Genetic Diversity Of Plasmodium Vivax Merozoite Surface Protein 1₄₂ And 7(MSP1₄₂ And MSP7) In Field Isolates From Great Mekong Subregion

Objective:To identify the polymorphism of the C-terminal 42kDa of the merozoite surface protein 1 and merozoite surface protein 7 gene of Plasmodium vivax in the Greater Mekong Sub-region which considered as candidate for blood-stage vaccine,and fully understand the characteristics of potential vaccine genes to provide information for the development of effective malaria vaccine.Methods:After obtaining the patient's consent,take the fingertip blood from P.vivax infection patients who were microscopy-positive diagnosis and make dry filter paper blood slices.The samples were extracted with gDNA according to the instructions of the dried blood spot reagent,and the positive samples of Plasmodium vivax were identified by nested PCR using the extracted gDNA as templates.The Msp3?fragment was amplified and identified by enzyme digestion to ensure that the strain was monoclonal.A total of 143 samples were obtained,including 45 in Anhui,China,50 in China-Myanmar borders,and 48 in Thailand.The Pvmsp1₄₂ gene fragment and eight Pvmsp7 genes were amplified by nested PCR and sequenced.Polymorphism analysis,neutrality test,and F_STT test were performed on the sequences in this study and corresponding gene sequences in other popular regions downloaded from Genbank using MEGA7.0 and DnaSP6.10.1;Network5.0 and PHYLOVIZ 2.0 software were used for haplotype analysis.MEGA7.0and online software iTOL are used for evolutionary tree analysis,and STRUCTURE v2.3.4 software is used for population genetic structure analysis.Results:1.Pvmsp1₄₂:The?values of Pvmsp1₄₂ gene in China-Myanmar border,Thailand and Cambodia were 0.01416,0.02405 and 0.02306,respectively.The results of neutral test indicated that the genes in all three regions were in balance selection and the central region of Pvmsp1₄₂2 gene was statistically significant?P<0.05?in Thailand and Cambodia;Higher genetic differentiations were observed in China-Myanmar border than roPetvghimeors npts7w.Co,2 r.e P gPviovmnmssps.7pT7E:h,e rPAev mmwosanpsg 7 n Hto,h o ea b nvedii go huPtsv gmgeseopng7ersI a,p atrhheie c s ciplgounlsiytfeimrcioanrngpt lhoyif s thmhisge h hoeafr p lttohhtaey n p f eoPsu vri mn sgtpeh7nreeAes,Pvmsp7F,Pvmsp7K,and Pvmsp7L,and they are relatively conserved in the C-terminal region;different genes have different degrees of neutral selection,Z-test results of synonymous mutation and non-synonymous mutation site analysis showed that Pvmsp7A positive selection hypothesis was 1.883,which was statistically significant?P<0.05?;Pvmsp7E,Pvmsp7H,and Pvmsp7I purification selection hypotheses were 2.855,2.264,and 3.631,which were statistically significant.?P<0.01,P<0.05,P<0.001?;phylogenetic tree analysis of eight genes in four regions including Colombia showed no obvious clustering,and population structure analysis revealed parasites in dour region with mixed haplotypes,and there is no obvious difference between four low polymorphisms,the genes with higher polymorphisms have obvious dominant strains in different countries;It can be seen from the haplotype network structure diagram from three regions that Pvmsp7C,Pvmsp7E,Pvmsp7H,and Pvmsp7I with high polymorphisms have a certain aggregation in Thailand,Pvmsp7A,Pvmsp7F,Pvmsp7K and Pvmsp7L have commom haplotypes in three regions.Conclusions:1.The Pvmsp1₄₂ showed genetic diversity and experienced the balanced selection.The central region with obvious balanced selection may be related to human immunity,which can be used as a target for vaccine development.It also provides genetic information for Pvmsp1₄₂ gene as an effective candidate for blood-stage vaccine of vivax malaria.2.Due to the low polymorphism of Pvmsp7A and low differentiation in different regions,it can be regarded as a candidate vaccine target;the C-terminal conserved regions of other genes can also be formulated according to the different epidemic strains in different regions.