Cloning And Identification Of Microtus Fortis HSP90α As A Molecule Against Schistosoma Japonicum

Microtus fortis has been proved to be a naturally resistant vertebrate host of Schistosoma japonicum by preventing completion of the parasite's life cycle both through epidemiological investigation and artificial Infection in lab. M. fortis sera were found to have anti-schistosome characteristic in vitro and transfered into mice. We have found that there was no significant difference between the tissues homogenate supernatant of M. fortis and those of the mice in schistosomula-killing effect in vitro. Then we constructed a cDNA library of M. fortis marrow and screened a gene fragment with anti-schistosomula function in some extent by expression cloning. In this study, a 331-bp clone gC 14.75 was screened from M. fortis marrow cDNA library by expression cloning. After searching with BLASTN at the NCBI database, we found cDNA gC 14.75 was homologous with HSP90a. Then we amplified the full-length of M. fortis HSP90a (Mf-HSP90a) and cloned it into the eukaryotic expression vector pcDNA1.1/Amp to test the schistosomula-killing effect of eukaryotic expression products in vitro. We transferred Mf-HSP90a by retroviral expression vector into mice to investigate its function in vivo. Results showed that it had significant effect of killing S. japonicum both in vitro and in vivo.Chapter 1 Screening of resistance-associated gene against Schistosma japonicum gC14.75 from Microtus fortis The cDNA expression library of M. fortis marrow was initially screened by testing the effects of conditioned media from transfected 293T cells for their capacity of killing schistosomula in vitro. Conditioned media (50%) of gene pool were added to schistosomula cultured with DMEM in vitro, observed the schistosomula in 96 h. The number of the schistosomula was counted in 96 h and compared with control group, calculated death rate of each group of the experiment. The highest death rate of the 8 gene pools (from gA to gH) conditioned media is gC (10.9%) which was significant different from the negative control group (1.7%). Then further the screen as gene pool. Gene pool C was separated into 8 subpools (from gCl to gC8), in which gC1 has the highest schistosomula-killing rate of 11.5%. Then divided gC1 into 8 secondary subpools (from gC11 to gC18), tested the schistosomula-killing effect of their conditioned media, we screened the highest schistosomula-killing secondary subpool gC14 with killing rate of 15.9% compared to 0.3% of the negative control. Thirty-two clones with different inserted fragments were selected from the gC14 with the highest schistosomula death rate, and then tested the schistosomula-killing capability in vitro of their conditioned media. A clone (gC 14.75) with the most significant schistosomula-killing effect was selected and sequenced by standard techniques.Chapter 2 Cloning and functional analysis of the full-length of gene gC14.75After searching with BLASTN at the NCBI database, we found cDNA gC 14.75 was homologous with HSP90α. It had 94% identities with Cricetulus griseus HSP90α(L33676), the identities lied between 1042-1373 neucleotides of C. griseus HSP90α. HSP90s are the most phylogenetically conserved proteins present in all prokaryotes and eukaryotes. So we designed primers according to gC 14.75 sequence and C. griseus HSP90α, tried to amplify the full-length of gC14.75. The amplication product was cloned into pGEM-T easy and sequenced it to ascertain its sequence. Searching with BLASTN at NCBI database indicated that it was a homologue of HSP90α, we named it Mf-HSP90α. Bioinformatics analysis results showed there were no significant difference of HSP90αbetween M. fortis and other species in nucleotide and amino acid sequence. The stereochemical structure of HSP90αbetween M. fortis and mouse was significant different. We cloned HSP90αof M. fortis (Mf-HSP90α) and mouse (Ms-HSP90α) into eukaryotic expression vector pcDNA1.1/Amp to test their schistosomula-killing effect of the eukaryotic expression products in vitro, respectively. The schistosomula-killing rate of Mf-HSP90αwas 20.3% while the schistosomula-killing rate of Ms-HSP90αwas 4.4%. There was significant difference between them. To identify the anti-schistomosome mechanism of Mf-HSP90α, we analyzed the expression abundance of the tissues by RT-PCR and Western-blot in 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.Chapter 3 Anti-schistosome effects of Mf-HSP90αon mice infected with Schistosoma japonicum and analysis of the expression levelsTo confirm the anti-schistosome function of Mf-HSP90α, we transfered it into mice infected with Schistosoma japonicum by retroviral expression vector pLXSN. Cloned Mf-HSP90αinto pLXSN, and packaged recombinant virus with PA317 cells. Infecting NIH/3T3 cells cultured with polybrene in six 10-fold serial dilutions and selected by G418 to determine virus titers. The titers of pLXSN and pLXSN-HSP90a retrovirus were 1.6×107 cfu/ml and 4×106 cfu/ml respectively. Mice infected with Schistosoma japonicum were administrated systemicly with DMEM, pLXSN and pLXSN-HSP90αretrovirus respectively. In all treatments, adult worms were quantified after perfusion of mice 42 d after infection. Worms recovered from pLXSN-HSP90αretrovirus treatment were stunted relative to worms from DMEM and pLXSN retrovirus treatment. Worm burden and egg reduction of pLXSN-HSP90αretrovirus treatment were statistically different from those of DMEM and pLXSN retrovirus treatment (P<0.05). The expression levels of HSP90αwere different among tissues and between DMEM and pLXSN retrovirus treatment in mice.In summary, we screened the Mf-HSP90oαgene against Schistosoma japonicum from Microtus fortis by expression cloning, analyzed its bioinformatical structure and function, and compared the expreeion levels among tissues and between Microtus fortis and mouse in vivo. And then we testified its anti-schistosome function in vitro and in vivo. All the results suggest Mf-HSP90αas a novel anti-schistosome molecule.