Genetic Study Of IL-15 In All And Generation Of Anti-IL-1α Antibody And Related Study Of IL-1α

Objective: Interleukin-15 (IL-15) plays important roles in immune system and in development of hematopoietic cells. Previous studies revealed that five SNPs (Single Nucleotide Polymophism) in IL-15, rs10519612, rs10519613, rs35964658, rs17007695 and rs17015014, were significantly associated with childhood Acute Lymphoblastic Leukemia (ALL) treatment response. In adult ALL, the expression of IL-15 was also correlated with the immunophenotypes of ALL. Therefore, we hypothesize that SNPs of IL-15 might also be associated with adult ALL.Methods: We genotyped the above five SNPs of IL-15 gene by PCR-RFLP assays in adult ALL case-control studies. The current study included 121 adult ALL patients and 263 healthy controls. IL-15 genotypes and haplotypes were determined and the associations with the risk of ALL were analyzed by logistic regression.Results: SNPs rs10519612 and rs17007695 were significantly associated with ALL (P=0.013 and P=0.001). We observed a 2-fold and 2.4-fold excess risk of developing ALL for the rs10519612 CC and rs17007695 TC genotype carriers compared with non-carriers, respectively. Haplotype analysis revealed that haplotypes ACAC, CAGT and CCAT were significantly associated with adult B-ALL, while haplotype CCAT conferred susceptibility to T-ALL.Conclusion: These findings suggest that IL-15 gene polymorphisms are significantly associated with ALL in adult Chinese population. Objective: IL-1αis a pro-inflammatory cytokine, which can stimulate the gene expression associated with inflammation and immunology. IL-1αhas cardinal effects on malignant process. IL-1αis mainly active in cell-associated forms (membrane and nuclear), and rarely as secreted cytokine digested by Calpain. Nuclear localization of IL-1αcan affect the transcriptional activity, membrane-bound IL-1αcan stimulate immune response, and released IL-1αfrom necrotic cells may promote tumor invasion and metastasis. Therefore, membrane or released IL-1αmay play important role in inflammatory disease and tumor. In our study, we intend to construct the mouse and human IL-1αprokaryotic expression vectors, expressing IL-1αprotein and prepare anti-mouse IL-1αpolyclonal antibody and anti-human IL-1αmonoclonal antibody. Another goal is to construct four different types of IL-1α(Wildtype, Propiece, Secreted and Membrane) expression lentiviral vectors and construct the stable transfected cell lines.Methods:(1) The cDNA were obtained from the spleen cells of BALB/c mice and PBMC of human, the full length of mouse and human IL-1αgene were amplified by RT-PCR. Then it was recombined into prokaryotic expression vector pET32a(+) and transformed into E.coli BL21(DE3). After induced by auto-induction, the recombinant protein was expressed and purified using protein electroelution purification.(2) The polyclonal antibody was obtained from New Zealand rabbit with the recombinant mouse protein immunization and the titer was determined by ELISA. The polyclonal antibody was confirmed by Western blot and Flow Cytometry assays.(3)Immunized BALB/c mice with the recombinant human protein, the MAb against IL-1αwas prepared using hybridoma technique. The positive hybridoma was selected by ELISA and Flow Cytometry. Ascites MAb were obtained after inoculation of isolated hybridoma into BALB/c mice. The ascites MAb were used for ELISA and Flow Cytometry.(4) The genes of four types of IL-1α(Wildtype, Propiece, Secreted and Membrane) were amplified by PCR and site-directed mutagenesis PCR. The four types genes were subcloned into the transfer plasmid of the lentiviral vector, which was transfected together with the packaging plasmids into 293T cells. Transfected the Jurkat cell with the lentivirus and obtained the stable transfected cell lines by flow sorting which positive rate was about 100%. Results:(1) The mouse and human recombinant prokaryotic expression vector pET32a(+)-IL-1αwere constructed and the recombinant protein was purified successfully. The rabbit anti-mouse polyclonal antibody and mouse anti-human monoclonal antibody were prepared successfully. ELISA analysis showed the titer of the generated antiserum was 1: 25600. Western blot and FCM analysis demonstrated that this polyclonal antibody bound specifically to IL-1α. Hybridoma L-1F12 cell line were screened and the subtypes of the antibody was IgMκ. Study showed that the MAb can be used for FCM analysis.(2) The lentiviral vectors containing the four types of IL-1αgenes were constructed successfully. We also obtained the stable transfected Jurkat cell lines.Conclusions: The polyclonal anti-IL-1αantibody from the rabbit was obtained with high titer and the specificity had been tested with the recombinant IL-1αas antigen, which could be used for neutralization in vivo to study the functions of released IL-1αin the process of liver chronic inflammation to to tumor. We examined the specificity of the anti-IL-1αMAb, which can be used to test the serum level of IL-1αof the patients. We also constructed four different stable cell lines expressing different forms of IL-1α. They could be used to study the function of IL-1αin inflammatory diseases and tumor.