The study on venom proteins of Lapemis hardwickii by cDNA phage display

Abstract

In this study, a cDNA T7 phage display library was constructed from sea snake Lapemis hardwickii venom gland mRNA to analyze the components in venom and find new toxins. All the venom gland cDNA-encoding proteins, including housekeeping proteins and venom proteins were expressed on the surface of bacteriophage T7. This library was then panned with rabbit anti-sea snake venom IgG. Phage particles displaying venom-components with interaction with the antibodies were enriched. Thus, phage-carrying venom proteins, such as short chain neurotoxin, long chain neurotoxin, PLA2-like toxin, and c-type lectin-like protein were found, some of them were never reported previously. Four different short chain neurotoxins (SNT-1, 2, 3, 4) were cloned and expressed in Escherichia coli. The LD50 and analgesic activities of their purified forms were determined. Their structure–function relationship were studied with the aid of a toxin–nAChR complex model constructed by ourselves. Among them, SNT-4 was a new neurotoxin identified in this study and showed potential as pain killer. These results prove that cDNA phage display technique has great advantage than traditional cDNA library method because of the linkage between phenotype and genotype of phage, which provided an effective means to study unknown proteins with target functions.