| Interleukin-17A(IL-17A) is usually known as IL-17, and a family member of IL-17. IL-17 A has been classified as a proinflamatory cytokine because of its ability for inducing expression of proinflammatory genes such as cytokines. As a typical regulatory cytokine, IL-17 A plays double roles in host defense responses against microbial invasion, as well as in inflammatory damage. Echinococcus multilocularis larvae can cause alveolar echinococcosis, which is one of the most serious and life-threatening zoonoses in the world. The vital pathogen can induce humoral and cellular immune responses in the host while immune tolerance and immunopathological change can be observed in alveolar echinococcosis. In order to explore the immunoregulating effect of mIL-17 A on BALB/c mice experimentally infected with E. multilocularis larva, the following 4 aspects were performed in this study.(1) The complete IL-17 A gene was chemically synthesized, and inserted to the eukaryotic expression vector of pcDNA3.1(+) to construct a pcDNA3.1-mIL-17 A expression plasmid. The recombinant plasmid was used to transfect 293 T cells, and the cells were collected after 48 h and 72 h to extract RNA for real-time PCR. Recombinant mIL-17 A was efficiently expressed in 293 T cells.(2) Four small interfering RNA(siRNA) were designed according to mIL-17 A gene, and DNAs for siRNA were synthesized for acquire double-stranded DNA by nucleation annealing. The four double-stranded DNAs were linked to pGPU6/GFP/Neo vectors to construct to pGPU6-sh RNA-136, pGPU6-shRNA-180, pGPU6-sh RNA-288 and pGPU6-sh RNA-381 recombinant interfering plamids. These four plamids and pcDNA3.1-mIL-17 A were cotransfected into 293 T cells. The mIL-17 A gene and its encoded protein were detected with real-time PCR and direct immuno-fluorescent antibody assay showing that the transcription of mIL-17 A was interfered by the four siRNA, of which mIL-17A-136 had the most efficent interfering capacity, and the mean eliminating rate of mIL-17 A was 83.5% at the time points of 24 h and 48 h.(3) AD-pAVT1502 adenovirus for interfering mIL-17 A and AD-pSB2000 adenovirus for expressing mIL-17 A were packaged and coinfected HEK293. Virus titer was measured to be 7.58×1010 PFU/mL for AD-pAVT1502 and 5.85×1010 PFU/mL for AD-pSB2000 after virus proliferation and purification. The mIL-17 A gene and the protein were determined using real-time PCR and direct immuno-fluorescent antibody assay to confirm the efficient interfering and expressing capacity of the adenoviruses. The growth rate of mIL-17 A expressed by AD-pSB2000 and the eliminating rate of mIL-17 A interfered by AD-pAVT1502 were 842% and 55% respectively in the RT-PCR detection.(4) BALB/c mice experimentally infected with E. multilocularis larvae were injected with 1×1010 PFU/mL AD-p AVT1502 or AD-pSB2000 by tail vein, and their sera and spleens were collected at different time intervals. The result of ELISA indicated that concentrations of mIL-17 A in the sera were regulated significantly by AD-pAVT1502 or AD-pSB2000 infection(P<0.05). The concentrations of some cytokines were significantly different from that of the same cytokines in another group(P<0.05), and the immunoregulating effect of mIL-17 A on CD30 L, Fas L, GM-CSF, IFN-γ, IL-1β, IL-2, IL-3, IL-5, IL-6, IL-7, IL-13, IL-21 and TNF-α was obvious. |
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