| Schistosomasis is one of the world-wide spread parasitic zoonosis that causes serious healthy problem to both human and animals. It was reported that there are 40 species of mamals in the endemic area could be naturally infected with Schistosoma japonicum (S. japonicum),and Microtus fortis (M. fortis) is the only mamalian animals with inborn resistance to S. japonicum. Exploring the anti-schistosoma mechanism of M.fortis, screening the genes of M. fortis or target genes of S.japonicum that related to the resistance could provide some useful information for the development of anti-schistosomula vaccines and drugs.In this paper, the killing effects of the extractions from different tissue or organ, and sera, inactivated sera of M. fortis to schistosomula were compared by cocultivation in vitro, and then, the extractions of liver and lung, which could induce higher schistosomula-morality, were choosed to screen a phage display cDNA library of S. japonicum. The positive phage clones picked randomly were sequenced and analysed by bioinformatic, and some of them was selected to vaccinate BalB/c mice for evaluation of the immunoprotecive efficacy. At the same time, cloning and expression of the complete ORFs of some obtained clones were carried out.1.In the cocultivation system of schistosomula japonicum with complete RPMI1640 medium, the schistosomula morality induced by the extractions of liver, lung, spleen, muscle and sera as well as inactivated sera of M.fortus were respectively 33.51%,36.33%,3.92%,16.31%,27.06% and 29.32% after 22 hours, and 83.07%,69.25%,28.34%,35.91%,86.14% and 83.14% after 48 hours incubation. The results showed that the homogenate supernatant of liver, lung which are the tissues provide for S.japonicum moving and development also have excellent killing effect, excluding the serum reported.2. After three rounds of biopaning of Sj44d phage display cDNA library with extractions of M.fortis liver and lung, total 168 phage clones picked at random were sequenced and further bioinformatically analysed, and 32 effective displayed ESTs were obtained. Among them, 22 ESTs have homology to the schistosoma genes or ESTs deposited in Genbank, 1 EST are new genes that only can be found in other beings, and the 9 left are totally new ESTs which have no homology with known genes or ESTs. Function analysis of the encoded proteins of the 23 ESTs with homological sequceses and background revealed that the ESTs encoded proteins were mainly involved in the regulation of gene expression and protein processing or modification. The results hint that M.fortis could directly or indirectly influence the gene expression in S. japonicum, making the worms malformed or development delayed, which might be a base of the molecular mechanism to the schistosoma resistance of M.fortis .3. After immunization of BalB/C mice with some of the positive phage clones obtained by screening with liver extraction, the highest reduction rates of worm counts, female counts, faecal egg counts and liver egg cousts were 32.91%,33.33%, 95.49%, 87.41% respectively. The results of ELISA also revealed that all vaccinated groups showed significant (P < 0.05) increase in total IgG antibody against soluble worm antigen before challenge. The result proves that the most ESTs screened can offer a key resourses for finding the candidate vaccination antigens or target drugs. 4. Based on the results of sequencing and further bioinformatical analysis, the complete ORF sequences of the genes were cloned with PCR and SMART RACE techniques. We cloned two genes that may related to M.fortis resistance whose function are dividely phosphodiest, hydrolase activity and structural constituents of ribosome, successfully constructed a recombinated expression plasmid and expressed it in E.coli with a 19.4KD fusion protein, that could be have a significant academic value and application capacity.It our studies, the excellent killing-schistosomula effect of the extractions from M.fortis liver and lung was firstly demonstrated and the extractions of M.fortis liver and lung were firstly used to screen the target schistosoma genes related to the nature schistosoma-resistance of M.fortis, and secveral molecules, ESTs or full-length cDNA which were worthful for further studies were obtained. The results reported here were meaningful for exploring the molecular mechanism of schistosoma-resistance of M.fortis and bringing out a new way to develop anti-schistosomula vaccines and drugs.
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