Analysis Of Genetic Polymorphism In Duffy-binding Protein Among Chinese Plasmodium Vivax Isolates

OBJECTIVETo investigate the genetic polymorphism of PvDBP RⅡin chinese yunnan province.METHODSStudy sites of Yunnan province is a malaria year-round transmission of P.vivax and P.falciparum.P.vivax genomic DNA was extracted from dried filter paper blood spots which obtained by using QIAamp DNA mini kit.The full-length open reading frame of the PvDBP RⅡfrom P.vivax genomic DNA was amplified with a polymerase chain reaction.The PCR products were directly sequenced.Computer-based algorithms were used to analyze neucleotide and amino acid sequences and the sequence alignments were done with BioEdit software.Intrapopulation and interpopulation diversity indexes were analyzed by DnaSP version 4.5 and MEGA 4.1 software.RESULTS1.Nineteen samples of PvDBP RⅡfrom P vivax genomic DNA was amplified successfully with the full-length 600bp(853-1452bp),which coded 200 amino acid(codon285-484).2.Compare to DBP Sal-Ⅰsequence,there were 13 variant sites in isolates from Yunnan province,with the frequency of 2.17%(13/600),accordingly,12 variant amino acids were found,with the frequency of 6.00%(12/200).Among all the variant sites found,one was synonymous,i.e.A1134G.There were 9 kinds of haplotype found,also 9 amio acid types,A1151G-G1169A-T1270A(D384G-R390H-L424I)was the most predominant one(26.3%).No insertion or deletion was found in both levels of base pair and amino acid.3.Compare with foreign isolates,the variant amino acids sites of DBP found in isolates from Chinese Yunnan were among those foreign ones,no geographic specificy was found.4.Data of nucleotide diversity showed thatπ=0.0091(SE:0.0028);dn/ds=6.168＞1(P＜0.05);Tajima's D:1.21360,P＞0.10.Fu and Li's D~* test statistic:1.48091 (P＜0.05),Fu and Li's F~* test statistic:1.62640(P＜0.05).DISCUSSIONCompare the genetic polymorphism of PvDBP RⅡamong China,South America, Africa and region of Middle East,variant sites of Chinese isolates were relatively limited,suggesting relative conserve founction of PvDBP antigen in China.From our results,we found 3 new sites in Yunnan isolates compare to Zhejiang and Hubei ones,although the low frequency,there was some suggestion on the studies of genetic polymorphism of PvDBP RⅡamong our country.Also,we found 2 sites occurred frequently in association,i.e.N417K and W437R,which implied that vaccine developed targeting these sites might be available to malaria endemic regions in our country.Following the statistic tests departure from neutuality showed evidence that the observed polymorphism might be trending natural selection,dn/ds predicting that positive selection may be occurring in regionⅡof PvDBP favouring the fixtion of amino acid replacement in isolates from Cinese Yunnan.Being the limitation of samples and analysis method,this is only a glimpse at the overall population in our country.CONCLUSION1.Compare the results of South America,Africa and region of Middle East,variant sites of Chinese isolates were relatively limited,there were some similar with isolates in South Korea located in the same continent of Asia.In China,there were some difference in variant sites between Yunnan and Zhejiang,Hubei province. 2.Our analysis showed evidence that the observed polymorphism might be trending natural selection,dn/ds predicting that positive selection may be occurring in regionⅡof PvDBP favouring the fixtion of amino acid replacement in isolates from Cinese Yunnan.