Expression Of Diapause Hormone Gene Of Oriental Tobacco Budworm (Helicoverpa Assulta Guenée) In Escherichia Coli

With primers designed according to sequence(AY052417) published on Genbank and diapause pupae of oriental tobaceo budworm(Helicoverpa assulta Guenee). a double-strand cDNA fragment of DH(diapause hormone) gene was obtained from KT-PCR amplification.The fragment was digested by two restriction enzyme Nde 1 / EcoR 1 and then was joined with pET-30a(+) plasmid which was digested by Nde 1 / EcoR 1 too. Then the outcome was transformed into Escherichia coli BL21. By cheeking the transformed bacterial colonies, we had filtrated the masculine clone successfully. The sequencing result showed that the inserting fragment contained intact coding sequences of DH. PBAN (pheromone biosynthesis activating neuropeptide) and three SGNPs(suboesophageal ganglion neuropeptide) and the leading frame of it was correct.After induced by IPTG and cultured for some lime, the pDH/BL21 bacterial strain was cheeked by SDS-PAGE(SDS polyacrylamide gel electrophoresis). We found that the recombinant vector expressed a new protein of which the molecular weight was about 21 kDa. Based on the sequencing result of the recombinant plasmid ,we confer that this protein consists of 182 amimo acids and is integrated with His .Tag that is a purifing tag in its N-terminal, which provide a convenient way for seperation and purification of this protein.We integrated DH gene into procaryotic expression vector for the first time,which establish foundation for obtaining DH recombinant protein to research the structure and function of DH.