title: [Cloning and expression of var2csa DBL domains from Plasmodium falciparum hainan isolate and functional analysis of the recombinant protein].
abstract: OBJECTIVE
  To clone and express three VAR2CSA duffy antigen-binding ligand (DBL) domains (DBL4/ 5/6) encoded by var2csa gene of a Hainan isolate of Plasmodium falciparum, and study the difference of chondroitin sulfate A (CSA)-binding activity among them.
  
  
  METHODS
  Three DBL domains was amplified by PCR and cloned into the vector pMD18-T. The recombinant plasmids were identified by enzyme digestion and sequencing, and then subcloned into the prokaryotic expression vector pET-22b. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was purified with His GraciTrap kit and identified by SDS-PAGE and Western blotting. CSA-binding activity of the three recombinant DBL domains was assayed by ELISA.
  
  
  RESULTS
  The target genes were amplified with the length of 996 bp, 859 bp and 894 bp. The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing. The recombinant proteins (DBL4/5/6) were purified, the relative molecular mass of DBLfA, DBL5 and DBL6 was Mr 439 800, Mr 34,500 and Mr 36,000, respectively. The purified protein has been confirmed with immunogenicity by Western blotting. The results of adhesion experiment indicated that A405 values of DBL5 domain with different concentration were significantly higher than that of DBLA and DBL6.
  
  
  CONCLUSIONS
  The three recombinant proteins (DBLA/5/6) of VAR2CSA DBL domains were expressed, and DBL5 domain has high binding affinity with CSA.
authors.affiliation: Key Laboratory of Zoonosis, Ministry of Education, Jilin University, Changehun 130062, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Kang
authors.forename: Zhi-guang
authors.initials: ZG
authors.lastname: Chang
authors.forename: Hui-jun
authors.initials: HJ
authors.lastname: Lu
authors.forename: Ning
authors.initials: N
authors.lastname: Jiang
authors.forename: Ji-gang
authors.initials: JG
authors.lastname: Yin
authors.forename: Qi-jun
authors.initials: QJ
authors.lastname: Chen
mesh_terms: D000953:Antigens, Protozoan / Q000235:genetics* / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000378:metabolism; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D010963:Plasmodium falciparum / Q000145:classification* / Q000235:genetics* / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000953:Antigens, Protozoan; D016054:DNA, Protozoan; C066001:Duffy antigen binding protein, Plasmodium; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D002809:Chondroitin Sulfates