| Microtus fortis (M.fortis) is known as the only rodents mammalian in China, which is the highest ability of natural resistance to Schistosoma japonicum (S.japonicum) by preventing completion of life cycle of parasite. It was hypothesized that the anti-schistosome characteristic of M. fortis was due to some kind of unknown but heritable materials against S. japanicum. In previous study, we obtained a secondary subpool gE76 by expression cloning which had considerable anti-schistosomula effects in vitro. Therefore, the goals of this study were to screen and identify the anti-schistosome materials from the secondary subpool gE76 by expression cloning and to understand their potential mechanisms of anti-shcistosome effect.Chapter 1 Screening of resistance-associated gene gE76.44 against Schistosma japonicum from cDNA of Microtus fortisIn our initial study we constructed a cDNA expression library of M. fortis marrow, and screened by expression cloning obtained a secondary subpool gE76 which had considerable anti-schistosomula effect.246 clones were randomly picked out from the gene pool gE76 and selected 153 clones according to the length of inserted gene fragments after EcoR I digestion. A total of 37 clones (?800bp) was selected to further tested the schistosomula-killing capability of the conditioned media in vitro. Plasmids were extracted with High Pure Plasmid Isolation Kit, and transiently transfected into 293T cells cultured in 6-well plates using LipofectamineTM 2000. Expression supernatant (conditioned media) of each plasmid was collected 36?48 h later and tested at a concentration of 50% for their capacity of anti-schistosomula effects in vitro. The death rate of schistosomula was observed and calculated in 96 h. The plasmids with a higher death rate of schistosomula were selected.We selected and sequenced four clones bearing the most significant anti-schistosomula effect (16.7%?16.5%?10.4% and 11.3%, respectively). Their length of EST sequence were 2008 bp?1626 bp?1420 bp?828 bp respectively. Finally, clone gE76.44 with the most significant anti-schistosomula effect was selected. The average death rate of gE76.44 was 14.0% which was significantly higher than that of other plasmids and the negative control (P<0.05). After searching with BLASTN at the NCBI database, we found cDNA gE76.44 was homologous with karyopherin alpha 2 (KPNA2), so we named it Mf-gE76.44 for the following experiment.Chapter 2 Bioinformatics and functional analysis of the full-length of gene gE76.44After searching with BLASTN at the NCBI database, we found cDNA gE76.44 had 89.8% identities with mouse KPNA2 (NM-010655).Their full-length of gE76.44 cDNA was 2008 bp with ORF of 1590 bp encoded a polypeptide of 529 amino acid residue. Bioinformatics analysis results showed there were no significant difference of KPNA2 between M. fortis and other species in nucleotide and amino acid sequence. But the predicted stereochemical structures of KPNA2 between M. fortis and mouse was different. In order to confirm anti-schistosomula effect of Mf-KPNA2 in vitro, the mice KPNA2 cDNA were PCR-amplified and inserted into eukaryotic expression vector pcDNA1.1/Amp as a negative control to test its schistosomula-killing effect of the eukaryotic expression products in vitro. Conditioned medium of Mf-KPNA2 and Ms-KPNA2 were added to cultured schistosomula at a concentration of 50%. We observed and calculated death rate of the schistosomula in 96 h. Results showed that the average schistosomula-killing rate of Mf-KPNA2 was 15.8% which was significantly higher than that of the Ms-KPNA2 (2.80%), (P=0.003).We analyzed the expression abundance of the tissues by RT-PCR between M. fortis and mouse. The expression levels were different among tissues in each kind of animal and there was difference between two kinds of species, too. Besides the Mf-KPNA2 mRNA were higher expressed in liver after 12 days post-infection in M. fortis than that in mice.Chapter3 Recombinant retrovirus pLXSN-KPNA2 gene therapy and the expression levels analysisTo confirm the anti-schistosome effect of Mf-KPNA2, it was inserted into retroviral expression vector pLXSN. We transfected recombinant pLXSN-KPNA2 into packaging cell PA317 to prepare the recombinant retrovirus. The biological viral titer was determined by the number of colonies presented at the highest dilution that containing positive colonies, multiplied with the dilution factor. Mice infected with S.japonicum cercariae were administrated by intravenous injection through tail vein with DMEM, pLXSN and pLXSN-KPNA2 retrovirus respectively. In all groups, adult worms were counted after heart perfusion of mice 42 d after infection. Compared with DMEM and pLXSN treatments, Mf-KPHA2 retrovirus treatment had 39.42%(P=0.006) and 38.97%(P=0.007) worm burden reductions and had 76.50%(P=0.000) and 75.04%(P= 0.000)egg reduction, respectively. We observed no statistically significant difference between DMEM and pLXSN retrovirus treatment in all examined assays (P=0.321). Also the mRNA expression levels of KPNA2 in pLXSN-KPNA2 treatment group mice was high in liver, kedney and mustle compared with DMEM and pLXSN treatment mice.Chapter4 Immortalization of M.fortis embryonic fibroblastsOur studies suggested that Mf-KPNA2 had anti-schistosome effect both in vitro and in vivo. It may be as a novel anti-schistosome molecule. The mechanisms how Mf-KPNA2 could killed schistosome were unclear. In order to understand the potential mechanisms and to get more clear picture, the M.fortis embryonic fibroblasts (MFEF) were successfully isolated and cultured and mammalian expression vector (pSV3 neo) containing the SV40 large T antigen was used to transfect the 3 rd passage MFEF using LipofectamineTM 2000. The immortalized cells of M fortis embryo fibroblasts are successfully established. It layed the foundation for further studies from cellular level on M. fortis against S. japonicum infection and for comparative fibroblast study between different animals.In summary, we screened M. fortis bone marrow cDNA expression library by expression cloning and identified a clone named Mf-KPNA2 and observed its anti-schistosome effect both in vitro and in vivo. We analyzed its bioinformatical structure and function, and also compared the expression abundance of the tissues by RT-PCR beween M. fortis and mouse. Besides the immortalized embryo fibroblasts of M.fortis are successfully established. All the results suggest KPNA2 as a novel anti-schistosome molecule.Although the mechanism by which the Mf-KPNA2 serves the anti-schistosomula effect is not clear, it sets the stage to further explore the role of KPNA2 in the natural resistance to S.japanicum. |
PDF Full Text Request