The Study Of Plasmodium Yoelii's Protein In Exoerythrocytic Stage

Malaria, a disease caused by the protozoan parasites of the genus Plasmodium, remains the most important parasitic infection of man, causing 300-500 million clinical cases and 1-3 million deaths each year. The parasites persist in people by employing the immune evasion mechanism of antigenic variation, which extends both the disease and opportunities for transmission to new hosts via mosquito vectors. After the bite of an infected female Anopheles mosquito, the Plasmodium sporozoites enter the mammalian host. Then the sporozoites move through the dermis until they come into contact with a blood vessel and move into the circulatory system. Once inside the circulatory system, sporozoites rapidly reach the liver sinusoids, then crossing the Kupffer cells, the endothelial cell and several hepatocytes, at last reaching the hepatocyte in which they begin to develop into merozoites. After the merozoites matured, they will be released into the bloodstream. Each merozoite will invade an erythrocyte, initiating a replication cycle that ends with the release of new merozoites from the mature infected erythrocyte(schizont), which go on to infect other erythrocytes, and cause the clinical symptome. The exoerythrocytic stage refers to the developmental stage of the malaria parasite (Plasmodium) in liver parenchyma cells of the vertebrate host before erythrocytes are invaded, also called liver stage. It is widely acknowledged, based on experimental data, that a vaccine arrested parasite development in the liver could be highly efficacious. These experimental findings can be summarized as follows: First, animals and humans exposed to radiation attenuated sporozoites can be consistently protected against subsequent infection with viable parasites; Second, protecptive immunity in these models is mediated principally by CD4+ and CD8+ T cells, this does not rule out a role for antibody directed against sporozoites, but highlights the importance of cellmediated responses against intrahepatocytic parasites; Third, epidemiologic studies also support the role of a protective cellular immune response directed against antigens expressed by parasites in the liver. Malaria research entered the postgenomic era in 2002, when the Plasmodium falciparum, Plasmodium yoelii yoelii and Anopheles gambiae genomes joined the assembled human genome in the published literature. These achievements are the basis and primer for a broad range of future experimental designs in the malaria research community for years to come, including analyzing malaria stage-specific gene expression/regulation, protein expression/interactions, and identifying novel vaccine targets.Current efforts in characterizing malaria liver-stage development have successfully stemmed from the use of the rodent malaria model systems of P.yoelii and P.berghei. These model systems circumvent the need for infected human or monkey liver samples in studying human malaria liver forms. The whole-genome shotgun sequence of Plasmodium yoelii yoelii and Plasmodium falciparum clone 3D7 show that of about 5,300 P. falciparum genes, more than 3,300 P. y. yoelii orthologues of predominantly metabolic function were identified. Over 800 copies of a variant antigen gene located in subtelomeric regions were found. The rodent and human malaria parasite genomes are remarkably conserved, and P.yoelii can be used as a good model for the human malaria studies.The changes in gene expression, although crucial to the development and survival of the parasite, have remained largely uncharacterised in the liver stage. Several factors are responsible for this situation, with the primary impediment being the relatively small number of parasite infected hepatocytes that are found against the large background of uninfected hepatocytes. This has made studies of gene and protein expression occurring during liver stage development nearly impossible. The important next step has become the identification of the proteins that are expressed during this stage of development and the determination of their role in both the biology of the parasite and the host immune response. The percentage of infected cells, in the liver, has been very low, approximately 0.3%. Even by utilizing laser capture microdissection to provide a high quality source of parasite, the limited number of parasites is only fit for the use of gene analyzing. All of this call for an effective way to identify the protein expressed in liver stage. We know that some antigens expressed in several stages such as CSP and TRAP emerged both in sporozoite and in liver stage, PyHEP17 expressed in hepatic and asexual erythrocytic stages. For the sporozoites are relatively easy to be separatied and purified, we immunized rabbit with P.yoelii sporozoites, and using the producing sera to recognize liver stage parasites after 43h of invasing rat hepatocytes by the Western blotting. The recognized proteins were analyzed by LC/MS and MALDI-TOF-MS. In the analyzed proteins, we are interested in the YIR protein and plan to further study. So the YIR protein expression plasmid was constructed. The experimental concluding two main steps:1. Analyze the protein of P.yoelii exoerythrocytic stage recognized by salivary sporozoite immured rabbit's sera in Western Blotting with mass spectrum technology such as LC/MS and MALDI-TOF-MS. The protein's separated means includes SDS-PAGE and two-dimension electrophoresis. The MS results were submitted to the Mascot online tool for analyze. From the Mascot, we get the matched protein's information about YIR protein. YIR protein has been thought as a surface protein expressed on the surface of the P.yoelii infected red blood cell, and it acts as a'smoke screen'for the adaptive immune system in the mammalian host. In this experimental, we detect the YIR protein in exoerythrocytic stage for the first. And the result also confirmed that immuning rabbit with one stage parasite can produce sera to recognize the neighbor stage parasite.2. Detecting yir gene and construct the plasmid pQE80L/YIR. Since we know that the YIR expressed in exoerythrocytic stage, the relative gene should be active and the mRNA will persistent in this stage. So we extract the mRNA, amplified yir gene with RT-PCR to confirm the MS results. After we get the gene, we insert this part cDNA into the pQE80L vector to express the YIR protein in the host E.coli DH5α. With the positive immunoblotting result, we think we get the YIR protein expressing in E.coli.Above all, we design this experiment which combined 2-DE with Western Blotting and mass spectrum to identify the P.yoelii proteins expressed in liver stage which difficult to research in common way. Then amplifying the yir gene in the same stage according to the protein result, confirmed the previous protein result. To deeply study the YIR protein, we construct an expression plasmid contains yir gene called pQE80L/YIR, and put this plasmid into the host DH5α, inducing the YIR with IPTG. The Western Blotting result shows that the YIR has been expressed. The successful detecting and expressing YIR protein in exoerythrocytic stage will help us to know more about the biology of Plasmodium.