The Study On NIF4 Protein Expression And Subcellular Localization And The Correlation Between NIF4 And Artemisinin-based Combination Therapy Resistance

Objective:The artemisinin-based combination therapy?DHA-PPQ?as first-line antimalarial drug was first used in Cambodia and other neighboring countries.The resistant isolates of Plasmodium falciparum have been found in Southeast Asia in recent years and have a significant tendency to spread.Over the past few years,protein kinases and phosphatase have become hot targets for drug discovery.In the gene bank of Plasmodium falciparum,Nif4 is adjacent to the position of ATG18 which has recently been considered as a potential art target.ATG18 encodes a phosphatidylinositol-3-phosphate binding protein necessary for autophagy.NIF4 FCP1 like NIFs can catalyze the dephosphorylation of?RNA polymerase II?CTD.The role of RNA polymerase II in transcriptional regulation has been a hot research in recent years,but little research has been done on Plasmodium falciparum.The purpose of this study was to explore whether NIF4 could be a new target for the study of ACTs resistance and to understand the process of NIF4 action.The purpose of this study was to help monitor,slow down and prevent the spread of drug resistance.Methods:Bioinformatics was used to analyze PF3D7₁012700 NIF4 protein domain.We do Phylogenetic analysis of the important functional structure CPDc and prediction of transmembrane region,signal peptide,also protein 3D structure.The recombinant nif4protein was obtained by construction of prokaryotic expression vector.The NIF4 immune serum was obtained by immunizing Japanese rabbits.ELISA was used to detect antibody titer.We Divided 3D7 isolate into five periods by percoll separation method.Subcellular localization and expression analysis of NIF4 were by Western blot and IFA.The drug sensitivity values of wild isolates were measured by in vitro drug-sensitive experiments?SYBR green?for DHA and PPQ.IC₅₀0 was calculated by Prism software.The specific fragment of NIF4 gene was amplified by PCR,sequenced and compared by Mega software.The results of NIF4 SNP were obtained.Statistical significance test of data difference by t-test method The data significant difference statistically was tested by T-test method.P<0.05 was determined to have a significant difference statistically.The Pearson correlation coefficients was used to determine the correlation of data.The criteria:The correlation coefficient is equal to 0.8-1.0,extremely strong correlation;0.6-0.8 strong correlation;0.4-0.6 medium degree correlation;0.2-0.4 weak correlation;0.0-0.2 extremely weak correlation or no correlation.Results:1.Bioinformatics analysis predicted that the domains of PF3D7₁012700including DXDX?T/V?and CPDc relatively conservatived.Dephosphorylation of RNA polymerase II CTD catalyzed by CPDc.Without transmembrane region and signal peptide,NIF4 may be expressed in cytoplasm.2.We successfully prepared NIF4 poly-antibody serum which is 10 mg/ml and with 95%purity.Serum titer is over 1:50k.3D7 strain were divided into five stages by percoll method.The results of western blot and IFA showed that NIF4 was expressed in all stages of Plasmodium falciparum of erythrocytic stage and expression of NIF4 is the most abundant in trophozoites.Also we found that NIF4 was expressed in cytoplasm.3.In vitro drug sensitivity tests were performed on 18 clinical isolates of Sino-Myanmar border.The results showed that PPQ IC₅₀=25.20±26.42 nM was much larger than the PPQ threshold value and the percentage of resistant isolates is9/18.DHA IC₅₀=9.37±13.23 nM was much larger than the DHA threshold value and the percentage of resistant isolates is 5/18.4.The NIF4 specific fragments of 18 clinical isolates were amplified and sequenced successfully.The amino acids at the 1133 and 1157sites were mutated at the same time in 5 samples and the mutation rate was 27.8%.The mutation patterns were Y1133N and V1157L.There was a moderate correlation between DHA-PPQ resistance and NIF4 mutation.The Pearson correlation coefficients showed moderate correlation between DHA-PPQ resistance and NIF4 mutation.Conclusion:1.The expression and subcellular localization of NIF4 protein were studied in all stages of P.falciparum.NIF4 was observed in all erythrocytic stages of P.falciparum and mainly expressed in trophozoites and early merozoites.It is expressed in P.falciparum cytoplasm.We preliminarily understand the role of protein phosphatase NIF4 in transcriptional regulation.2.The Pearson correlation coefficients showed moderate correlation between DHA-PPQ resistance and NIF4 mutation,that could provid the theoretical principle for NIF4 to become a new molecular marker for ACTs resistance.