Primary Investigation Of The Function Of T Cell Immunomodulatory Like Protein In Plasmodium Induced Immune Suppression

Malaria is number one killer of parasitic diseases, with estimated100million cases ofhuman malaria each year. Plasmodium exists and proliferates even under the conditions ofacquired immunity developed after malaria infection, which is called Plasmodium immuneevasion. Multiple factors including variable antigen, antigen diversity/polymorphismderived from complex life cycle, and immune suppression contribute to Plasmodiumimmune evasion, resulting in instable host immune response. However, the explicitmechanism still remains uncovered, which brings great difficulties to the Plasmodiumresearch and development of malaria vaccine.T cell immunomodulatory protein (TIP), a cross membrane protein composed by612amino acids, is a new discovered immunomodulatory molecule in human and manyanimals, which exhibited a protective effect against acute graft-versus-host disease(GVHD) mode as evidenced by increased survival rate after allogenetic graft transplant.We have previously identified protein analogues to TIP existed in a variety of Plasmodiumwith bioinformatic tools, which all showed similar amino acid sequence, function domainand tertiary structure to TIP and named them Plasmodium TIP like proteins.According to these findings, we proposed Plasmodium TIP like proteins to be a potential candidator insuppression of immune responses during malaria infection.To evaluate the function and mechanism of Plasmodium TIP like proteins in immuneevasion during malaria infection, the following experiments were performed.1. Plasmodium TIP like proteins expression feature and anti-PbTIP can inhibitPlasmodium proliferationWe constructed a Plasmodium berghei TIP like protein (PbTIP) prokaryoticexpression vector with GST tag according to the PbTIP partial gene sequence in thePlasmodb database. The recombinant protein was then purified and was used for rabbitimmunization to harvest anti-PbTIP-GST antibody. Indirect immunofluorescence wasperformed by this antibody in red blood cells infected by Plasmodium berghei. The resultsindicated that PbTIP was evenly expressed in the cytoplasm of the Plasmodium berghei,but neither in the nuclei of the Plasmodium berghei nor on the membrane of infected redblood cells.With the limitation of in vitro Plasmodium berghei culture, Plasmodium falciparumwas chosed for plasmodium TIP like proteins expression in its erythrocytic stage.Quantitive PCR results revealed thtat late trophozoite stage, rather than ring stage orschizont stage, exhibited6or5fold higher mRNA level of Plasmodium falciparum TIPlike protein (PfTIP), indicating that PfTIP is highly expressed and functional in the middleof erythrocytic stage and declines to a comparable lower level with development intoschizont stage. These findings implicate that PfTIP is not a key molecular regulator duringPlasmodium falciparum merozoite invasion process. In addition, this finding was inaccordance with the result that rabbit anti-PbTIP sera exhibited weak inhibitory effect onPlasmodium falciparum growth.The important finding is that recombinant PbTIP immunization protected mice fromPlasmodium berghei attack. A possible explanation for this result is that PbTIP anti-PbTIPantibody inhibits the immunosuppression negative immunoregulatory effect ofendogenous PbTIP expressed by Plasmodium berghei, results enhanced immunoresponseto Plasmodium berghei attack. 2. The effect of PbTIP extracellular domain on T cellsTo further explore the function domain of PbTIP, we constructed a PbTIPextracellular domain prokaryotic expression vector according to the PbTIP whole genesequence in the Plasmodb database. Since recombinant PbTIP-GST expressed by apGEX4T-1vector showed a low affinity to chromatography column, pET32a vector wasused for PbTIP extracellular domain expression.The results showed that recombinant PbTIP extracellular domain was expressed inthe form of inclusion body, and denatulized recombinant protein showed a high affinity toNi-NTA Agarose. However, purified soluble recombinant protein neither inhibited theproliferation of CTLL-2cells nor influenced the secretion of cytokines including, IL-4,TGF-β and IL-10. The possible reason is that natural conformation of PbTIP extracellulardomain could not be recovered completely after prokaryotic expression and purification,or high level proliferation of CTLL-2cells activated by IL-2covers the effect of PbTIPstimulation.3. Knocking out of PbTIP geneTo further investigate whether PbTIP is a nonspecificly inhibitory regulator in hostimmune response, especially the role of it in plasmodium immune evasion, PbTIP geneknock-out experiment was ferformed based on homologous recombination theory.Homoogous recombination vector Pl0018/PbTIP with tg-dhfr/ts antibiotic resistance tagwas constructed and linearized, which was thereafter electrotransfected into Plasmodiumberghei with transcription activator-like effector nucleases (TALENs) vector.But the results revealed failure in knocking out of PbTIP due to different features ofgene expression between and regulation plasmodium and mammal. Only exogenouslinearized vector fragment could be detected as episomes in Plasmodium berghei, whichdisappeared with proliferation and passage of Plasmodium berghei. Besides, anexplanation that knocking out of PbTIP could be lethal for Plasmodium berghei, resultingnone harvesting of successful Plasmodium berghei recombinant.Due to limited time, experience and technique problem, the thesis stopped here butnot the research work.