Evaluation Of Recombinant Signaling Protein 14-3-3 Of Schistosoma Japonicum On Immunodiagnosis Of Schistosomiasis And The Construction Of S.japonicum Eggs CDNA Library

Objective: To approach the potential of the recombinant signaling protein 14-3-3 (rSj14-3-3) of Schistosoma japonicum in the diagnosis of schistosomiasis. Methods: The rSj14-3-3 were expressed in E. coli and purified through the collumn of affinity chromatography with Ni2+ (nickel sulfate, NiSO4),then the recombinang protein was subjected to the serological diagnosis in the patients with acute and chronic Schistosomiasis japonica using indirect ELISA. The sensitivity and specificity of the recombinant antigens used in ELISA (rSj-ELISA) were compared in parallel with the traditional methods of soluble egg antigen(SEA)-ELISA ,SEA-IHA and COPT. In addition to the sera of patients with acute and chronic schistosomiasis, the detected samples include the sera of individuals with clonorchiasis and nematode infections and those from the areas without schistosomiasis. Results:. The expressed rSj14-3-3 was verified with monoclonal antibodies by SDS-PAGE and Western blotting. Parallel detections using rSj-ELISA together with SEA in ELISA (SEA-ELISA), SEA in indrect heamagglutination assay (SEA-IHA), and circumoval precipitation test (COPT) for the detection of antibodies against S. japonicum in the patients with Schistosomiasis japonica, individuals infected with Clonorchis sinesis and nematodes, and the normal control. Conclusion: Our results demonstrate that using recombinant signaling protein 14-3-3 as probe antigens in ELISA (rSj-ELISA) for the detection of sera of both acute and chronic schistosomiasis has similar sensitivity and specificity to the crude soluble egg in ELISA. The rSj-ELISA has equal practical value compared with traditional SEA-IHA but is obviously superior to COPT as to sensitivity for the diagnosis of chronic schistosomiasis, suggesting that it may be a promising tool for large scale serological surveys in schistosome endemic areas. The recombinant antigen is easy to standardize, economic to prepare, and have the potential of practical use for the immunodiagnosis of schistosomiasis. Objective: Constructing a cDNA library of Schistosoma jiponicum (S.j) eggs and screening it using the sera of acute patients infected with S.j in order to look for new vaccine molecular and earlier period diagnostic reagents .Methods: The 42 days of S.j eggs were collected. The first strand cDNA was synthesized immediately using total RNA extracted from the eggs by Reverse Transcriptase PCR and then synthesized the double strand cDNA by Long Distance PCR. The cDNA was recombinant oriented into phage vector with sfi I enzyme. A S.j eggs cDNA library was constructed after packaging in vitro. The sera of acute patients infected with S.j were collected and eliminated anti-E.coli antibodies by E.coli lysates, used as antibody to screen the constructed cDNA library. The fourteen positive clones from rescreening were sequenced. The obtained genes were compared to GenBank datebase by BLASTN and BLASTP. A new gene which has a complete 432bp open reading frame(ORF) was analysed by bioinformatics methods. Result: The volume of the constructed S.j eggs unamplified cDNA library was about 2.7×106 recombinants. After amplification the library's titer reached nearly 1010 pfu/ml. The recombination rate was 85% measured by plate with IPTG and x-Gal. Seven recombinant clones were picked out randomly and were self-cycled into plasmid. The plasmids were digested by two enzymes. The rangement of cDNA molecule weight from 400~1500bp was identified with agar electrophoresis. About 7.2×103 recombinant phages were screened by using the positive sera , twenty-eight positive clones were obtained in which fourteen continuing positive clones were confirmed. The sequence data of positive clones were analyzed in GenBank datebase. The sequence of No.2 clone has no significant similarity and may be a gene to be recognized. The new gene has a 432bp-size complete open reading frame. The bioinformatics analysis predicted that the gene is to be an encoding transmembrane protein. Conclusion: A cDNA library of S.japonicum eggs has been successfully constructed. Some positive clones were obtained by immunoscreening from the cDNA library. On the base of this research , taking the gene as a potential vaccine molecule or diagnostic antigen may be possible.