Cloning And Immunoprotection Of Schistosoma Japonicum(Chinese Mainland Strain)Mito Chondria Related Protein And Its Antigenic Epitopes

Cloning and Immunoprotection of Schistosomajaponicum (Chinese Mainland Strain) MitochondriaRelated Protein and Its Antigenic EpitopesPh.D. CandidateHU XuemeiSupervisorZHANG ZhaosongInstitute of Medical Mocular BiologyNanjing Medical University, Nanjing, 210029, ChinaAbstactSchistosomiasis is a serious threat to the public health. The main control strategy is mass chemotherapy with praziquental, combined with snail control in easily-areas. With the development of molecular biology, the development of a vaccine for the control of Schistosomiasis has been targeted as a priority by WHO/TDR. Up to now, a number of the parasitespecific antigens have been cloned and characterised, and their protective activity assessed by passive transfer of the corresponding antibodies or by direct immumisation with purified antigenic preparations is in a range of O-<40% among rodents and/or domestic animals. One hypothesis is that a number of antigens should be combined to elicit an ideal protection. Therefore, gene cloning and expression of novel Schistosoma japonicum antigens is an important strategy in developing Schistosomajaponicum vaccine.9l. Cloning and immunoprotection of the recombinantprotein rS j338 and its antigenic epitopesOne gene was obtained by screening the cDNA library ofSchistosomejaPonicum. Homology comparisions with GenbankDatabase showed that the clone (Sj338/24) was homologous tothe gene encoding mitochondrial protein of human and Rattus.In order to obtain the SJaPonicum mitochondria-related proteinand evaluate its immunoprectective effect, a serial of researchesabout this gene was taken. To analyse the open reading frame ofthe fragment (Sj338/24), at both upstream and downstream ofthe open reading fram (ORF) primers A and B were designedrespectively, and the cDNA fragment was used as PCR template.The Sj338 gene fragment was obtained and amp1ified by PCRmethod, then subcloned into pGEM-T vector fOr sequencing.The sequence of nucleotides and the characteristics of theencoded protein were analyzed by DNASIS Program andGoldkey DNA and Protein Analytical Program, and then thehomology of the amino acid sequence was searched on theBLAST net. The target fragment was restrictedly digested andsubcloned into expression vector pGEX-6P-l. The expressedrecombinant protein was characterized. It was demonstratedthat the cloned Sj338 gene has 487bp containing one ORF with459bp long, which codes for a protein consisted of l53 aminoacids with a molecular weight of l7.6 kDa. The amino acidsequence of the recombinant protein rSj33 8 shared 46% identitywith that of the corresponding part of human mitochondriall 0import receptor and 44% identity with that of the Rattus sp.mithochondrial precursor receptor. The possible antigenicepitopes were predicted within the peptide fragments of 26-32aa, 37-46 aa and l47-l5l aa. The recombinant construct ofpGEX-6P-l/Sj338 could be expressed efficiently and theantigenicity of its product rSj338/26GST was demonstrated byWestern B1ot.A purification procedure for the recombinantmitochondria-related protein of S jaPonicum expressed inEscherichia coli was carried out as follows to obtain this idealprotein. After centrifugation of the bacterial culture, thebacterial pellet was resuspended and lysed by freezing-thawingtreatment and u1trasonication. The inclusion bodies werepurified, and further purified by Gel filtration chromatographyand caracterized by Western-blot. The purity of the fusionprotein rSj338/26GST was verified by using SDS-PAGE and itcould react with the serum of the rabbit heavily infected withSjaPon icum.To obtain the antibody against rSj338, the purified proteinwas injected twice into rabbits. Dot-ELISA was used to detectthe antibody titer of immunized rabbit's sera. The inclusionbodies and the purified protein from S-12 gel filtration couIdstimulate the rabbits to produce high level of anti-rSj338...