Bacterial Expression And Activity Analysis On Neurotoxin Ar1b From Spider Atrax Robustus And Construction Of Recombinant HearNPV With Ar1b

Nucleopolyhedrovirus (NPV) is belonged to the family Baculoviridae that infect invertebrates, particularly insects of the Lepidoptera, and is a natural control agent of insect pests in agriculture and forestry. Helicoverpa armigera nucleocapsid nucleopolyhedrovirus (HearNPV) is belonged to the family Baculoviridae, Nucleopolyhedrovirus and the ability of baculovirus to infect insects has many applications, is essential to use widely as bio-insecticides to control heliothine insects in cotton and vegetable crops around the world. Nevertheless, Baculoviruses are pathogens that only infect certain specific arthropods and a critical drawback of these baculoviruses, delayed effectiveness due to a lag period from initial infection to ultimate appearance of larval mortality, evidently impedes their applications. To overcome this impediment, we have been genetically modified to HearNPV with the insect specific toxins for the enhancement of insecticidal efficacy.We have selected the Australian funnel-web spider neurotoxinsω-atiacotoxin-Ar1b (ω-ACTX-Ar1b) which are candidates for development as biopesticides.ω-ACTX-Ar1b is toxic to a wide range of insect, including Leptidoptera, Diptera, and Orthoptera, but it has no affect on vertebrate.ω-ACTX-Ar1b was synthesized and were cloned into pET-32a(+)to allow expression of Ar1b as a fusion protein with thioredxin and the His-tag in E. coli BL21(DE3). The expressed 28.4 kDa fusion protein was corresponding to the predicted size. The fusion protein rTrx-Ar1b was expressed in soluble form and was purified effectively by HisTrap HP,the purity increased by rpHPLC and up to 91% purity was achieved finally. The LD50 for Mythimna separate was 111.66μg g-1 and for Tenebrio molitor was11.04μg g-1.We have constructed a donor plasmid HtEC contained a mini-F replicon, the chloromycetin resitatnee gene, the polyhedrin promoter. The neurotoxins gene (Ar1b) was inserted into the donor plasmid HtEC to construct the recombinant HtEC-Ar1b. The recombinant donor plasmid HtEC-Ar1b have transformed into the recombinant HaBacmid-HZ8 (HearNPV genome was cloned and maintained as 132 kb bacterial artificial chromosome (Bacmid) in E. coli) to obtain the recombinant HearNPV Bacmid contained the gene Ar1b. The recombinant HearNPV Bacmid is about to transfect HzAm1 cells to obtain recombinant virus.