Prokaryotic Expression And Characterization Of Prophenoloxidase From The Asian Corn Borer, Ostrinia Furnacalis (Lepidoptera:Pyralidae)

Prophenoloxidase (PPO) is an important immune protein in insects, which is involved in insect humoral immunity and cellular immunity. In our study, the prokaryotic expression system was applied to express soluble recombinant PPO with activity on a large scale. The recombinant PPO was used to screen various phenoloxidase (PO) inhibitors so as to create novel insecticides targeted on the insect immune system. Using the PPO gene cloned from the 5th instar larvae of the Asian corn borer, Ostrinia furnacalis (Guenee), the prokaryotic expression vector, pET-28b-PPO, was constructed, and the recombinant PPO protein was expressed in Escherichia coli. The fusion protein was purified from the supernatant of the lysis of E. coli cells with Ni-NAT affinity chromatography column, and identified with Western blotting. Enzymatic characteristics of phenoloxidase (PO) activated from the recombinant PPO with cetylpyridinium chloride (CPC) and the effects of metal ions, such as Mg+, Cu2+ and Fe+ on the secondary structure of the recombinant PPO were also assayed and analyzed.The main results were showed as follows:(1)Construction of prokaryotic expression vector of full length cDNA encoding PPO. A pair of specific primer was designed according to the cDNA sequence of O.furnacalis PPO(Of-PPO), and PCR amplification was performed with cDNA as template, then the Of-PPO PCR products was recovered and connected with the pMD18-T vector. The recombinant vector was transformed in to bacterial strain TOP10. The positive clone bacterial colony was identified with PCR, and target band with the 2686bp showed that we had successfully obtained pMD18-T-PPO cloning vector. The constructed clone was sequenced, which demostrated that we had obtained complete open reading frame (ORF)of Of-PPO.The plasmid DNA was extracted, and then pET-28b vector and pMD18-T-PPO were digested with Nde I and Xho I, and the recombinant vector was reovered and transformed into cells of E. coli RIL BL21(DE3). The PCR identification result showed that we had successfully constructed the pET-28b-PPO expression vector.(2) Expression of recombinant pET-28b-PPO in vitro. E.coli BL21(DE3) which containing recombinant plasmids was induced under the condition of 16? temperature,0.4mmol/L, 0.2mmol/L, 0.1mmol/L,0.05mmol/L,0.025mmol/L IPTG, for 16 hours respectively. SDS-PAGE analysis showed that the 75 kDa recombinant proteins was mainly expressed in supernatant and some target proteins in inclusion body after 0.1mmol/L IPTG induction.(3) The recombinant PPO protein was expressed and purified. The suitable temperature for PO obtained through the activation of the recombinant PPO was 30?, and the suitable pH was 7.2. The kinetic parameters calculated for substrate oxidation were Vmax=140.8 U/mgmin and Km=0.92 mmol/L for L-DOPA. The content of ?-sheet in the recombinant PPO increased dramatically to 53.7%±4.6% when Fe2+ ions existed. However, the content of ?-helix decreased to 2.6%±1.2%(P<0.05) significantly. The content of ?-sheet in the recombinant PPO reduced clearly and the content of ?-helix increased slowly when Mg2+ ions existed. The content of ?-sheet in the recombinant PPO declined remarkably to 10.0%±1.6% when Cu2+ ions were present, but the content of ?-helix increased to 35.3%±6.9% significantly. The results suggest that the secondary structure of the recombinant PPO is affected notably by different metal ions.