Cloning, sequencing and prokaryotic expression of cDNAs for antifreeze protein family from Beetle

Abstract

Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR. Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank. The recombinant pGEX-4T-1-tmafp-XJ430 was introduced into E. coli BL21 to induce a GST fusion protein by IPTG. SDS-PAGE analysis for the fusion protein shows a band of 38 kDa. pCDNA3- tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA. The titer of the antibody was 1:2000. Western blotting analysis shows that the antiserum was specifically against the antifreeze protein. Our results laid the foundation for further studies on the properties and functions of insect antifreeze proteins.

Keywords

Tenebrio molitor / antifreeze proteins / cDNA fragment / sequence analysis / prokaryotic expression