[Cloning and prokaryotic expression of Anopheles sinensis defensin gene and preliminary bioactivity evaluation of the recombinant product].
OBJECTIVE
To clone the defensin gene of Anopheles sinensis for its prokaryotic expression and preliminary bioactivity evaluation of the recombinant protein.


METHODS
Two pairs of primer were designed and synthesized based on the published defensin sequences, and PCR amplification was performed using the cDNA of An.sinensis as the template. The PCR products were cloned and sequenced. A truncated defensin fragment was cloned into pET-32(a) plasmid and expressed with the induction by IPTG. The expressed product was purified and its bioactivity evaluated with agarose diffusion assay.


RESULTS
The PCR product 270 bp in size was identified as the coding sequence for defensin after sequence analysis, which had 85%; homology with the defensin sequence from An.gambiae. The truncated defensin (162 bp) cloned was subcloned into the expression plasmid pET-32(a) and the molecular weight of the target fusion protein was 26 000 after IPTG induction. Preliminary experiment, however, failed to demonstrate bacteriostatic effect of the purified recombinant protein.


CONCLUSIONS
The coding sequence for defensin gene of An.sinensis has been successfully cloned and the truncated fraction can be highly expressed as a soluble fusion protein, which, however, does not possess bacteriostatic activity.
Yan
Xiao-Yu
XY
Department of Porositology, Southern Medical University, Guangzhou 510515, China.
Chen
Xiao-Guang
XG
Peng
Hong-Juan
HJ
Zheng
Xue-Li
XL
D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles / Q000235:genetics*; D000890:Anti-Infective Agents; D001483:Base Sequence; D003001:Cloning, Molecular; D023082:Defensins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017421:Sequence Analysis
D000890:Anti-Infective Agents; D023082:Defensins; D011993:Recombinant Fusion Proteins