Construction Of Full-length CDNA Library Of Plasmodium Vivax, And PvGDH Cloning, Expression, Epitope Identification And McAbs Preparation

Background: Malaria still remain one of most serious vector-born diseases, it's one of most important disease threat to health in China as well. Plasmodium vivax is the most wide-distributed of human malaria parasites, and contributed as major species of malaria parasite in China, although it's not as severe as another Plasmodium falciparum. The burden of vivax malaria is always under-estimated, and the research about vivax was neglected comparing with falciparum. Diagnosis is essential for malaria control, however, the malaria diagnostic technique is still lack with the demand of disease control. The rapid diagnosis test based on specific Plasmodium antigen detection was applied in field and showing promising results. But most of them can not specific detect Plasmodium vivax, the vivax specific RDT is urgent needed. To address it, a full length cDNA library of erythrocytic stage Plasmodium vivax was constructed for vivax specific diagnostic antigen screening.Glutamate dehydrogenase (GDH) is an essential enzyme for glycolysis in Plasmodium, is a potential diagnostic target as well, the established immunology diagnosis test based on Plasmodium falciparum glutamate dehydrogenase (PfGDH) shows satisfactory results, however, no report revealed about Plasmodium vivax glutamate dehydrogenase (PvGDH) so far. Therefore, the PvGDH was cloned and expressed, and monoclonal antibody against recombinant PvGDH was raised for further Plasmodium vivax specific diagnosis test development.PART 1 Construction and Identification of Full-length cDNA Library of Erythrocytic Stage Plasmodium vivaxObjective: To construct a full-length cDNA library of erythrocytic stage Plasmodium vivax of China isolation.Methods: The blood was collected from confirmed vivax malaria patients before treatment, and WBCs were removed using Plasmodipur filter, and Plasmodium vivax infected RBCs were enriched by Porcoll gradient purification. Total RNA was extracted using TRIZOL reagent,first-stranded cDNA was synthesize by reverse transcription polymerase chain reaction ( RT-PCR ) method. Then, the second-stranded cDNA and double-stranded cDNA was synthesized using long distance polymerase chain reaction (LD-PCR) method. The ds-cDNAs were digested by proteinase K and Sfi I restriction enzyme, to ensure the length of inserts, fragments less than 400 bp were separated by CHROMA SPIN-400 column, meanwhile the cDNA was ligated to the Sfi I digested vector and transformed into E. Coli DH5αcompetent cells. Then the titration and the recombinant rate of the cDNA library were evaluated, and 48 colonies were choose randomly and identified the size of the inserted cDNA using PCR with M13+-primers.Results: The full-length cDNA library was constructed, with 1.14×106 independent clones, a recombinant rate of 97.2%. The lengths of the inserted cDNA fragments ranged from 900 to 2500bp.Conclusion: A high-quality full-length cDNA library of erythrocytic stage Plasmodium vivax of China isolation was successfully constructed.PART 2 Cloning and Expression of Plasmodium vivax Glutamate DehydrogenaseObjective: To clone and express Plasmodium vivax Glutamate Dehydrogenase (PvGDH)Methods: The primers were designed based on putative Plasmodium vivax Glutamate Dehydrogenase gene, then PvGDH coding sequence was amplified from emplicationcon was the full-length cDNA library of erythrocytic stage Plasmodium vivax, and inserted into pMD18-T vector. The sequence was confirmed by sequencing after identification using PCR and restriction enzyme digestion, then sub-cloned into pCOLD II vector for expression. The immunoreactivity of the serum from vivax malaria patients , falciparum malaria patients and health volunteers to recombinant PvGDH (rPvGDH) was tested using ELISA and Western-blot. The serum was collect from mice after immunization using rPvGDH, and its immunoreactivity to rPvGDH was tested using ELISA, as well as the immunoreactivity to antigen of Plasmodium vivax, Plasmodium falciparum, and normal human RBC was tested using Western-blot.Results: A 1442 bp gene was amplified by PCR, and cloned into vector, the sequence has 3 bp difference with the coding sequence of PvGDH from sal-I strain genomic database, but without encoding amino acid changing. The recombinant PvGDH was expressed as soluble form, and could be recognized by the serum from vivax malaria patients or falciparum malaria patients , but not be recognized by the serum from health volunteers. The serum was collect from mice after immunization using rPvGDH could recognize rPvGDH, both antigen of Plasmodium vivax and Plasmodium falciparum, but not normal human RBC.Conclusion: PvGDH was successful cloned and expressed with immunogenicity.PART 3 Prediction and Identification of B Cell Epitope of Plasmodium vivax Glutamate DehydrogenaseObjective: To predict and identify B cell epitope of Plasmodium vivax Glutamate Dehydrogenase (PvGDH)Methods: The B cell epitope of PvGDH was predicted through the sequences analysis and comparing of PvGDH and PfGDH using bioinformatics software. The peptide was synthesized according to the predicted linear epitope, and for raising anti-serum by mice immunization. The immunoreactivity of serum to rPvGDH was tested using ELISA and Western-blot, as well as the immunoreactivity to antigen of Plasmodium vivax, Plasmodium falciparum, and normal human RBC.Results: A specific linear B cell epitope of PvGDH was predicted, and the serum against the peptide could recognize rPvGDG and antigen of Plasmodium vivax, but either Plasmodium falciparum or normal human RBC.Conclusion: A specific linear B cell epitope of PvGDH was successful predicted and confirmed.PART 4 Study of Monoclonal Antibody against Plasmodium vivax Glutamate DehydrogenaseObjective: To produce monoclonal antibodies against PvGDH using recombinant PvGDH and predicted PvGDH B cell epitope.Methods: Monoclonal antibodies against PvGDH were produced using recombinant PvGDH and predicted PvGDH B cell epitope for mice immunization. The titer and subtype of monoclonal antibodies were tested, the immunoreactivity of monoclonal antibodies to rPvGDH was tested using ELISA and Western-blot.Results: 4 and 6 monoclonal antibodies were produced respectively using rPvGDH, and predicted PvGDH B cell epitope. All of them can specifically recognized rPvGDH with titer higher than 1: 2430 Out of total 10, 2 monoclonal antibodies rose using predicted PvGDH B cell epitope are IgG1, others all are IgG2b.Conclusion: 10 monoclonal antibodies against PvGDH were successfully produced using rPvGDH and predicted PvGDH B cell epitope.