Expression And Protective Immunity Effect Study Of Large Hydrophobic Domain(HD) Of Schistosoma Japonicum Tetraspanin 2

Schistosomes are parasitic blood flukes, which prevail mainly in tropical and subtropical areas and threat about 600 million people. Approximately 200 million people were infected and about 20 million patients had severe symptom all over the world. China is one of countries prevailing with schistosomiasis japonicum seriously. At present, it is still the primary means of treating schistosomiasis by praziquantel. Although drug treatment could reduce the infection, it couldn't control the transmission of schistosomiasis. The re-infection will be happened when people contact with infectivity water again. Due to repeated chemotherapy, the resistance had detected in Schistosoma mansoni, it still can not rule out the possibility that praziquental resistance presenting in Schistosoma japonicum in future. The repeated chemotherapy and long-term monitor cost a lot of human, material and financial resources, so it is emergent for developing an effective method to prevent schistosomiasis.Vaccines are effective means of prevention and treatment of infectious diseases, such as the control of smallpox and Polio dependent on the application of efficient vaccine in history. As a relatively cheap and long-term control measure, developing schistosomiasis vaccines has become a mainly goal of science and technology workers. In the 1990s, schistosomiasis vaccine development has been identified as priorities in schistosomiasis prevention and treatment strategies by the World Health Organization.The development of molecular biology, molecular immunology and bioinformatics provides us an extremely effective means of research. The vaccines research has changed from the traditional attenuated vaccine or dead vaccine to genetic engineering vaccine, and then to DNA vaccine in recent years.Through long-term unremitting effort, researchers had finished a large number of experimental studies on schistosomiasis vaccine candidates by using molecular biologic and immunologic methods, some effective candidates were identified, such as GST,PM,TPI,23 kDa membrane protein,FAEP,Irv5 and Signal protein 14-3-3. WHO had evaluated the immune protective effect of six candidates among them in mice model, but the results were not ideal. The worm reduction rate of the six candidates was lower than 40 percent when they were used to immune mice alone, so these vaccine molecules can not meet the need of preventing schistosomiasis. Searching for new and efficient vaccine candidates is still the main job of vaccine development in the future for a long time.Secreted and membrane-binding proteins, which related to cell development, proliferation, activation and migration, are potential target molecule of vaccines and new drugs development. We had screened a number of secreted protein and membrane-binding protein genes of Schistosoma japonicum using signal sequence trapping method. In this study, the gene sequence of large hydrophobic domain (HD) of tetraspanin 2 of Schistosoma japonicum was synthesized and expressed, the immune protective effect against schistosome infection of recombinant protein TSP₂HD was also investigated.The study includes three parts as fellows:1. Gene synthesis and identification of the tetraspanin2 large hydrophilicdomain of Schistosoma japonicum (Sj TSP₂HD)The DNA sequence of open reading frame of tetraspanin 2 (TSP2) was obtained by matching the EST database of Schistosoma japonicum with partial DNA sequence of TSP2 captured by signal sequence trapping method previously. Eight oligonucleotides segments (each of 50 bp) were designed according to the DNA sequence of Sj TSP₂HD, and synthesized by chemical method, then, the whole DNA fragment of Sj TSP₂HD ( about 246 bp) was produced by overlapping polymerase chain reaction (overlapping PCR). The synthesized gene fragment of TSP₂HD was inserted into pGEM-T vector and sequenced. The sequencing data showed that the DNA sequence of synthesized TSP₂HD is the same to the one of natural TSP₂HD completely, which means that the gene synthesis of Sj TSP₂HD was successful in this study.2. Construction of prokaryotic expression plasm id of Sj TSP₂HD, protein expression and immunogenicity analysis of GST - TSP₂HDThe gene of Sj TSP₂HD was cloned into downstream of glutathione-s-transferase (GST) of expression vector pGEX-4T-3 directionally to construct a recombinant expression plasmid TSP₂HD/PQ then the recombinant expression plasmid was transformed into host bacteria E.coli BL21 (DE3). The single colony bacteria containing recombinant expression plasmid, was induced by IPTG and could express a 34 kDa recombinant GST-TSP₂HD fusion protein. The GST-TSP₂HD fusion protein could be purified through affinity chromatography of Glutathione sepharose 4B gel, the recombinant TSP₂HD protein could produce by cleaving the GST-TSP₂HD fusion protein with thrombin on the Glutathione sepharose 4B gel. The western blotting result showed that the TSP₂HD protein could be recognized by the sera of schistosomiasis japonica patient and serious infected rabbits, but not be recognized by the sera of heathy persons and rabbits. The protein could also induce high level specific IgG when GST-TSP₂HD fusion protein was used to immunize C57BL/6J mice. These results indicated that the recombinant TSP₂HD protein has good immunogenicity.3. Study on the immune protective effect of recombinant GST-TSP₂HD fusion proteinThirty female C57BL/6J mice of six weeks old were randomly divided into three groups: A, B, C. In group A, each mice immunized with 100μl of PBS-adjuvant in back subcutaneous; In group B, each mice were immunized with 100μl of GST-adjuvant antigen (including 100μg GST protein) in back subcutaneous; In group C, each mice was immunized with 100μl of GST-TSP₂HD-adjuvent antigen (including 100μg GST-TSP₂HD protein) in back subcutaneous. The Freund's complete adjuvant (FCA) was used in first immunization at week 0, and the Freund's incomplete adjuvant (FIA) was used in boost immunization at weeks 2, 4. Two weeks after the last immunization, all mice were challenged with 45±2 cercariae of Schistosoma japonicum by abdominal skin. Forty-two days post-challenge, the mouse were sacrificed and perfused, and the numbers of recovered worms and eggs in liver were counted. The blood was collected from the tail veins of all mouse two days before the first immunization and the challenge respectively, then sera were prepared and the level of IgG and its subclass IgG1,IgG2a,IgG2b,IgG2c and IgG3 against recombinant TSP2HD fusion protein were detected by enzyme-linked immunosorbent assay (ELISA). The tissue sections of mice liver were prepared and its pathological changes were observed by microscopic after H&E staining. By comparing with the group A and B, the worm reduction rates in group C were 27.01% and 39.57%, the egg reduction rates in group C were 43.53% and 32.30%, the reduction rate of single egg granuloma even diameter in group C were 27.08% and 40.53%. The level of IgG against recombinant TSP₂HD protein specifically of group C were higher than the one of group A and B. In particular, the levels of IgG1,IgG2b,IgG2c were much higher than the one of group A and B. These results suggested that the recombinant TSP₂HD protein can induce higher level immune protective effect against Schistosoma japonicum infection.