Functional Verification Of The Prokaryotic Expression Product Of The Enhancin Gene From TnGV And Eukaryotic Expression

ENHANCIN is a metalloproteinase, mainly encoded by baculovirus, and the molecular is about89-110KDa. It accelerates the invasion of the virus particles by destroying the complete structure of the insects peritrophic membrane, so as to improve the biological pesticides to pests infection efficiency.For the purpose of finding and ensuring the target protein in the insect midgut peritrophic membrane of the efficiency protein, and further reveal the mechanism of protein Enhancin synergistic, these experiments were conducted as followed:clone and obtain the En gene from TnGV, and express it in prokaryotic. We use the technology of SDS-PAGE、Western blot and Ni+affinity chromatography to verify the expression of the target protein. The expressed product and HaNPV was used to conduct the experimentof preliminary bioassary on Helicoverpa armigera. Peritrophic membrane was dissected from the fifth-instar H.ar larvae, which was stored for the in vitro enzymatic by the prokaryotic expression product of Tn-En. The gene was cloned into the baculovirus eukaryotic expression vector Bac-to-Bac; recombinant expression vetors Bacmid-pFastBac-En and Bacmid-pFastBac-En-EGFP then transfected into the insect cells Sf9, fusion proteins were detected by SDS-PAGE and Western blot.The Tn-En protein can express in the prokaryotic system. Bioassay experiments shows that, the fusion proteins exprssed by Tn-En gene can increase larval mortality18.89%,22.22%and1.67%respectively, which in the treatment group3VE、4VE and5VE. In vitro degradation experiment, we found a high molecular weight protein of the peritrophic membrane can be degraded by the Tn-En protein. With the fluorescence microscope, we can observe the green fluorescent from the cells which were transfected by Bacmid-pFastBac-En-EGFP. The fusion protein was detected by SDS-PAGE and Western blot. From the result, it can testify that the protein was expressed and existed in the cells. With the same methods, the recombinant expression vetors Bacmid-pFastBac-En can expressed in the insect cells successfully.