Immunohistochemical Analysis Of Plasmodium Falciparum Merozoite Surface Antigen

Malaria caused by Plasmodium falciparum is an extremely important global disease, causing high levels of mortality and morbidity worldwide. Lacking of efficient malaria vaccine, there were more than1200000malaria deaths worldwide. There are two hosts during malaria life cycle, including human and Anopheles mosquitoes. The most pathogenic period during the human host is invading the erythrocytes. The relationship of P. falciparum merozoite proteins and erythrocytes become significant research field.The research object of our experiment is targeting P. falciparum merozoite surface proteins. Screening the immunoreactivity of merozoite surface antigens by protein array provides the convenient for the research of antigen function and antibody preparation. Using the In-Fusion cloning method, wheat germ cell-free system （WGCF） and protein array technology, the antibody profiles of human serum against P. falciparum merozoite proteins were screened that some highly immunoreactivity antigens were identified. The obtained experiment results were as follows:（1） A total of112unique genes encoding P. falciparum merozoite proteins were selected from the Plasmodium database. There were192ORFs fragments for PCR amplification and In-Fusion cloning in total. These merozoite proteins are selected according to specific sets of criteria, includingâ‘ proteins of schizont stage-specific expression,â‘¡proteins associated with P. falciparum merozoite invasion processâ‘¢proteins with signal peptides, andâ‘£proteins with one or more transmembrane domains.（2） From a total of192P. falciparum ORFs,152ORFs were amplified successfully by PCR. To evaluate the efficiency of In-Fusion cloning, we picked four colonies per transformation at random which were screened by colony PCR. The cloning results showed that of the152ORFs screened,138were successfully cloned （90.8%）. Of the97cloned genes corresponding to138ORFs fragments,79and53genes have signal peptides and transmembrane domains, respectively.（3） P. falciparum merozoite proteins were expressed by wheat germ cell-free system. Of the138ORFs,42expression products were selected at random for western-blot analysis. There were37proteins expressed successfully which are all solubility （100%,37/37）.（4） The expression level of P. falciparum merozoite proteins were screened by protein array with Penta-His antibody specific to the His6-tag epitope of the fusion proteins.85.5%of the merozoite proteins tested positive.（5） The merozoite protein arrays were probed with sera samples from16subjects who were naturally exposed to P. falciparum and16unexposed subjects.30（21.7%,30/138） merozoite proteins were detected with highly immunoreactivity. Of the30highly immunoreactivity antigens,14proteins showed immunoreactivity with more than50%（8/16） patient serum samples. Pf34and GAMA were the most immunogenic merozoite protein recognized by93.8%（15/16） of malaria serum samples. In addition, there were7proteins, including MSP3.5, ETRAMP11.2, ETRAMP14.1, MRSP2, and RALP1together with two hypothetical proteins PFA0210c and PF14₀572, were described as immunologically reactive for the first time. Among the average signal intensity of merozoite proteins against malaria-exposed subjects and unexposed subjects,5merozoite proteins, including Pf34, GAMA, MSP3.1, ETRAMP14.1and PF10₀138, had signal intensity higher than4000. The average signal intensity of a hypothetical protein （PF10₀138） was higher than7000. In a previous study, this protein was detected in83.3%（10/12） of P. falciparum-exposed individuals.This study shows P. falciparum merozoite immunoproteome profiled by high-throughput screening assays. Some of the highly immunoreactive merozoite proteins played a role in invading the erythrocytes. These novel merozoite proteins may be the potential vaccine candidates for blood stage malaria, and need to research deeply in future.