.coli BL21(DE3)host cell and the protein was induced to optimally with lmM IPTG for 6h at 28℃.The expression of recombinant LFn-PSCA was secretory,and was about 5%of total protein.The fusion protein was purified by chromatography.The cytotoxic activity and the binding activity of the LFn-PSCA were tested in macrophage cell line J774A.1 cells,which is sensitive to LeTx,It showed that it is nontoxic and can compete with LF for binding to PA.To test whether PA/LFn-PSCA could induce PSCA specific immune response,we used of 5μg PA and 15μg LFn-PSCA were selected to immunize female BALB/c mice. The antibody response against PA,LF and PSCA was measured by ELISA.The result showed that there was a significant antibody response after primary immunization with PA/LFn-PSCA.There was a significant difference of PSCA-specific antibody level in mice between PA/LFn-PSCA and LFn-PSCA alone group(p＜0.05), respectively.PA may enhance humoral immune response against PSCA.To study the PSCA mediated cellular immune response in mice,proliferation of splenocytes and CTL response were tested respectively.The proliferation of splenocytes was examined by MTT.The activity assay suggested that there was a low level stimulating index and no significant difference between PA/LFn-PSCA and LFn-PSCA groups.The PSCA specific CTL activity of the two groups was dominantly higher than that in the other control groups,but no significant difference was found between PA/LFn-PSCA and LFn-PSCA alone group.The results provide the basis for exploring the possibility of using PA/LFn system in inducing specific immune response and developing tumour vaccine,and was contributed to understanding the mechanism of anthrax toxin pathogeny.