| Backgroand: Schistosomiasis, an important verminosis caused by schistosome, is widely and severly hamful to human beings and livestocks. WHO statistics indicated that about 200 million people are infected in 76 countries and prevailing area, about 600 million people are threatened by this parasitic zoonosis. There are many kinds of Schistosomes that can parasitize on human body, such as Schistosoma japonicum, S. haematobium, S. mansoni S. intercalatum, S. mekongi S. malayensis. Schistosoma japonicum is endemic in Southeast Asia including China and Philipine. Schistosomiasis had been epidemic in 12 provinces in the south of China, there're still 22 million people at the risk of suffering from this disease in 8 provinces and 127 counties. Although chemo and snail control had been applied to prevent this verminosis, it is still insufficient to controll this disease. Consequently, the study of schistosomiasis diverstion to research on foundation principle of schistosome, especially on genome of schistosomiasis. Schistosoma Genome Project(SGP) began in 1992, "Schistosoma Genome Discovery Project" is a part of SGP, this project is to find new drug and vaccine targets through finding and identification of novel genes of S. mansoni and S. japonicum, collecting the datas for physiological functions, drug resistance mechanism and immune evasion. Schistosomiasis prevention and therapy is major through chemocontrol and snail controll, but it is still far away from getting good result, it may ascribe to drug toxicity, administration poverty, drug-resistance, frequent re-infection, the restriction of using and developing effective vaccine, the shortage of deep and wide knowledge about schistosome biology. Therefore, in this hard time for schistosome control, a large scale research on basic schistosome biology, drug-resistance and immune evasion in gene level is very important.There're not many reports about specific genes of female S. japonicum, these genes were identified one by one in traditional way, it was not only waste of time, but also impossible to get the imformation about the interaction of every single gene. For this reason, it's important to research on the S. japonicum female-specific gnes by high throughput technique to screen and identify sex-maturity related genes, egg-laying related genes, signal transmission of paroecious embracing genes. It's ergent to screen and identify the related functions and structures of these genes. It is helpful to understand the growth and development rationale of S. japonicum, and also provide a substantial way to devise reasonable anti-disease vaccines and new drugs, then in the end it will be helpful to expand a new way to prevent and cure schistosomiasis.This study applied Suppression Subtractive Hybridization (SSH) and Expression Sequence Tag (EST) method to screen female specific genes of S. japonicum. SSH is a new and highly effective method, which has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. We constructed the female S. japonicum substrative cDNA library by SSH to screen the female specific genes. The ultimate objective is to find some valuable female specific genes. It's significant for vaccine development and schistosomiasis control.Secreted antigens of S. japonicum is excreted by development process of schistosomulum after invading human body, including the secreta of schistosomulum, adult worms and development stage of eggs. Host's immunologic system response to the E-S antigens, it's related with the provocation of antibody, immunoregulation and the forming of granulomatous. For these reasons, it's good to palmar grasp much more molecules for vaccine and diagnosis, and understand overall the immune relationship between S.j and host, such as immunoreaction, the mechanism of granuloma and so on. We collected E-S antigens of schistosomulum, adult worms and eggs spawned in vitro, after two-D SDS-PAGE and then got the target bands of protien to sequencing polypeptide by LC-MS/MS. The polypeptide sequence blasted by NCBI for searching the homology. We analyzed the spectrotype of differents stages of E-S antigens. This study of identifying and analyzing protein-spectrum of S.j E-S antigens is never reported at present. It's simple and easy to manipulate by culture in vitro, and it can get high purity E-S antigens without the effective of host ingredient and avoid contaminating.Objective:1. To discover the female specific genes of S.japonicum, after homology search through Blast program in GenBank, probably get the message of the gene functions; and to find and authenticate reproductive development gene and egg-laying gene.2. To gain spectrotype of E-S antigens as perfect as possible, we utilized technology of proteome to identify and analyze E-S antigens of S.j. This basic work provided datas for discovering new molecules for vaccine.Methods:1. Constructing the female S. japonicum subtractive cDNA library with CLONTECH PCR-SelectTM cDNA Subtraction Kit. This cDNAs were linked with T vector to form a plasmid, and transformed to E. coli, then these plasmids were used to screen the target genes.2. Some positive clone plasmids were randomly selected, the insers were amplified by PCR, then transferred to nylon membrane after agarose gel electrophoresis, and used to hybridize with the first cDNA chain of male and female worms marked with 32p. Then the female specific genes were screened and identified.3. The female specific genes were sequenced and searched through Blast X and Blast N (http://www.ncbi.nlm.nih.gov/blast/) program to predict the gene function.4. Gathering E-S antigens by founding S. japonicum adult and ovum culture in vitro process.Results:1. The female S. japonicum subtractive cDNA library was constructed.2. Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. After dot-blot hybridization, 50 clones were tested to be the potential femal differentially expressed genes and were sequenced.3. 50 randomly selected clones were hybridized with female subtractive and male subtractive libraries, the former hybridization signal were stronger than the latter ones, then 42 expressing sequence tags were got. After bioinformatics analysis, 17 fragments showed high identity with the S. japonicum egg-shell protein genes, 17 sequences were highly homologous to unknown S. japonicum genes and partly homologous to female specific 800 protien. 8 fragments showed high identity with other S. japonicum unknown genes.4. S. japonicum adult and ovum culture in vitro process is founded, and has gained the E-S antigen.Conclusion:1. Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. A number of specific genes have been found by SSH.2. E-S antigens were analyzed by SDS-PAGE, showing 7 relative evident protien bands. There were 4 main bands among them and the corresponding molecular weight of the protein bands respective are 20.1DKa,31.0DKa,43.0DKa,90.0Dka, meanwhile they were tested to be the specific protein bands. This work settled substantial foundation to forward study. |
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