Cloning And Prokaryotic Expression Of Carboxylestesrase From Helicoverpa Armigera

Helicoverpa armigera (Hiibner), (Lepidoptera Noctuidae), is one of the most serious agricultural pests in the world. It hosts many plant.Cotton bollworm control mainly rely on chemical pesticides, inevitably lead to a stronger resistance on the pesticide.So we must cost more and control more difficultly. Carboxylesterase (CarE) play an important role in the detoxification enzymes in insects, providing key mechanism of resistance to insecticide.The study used molecular cloning technique.After PCR amplification, we cloned 4 full-length carboxylesterase gene sequence in cotton bollworm. Through double restriction digestion, the obtained gene was ligated with pET-32a(+) expression vector. They were transformed into E. coli BL21(DE3) strain to overexpress. The recombination expression low-temperature-induced by IPTGAt last, SDS-PAGE analysis showed that the recombinant protein was correctly expressed in E. coli.Then it purified by immobilized Ni2+-NTA affinity chromatograph column.The result demonstrated that the WHOOld zymoprotein was more pure. After optimizing prokaryotic expression conditions, soluble esterase expression level improve.Through esterase directed evolution in vitro,improve the activity of OPs detoxification and construct detoxifying enzyme engineering bacteria.The main results were as below:1. Through TA cloning,we get 4 cotton bollworm full-length gene,they were WH001a, WH001d, WH001h and WH001g.The open reading frame were 1665 bp,1662 bp, 1665 bp,2241 bp respectively,and encoded 555 amino acid,555 amino acid,554 amino acid,747 amino acid respectively. In addition, WH001b and WHOOlj were 1762 bp and 1804 bp in length.With a predicted protein molecular mass, their molecular mass were 62.78 kD,62.59 kD,61.7 kD,77.18 kD, and their isoelectric point were 5.56,5.27,5.80, 4.34,respectively.They potentially comprises 17,20,31,28 phosphorylation sites, respectively.2. Signal peptide prediction analysis revealed WHOOla, WHOOld,WH001h, WHOOlg probabely contain 16,16,16,17 amino acids signal peptide, so they possibly were secretory protein.Mutiple sequence alignment analysis with Lucillia cuprina dorsalis esterase E3 displayed all of them have the structure characteristics of insect esterases.Catalytically active members of the carboxlyesteras family have catalytic triad of Ser- His-Glu and suggest that it have a catalytic activity of hydrolytic enzymes. The other features include the the conserved pentapeptide Gly-x-Ser-x-Gly.3. We cloned WH001d genes again with the restriction enzyme site primer after removed the signal peptide to construct the recombined plasmid with pET32a(+) vector. The correct recombinant plasmid WH001d-pET32a(+) was transferred into E. coli BL21.The correct recombinant protein was induced by O.lmM IPTG and incubated at 18 ℃ for 48 h. SDS-PAGE analysis showed that the gene was correctly expressed in E.coli. The recombinant protein was purified by immobilized Ni2+ affinity chromatograph column with His-tag. The purified protein was about 70 kD 。 The purified enzyme concentration was 0.284 mg/mL, by Bradform method.