Construction Of Ph-Hv1a Expression Vector And Pesticidal Activity Of Ph-Hv1a Fusion Protein

Spiders are important natural enemies for insects because their toxins most of which are innocuous to human make insects paralytic and dead.ω-ACTX-Hv1a, an inhibitor of non-vertebrate Ca2+ voltage-gated calcium channel, can be isolated from the Australian funnel-web spiders.ω-ACTX-Hv1a and AcNPV polyhedrin genes were ligased and inserted into pET-30a to construct fusion protein expression vector pET-30a-ph-Hv1a. The fusion protein was prepared and purified to feed larvas of Prodenia litura and Phanerotoma planifrons to evaluate its insecticidal effects. Preparation ofω-ACTX-Hv1a was divided into two steps: synthesis of the central part of the DNA fragment as template and amplification to obtain the 3'and 5'terminal cleavage sites. Polyhedrin gene was amplified from vector PAcUW21. Two DNA fragments were ligased into PCR Blunt vector respectively and sequencing results showed that sequences of the two vectors PCR Blunt-Hv1a and PCR Blunt-Ph were completely identical to the sequences in the report. For construction of the protein expression vector pET-30a-ph-Hv1a: first, ph fragment from PCRBlunt-ph Nde I / EcoR I endonuclease digestions was ligased with pET-30a, and then Hv1a from PCR Blunt-Hv1a EcoR I / Xho I endonuclease digestions was inserted into pET-30a-ph. The vector PET-30a-ph-Hv1a was transducted into BL21 (DE3) to express the recombinant protein. 34.5kD band was observed using the SDS-PAGE analysis after 1mmol/L IPTG induction for 4 hours at 37℃. The induction temperatures (28,32,35,37,39℃), the induction times(1,2,3,4,5h) and IPTG concentrations (0.2,0.4,0.6,0.8,1.0,1.2mmol/L) have been tested to figure out the optimum induction conditions of the target fusion protein. It was found that 1 mmol/L IPTG induction for 4 hours at 37℃was optimal. Purified fusion protein was obtained through ultrasonic fragmentation and purification using inclusion body washing buffer. Larvas of P. litura and P. planifrons were fed with leaves sucked in purified fusion protein solution to observe the pesticidal activity. Group 1 and 2 were larvas of P. litura from purchasing and field.Group 3 and 4 were larvas of P. planifrons from non-fruit-bearing field and fruit-bearing field. Each group was composed of 3 parts in terms of different concentrations of purified fusion protein 1:10, 1:20 and 1:40. Mortalities of Group 4 were 80%, 78.33% and 71.67%; Mortalities of Group 3 were 35%, 33.33% and 26.67%; Mortalities of Group 2 were 25%, 10% and 15%; Mortalities of Group 1 were 11.66%,6.67% and 5%.