Screening The Receptor On The Surface Of Erythrocyte Interacting With Plasmodium Falciparum And P.vivax GPI-anchored Micronemal Antigen(GAMA)

Malaria is one of the important infectious diseases that endanger human health and life seriously.According to the World Malaria Report 2019,a total of 228 million malaria cases and 405,000 deaths occurred globally.Most malaria cases in 2018 were in the World Health Organization(WHO)African Region(213 million or 93%),followed by the WHO South-East Asia Region with 3.4% of the cases and the WHO Eastern Mediterranean Region with 2.1%.Children aged under 5 years were the most vulnerable group affected by malaria.In 2018,they accounted for 67%(272 000)of all malaria deaths worldwide.Plasmodium falciparum is the most prevalent malaria parasite in the African Region,accounting for 99.7% of estimated malaria cases in 2018.Globally,53% of the P.vivax burden is in the South-East Asia Region,with the majority being in India(47%).P.vivax is the predominant parasite in the Region of the Americas,representing 75% of malaria cases.Previous study related to Plasmodium vivax and Plasmodium falciparum prove that attenuated sporozoites can produce good protection in humans and experimental animals.However,problems such as large-scale production,preservation,and transportation have not been solved,which has affected development and promotion.Plasmodium vivax GPI-anchored micromitochondrial antigen(PvGAMA)can bind to erythrocyte of Duffy-positive and negative blood groups,and the homologous protein of the Plasmodium falciparum GPI-anchored micromitochondrial antigen(PfGAMA)can also bind erythrocytes.Its specific antibodies can successfully inhibit the invasion of P.falciparum to erythrocyte,but its receptors on erythrocyte have not been revealed.Therefore,searching for erythrocyte membrane surface receptors of Plasmodium adhesion molecules not only helps elucidate the mechanism of Plasmodium invasion,but also plays a vital role in the development of a malaria vaccine.This study revealed that P.falciparum,P.vivax mechanism of how to invade erythrocyte as the basic goal.By elucidating the functions of PfGAMA and PvGAMA proteins binding to the key molecules of erythrocyte and their complexes,we will ultimately provide a theoretical basis for the development of strategies for the development of vaccines for P.falciparum and P.vivax.The study includes three parts:Part Ⅰ,The construction of prokaryotic expression plasmids of erythrocyte binding regions of GAMA and the expression and purification of recombinant proteins.The erythrocyte binding regions of the PfGAMA and PvGAMA(PfGAMA Tr3 and PvGAMA F2)gene were optimized into the E.coli expression system.We used the Infusion cloning technology to construct the PfGAMA and PvGAMA genes into the E.coli expression vector(pET30a);The plasmids were induced and expressed by prokaryotic expression system,and the crude protein was purified by nickel column affinity chromatography.Finally,PfGAMA and PvGAMA protein were prepared at a concentration of more than 1 mg/ml and purity of more than 80%.Part Ⅱ,GAMA interacted with human erythrocyte membrane proteins.We extracted human erythrocyte membrane proteins by lysing erythrocytes with hypotonic Tris-HCl buffer,and the protein concentration was quantified using a BCA protein content detection kit.Next,nickel column tandem affinity chromatography was used to screen human erythrocyte membrane proteins bound to PfGAMA,and the binding protein complex eluted from the nickel column was subjected to silver staining to identify differential bands.The difference bands were cut out for decolorization,enzymolysis and desalting steps.Finally,the peptides were detected by mass spectrometry to obtain the protein located on the surface of the erythrocyte membrane.Only Ankyrin-1 protein was identified,and mass spectrometry predicted that Ankyrin-1 protein had the most peptides(7 segments)and the highest comprehensive score(154.62).Part Ⅲ,GAMA interacted with Ankyrin-1.The functional region of Ankyrin-1 was divided according to the published article.The membrane-bound region at the Nterminus was divided into three regions by partial overlapping,and three segments of genes were cloned into the pET30 a expression vector by In-fusion cloning technology.The crude protein was purified by nickel column affinity chromatography.Ankyrin-1-D1(46 kDa),D2(47 kDa),and D3(48 kDa)were prepared with a concentration of more than 0.9 mg/ ml and purity of more than 80%.The binding regions of Ankyrin-1 to PfGAMA and PvGAMA were screened by Flag Pull-down assay.Pull-down results showed that the three binding elution buffers of PfGAMA and PvGAMA were all confirmed to bind Ankyrin-1-D2.The gray value of the bands in the results of western blot was calculated.The highest gray value of Ankyrin-1-D2 was found in the HA-tag antibody binding protein elution buffer.Ankyrin-1-D2 protein was the highest in the protein elution buffer.Next,the results of the interaction between the extracted Plasmodium falciparum protein and Ankyrin-1-D2 showed that Ankyrin-1-D2 could interact with native PfGAMA.Enzyme treated Ankyrin-1-D2 combined with GAMA confirmed that Ankyrin-1 is a chymotrypsin-dependent receptor.Cell experiments confirmed that Ankyrin-1-D2 antibody could effectively inhibit the binding of GAMA to erythrocytes.Finally,the gold standard "surface plasmon resonance technology" to verify protein interactions was used to detect the interaction affinity between PfGAMA,PvGAMA and Ankyrin-1-D2,which further confirmed the interaction between PfGAMA,PvGAMA and Ankyrin-1-D2.In summary,our research screened the specific binding receptors of Plasmodium GAMA on erythrocyte as Ankyrin-1.It revealed the important function of GAMA in the process of Plasmodium invasion to erythrocyte,and provided a theoretical basis for improving the vaccine development of Plasmodium falciparum and Plasmodium vivax,especially the development of recombinant subunit vaccines.