Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells.
The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.
Ji
Sheng Jian
SJ
The National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, China.
Liu
Feng
F
Li
Er Qiu
EQ
Zhu
Yu Xian
YX
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D002460:Cell Line; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007313:Insecta; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012604:Scorpion Venoms / Q000494:pharmacology* / Q000633:toxicity*; D020539:Sequence Analysis, Protein; D012964:Sodium / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
D010455:Peptides; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C065634:AaIT neurotoxin, Androctonus australis; D012964:Sodium