Cloning And Expression Of6-Tox Gene From Antheraea Pernyi In E.Coli

Lepidopteron have strong innate immune ability, which could resist infection of foreign microbe as higher animals. In humoral immunity, the immune system includes immune factors, such as lysozyme, antibacterial peptide and antibiotic protein, were produced to against the microorganism’s invading. Antibacterial proteins have the characteristics of broad antibacterial spectrum with small molecular weight, which have been identified in plants and animals and shown to play a fundamental role in host defence against bacteria and fungi.6-Tox is one of the immune-related protein, which was only found in Lepidopteran species, such as Spodoptera frugiperda, Helicoverpa armigera and Bombyx mori. In this study, Real-time PCR were performed with cDNA from infected fatbody which infected with Bacillus subtilis, Escherichia coli Top10, S. cerevisiae W303-1A and Antheraea pernyi nuclear polyhedrosis virus and the expression level in different tissuses was evaluated. The6-Tox gene of Antheraea pernyi was amplified by PCR with specific primers designed according to6-Tox gene sequences based on cDNA from pupa fat body induced by E. coli. Expreession vector of pGEX-6P-1-6-Tox was constructed and optimal conditions were set up after concentration of IPTG, temperature and time were selected, and recombinat protein was purified by using of affinity chromatography.In this study, we successfully constructed the expression vector pGEX-6P-1-6-Tox and obtained GST-6-Tox fusion protein by affinity chromatography. This would lay a good foundation for the study and application of6-Tox protein.