Cloning Of Full-Length Ascaris CPI Gene And Preliminary Study On Immune Function

It has been widely reported that helminth infection can inhibit the immune function of their hosts, however, little is known about the immunological regulation factors in these parasites. Recently, many investigations have demonstrated that cysteine protease inhibitors (CPI), which were found in excretory-secretory products of helminth, can significantly impair the host immune function. However, the immunoregulation mechanism and application value of CPI still need to be studied.The pseudocoelomic body fluid (ABF) and extract of Ascaris lumbricoides (Al), an intestinal parasite that is widely existed, have anti-allergic and anti-tumor activity. However, the immunoregulatory molecules in Ascaris are unknown. Therefore, here we studied the cysteine protease inhibitors in Ascaris.Afer a pair of degenetate primer was designed according to the conserved sequences of CPI gene in known helminth, the conserved sequence of Al-CPI was amplified by PCR. Then full-length cDNA of Al-CPI gene was acquired using RACE method. The coding sequence of Al-CPI was cloned into the expression vector pET-32a, then recombinant vector was further transformed into E.coli origami and induced to express a fusion protein by IPTG. The recombinant Al-CPI protein was purified by Ni-NTA affinity chromatography.The fact that recombinant Al-CPI can inhibit the hydrolysis activity of cathepsin B, L, S, C indicated that the protein has biological activity.Further studies on immune function of this protein indicated that CPI can inhibit the proliferation induced by ConA, PPD and other stimulators in human PBMC. IL-4 and IFN-γsecreted by ConA-stimulated PBMC were also reduced. When the surface markers of different T cell subpopulations were assayed with flow cytometry, significantly decreased expression of CD25 and HLA-DR on both CD4~+ T cells and CD8~+ T cells was found. These results implied that Al-CPI has an immunosuppressive effect on human immunological cells.In mixed lymphocyte reaction (MLR), Al-CPI can inhibit the proliferation of CD4~+ responder T cells. The expression of co-stimulatory molecules CD40, CD80 and CD86 on monocytes in MLR can also be downregulated by Al-CPI. Simultaneously, IL-12 and IFN-γsecretion levels were decreased in Al-CPI-treated group. These results revealed that Al-CPI can suppress the allograft rejection caused by alloantigen. This may endow Al-CPI rather high medical value.