Cloning, Expression And Identification Of Duffy Binding Protein Region Ii Of Plasmodium Vivax Isolated From Central China

Purpose：(1) To clone Duffy Binding Protein region II of Plasmodium vivax fromanhui, China;(2) To express and identificate of recombinant PvDBPII protein.Method：(1) The primers were designed according to PvDBPII gene of Plasmodiumvivax Sa1-1strain (GI:156081788). And then PvDBPII gene was amplificated. fromPlasmodium vivax genomic DNA extracted from vivax-malaria patient blood samplefrom Anhui by PCR The PCR product incubated with enzymes Nco I and Hind III wasconnected with pET28a(+) prokaryotic expression vector which incubated withenzymes Xho I and Nhe I by T4DNA Ligase to construct pET28a(+)-PvDBPIIrecombinant plasmid. The recombinant plasmid was transformed into Ecoli BL21(DE3)and the expression conditions of the recombinant PvDBPII protein fused with His tagwere optimized. The expressed product purify the protein by His-NTA affinitychromatography, and identified by SDS-PAGE and Western Blotting.Results：The PCR product of PvDBPII gene was about1.1kb, meet the expectation ofprediction in size. The recombinant pET28a(+)-PvDBPII plasmid was verified bysequencing that the insertion was correct both in direction and in frame, but with4non-synonymous mutations（R319G、D384G、R390H和L424I）compared to referenceP. vivax strain Sal-I. SDS-PAGE and Western Blotting analysis showed that therecombinant PvDBPII protein was about44kDa, and can be recognized by pooledserum from vivax malaria patients. After4h induction with0.5mmol IPTG at37℃, theexpression of the44kd insoluble fusion protein (inclusion body) may reach a maximumamount of expression．Conclusion： The PvDBPII gene of Anhui isolate was successfully cloned, andrecombinant PvDBPII was expressed in E. coli，which provides the basis for its furtherstudy and application.