Expression And Function Analysis Of Plasmodium Falciparum Merozoite Protein PF3D7₀811600

Malaria is a serious infectious disease caused by malaria parasites,which can be spread by female anopheles mosquito. During the past 15 years, under the active prevention and treatment, malaria was well controlled. The World Health Organization reported that there were 198 million cases and approximate 36-75 deaths worldwide in 2013. It is expected that the addition of an effective malaria vaccine to the battery of malaria control strategies would accelerate the decline in disease and promote long-term sustainable control. Plasmodium falciparum is the main pathogen of malaria, which could cause severe clinical symptoms or even death by the invading and dividing of merozoites. While the proteins and secretions ofparasite are key pathogenic factors in this course. Therefore P falciparum merozoite proteins have long been considered to be the attractive targets for malaria vaccine development.P. falciparum merozoites invaded to host red blood cells through specific identification of red blood cell surface receptors, and heparan has been identified as important receptor for invasion. To date, only a limited number of heparin-binding proteins derived from P. falciparum has been revealed. Previous study showed that some P. falciparum proteins could specifically bind to heparin receptor by affinity chromatography and high-throughput proteomic analysis. PF3D7₀811600 was shown to has more peptides in combination with heparin, so it might mediate adhesion and invasion.To understand the roles during the invasion. First, the transcription of PF3D7₀811600 gene in the P. falciparum 3D7 clone was detected by quantitative PCR. Through the sequence analysis, hydrophilic sequences in the C-terminal and N-terminal were selected respectively, the fragments were amplified by specific primers, and then cloned to p MD18-T. After restriction enzyme digestion by Eco R I and Xho I, the targetted fragments were inserted into expression vectors p ET-28 a and p GEX-4T-1, then His-tagand GST-tag recombinant proteins were expressed and purified. His-tag fusion proteins were used to immune the New Zealand rabbits to prepare polyclonal antibody. The expression of the native protein of parasite wasdetected by Western blot. The subcellular localization of PF3D7₀811600 was assessed by immunoelectron microscopy assay and indirect immunofluorescence assay. GST-tag and His-tag recombinant proteins were used to detect the heparin binding ability and red blood cell adhesion property, respectively.The invasion inhibition assay was carried out with PF3D7₀811600 specific antibodiesto further explore the function of PF3D7₀811600 during the invasion. The malaria patient serums were detected by ELISA to evaluated the immunogenicityduring the natural infection.The result of Q-PCR showed that PF3D7₀811600 gene was transcribed during the erythrocyte stage, and primarily expressed at 40 hours post-invasion. The recombinant proteins were purified with high purity. The result of Western blot showed that the polyclonal antibody against PF3D7₀811600 was able to identify the natural protein of parasites at the late schizont stage. The results of localization analysis showed that PF3D7₀811600 was expressed on the surface of merozoites,which speculated that it is a merozoite surface protein. The peptide fragments of PF3D7₀811600-N and PF3D7₀811600-C could bind to heparin, while only PF3D7₀811600-C peptide could adhere to erythrocyte specifically. The anti-PF3D7₀811600 specific antibodies, especially the Ig G of antiPF3D7₀811600-C, could inhibit the invasion of parasites obviously, which suggested that PF3D7₀811600 played an important role in invading the host red cells. And PF3D7₀811600-C could specificially react with the serums of malaria patients.Therefore, PF3D7₀811600 was believed to be a potential malaria vaccine candidate.