Study On Molecular Details Of The Interaction Between Erythrocyte Binding Antigen-175 (EBA-175) Of Plasmodium Falciparum And Its Receptor

Background and Objective:Malaria remains one of the leading causes of morbidity and mortality in the developing countries in tropical and subtropical regions. For the past few years, the chemical resistance of plasmodium and the gnat's chemical spoofs increasingly intensifications spread out more faster and larger, which together with some modern economic and social factors, malaria (especially Plasmondium falciparum) recrudescence were seriously for the human being. However, with the increasing number of acute infection patients, there are less cheap and effective chemotherapeutics to be selected than before. Thereby, prevention and treatment of malarial are facing with rigorous challenge.The erythrocyte binding antigen-175 (EBA-175) was reported in 1985 by Camus and Hadley. After a large number of immunology empirical studies and epidemiological investigations, it has been proved that EBA-175 is not onlycandidate antigen of malaria vaccine but also specific diagnostic antigen. EBA-175 on Plasmodium falciparum merozoites mediates sialic acid dependent binding to glycophorin A on host erythrocytes and, therefore, plays a crucial role in cell invasion. EBA-175 bound to erythrocytes with receptor-like specificity and was saturable . Most Neu5Ac on human erythrocytes is linked to galactose by a2-3 linkages on glycophorin A. Within the 5'cysteine-rich region II(RII) in EBA-175, there are two domains, designated F1 and F2, the red blood cell binding function is located primarily within the F2 domain. The studies indicate that antibodies to RII of EBA-175 are associated with protection against clinical disease. These studies not only provide a novel theory to the pathogenesis of malaria, but also develop a new clinically relevant therapeutic target for malaria.Since 1950's, many remarkable achievements, especially application of Artemisinin, have been made in malarial control by traditional Chinese medicine. Nowadays, with the development of biotechnology, scientists focus their studies on molecular level of Chinese herbs in treating disease. This will lead the study of Traditional Chinese Medicine to a more essential way. In this study, we not only try a new way in study of compounded Chinese herbs, but also develop new clinical therapeutic targets for traditional Chinese medicine on gene therapy.Methods:A pair of primers was designed according to the published P. falciparum FCC1/HN strain genemics sequence in NCBI. Then the gene encoding EBA-175 was amplified by PCR. The PCR products were purified and cloned into pMD18 T vector. The pMD-EBA was sequenced and the result of sequencing was analyzed. Then the gene of EBA-175 was sub-cloned into pET23a(+) to construct prokaryotic expression vector. The pET23a-EBA was transformed into E.coli BL21(DE3) and the proteinexpression was induced.The best expressing clone was screened. To seek the most expressing quantities, we optimized inducing conditions. The recombinant protein was purified by preparative SDS-PAGE and used to immunize mice for the preparation of polyclonal antibodies. BALB/ c mice were immunized with purified recombinant EBA-175 and McAbs against EBA-175 were prepared according to the protocol of hybridoma technique. The McAbs were characterized by EL ISA and Western-blot. The recombinant protein of EBA-175 was chosen as target molecule to screen mimetic peptides of GPA from a 12-mer random peptide library and disulfide constrained heptapeptide library. Three rounds of biopanning were carried out and then ELIS A, competitive ELIS A, Dot-ELIS A and western blot were used to evaluate the binding character between pHage-borne peptides and EBA-175. The insert DNA sequences of positive clones were determined and their amino acid sequences were deduced.Results:1. The gene of EBA-175 RIIF2 was successfully amplified by PCR. The prokaryotic expression plasmid pET23a-EBA was constructed using the sub-cloning technique from the TA clone pMD18-EBA. The sequencing result of cloned EBA-175 gene showed the correct reading frame by the biological software, DNAMAN, and 99.29% identity to P. falciparum FCC1/HN strain genome individuals by BLAST.2. The protein accounted for about 13.6% of the total bacterial protein. The protein was expressed best when OD6oonm equaled to O.8,the inducing time was 5 hours and the concentration of IPTG was 1.0 mmol/L. The purity of recombinant protein was about 100% and can be used for the follow study.3. Six McAbs against EBA-175 antigen were obtained, five ones of them wereIgGl and one was IgG2a. The titer of these McAbs was 1:12800 to 1:25600 in the ascites and 1:256 to 1:512 in supernatant. The result of EL ISA indicated that four McAbs(1F3,2H5,4AK 4H9)reacted with P. falciparum . Three of them recognized a 36 KDa protein which was defined as EBA-175 by Western blot.4. Fifty clones from the third round were randomly selected, and thirty-six ones among them were detected to be positive by sandwich ELISA, concluding nine ones of disulfide constrained heptapeptide (9/20,45%) and twenty-seven ones of 12-mer random peptide (27/30, 90%).The competitive ELISA tests proved that most phage-borne peptides could competitively inhibit the binding of antibody (EBA-175 Ab) with EBA-175. Analysis of DNA and amino acid sequences indicated that three sequecces of disulfide constrained heptapeptide and four sequences of 12-mer random peptide be showed. Peptide MLLITTR was the most frequently appeared peptide of disulfide constrained heptapeptide, which have the conserved sequence ? ? LH ? ? showed high homogeneity to 108-110 aa of GPA. Peptied RNNTIRRHRLMMP and KPRTPNSIIIRR have the conserved sequence ? ? IRR ? ? showed high homogeneity to 114-116 aa of GPA.Conclusions:1. The prokaryotic expression plasmid pET23a-EBA was successfully constructed.2. The EBA-175 protein was highly expressed as non-fusion protein and was successfully purified, which has good antigenicity.3. Several hybridoma cell lines secreting high titer of McAbs against EBA175 with high specificity were established.4. With chloroquine, HuangQI and CiWuJia could enhance immunity and facilitate producing antibody.5. These peptides displayed by pHage may be analogs of EBA-175, LLI and IRR probably play a significant role on the binding reaction ofEBA-175 and GPA.6. We not only try a new way in study of compounded Chinese herbs, but also develop new clinical therapeutic targets for traditional Chinese medicine on gene therapy.