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Cloning, Expression and Protective Immunity Evaluation of the Full-length cDNA Encoding SjHGPRT and SjSDISP

Author: YuJunLong
Tutor: WangShiPing
School: Central South University
Course: Pathogen Biology
Keywords: Schistosoma japonicum Hypoxanthine - guanine phosphoribosyl transferase (HGPRT) Succinate dehydrogenase iron -sulfur protein ( SDISP ) Gene Cloning Sequence analysis Recombinant expression Protective immunity
CLC: R383
Type: PhD thesis
Year: 2006
Downloads: 139
Quote: 3
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Abstract


Schistosomiasis is a parasitic disease transmitted to man and othermammals. As we know, the main pathologic lesions in patients or animalswith schistosomiasis japonica are the granuloma formation around eggsdeposited in the host tissues and the subsequent liver and intestinesfibrosis. Many scientists have been trying to make anti-schistosomiasisvaccines to relieve the pathologic lesions. It was reported that the solubleimmature egg antigen of S.japonicum (SjSIEA) inducedanti-embryonation and anti-fecundity immunity, and the 26-28KDacomponents extracted from SjSIEA were the major antigens responsiblefor both anti-fecundity and anti-egg immunity. In China NationalSchistosome Vaccine Unitive Test carded out in 2000, SjSIEA26-28KDa(native molecular vaccine or vaccine No 1) demonstrated the bestimmunoprotective results in the experimental animals (with a pairingreduction rate of 53.9%and liver eggs per gram (LEPG) reduction rate of89.3%). The SjSIEA26-28kDa is therefore regarded as a candidatevaccine to be used for the induction of anti-embryonation andanti-fecundity immunity. By means of two dimensional gelelectrophoresis and immunoblotting, we found that the SjSIEA26-28kDacomponents consist of more than 10 different protein spots, in whichthree protein spots, designated as SIEA-2D66, SIEA-2D71 andSIEA-2D73, were detected fairly strong immunoreaction. Peptide massfinger print maps and Peptldent software analyses showed that the threeprotein spots were significantly homologous to hypoxanthine-guaninephosphoribosyltransferase (HGPRT), succinate dehydrogenase iron-sulfurprotein (SDISP) and glutathione-S-transferase (GST), respectively.Whether they are the major target antigens responsible for theanti-embryonation and anti-fecundity immunity induced bySjSIEA26-28kDa component remains an interesting topic. As we known, S. japonicum GST was one of the six most potentialSchistosomiasis vaccine candidates proposed by the Special Programmefor Research and Training in Tropical Diseases (TDR). However, noinvestigation about SjHGPRT and SjSDISP in immunoprotection asvaccine candidates for schistosomiasis has been found. It was necessaryto evaluate their possibility as vaccine candidates against S. japonicum.Objective Searching in NCBI GenBank nucleotide database showedthe 3′end and 5′end of the expression sequence tags (EST) forSjHGPRT and SjSDISP were all incomplete. By means of the techniqueand strategy in Molecular Biology, to clone the full-length eDNA ofSjHGPRT and SjSDISP; to construct prokaryotic single- and multi-valentrecombinant expression plasmid; to investigate the antigenicity andimmunoprotectivity of the recombinant proteins; to detect the levels oftarget genes in adult worm; to construct the eukaryotic recombinantexpression plasmid of target genes and evaluate the immunoprotectivityas vaccine candidates for schistosomiasis by immunization with nakedeukaryotic recombinant expression plasmids and co-immunizationcombined with their recombinant proteins.Methods 1) The 5′and 3′-terminals of EST of SjHGPRT andSjSDISP were amplified from S.japonicum adult worm cDNA library bythe anchored PCR with general primers designed according to thecontiguous nucleotide sequence of the multielone sites of cDNA libraryvectorλgt11 and special primers designed by the EST of SjHGPRT andSjSDISE After sequencing, the target gene full-length cDNA wereobtained by spelling ESTs, the PCR extension of the 3′and 5′endswith electronic software.2) The coding sequences(CDS) of SjHGPRT and SjSDISP wereamplified from S.japonicum adult worm cDNA library by PCR with theprimers designed by the full-length cDNA obtained above, and weredirectionally subcloned to prokaryotie expression plasmids pQE30 andpGEX-4T-1, and eukaryotie expression plasmid pcDNA3 to constructrecombinant expression plasmids. The prokaryotic recombinantexpression plasmids were transformed into E.coli and expressed inprokaryotic system and purified in large quantity. The expression products were analyzed by SDS-PAGE and Western blot. The expressionlevel of target genes in adult worm were demonstrated with indirectimmunofluorescence; With the same procedure done to the prokaryoticrecombinant expression plasmids, the eukaryotic recombinant expressionplasmids were transformed into E.coli, copied and extracted in largequantity.3) The Kunming female mice were immunized by using prokaryoticrecombinant proteins and eukaryotic recombinant plasmids (SjHGPRTand SjSDISP). The experiment animals were grouped as follows: Controlgroups: FCA, pcDNA3, rSjGST. Prokaryotic single valent groups:pQE30/SjHGPRT; pQE30/SjSDISP. Prokaryotic multivalent groups:pGEX-4T-1/SjHGPRT;pGEX-4T-1/SjSDISP. Eukaryotic recombinantplasmid groups: pcDNA3/SjHGPRT;pcDNA3/SjSDISP.Co-immunization with recombinant protein and recombinant plasmid:(pQE30/SjHGPRT+pcDNA3/SjHGPRT);(pQE30/SjSDISP+pcDNA3/SjSDISP).The immunoprotections were assessed by worm burden, liver eggsper gram (LEPG), fecal eggs per gram (FEPG) and Intrauterine eggreduction percentages.Results 1) Two fragments of about 160bp and 600bp were obtainedafter the 5′and 3′ends of EST of SjHGPRT were amplified fromS.japonicum adult worm cDNA library by the anchored PCR. There wererespectively 76bp and 84bp nucleotide overlaps with the 5′and 3′terminal of EST of SjHGPRT (BU803192) in 160bp and 600bp fragments,indicating that the two fragments amplified were needful. After the threefragments mentioned above were pieced together with electronicalsoftware, a 1270bp full-length cDNA was obtained. Sequence analysis ofthe full-length cDNA for SjHGPRT indicated that the biggest ORF is696bp and encodes 231 amino acid residues, beginning with the initiationcodon ATG at position 17 to 19 and ending with a TAA terminationcodon at position 710 to 712, followed by a strong termination codonTAA at position 737 to 739. The putative protein molecular weight(M.W.) is 26kDa and pI is 5.80. The nucleotides around the start codon(ACGACATGTC) fulfill Kozak’s criteria for C at the -1 position and Gat the -3 position. The ORF is preceded by a 16bp 5′untranslated region and followed by a 558bp 3′untranslated region in addition to the polyAtail, and two polyadenylation addition signals ATTAA and AATAA arepositioned at 1216 and 1236 respectively, indicating the 1270bp fragmentwas the full-length cDNA of SjHGPRT. The deduced amino acidsequence from the full-length cDNA of SjHGPRT exhibits identity of83%, 49%, 47%, 46%, 46%, 46%, 46% to that of corresponding genes ofSchistosoma mansoni, Gallus gallus, Rattus norvegicus, Mus musculus,Chinese hamster, Sus serofa and Chinese hamster respectively. Thenucleotide sequence date of SjHGPRT has been submitted to GenBank,the accession number is AY841891.After the 5′and 3′ends of EST of SjSDISP were amplified fromS.japonicum adult worm cDNA library by the anchored PCR, twofragments of about 350bp and 150bp were obtained, Omitted nucleotideoverlaps with the 5′and 3′terminal of EST of SjSDISP(BU804141) in350bp and 150bp fragments, the three fragments mentioned above werepieced together with electronical software, a 1071bp full-length cDNAwas obtained. Sequence analysis demonstrated that the biggest ORF is837bp and encodes 278 amino acid residues, beginning with the initiationcodon ATG at position 106 and ending with a TGA termination codon atposition 942, followed by a strong termination codon TGA at position949. The putative protein M.W. is 30kDa and pI is 8.57. The nucleotidesaround the start codon (TCAGAATGCT) fulfill Kozak’s criteria for C atthe -4 position and A at the -3 position. The ORF is preceded by a 105bp5′untranslated region and followed by a 118bp 3′untranslated regionin addition to the polyA tail, and also a polyadenylation additional signalsATTTAA is positioned at 1036. Furthermore, another terminator codonof ORF was found at position 7 before initiation eodon ATG. Allmentioned above indicated that the 1071bp fragment is the full-lengthcDNA of SjSDISP. The deduced amino acid sequence from thefull-length cDNA of SjSDISP shows identity of 73%, 72%, 74%, 70%,68%, 73% to that of corresponding genes of Mus musculus, Homosapiens, Drosophila melanogaster, Saccharomyces cerevisiae,Caenorhabditis elegans and Rattus norvegicus respectively. Thenucleotide sequence data of SjSDISP had been submitted to GenBank, the accession number is AY841892.2) The coding sequences (CDS) of SjHGPRT and SjSDISP weredirectionally cloned to prokaryotic expression plasmids pQE30 andpGEX-4T-1, eukaryotic expression plasmid pcDNA3 to constructrecombinant expression plasmids. Confirmed by restriction endonuclease,PCR and sequencing, the recombinant expression plasmids were allsuccessfully constructed. Induced by IPTG, the recombinant fusionproteins, whose molecular weights were identical with the bioinformaticspredicted protein MWs, were proved to be efficiently expressed and havea satisfactory antigenicity by SDS-PAGE and Western blot. SjHGPRTand SjSDISP were all immuolocalized in body wall of Sj adult worm, andthe expression level of SjHGPRT gene was obviously higher than that ofSjSDISP.3) Immunized with prokaryotic recombinant proteins, nakedeukaryotic recombinant plasmids and their co-vaccination, the mice werechallenged with Sj cercariae. The results suggested that, except thepcDNA3/SjSDISP group in worm reduction and prokaryotic multivalentgroups in worm and egg reduction, the worm and egg reduction rateswere all significant in the other groups, especially in the co-immunizationgroups.Conclusion 1) Two full-length cDNA of SjHGPRT and SjSDISPwere obtained by anchored PeR. The full-length cDNA of SjHGPRT is1270bp, and the full-length cDNA of SjSDISP is 1071bp. 2) Theprokaryotic single-and multi-valent recombinant expression plasmidsand the eukaryotic recombinant expression plasmid were successfullyconstructed. The prokaryotic recombinant fusion proteins were efficientlyexpressed in E.coli and highly antigenic. SjHGPRT and SjSDISP wereboth localized in body wall of Sj adult worm, and the expression level ofSjSDISP gene is lower than that of SjHGPRT. 3) The recombinants ofSjHGPRT and SjSDISP could induce partial protective immunity againstS.japonicum chellenge infection and the protection could be enhanced viaco-immunization of the prokaryotic recombinant protein and itseukaryotic recombinant plasmid. Higher egg reduction percentages thanworm reduction rates in all groups suggest that SjHGPRT and SjSDISP could play a role mainly in anti-fecundity or anti-egg immunity againstSchistosoma japonicum.

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