Polymorphism And Humoral Immunity Characteristics Of Type A RIFIN Protein Of Plasmodium Falciparum

Objective:To further understand the host selection of Plasmodium falciparum,observe and analyze the differential relationship between the selection of rif genes associated with severe malaria and immune evasion,the response of wild-type,mutant RIFIN peptides to host humoral immunity（IgG）and inhibitory receptor LILRB1,and explore the key fragments and binding sites of RIFIN binding to their antibodies and inhibitory receptors,so as to provide a theoretical basis for the study of the structure and immunological function of Plasmodium falciparum.Methods:After DNA extraction,the collected samples were PCR confirmed as single P.falciparum infection,and genome DNA were broken into 500bp fragment using Covaris S2 instrument to build libraries.All genome libraries were re-sequenced using an Illumina X-10 sequencer.We downloaded the reference sequence of PF3D7 from the PlasmoDB website,the SNP data of the Ghana native P.falciparum isolates in 2013 were downloaded from Pf3k project to build the reference data set.For all of the 9 isolates,we performed PCA and STRUCTURE analysis to reveal their population structure,and calculated the nucleotide polymorphisms（π）and population genetic differences（FST）between two subgroups to check their DNA diversity.Meanwhile we performed the Tajima’s D and iHS tests on the entire genome to identify those selection signature and check the natural selection degree.We focus on the differences in the selection of A-type rif genes in the two populations,to determine the selection pressure of A-type rif between different hosts.After combining the whole genome SNP data,we picked out A-types rif gene sequences using MEGA6 and DNAsp software.The wild-type and mutant peptides were determined based on the results of the analysis combined with the predicted B cell binding region rif,to detect the humoral immune（IgG）response of peptides to the whole blood of P.falciparum by indirect ELISA experiments.And then immunize wild-type and mutant rif peptides against New Zealand rabbits.Analysis of immunogenicity and immunoreactivity of wild type and mutant by ELISA experiments.Through molecular interactions,peptides react ELISA LILRB1 inhibitory receptors.To design mutant polypeptides with a single locus gene mutation based on the response,re-experimental observation of its binding to inhibitory receptors,to judge the key sites for binding rif peptides and LILRB1 inhibitory receptors.Results:A total of 9 samples were selected for genome sequencing and further analysis.Our results showed that most of the imported samples are single infection of P.falciparum.In the global population structure,samples were clustered according to their geographic origin,and imported isolates,which could be regarded as part of the Ghanaian population,maintained slight diversity to the reference sequence.It was quite unexpected that overall genetic diversity was lower in imported samples（the mean π values in the imported and native samples were 0.0013 and 0.0017,respectively）;even though we assumed low selection pressure from Chinese hosts would enable parasites to mutate faster.However.when it comes to genes associated with RBC invasion and immunity,greater diversity was exhibited in imported samples than local ones,such as rif（0.0116）,var（0.012）,and stevor（0.013）.The VSA gene families exhibited the greatest diversity in the parasite,and our observations were consistent with previous studies.In the natural selection test section,the host immune response along with its genetic background is essential for malaria prevention.Clinical immunity to malaria is definitely acquired,even though the process might be slow in areas of stable transmission.The significantly higher average Tajima’s D values（P<0.0001,z-test for paired samples）of imported samples demonstrated a lack of acquired immunity in Chinese citizens,while 3,934 genes showed negative values.However,in local samples,almost all genes were under directional selection（5.253 genes with negative values）.This difference revealed that continued exposure to malarial antigens maintains immunity.We also performed single gene analysis to find mutant peptides from 3 A-type rif gene.Three peptides 1-RIFIN、2-RIFIN、3-RIFIN,from 234 to 254 were selected from the PF3D7₁254800 sequence two polypeptide 4-RIFIN、5-RIFIN,from 252 to 272 positions were selected from the PF3D7₁100400 sequence.Two peptides 6-RIFIN、7-RIFIN,from position 182 to 205 were selected from the PF3D7₀900200 sequence From 243 to 2678-RIFIN、9-RIFIN,of two peptides The results of whole blood experiments of 9 peptides and P.falciparum showed that there was significant difference in the reaction between whole blood and normal blood serum of each polypeptide,Only wild type 1-RIFIN and mutant type 2-RIFIN,between wild type and mutant type Wild type 1-RIFIN,mutant 3 There was a significant difference RIFIN total blood IgG between mutant 9 and patient,In response to the corresponding rabbit resistance,Wild type 8-RIFIN and mutant 9-RIFIN have good immunogenicity and immunoreactivity;The molecular interaction LILRB1 inhibitory receptors shows that only 8 of the 9 peptides can RIFIN react with them,while the mutant 9-RIFIN,single point mutant 10 can not react with them.Conclusion:Through the whole genome sequencing analysis of P.falciparum,it is considered thatthe acquired immunity to P.falciparum obtained through continuous contact with pathogens was the main source of population difference.The difference of gene selection between P.falciparum from imported and Ghana native cases mainly comes from the difference of host immunity or transmission level.P.falciparum samples can be clustered by geographical source and showed little difference in the population structure,but theselection intensity of genes from different hosts was significantly different in those associated with erythrocyte invasion and immune evasion,and the selection presure of VS As families was scattered in different individual genes.On the basis of the results of rif single gene analysis and the three rif:PF3D7₁254800、PF3D7₁100400、PF3D7₀900200,selected from previous studies,our results are consistent with previous studies.The proteins corresponding to the first two genes are mainly immunoreactive with inhibitory receptors.It is also proved that mutations in the key sites of PF3D7₀900200 corresponding proteins lead to a decrease in immune response level.However,wild-type and mutant peptides can be found to have strong immunogenicity and immunoreactivity to experimental rabbits;The molecular interaction LILRB1 PF3D7₀900200 the corresponding protein and the inhibitory receptor reflects a certain degree of binding ability,and determines one of the specific sequences in the protein that can react with the LILRB1,that is,the 8-RIFIN sequence.Key mutation sites that can react with inhibitory receptors are further identified.