The Screening Of Protective Antigen Genes Against Schistosoma Japonicum Using Expression Library Immunization And EST Strategies

Schistosomiasis, caused by the helminth blood-fluke, Schistosomes, is one of the major public health problems in developing countries like China, Philippines and Kenya, it is also hazard for both progress on life quality of rural inhabitants and development of agricultural economy in endemic areas. Chemotherapy remains the cornerstone of intervention, but the need for re-administration of treatment for both humans and domestic animals (a major resource of transmission in China) as well as the possibility of drug resistance makes chemotherapy an ineffective long-term measure. Therefore, the application of an effective vaccine against this parasite would be a long-term measure. However, protective effects of all available antigens obtained by conventional gene engineering techniques are unsatisfactory, suggesting that the devoid of the more effective protective antigens for the use of schistosomiasis control is becoming the bottleneck to limit the progress of the vaccine development. The establishment of new techniques and strategies to screening of effective protective antigens against schistosomiasis would be an alternative to resolution of the above problem.The objectives of present study are to construct an expression library containing partial cDNA from Schistosoma japonicum , to evaluate the protective effect against Sj cercariae challenge in mice immunized with above eukaryotic expression library as well as the feasibility of expression library immunization(ELI) used in the screening of protective antigens. Meanwhile, to identify the features of DNA fragments from the Sj partial cDNA expression library by EST sequencing and bioinformatical analysis, and elucidate motifs and domains of above DNA fragments through protein prediction program, so as to give valuable information for further searching protective antigens.The methods includes①amplifying cDNA fragments of interest using SjλZipLox library of S.j as the templates by PCR and constructing a partial cDNA expression library of S.j, and partitioning it into smaller groups of clones(sublibraries), mice were immunized using above libraries and challenged on day 35. Protection was assessed as the percentage reduction in worm burden in the vaccinated animals compared with the control.② amplifying cDNA fragments of interest as described①, constructing a directional cDNA library used for the generation of expressed sequence tags (EST), the re-plasmids were obtained from randomly selected clones and sequenced in the forward or reverse directions by Sanger's method, ESTs were produced by single passes on automated sequencer. Analysis of EST sequences by Blast, Genescan and protein-prediction programs/servers.Results are as follows: (1) A partial cDNA expression library of S.j (L-CMV-SjR) with～10~5 clones was constructed and further divided into three sublibraries (L-CMV-SjR1, L-CMV-SjR2 and L-CMV-SjR3 ) , each containing～3.3×10~4 clones, mice immunized with L-CMV-SjR, L-CMV-SjR1 and L-CMV-SjR2 induced a significant protective effect after i.m. immunization, compared with control mice immunized with the empty vector. The rate of worm reduction was about 30%. (2) a directional cDNA library with～10~4 clones was constructed and a total of 26 usable sequences was obtained after partial sequencing from one end of 32 clones. Of these, six clones that had homology with genes previously characterized in Schistosomes corresponded to six different genes. eight clones, corresponding to eight distinct genes, showed homology with non-schistosomes entries of public databases. Eight clones represent six different rRNA genes. One clone had no significant database matches. Most of EST sequenced showed the features of protein-coding sequences, which supported the results of alignments by Blast searching as well as the specificity of cDNA fragments amplified by PCR. The results of putative protein prediction showed that motifs and domains involved in the ESTs were correlative with metabolism and transcription of this parasite.Conclusions: Data presented in this study preliminarily demonstrate that screening of effective protective antigens against schistosomiasis using cDNA expression library immunization is feasible. Furthermore, bioinformatical analysis of ESTs from cDNA expression library could give valuable information for further searching protective antigens. Therefore, this study has provided the theoretical and methodological basis for further screening of protective antigens against Schistosomiasis using ELI and EST strategies.