Species Differentiation Of Malaria Parasites And Function Analysis Of Plasmodium Falciparum Sporozoite Surface Protein

More than 230 million individuals in worldwide are infected with Plasmodium spp, and 2.5 million people per year, most of whom are children, die from the infection. (2009.03, WHO). Approximately 90% of the malaria deaths occur in Africa. Despite continuing efforts in vaccine development, malaria prevention is difficult, and no drug is universally effective. In this study, two aspects of the work have been done for detection of malaria parasites and function analysis of Plasmodium faliparum sporozoite surface protein respectively.Part one:Species differentiation of malaria parasites.Of the 4 most common species that infect humans are P.vivax, P. falciparum. P.malariae and P.ovale. As recently as 2004, the fifth malaria was implicated in human disease; Plasmodium knowlesi, a malaria parasite of long tailed macaque monkeys, has been confirmed in several human cases from Malaysian Borneo, Thailand and the Philippines. It is now well established that P knowlesi is emerging as an important zoonotic human pathogen.In 2008, one hundred and forty-six blood samples were obtained from randomly selected patients with uncomplicated malaria in southern Myanmar near China, in 2008.Using a nested PCR method is according to a previously reported. The five Plasmodium species that infect humans are detected. It was the first time to detect P. knowlesi infecting people in this region. Results were important for the development of malaria prevention and control measures in China.Because of the found that the prevalence of P. knowlesi in the border area between China and Myanmar. Forty-five blood samples were obtained from Macaques in the same region and use the same method to detect. In this study, we did not find any malaria parasites in these samples.while we found a malaria-like parasite-Hepatocystis spp. Our studies revealed that geographical localities and host specificity may reflect phylogeny of Hepatocystis sp.Part two:function analysis of Plasmodium falciparum sporozoite surface protein. Malaria infection starts when infected Anopheles mosquitoes take their blood meal and inject sporozoites into the host skin. Sporozoites enter blood vessels, are transported to the liver, invade hepatocytes. and developed into liver stages. The mechanism of sporozoite invading liver cells has not yet been fully clear. But the import step is the specific binding between the protein of sporozoite and the protein of hepatocytes.Circumsporozoite protein (CSP) and thrombospondin-related anonymous protein(TRAP) were considered as ligands for sporozoite invading liver cells. Because of the invasion of cultured human hepatocytes by sporozoites was inhibited in the presence of antibodies to TRAP and CSP. CD81. a member of the tetraspanin superfamily was considered as a receptor for sporozoite invasion liver cells. Because of sporozoites of P. falciparum and P. yoelii do not infect CD81-deficient hepatocytes and mouse.Then the function of Plasmodium falciparum sporozoite surface protein was analysied. We studied the immunogenicity of the Plasmodium falciparum sporozoite surface protein and the interaction of receptors and ligands during the process of the sporozoite invasion to liver cells.Concerning the the structures and functions of Plasmodium falciparum sporozoite surface protein CSP and TRAP. using PCR method. we divided CSP into three segments(C1, C2 and C3) and TRAP into two segments(T1 and T2). And prokaryotic expression vector pGEX-4T-1-C1. pGEX-4T-1-C2, pGEX-4T-1-C3, pGEX-4T-1-T1 and pGEX-4T-1-T2 were constructed, respectively. The above plasmids were transformed into BL21-CodonPlus (DE3)-RIPL. According to the sequence of CD81 large extracellular loop (CD81-LEL) to design specific primers, the prokaryotic expression vector pET22b-CD81-LEL was constructed and transformed into Rosetta (DE3). These recombinant strains were induced by IPTG and identified by Western blot and SDS-PAGE. Glutathione SepharoseYM 4B and His GraviTrap were used to purify, BCA method for the determination of the concentration. Recombinant proteins with high concentration and purity were produced for the further study.Wistar rats were immunized respectively with recombinant sporozoite surface protein (C1, C2, C3, T1 and T2) emulsified with Freund's adjuvant, then the dynamic and longevity changes, antibody titer, and IgG subtypes of antibodies specific to the different recombinant protein were monitored and compared by indirect ELISA to determine the strength of their immunogenicity. Rats immunized with recombinant sporozoite surface protein had a significant amount of antibodies. The antibody titers reached the highest value and could maintain several weeks after the third immunization. The ratios of antibody subclass IgG1/IgG2a that was induced after immunization were less than 1, but close to 1. The results showed that immune responses against recombinant sporozoite surface protein were mainly Th2-type and Th1-type mixed. The research was a foundation for the Plasmodium falciparum sporozoite recombinant surface protein vaccine.The adhesion character of recombinant Plasmodium falciparum sporozoite surface proteins C1, C2, C3, T1 and T2 with HepG2 were identidied by adhesion assay, and the combination C3 and T2 with HepG2 cells were relatively strong. GST-pulldown and His-pulldown assay were carried out to determine whether these proteins binding purified surface protein CD81-LEL of liver cells. Reaults proved that the recombinant sporozoite surface protein C2 has the ability of binding to CD81-LEL Therefore, our study determined the binding capacity between C2 and CD81-LEL protein at the protein level, and provided new clues for the mechanism of sporozoite invading to liver cells.