Constructed The System Of A High Level Expression Of Recombinant Baculovirus And Synthesized And Expressed The Gene Of Spider Toxin And Evaluated Its Effect Of Killing Insect

New methods of insect control are urgently required due to the evolution of insectresistance to classical chemical pesticides, growing appreciation of the environmental damagecaused by many agrochemicals, and increased public concern about the human health risksassociated with prolonged insecticide exposure. The transgenic strategy has the potentialproblem of inducing accelerated resistance because insects are continuously exposed to theconstitutively expressed toxin. A potentially more selective method is to use insect-specificbaculoviruses as vectors to deliver insecticidal neurotoxins to a restricted number of targetinsects without harming non-target animals.The toxin(ω-ACTX-Hv2a) are the most potent blocker of insect voltage-gated calciumchannels reported to date, but they are virtually inactive on vertebrate ion channels, makingthem ideal biopesticide candidates. Whereas the toxin was inactive in all vertebrates tested inthis study, we have found thatω-ACTX-Hv2a is toxic to a wide range of insect orders,including Leptidoptera, Diptera, and Orthoptera.ω-ACTX-Hv2a has a neural site of actionand it is orally inactive. Thus, expression of the toxin in a transgenic plant is unlikely to be aviable approach to pest control. Incorporation of toxin genes into a baculovirus vector is likelyto be a more feasible method of deliveringω-atracotoxins to target insects.However, in general insect baeulovirus vector system, the expression of extrinsic gene isinfluenced by the delitescence and replication of virus, which results in the instability ofexpression, and which affect the pesticidal effect severely. In this study, we construct a kind ofrecombinant insect baculovirus model. Using the baculovirus as carder to the cells of insects,and carrying operator recognized to insect cells, the expression of extrinsic gene is no morecontrolled by baculovirus reproducing, but controlled by insect cells, which recognize theoperator in the vector. Theoretically, after the recombinant baculovirus infecting the insectcells, it can enter the cells infected, and can deliver the specific expression cassette of insectto the nucleus of insect cells, because there isn't insect baculovirus specific acceptor in insectcells. When expression element enters the nucleus, the operator is activated by regulation ofinsect specific factors, and starts the transcription, and drives the extrinsic gene expression, but the expression has not direct relationship with the replication of insect baculovirus. Hence,the expression model we constructed may find out a new method, which may settle thequestions that insecticidal speed is slow and host range is cabined of insect baculovirus as apesticide.AcNPV is a kind of Nuclear Polyhedrosis Virus, and pAcE1 contains insect specificelements. In this study, we used pVL1392 and pAcE1 as vectors, and successfully constructedrecombinant virus expression models, rbpAcE1-Lu and rbpVL-Lu, which took Luciferase asreport gene. Expression result in insect sf9cell and SL-2 cell indicates that, after infecting sf9cell and SL-2 cell 24 hours, the expression quantity of extrinsic gene in recombinant virusrbpAcE1-Lu is quite high, whereas, the expression quantity of extrinsic gene in recombinantvirus rbpVL-Lu is very low.On the base of above study, (1) adopting subsection synthesis, we designed andsynthesized artificially an insect toxicity geneω-ACTX-Hv2a that is excretory to the outsideof cell, and cloned it into PCR-Blunt vector. Approved by DNA sequence analysis, comparingwith designed sequence, synthesized sequence lacks five bases. Renovating it with pointmutant, ultimately we made it consistent with designed sequence. (2) using pAcE1 as transfervector, we cloned insect-specific toxin geneω-ACTX-Hv2a into it and constructed transfervector pAcE1-Hv2a, and then constructed recombinant baculovirus rbpAcE1-Hv2a. (3) Wemade rbpAcE1-Hv2a and wild insect baculovirus (AcNPV) infect 2-age pine caterpillar(Dendrσlimus spp.) grub by injecting. As a result, rbpAcE1-Hv2a can kill the host insects, andthe symptom of dead insect is retractile, hardened and present typical poisoning symptom.Unlike it, the symptom of dead insect that infected wildtype baculovirus presents aliquefaction tissue. Above results suggested that, the insect specific expression element inrecombinant virus had worked, and the synthetical toxin geneω-ACTX-Hv2a expressedcorrectly in the body of test insects, and the toxin works important in killing the insect. ItsLT50 was shorter 50h than wildtype baculovirus(AcNPV). This research provided importanttheory foundation and technique support, for studying novel and highly effective biologicalvirus pesticide to insects.Conclusion1) Using insect baculovirus as expression vector, we constructed transfer model vectorpAcE1-Lu whose report gene is luciferase gene. At the same time, we constructed transfervector pVL1392-Lu, and it also contains the luciferase gene. By Digestion and checkup, thesequence hadn't difference from designed sequence. 2) Having plasmid DNA pAcE1-Lu and pVL1392-Lu with linearity AcNPV DNArespectively co-transfect insect SF9 cell, we constructed recombinant virus rbpAcE1-Lu andrbpVL1392-Lu, and mensurated expression activity of the two recombinant viruses. Resultsindicated that, after infecting SF9cell 24 hours, the luciferase gene in rbpAcE1-Lu had a higheffective expression, while the luciferase gene in rbpVL1392-Lu had a very low expressionwhich was one in ten of rbpAcE 1-Lu.3) By subsection synthesis, artificially synthesizing spider toxicity geneω-ACTX-Hv2a,which was excreting expression, contained the signal sequence and prosequence of Hs2a.4) Five lacks of synthesized sequence were renovated by point mutant, after beingapproved by DNA sequence analysis, the sequence was consistent with designed sequenceultimately.5) With three segments linkage method, we cloned Hv2a gene into a new baculovirustransfer vector pAcE1 and constructed transfer vector pAcE1-Hv2a. By Digestion andcheckup, the sequence hadn't difference from designed sequence. And then we constructedrecombinant virus rbpAcE 1-Hv2a.6) Using AcNPV WT as control, we made insecticidal experiments preliminarily ofrecombinant virus rbpAcE1-Hv2a.Results suggested that recombinant virus rbpAcE1-Hv2acan kill insects tested, and the speed was obvious faster than the control virus AcNPV WT. ItsLT50 was shorter 50h than AcNPV WT; and the symptom of insect death was like poisoningobviously.