[Cloning, prokaryotic expression and antibacterial assay of Tenecin gene encoding an antibacterial peptide from Tenebrio molitor].
OBJECTIVE
To clone tenecin gene, an antibacterial peptide gene, from Tenebrio molitor for its prokaryotic expression and explore the molecular mechanism for regulating the expression of antibacterial peptide in Tenebrio molitor larvae.


METHODS
The antibacterial peptide was induced from the larvae of Tenebrio molitor by intraperitoneal injection of Escherichia coli DH-5α (1×10(8)/ml). RT-PCR was performed 72 h after the injection to clone Tenecin gene followed by sequencing and bioinformatic analysis. The recombinant expression vector pET-28a(+)-Tenecin was constructed and transformed into E. coli BL21(DE3) cells and the expression of tenecin protein was observed after IPTG induction.


RESULTS
Tenecin expression was detected in transformed E.coli using SDS-PAGE after 1 mmol/L IPTG induction.  Tenecin gene, which was about 255 bp in length, encoded Tenecin protein with a relative molecular mass of 9 kD. Incubation of E.coli with 80, 60, 40, and 20 µg/ml tenecin for 18 h resulted in a diameter of the inhibition zone of 25.1∓0.03, 20.7∓0.06, 17.2∓0.11 and 9.3∓0.04 mm, respectively.


CONCLUSIONS
Tenecin protein possesses strong antibacterial activity against E. coli DH-5α, which warrants further study of this protein for its potential as an antibacterial agent in clinical application.
School of Basic Medicine, Wannan Medical College, Wuhu, China. liuyingwuhu @wnmc.edu.cn
Ying
Y
Liu
Yu-xin
YX
Jiang
Chao-pin
CP
Li
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D019476:Insect Proteins / Q000096:biosynthesis / Q000235:genetics*; D007814:Larva / Q000737:chemistry; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D013712:Tenebrio / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; C091169:tenecin 1 protein, Tenebrio molitor