Identification Of The Genes Encoding The Antigens Related To Natural Resistance Against Schistosoma Japonicum In Microtus Fortis

Schistosomiasis japonica, a zoonotic parasitic disease, is still recognized as a major public health problem in China. Current strategy to control schistosomiasis japonica in China is an integrated and comprehensive approach based on control of the source of infection. Those measures rely predominantly on mass chemotherapy with the effective antischistosome drug praziquantel (PZQ). But schistosomiasis is spreading to new areas, in addition, PZQ chemotherapy does have limitations including the inability with mass treatment to prevent reinfection and the increasing possibility to develop resistance to PZQ. Consequently, vaccine strategy represents an essential component for the future control of schistosomiasis as an adjunct to chemotherapy. Genes encoding antigens including enzymes, muscles and teguments of S. japonicum have been cloned and expressed, but in general they did not exhibite the expected protective efficacy. It is still main task to seek and search for new vaccine candidates. Immunoscreening of S. japonicum cDNA library seems to be an efficient way to identify new molecules as vaccine candidates with higher immunogenicity and antigenicity.The main aim to develop antischistosome vaccine is to reduce worm burden and egg depositon in the liver tissue. Thus the vaccine should effect on the schistosomula and fecundity of adult worms. Microtus fortis is a non-permissive mammal host for S. japonicum. S. japonicum infection does not lead to Schistosomiasis in Microtus fortis. It seems that schistosomula may be a suitable target of the immune system of the host. If some specific antigen molecules derived from the larvae could be found, it will be possible to kill it off in this stage or stop it from growing, developing, maturing, egg depositing and so on. In this case, not only the pathologic lesion caused by Schistosome can be alleviated, but also the transmission of schistosomiasis can be effectively interrupted. Therefore, encouraging results may be obtained by immunoscreening of S. japonicum schistosomula cDNA library with sera of Microtus fortis. Meanwhile, this study may provide related information on the mechanism of natural resistance to S. japonicum in Microtus fortis.Protein arginine methylation play important roles in the modulation mechanisms of gene expression in Schistosome. It is a posttranslational modification involved in various cellular functions including RNA processing, signal transduction, protein subcellular localization, transcriptional regulation and DNA repairing.High mobility group box-1 (HMGB1) involves in gene transcription, replication, recombination and DNA repair. Extracellularly released HMGB1 has been recently identified as a delayed mediator of inflammation and is shown to be a potent inducer of pro-inflammatory cytokines including TNF-αand IL-13. Since TNF-αand IL-13-mediated inflammation around eggs is a major pathology process associated with this infection, SjHMGB1 may have an important immunomodulatory role in the pathophysiology of this infection. HMGB1 has been implicated in the pathogenesis of several inflammatory disorders.In this study, the genes encoding protein arginine methyltransferases 1 (PRMT1) and high mobility group box-1 (HMGB1) selected from the positive clones of above Schistosome larvae library were further studied. Firstly the open reading frame was obtained respectively by tools of bio-informatics. Then gene encoding SjPRMT1 and SjHMGB1 were cloned and expressed using molecular cloning technique. Subsequently, recombinant SjcHMGB1 as vaccine candidate was carried out in mice experiments to evaluate its protective efficacy. 1. Immunoscreening of natural resistant molecules to Schistosoma japonicum in Microtus fortisS. japonicum schistosomula cDNA library was screened using pre-absorbing sera of Microtus fortis, which shows natural resistance to S.japonicum. The positive clones after three rounds of screening were transformed into E.coli BM25.8. E. coli BM25.8 clones containing the plasmid were cultured in LB, and then selected for plasmid extraction. the plasmid DNA was digested by EcoR I and HindIII, and analyzed by agarose gel electrophoresis. Then the positive clones were sequenced and analyzed through BLASTn software of NCBI. As a result, 32 positive clones were obtained after three rounds of screening and their sizes ranged from 300 bp to 1 100 bp. There were 26 different genes identified by BLASTn and BLASTp analysis that may code 26 proteins of S.japonicum including high mobility group box-1(HMGB1), cytochrome b5, mitochondrion coding region, 16 function-unknown S.japonicum proteins and 6 new proteins. Generally, in this study, the genes encoding some molecules which may have potencial efficacy against schistosomiasis by immunization were identified by immunoscreening of S. japonicum schistosomula cDNA library with sera of Microtus fortis, thus to be of assistance of the development of protective vaccine and diagnosis of schistosomiasis japonica.2. Cloning, expression and analysis of the gene encoding protein arginine methyltransferases 1 of Schistosoma japonicumA couple of primers were designed according to electronic elongation SjPRMT1 sequence, with BamH I restriction endonuclease site introduced in forward primer, and Xho I in reverse primer. The gene fragment was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using the total RNA isolated from adult S. japonicum (Chinese strain) as template. The purified PCR product was ligated with pGEM-T easy vector and then transformed into JM109. After identified by endonucleases digestion and sequencing, the target DNA fragment was cut and purified, and then subcloned into the proper prokaryotic expressin vector and transformed into competent E.coli DH5α. After identification as above, plasmids isolated from positive recombinant clone were transformed into competent E.coli BL21 (DE3) and the recombinant protein was induced to express in the presence of Isopropyl-β-D-thio-galactopyranoside (IPTG). The purified recombinant protein was analyzed by SDS-PAGE, western blotting and tools of bio-informatics such as Gene Runner. As a result, a 1083 bp DNA fragment was acquired by RT-PCR. BLASTn results revealed that the identity of SjPRMT1 gene to SmPRMT1 gene is 87%, and their deduced amino acid sequence shows 95% identity. SDS-PAGE and Western blotting analysis revealed that the molecular weight of reSjPRMT1 is approximately 42 kDa (including 6 His) in the soluble form. The protein could be recognized by sera of mice immunized with cercariae of S. japonicum and anti-His G HRP antibodies. The bio-information analysis demonstrated that the protein has multiple potential antigenic determinants. The gene encoding high mobility group box-1 of S. japonicum was successfully cloned and expressed, and the result may facilitate for further investigation on its biological characteristics, and immunogenicity as well.3. Gene cloning, expression and immunological study on high mobility group box-1 of Schistosoma japonicumA couple of primers were designed according to published SmHMGB1, with BamH I restriction endonuclease site introduced in forward primer, as well as Sal I in reverse primer. The gene fragment was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using the total RNA isolated from adult S. japonicum (Chinese strain) as template. The purified PCR product was ligated with pGEM-T easy vector and then transformed into JM109. After identified by endonucleases digestion and sequencing, the target DNA fragment was cut and purified, and was then ligated with pET28a and transformed into competent E.coli DH5α. After identification as above, plasmids isolated from positive recombinant clone were transformed into E.coli BL21 (DE3) and the recombinant protein was induced to express in the presence of IPTG. The purified recombinant protein was soluble and analyzed by SDS-PAGE, western blotting and tools of bio-informatics such as Gene Runner. In the immunological study, female C57BL/6 mice were randomly divided into five groups, challenge control group, Freund's adjuvant control group, Montanide ISA 206 adjuvant control group, reSjcHMGB1 plus Freund's adjuvant, reSjcHMGB1 plus Montanide ISA 206 adjuvant. As for challenge control group, mice were not treated with recombinant antigen or adjuvant. In two adjuvant control groups, each mouse was injected subcutaneously with sterile normal saline emulsified in FCA/FIA or Montanide ISA 206 respectively. Each mouse in two groups of reSjcHMGB1 plus Freund's adjuvant or Montanide ISA 206 adjuvant was immunized subcutaneously with 20μg reSjcHMGB1 emulsified in FCA/FIA or Montanide ISA 206 respectively. All the mice were vaccinated three times at an interval of 2 weeks. Two weeks after final immunization, the mouse was challenged with cercariae of S. japonicum. At the sixth week after challenge, all mice were sacrificed and the worms and eggs in each mouse were counted. The sera collected from mice periodically before immunization, challenge infection and sacrifice were tested respectively using ELISA assay to detect specific anti-SjcHMGB1 IgG antibodies. As a result, a 531 bp in size of the DNA fragment was acquired by RT-PCR. The sequence analysis indicated that the fragment shows 86% in homology to that of SmHMGB1, and the deduced amino acid sequence shows to be 93% identical with that encoded by SmHMGB1. SDS-PAGE analysis revealed that the molecular weight of expressed protein re SjHMGB1 is approximately 26 kDa (including 6 His) in the soluble form. The protein could be recognized by sera of mice infected with S.japonucum, sera of mice immunized with recombinant antigen reSjcHMGB1 and anti-His G HRP antibodies. The bio-information analysis demonstrated that the protein had two conservative domains (A box and B box) and acidic C-terminal tails, as well as multiple potential antigenic determinants. In the immunological study, the results of ELISA showed that a higher level of specific IgG antibodies in two immunized groups with reSjcHMGB1 was detected in comparison with that of challenge group (P<0.05) . The worm reduction rate and egg reduction rate in mice immunized with reSjcHMGB1 plus Freund's adjuvant were 17.9% and 17.6% respectively, but there was no significant difference compared with that in challenge control group (P>0.05) . Results also showed that reSjcHMGB1 plus Montanide ISA 206 adjuvant group showed 33.2% worm reduction rate and 11.3% egg reduction rate, and the difference in worm burden was significant when compared to that in challenge control group (P<0.05) . In this study, The gene encoding high mobility group box-1 of S. japonicum was successfully cloned and expressed. In the immunological study, no significant protective immunity and anti-fecundate immunity were found experimentally in mice immunized with recombinant antigen against challenge infection of S. japonicum.