Objective: To clone a Plasmodium vivax Duffy binding protein critical functional region II (PvDBPII) gene of central China isolate, and to express and identify the recombinant PvDBPII protein in vitro.
Methods: PCR was performed to amplify PvDBPII from P. vivax DNA of a central China isolate and the PCR product was inserted into pET28a(+) vector. pET28a-PvDBPII recombinant plasmid was constructed and transformed into E. coli host BL21 (DE3+). IPTG was used to induce the recombinant PvDBPII protein fused with His tag, and the protein was purified by His-NTA affinity chromatography. The recombinant protein was identified by SDS-PAGE and Western blot.
Results: The PCR product of PvDBPII gene was about 1,1 kb, meeting the expectation of predicted fragment size. The recombinant pET28a-PvDBPII plasmid was verified by sequencing that the insertion was correct both in direction and in frame, but with 4 non-synonymous mutations compared to reference P. vivax strain Sal-I. SDS-PAGE, and Western blot analysis showed that the recombinant PvDBPII protein was about 44 kDa, and could be recognized by pooled sera from vivax malaria patients.
Conclusion: The PvDBPII gene of central China isolate is successfully cloned, and recombinant PvDBPII is expressed, thereby providing opportunity for further study on PvDBPII.