CN1205741A

CN1205741A - Prodn. of recombinant baculoviruses

Info

Publication number: CN1205741A
Application number: CN 96199244
Authority: CN
Inventors: B·F·麦库辰
Original assignee: EI Du Pont de Nemours and Co
Current assignee: EIDP Inc
Priority date: 1995-12-22
Filing date: 1996-12-16
Publication date: 1999-01-20

Abstract

This invention pertains to methods that facilitate production of recombinant baculoviruses that have been engineered for use as biological control agents. More specifically, this invention pertains to regulation of expression of genes encoded by recombinant baculoviruses in an insect cell or in an insect host.

Description

The production of recombinant baculovirus

Background of invention

Chemical insecticide is the moiety of modern agriculture, and is the effective means that reduces the crop loss by the control insect pest.But chemical reagent is owing to its potential environmental pollution effect, monitored to the selective action of the resistance colonies of Agricultural pests and to non-target organism such as beneficial insect, hydrobiont, animal and human's toxic action always.Therefore people are seeking effectively but non-target colony and the useful insect of environment are being controlled alternative strategy.

One of them strategy is the microorganism that is applied under the state of nature pathogenic agent that is target insect colony.But many candidate's insect pathogenss that can become promising insect control reagent lack characteristic such as the host specificity and the rapid action of the typical chemical insecticide that farmer and other people engaged in agriculture have been accustomed to.From the virus of baculovirus family be host specific and have inactive environmental characteristics, but in lacking before significant crop damage takes place rapidly with the ability of target colony.Fortunately, modern molecular biology technique provides and has produced by the engineered instrument that is used as the recombinant baculovirus of biological control reagent.

The attracting feature of baculovirus is their incomprehensive host specificity.These viruses only infect arthropods, even and also have narrow and small relatively host range in specific insect order.Host specificity has carried out detecting (reference 1-3) with electron microscope, DNA hybridization and recombinant DNA technology.These studies show that narrow and small host range be because, at least in part because, baculovirus can not shift virogene into mammiferous nucleus.

Baculovirus is divided into three subtribes, comprises non-occluded baculovirus (NOVs), granulosis virus (GVs) and nuclear polyhedrosis virus (NPVs).Though specific GVs and NOVs have obtained careful research, NPVs be study baculovirus subtribe the most completely.The example of NPVs comprises autographa california NPV, beet armyworm NPV, bollworm NPV, Helicoverpa zea NPV, autumn mythimna separata NPV, wild cabbage chi Qu NPV, lopper worm NPV, gypsymoth NPV, extra large spodoptera NPV, zinc Autographa spp NPV, dragon spruce volume noctuid NPV, pears beans noctuid NPV and Heliothis virescens NPV.

In part because can use the effective cell culture system cloning vector flexibly of unifying, NPVs has been used as carrier for expression of eukaryon and has synthesized required heterologous protein (4,5).A special viral autographa california NPV (AcNPV) is used as model virus and is used for importing and expression of heterologous genes at baculovirus expression system.Though this virus is conventionally used as the recombinant protein that a kind of important external means are used for providing at eukaryotic expression system high yield, and, be the lepidopterous insects of important economic insect but AcNPV can infect multiple thus for expressed albumen provides suitable posttranslational modification.

Although the insect control reagent based on baculovirus has the potential practical advantage, multiple unfavorable factor hinders them and is used for modern agriculture.The most tangible obstacle that these viruses are widely used in furrow crop agricultural is between using of they and the crop damage that effectively the control host insect causes obvious time lag to be arranged.Be unlike in viewed rapid effect when using typical chemical insecticide, significantly the insect control of the wild-type baculovirus mediation body in-group that is detected in virus has reached and has been enough to hinder after the active high level of host.This may see by several Zhou Caineng after the infection circulation that relates to two types of virosome.After the infected insect cell, (BVs or extracellular virus ECV) generate in the motion of endochylema film at nucleocapsid blastogenesis C-type virus C body.These viruses are sloughed its capsid from nuclear in endochylema, and pass the blastogenesis of endochylema film to the haemocoele of host insect.This process causes that the system of host insect infects.In the later stage of course of infection, virosome is surrounded (inclusion body virus) by the albumen substrate of being made up of polyhedrin basically, thus formation polyhedrin inclusion body (PIBs or inclusion body, OBs).These inclusion bodies are peroral infection forms of virus, and the horizontal transfer (6,7) of virus is provided between insect host.The larva of Gan Raning is not taken food on the matrix of virus pollution and digests PIBs.Albumen substrate is dissolved under the alkaline pH effect of the insect midgut that sees multiple lepidopterous larvae.The virus nucleocapsid that discharges contains the viral DNA genome, and virus nucleocapsid adheres to and infect the epithelial cell of larva midgut.Typically, when virus is exponential growth in the host, infected insect will continue to grow and consume plant material.At last, after several weeks or longer time, infected larva will be involved in fully and be dead usually.

By using recombinant DNA technology, NPVs has deleted gene and has carried out genetically engineered to improve their the insect rate (8-10) of killing by the gene that instructs insecticidal proteins to express by introducing or from viral genome.Two kinds of strategies can both produce the biotic pesticide that demonstrate than wild-type, not engineered NPVs insect control more rapidly.The most effective reorganization NPVs is by the engineered insect selectivity neurotoxin (11-18) of expressing.Expressed toxin can quicken the startup of cytotoxicity, causes insect control faster.These recombinant viruses kill their host in the time of lacking 20-30% than wild-type NPVs.

Preparing must mass production as the baculovirus of biological agent for controlling noxious insect pests.The mass production of virus can carry out producing in the body with the external insect culture systems of standard or at the insect larvae that infects to be finished.Virion output in two kinds of systems all depends on enough virus replications, and the latter depends on keeping of healthy cell or insect again.Too early cell toxicant or insect death will inevitably limiting virus duplicate, thereby reduce the quantity of the progeny virus that is produced.

By engineered come quickly in and the genetic modification baculovirus of target insect also may cause lower virus replication and cause each cells infected (external) or the lower progeny virus output of each infected insect (in the body).Therefore, the efficient that is used for improving the insect control reagent of baculovirus mediation makes them become the surrogate of work of traditional chemical sterilant or the method for adjuvant, has also in fact reduced the economic feasibility of this insect control strategy.Therefore, need a kind of method that can overcome effective production insecticidal baculovirus of too early cell toxicant or the too early obstacle that kills host cell or insect.

Summary of the invention

The present invention relates to promote by engineered method of producing for the recombinant baculovirus of biological control reagent.More particularly, the present invention relates to the regulation and control that the gene of recombinant baculovirus coding is expressed in insect cell or insect host.

In one embodiment, the present invention relates to a kind of control by the gene of recombinant baculovirus genome encoding in insect cell culture or the method for in the insect that lives, expressing, wherein said insect cell or insect are expressed a kind of protein of regulating the genetic expression of shaft-like viral genome coding by genetically engineered.

More particularly, the method that this control is expressed by the insecticidal proteins of the mosaic gene in recombinant baculovirus genome coding can be used to effectively produce reorganization pesticidal baculovirus in insect cell culture or the insect that lives, and the step of said method comprises:

(a) make up a kind of recombinant insect cell that contains first mosaic gene, mosaic gene contains first nucleic acid fragment of first promotor of encoding, and first nucleic acid fragment operably is connected in one of coding can be influenced on proteic second nucleic acid fragment of adjusting of genetic expression;

(b) make up a recombination rhabdovirus expression vector, this expression vector contains second mosaic gene, this mosaic gene contains the 3rd nucleic acid fragment of second promotor of encoding, second promotor is subjected to the adjusting albumen influence of step (a), and said the 3rd nucleic acid fragment operably is connected on the 4th nucleic acid fragment of a kind of insecticidal proteins of coding;

(c) recombination rhabdovirus expression vector of (b) is stably imported the recombinant insect cell of (a); And

(d) at the recombinant insect cell of supporting to keep under the environment of shaft-like virus replication (a) of the recombination rhabdovirus expression vector that contains (b);

Wherein the proteic expression of adjusting of step (a) influences the expression of the insecticidal proteins of step (b).

In another embodiment, the present invention relates to the method that a kind of insecticidal proteins of controlling the mosaic gene coding that exists in the recombinant baculovirus genome is expressed, comprising:

(c) recombination rhabdovirus expression vector with (b) imports in the recombinant insect cell of (a); And

(d) at the recombinant insect cell of supporting to keep under the environment of shaft-like virus replication (a) of the recombination rhabdovirus expression vector that contains (b); Wherein the proteic expression of adjusting of step (a) influences the expression of the insecticidal proteins of step (b).

(a) make up a recombination rhabdovirus expression vector that contains following ingredients, (1) first mosaic gene, this mosaic gene contains first nucleic acid fragment of first promotor of encoding, and first nucleic acid fragment operably is connected in one of coding can be influenced on proteic second nucleic acid fragment of adjusting of genetic expression; (2) second mosaic genes, this mosaic gene contains the 3rd nucleic acid fragment of second promotor of encoding, second promotor influenced by said adjusting albumen, and said the 3rd nucleic acid fragment operably is connected on the 4th nucleic acid fragment of a kind of insecticidal proteins of coding;

(b) recombination rhabdovirus expression vector with (a) imports in the insect cell; And

(c) at the insect cell of supporting to keep under the environment of shaft-like virus replication (b); The proteic expression of wherein said adjusting influences the expression of said insecticidal proteins.

In another embodiment, the present invention relates to the method for production pesticidal recombinant baculovirus, comprising:

(c) recombination rhabdovirus expression vector with (b) imports in the recombinant insect cell of (a);

(d) at the recombinant insect cell of supporting to keep under the environment of shaft-like virus replication (a) of the recombination rhabdovirus expression vector that contains (b); Wherein the proteic expression of adjusting of step (a) influences the expression of the insecticidal proteins of step (b); And

(e) collect progeny virus.

(b) recombination rhabdovirus expression vector with (a) imports in the insect cell;

(c) at the insect cell of supporting to keep under the environment of shaft-like virus replication (b); The proteic expression of wherein said adjusting influences the expression of said insecticidal proteins; And

(d) collect progeny virus.

In another embodiment, the present invention relates to the method for production pesticidal recombinant baculovirus, the insect cell that wherein contains recombination rhabdovirus expression vector maintains in the vitro cell culture.

In another embodiment, the present invention relates to the method for production pesticidal recombinant baculovirus, the insect cell that wherein contains recombination rhabdovirus expression vector maintains in the insect of complete work.

(a) make up a recombination rhabdovirus expression vector that contains following ingredients, (1) first mosaic gene, this mosaic gene contains first nucleic acid fragment of first promotor of encoding, and first nucleic acid fragment operably is connected on second nucleic acid fragment of coding tsiklomitsin trans-activator; (2) second mosaic genes, this mosaic gene contains the 3rd nucleic acid fragment that the one or more tsiklomitsins of coding are handled the site, these tsiklomitsins are handled the site and operably are connected on the minimal promoter sequence, and said the 3rd nucleic acid fragment operably is connected on the 4th nucleic acid fragment of a kind of insect selectivity neurotoxin of coding;

(c) in the presence of the tsiklomitsin of significant quantity or tetracycline analogue, keep the insect cell of (b), make the tsiklomitsin trans-activator not handle the site and combine, thereby can not induce by the sequence-directed genetic expression of minimal promoter that operably connects said manipulation site with the tsiklomitsin that exists on said the 3rd nucleic acid fragment; And

(d) collect progeny virus.

In another embodiment, the present invention relates to contain a kind of recombinant insect cell of mosaic gene, this mosaic gene contains first nucleic acid fragment of first promotor of encoding, first nucleic acid fragment operably is connected on second nucleic acid fragment, second a kind of adjusting albumen that can influence the genetic expression of second promotor guidance of nucleic acid fragment coding.

In another embodiment, the present invention relates to contain the transgenic insect of one or more recombinant insect cells, this recombinant insect cell contains a kind of mosaic gene, this mosaic gene contains first nucleic acid fragment of first promotor of encoding, first nucleic acid fragment operably is connected on second nucleic acid fragment, second a kind of adjusting albumen that can influence the genetic expression of second promotor guidance of nucleic acid fragment coding.

In another embodiment, the present invention relates to a kind of insect cell or contain the transgenic insect of one or more recombinant insect cells, said insect cell contains a kind of mosaic gene, this mosaic gene contains first nucleic acid fragment of first promotor of encoding, first nucleic acid fragment operably is connected on second nucleic acid fragment, second a kind of adjusting albumen that can influence the genetic expression of second promotor guidance of nucleic acid fragment coding, wherein regulating albumen is the tsiklomitsin trans-activator.

In another embodiment, the present invention relates to contain the recombination rhabdovirus expression vector of first mosaic gene, this mosaic gene contains first nucleic acid fragment of first promotor of encoding, this fragment operably is connected on second nucleic acid fragment of a kind of insecticidal proteins of coding, said first promotor is subjected to be influenced by the adjusting albumen that the recombinant insect cell that contains second mosaic gene is expressed, second mosaic gene contains the 3rd nucleic acid fragment of second promotor of encoding, and the 3rd nucleic acid fragment operably is connected on proteic the 4th nucleic acid fragment of the said adjusting of coding.

In another embodiment, the present invention relates to contain the recombination rhabdovirus expression vector of first mosaic gene, this mosaic gene contains first nucleic acid fragment of first promotor of encoding, this fragment operably is connected on second nucleic acid fragment of a kind of insecticidal proteins of coding, said first promotor is subjected to be influenced by the adjusting albumen that the recombinant insect cell that contains second mosaic gene is expressed, second mosaic gene contains the 3rd nucleic acid fragment of second promotor of encoding, the 3rd nucleic acid fragment operably is connected on proteic the 4th nucleic acid fragment of the said adjusting of coding, and wherein said first nucleic acid fragment contains one or more tsiklomitsins that operably connect a minimal promoter handles the site.

In another embodiment, the present invention relates to contain the recombination rhabdovirus expression vector of following ingredients, (1) first mosaic gene, this mosaic gene contains first nucleic acid fragment of first promotor of encoding, and first nucleic acid fragment operably is connected on a kind of proteic second nucleic acid fragment of adjusting that can influence genetic expression of coding; (2) second mosaic genes, this mosaic gene contain the 3rd nucleic acid fragment that coding is subjected to second promotor that said adjusting albumen influences, and said the 3rd nucleic acid fragment operably is connected on the 4th nucleic acid fragment of a kind of insecticidal proteins of coding.

In another embodiment, the present invention relates to contain the recombination rhabdovirus expression vector of following ingredients, (1) first mosaic gene, this mosaic gene contains first nucleic acid fragment of first promotor of encoding, and first nucleic acid fragment operably is connected on a kind of proteic second nucleic acid fragment of adjusting that can influence genetic expression of coding; (2) second mosaic genes, this mosaic gene contains the 3rd nucleic acid fragment that coding is subjected to second promotor that said adjusting albumen influences, said the 3rd nucleic acid fragment operably is connected on the 4th nucleic acid fragment of a kind of insecticidal proteins of coding, wherein said adjusting albumen is the tsiklomitsin trans-activator, and said the 3rd nucleic acid fragment contains one or more tsiklomitsins manipulation sites that operably connect a minimal promoter.

Accompanying drawing and sequence table summary

Fig. 1. from the reorganization of polyhedrin inclusion body (PIBs) form of Heliothis virescens larva and wild-type virus filial generation quantitatively.On behalf of the wherein expression of AaIT toxin, " AcAaIT/p10 " be subjected to the recombinant baculovirus of baculovirus p10 promotor control in late period.On behalf of the wherein expression of LqhIT2 toxin, " AcLqhIT/p10 " be subjected to the recombinant baculovirus of utmost point baculovirus in late period p10 promotor control.On behalf of the wherein expression of LqhIT2 toxin, " AcLqhIT/IE1 " be subjected to the recombinant baculovirus of early stage baculovirus IE1 promotor control." AcNPV " represents wild-type baculovirus.

Fig. 2. the diagram of a tsiklomitsin trans-activator Controlling System.

The plasmid map of Fig. 3 .TV3tTa.

Fig. 4. be used to make up the sequence of the synthetic oligonucleotide of LqhIT2 gene.Oligonucleotide Lq1 (SEQ ID NO:4) coding bombyxin signal peptide.Oligonucleotide Lq1 (SEQ ID NO:4) and Lq10 (SEQ ID NO:13) are as the primer of synthetic gene pcr amplification.

Fig. 5. be used to prepare the diagram of the strategy of LqhIT2 gene.Oligonucleotide Lq1 (SEQ IDNO:4) and Lq10 (SEQ ID NO:13) (with " X " mark) are as the amplimer of PCR reaction.Single restriction site is also marked.

Nucleotide sequence of Fig. 6 .LqhIT2 gene (SEQ ID NO:14) and amino acid sequence corresponding.Lowercase in the nucleotide sequence (Nucleotide 1-57, coding 1-19 amino acid) refer to the to encode Nucleotide of bombyxin signal peptide.

Fig. 7. the collection of illustrative plates of rhabdovirus expression vector TV3tTaTo-LqhIT2.

The aminoacid sequence of Fig. 8 .tetrIE1A (SEQ ID NO:21).The tetR of trans-activator and IE1A zone are represented with the lower case and upper case letter respectively.Two amino-acid residues (208 and 209) of indicating with black matrix are owing to existing the additional limits site to increase in the sequence, the additional limits site is used for making things convenient for tetR and IE1A to meet and reads the insertion of frame ground.

Fig. 9. indicated TATA box, early stage (E) and late period (L) transcription initiation site the nucleotide sequence (SEQ ID NO:23 and 24) of AcMNPV minimum p35 gene promoter of position.The restriction site that is used for foreign gene insertion is in the future also marked.

Figure 10. indicated TATA box, early stage (E) and late period (L) transcription initiation site the nucleotide sequence (SEQ ID NO:25 and 26) of AcMNPV minimum p35 gene promoter of modification of position.Single base-pair mutation among the p35m marks with black matrix.The restriction site that is used for foreign gene insertion is in the future also marked.

Figure 11. the diagram of the upstream of polyhedron gene and then among transfer vector pTV3 (M) lq+ and pTV3 (M) lq-.Trans-activator gene and the relative position that contains the box of p35/p35m minimal promoter upstream tet operon sequence and LqhIT have been marked.

Figure 12. the diagram of the upstream of polyhedron gene and then among transfer vector ptTA (M) lq+ and ptTA (M) lq-.Marked the relative position of trans-activator gene with the box of tet operon sequence that contains p35/p35m minimal promoter upstream and LqhIT.

OJ EPO replenishes 37 C.F.R.1.82-1.825 and the appendix A and the appendix B (" comprising the requirement of the application content of Nucleotide and/or aminoacid sequence ") of issue in the numbering 2 according in December, 1992, the annex I and the II of " expression Nucleotide and amino acid whose standard rule in patent application " and EPO chief's order, the applicant provides 26 sequence tables.

SEQ ID NO:1. makes up the Bgl II joint that uses among the plasmid TV3tTa.

The Xho I of the 470bp of SEQ ID NO:2. plasmid pF43h contains the minimum hGH promotor of handling the sequence fused in tandem with seven tsiklomitsins to the nucleotide sequence of the restricted fragment of BamH I.

SEQ ID NO:3. makes up the Not I joint that uses among the plasmid ptetophGH.

SEQ ID NOs:4-13. is used to make up the synthetic oligonucleotide of LqhIT2 gene.Oligonucleotide Lq1 coding bombyxin signal peptide.Oligonucleotide Lq1 (SEQ ID NO:4) and Lq10 (SEQID NO:13) are as the primer of synthetic gene pcr amplification.

The segmental nucleotide sequence of synthetic DNA of SEQ ID NO:14. coding bombyxin signal peptide, the signal peptide back is the coding region of LqhIT2 toxin.

SEQ ID NOs:15 and 16. be used to increase the PCR primer TETR1 of tetR part of tTA and the nucleotide sequence of TETR2.

The nucleotide sequence of SEQ ID NO:17. tsiklomitsin suppressor gene (tetR), it is to use oligonucleotide TETR1 and TETR2 to come through pcr amplification.

SEQ ID NOs:18 and 19. be used to increase from plasmid pIE1H/C corresponding to the 454bp segmental PCR primer I E1A1 of preceding 145 amino-acid residues of IE1 and the nucleotide sequence of IE1A2.

The nucleotide sequence of the activating area (IE1A) of SEQ ID NO:20.IE1, it is with oligonucleotide IE1A1 and IE1A2 pcr amplification.

The aminoacid sequence of SEQ ID NO:21.tetrIE1A.

SEQ ID NO:22. contains the segmental nucleotide sequence of BamH I of SV40 polyadenylation signal.

The nucleotide sequence of SEQ ID NOs:23 and 24. two complementary oligonucleotide P35PRO1 (SEQ IDNO:23) and P35PR02 (SEQ ID NO:24), after this two oligonucleotide annealing, represent in the p35 minimal promoter with respect to the translation initiation codon of p35 gene from-8 to-57bp.

The nucleotide sequence of SEQ ID NOs:25 and 26. two complementary oligonucleotide P35MPR01 (SEQ IDNO:25) and P35MPR02 (SEQ ID NO:26), after these two oligonucleotide annealing, the p35 minimal promoter that representative is modified, wherein integrate a single base pair in the promoter sequence and changed, thereby eliminated the TTAAG RNA initiation site in late period.

Detailed Description Of The Invention

The invention discloses a kind of in the body or the produced in vitro purpose NPVs that suppresses to recombinate express the method for insecticidal proteins.In one embodiment, with the proteic stable gene of a kind of coding and regulating be incorporated in a kind of genome for the insect of insect baculovirus natural host or insect cell.Then, will stably import in the genome of baculovirus corresponding to regulating near the one or more sites of proteic adjusting sequence guidance of heterologous protein expression promoter or enhancer element.When importing the host (using recombinate shape virus infection transgenic insect or transfection transgenic insect cell), the adjusting albumen of transgenic insect or insect cell expression will interact with the adjusting sequence that exists in the baculovirus genome, and influence is regulated the gene of sequence control or the expression of gene group by this.In addition, operably be connected in the proteic gene of coding and regulating of derivable, a quenchable or viral promotor, operably be connected in the gene corresponding to the coding heterologous gene interested of regulating proteic adjusting sequence, the both is present in the reorganization NPV genome.These recombinant viruses then increase in wild-type insect or insect cell.When suitable inductor that has the proteic genetic expression of adjusting and inhibition, the control of allogeneic gene expression will be affected.

For example, the structure gene of coding tsiklomitsin (tet) arrestin under constitutive promoter (as actin promoter) or the viral promotors promotor of nuclear polyhedrosis virus (preferably from) control can be inserted in the genome of lepidopterous insects.To be incorporated into corresponding to the adjusting sequence of tet inhibition near the viral genome control a kind of insecticidal proteins expression promoter and/or the enhanser zone.The tet inhibition with regulate the genetic transcription that sequence combined and hindered encoding insecticidal proteins.The expression that lacks this insecticidal proteins will postpone infected insect paralysis and dead startup, and cause the increase of PIBs output.Make in a like fashion, this strategy can be used for controlling makes virus unsettled cytotoxic protein or albumen group's the expression that becomes, thereby makes the output maximum of virus in the insect cell culture systems.

In context of the present disclosure, have many speech and abbreviation and be used to." NPV " represents nuclear polyhedrosis virus, and it is a kind of baculovirus." polyhedron disease " is meant and is characterized as histolysis and in the virus disease of several insect larvaes of polyhedron particle accumulation any one arranged in the liquid that is produced." PIBs " is the polyhedron inclusion body." AcNPV " represents the autographa california nuclear polyhedrosis virus of wild-type." LqhIT2 " representative is from the insect selectivity neurotoxin of Leiurusquinquestriatus hebraeus." AaIT " representative is from the insect selectivity neurotoxin of Androctonus australis." AcLqhIT2 " representative has been the shorthand form that is carried at the AcNPV that transcribes the LqhIT2 encoding gene under the control of baculovirus utmost point p10 in late period promotor by genetic modification.

" expression " is meant that structure gene transcribes and translate to produce coded albumen.Those skilled in the art should know, the expression level of structure gene is subjected to the influence of the used host cell of used adjusting sequence (promotor, polyadenylation site, enhanser etc.) and expression of structural gene.

Suitable " adjusting sequence " is meant the nucleotide sequence in the upstream (5 '), inside and/or the downstream (3 ') that are positioned at structure gene.The adjusting sequence combines with the albumen synthesizer of cell probably and controls transcribing and/or expressing of encoding sequence.These are regulated sequence and comprise promotor, operon, enhancer element, transcription termination sequence and polyadenylation sequence.

" adjusting albumen " thus be meant and can discern and influence transcribing of encoding sequence and/or expressed protein in conjunction with regulating sequence.Regulate albumen and can show negative sense influence to genetic expression (promptly reduce or eliminate and transcribe and/or translate), and be called as " arrestin " thus.On the contrary, specific adjusting albumen can show the influence of the forward of genetic expression (be initial or strengthen transcribe and/or translate), and therefore be called as " activator ", " trans-activator " or " inductor ".

" operon " thus be meant and can be conditioned the adjusting sequence that albumen identification changes the transcription initiation speed of adjacent promotor.

" promotor " is meant and generally sees the nucleotide sequence that structure gene 5 ' end instructs transcription initiation.Promoter sequence is to drive downstream gene expression necessity, but is not sufficient.Though when gene placed the promotor upstream, promoter activity also can show (on the expression level that reduces), the preferential downstream of promotor drives and transcribes usually.Therefore, when making up allogenic promotor/structure gene composition, thereby structure gene places the adjusting control of promotor to make expression of gene be subjected to the control of promoter sequence down.Promotor preferentially is positioned at the upstream of structure gene, and with transcription initiation site certain distance is arranged, near the distance between the gene of promotor in the natural alignment and its control.As known in the art, some variations in this distance are sustainable and do not lose promoter function.

" minimal promoter " is meant a kind of RNA polymerase II type promotor, it comprise usually one be called as the TATA box, with RNA polymerase bonded dna sequence dna and the short extension sequence of DNA on every side, this promotor can not be supported transcription initiation when lacking other promotor or operon element." omnipresence promotor " is meant organic all has the promotor of activity (promptly supporting its gene transcription that links to each other) in a organized way with in the organ." inducible promoter " is meant that function is subjected to the combination of trans-activating factor in the special born of the same parents or discharges the promotor of controlling.Inducible promoter is different from constitutive promoter, and constitutive promoter is non-modulated type and therefore always activated.

" 3 ' non-coding sequence " is meant that containing the polyadenylic acid signal can influence the gene DNA sequence part of the conditioning signal of mRNA processing or genetic expression with any other.3 ' the end that the feature of polyadenylation signal normally influences at the mRNA precursor adds the polyadenylic acid tail.

" gene " is meant the global DNA part that participates in protein synthesis.Gene comprises structure or the encoding part that begins to extend to the 3 ' terminator codon (Nucleotide TAG, TGA or TTA) of holding from translation initiation codon 5 ' end (being generally Nucleotide ATG).It also contains a promoter region, and promoter region is usually located at 5 ' end or upstream of structure gene, initial sum adjustment structure expression of gene.Also comprise 3 ' non-coding sequence in the gene." chimeric " gene is meant the artificial gene that contains allogenic adjusting sequence and encoding sequence." allogenic " gene is meant under a part of normal circumstances of gene or gene and does not see host's organism, but introduced by the method with transgenosis." nucleic acid fragment " is meant and the consistent nucleotide sequence of specific function in the gene.

" structure gene " is the Gene Partial that contains the dna fragmentation of encode a kind of protein, polypeptide or its part, and it has removed 5 ' and the 3 ' sequence that relates to gene expression regulation.Structure gene can be a kind of gene that just is being common in cell, also can be the gene that does not see the cell site that it is introduced under a kind of normal circumstances, and it is known as heterologous gene under latter event.Heterologous gene can be all or part of available from any source known in the art, comprises bacterial genomes or episome, eucaryon, nuclear or plasmid DNA, cDNA, viral DNA or chemical synthesising DNA.Structure gene can be in the coding region or non-translational region contain one or more modifications that can influence biological activity or chemical structure, the expression speed of expression product or express control mode.Such modification includes but not limited to sudden change, insertion, deletion and the replacement of one or more Nucleotide.Structure gene can be formed the encoding sequence that is not interrupted, and perhaps it can comprise one or more introns, and intron is not expressed, and by suitable montage binding substances combination.Structure gene can be from multi-source, the spontaneous or segmental molectron of synthetic.Structure gene can also comprise the fusion gene of encoding fusion protein.

" synthetic gene " is meant that the whole or major part of coding region is the dna sequence dna of the structure gene of chemosynthesis.As here for example, the oligonucleotide member synthesizes with method well-known to those having ordinary skill in the art, and connect and annealing to form the fragment of gene, the fragment enzymatic is assembled make up complete gene then.As known to those of skill in the art, can be with gene of equal value on site-specific mutagenesis or other this areas methods involving preparation of using and the synthetic gene function of introducing here and on the structure.

" operably connect " speech is meant that the nucleic acid fragment on single nucleic acid molecule connects each other and causes a segmental function to be subjected to another segmental influence.For example, when a promotor can influence the expression of structure gene, promotor was operably to be connected (being that structure gene is in transcribing under the control of promotor) with structure gene.

" transfection " is meant that the dna fragmentation that will carry a gene stably imports the organism that does not contain this gene in the past." cotransfection " is meant more than one dna fragmentation imported organism simultaneously.

" transgenic insect " is meant by containing the insect that one or more genetically modified cells are formed." transgenosis " is the gene that is imported into a cellular genome, cell can develop into the transgenosis organism thus, and gene keeps in ripe organism, and therefore instructs the expression of its coded product in organic one or more cell types of said transgenosis or tissue.

" chemosynthesis " when relating to dna sequence dna, its meaning is that the Nucleotide component is in external assembling.The manual chemosynthesis of DNA can be finished (19) with very sophisticated method, and a kind of the carrying out in the synthetic available multiple commercial machine of robotics.

Should be appreciated that one or more cells in the insect that " a kind of insect cell " be meant external one or more insect cells of keeping and see complete work.

" baculovirus " is meant that is only infected an arthropodan virus, and arthropods is to comprise the animal door that the squama wing belongs to.The pesticidal baculovirus is for providing a kind of method with environmental benefit that very big potentiality are arranged for the control of agricultural insect insect.But, need on efficient, be improved these reagent and present chemical insect control reagent are compared have competitive power.Carrying out a this improved approach is by virus is carried out hereditary change.For example, might revise viral genome improves the host range of virus, improves virus stability and persistence or viral infection and the transfer ability of raising in environment.In addition, the activity ratio that improves the infected insect of virus damage will obviously improve the pesticidal baculovirus as the subsidiary of chemical insect control reagent and the magnetism of surrogate.A kind of method that improves the speed of its insect host of virus infection is, foreign gene is imported baculovirus, the foreign gene coding is to the deleterious protein of insect, insect death or devitalization no longer only depend on virus infection like this, but obtain the auxiliary of foreign protein toxic level accumulation.The result who obtains is the pesticidal recombinant baculovirus.

Multiple arthropods produces a kind of insecticidal proteins mixture that is called as venom.These toxicants are synthetic in special gland tissue, these toxicants bite or the guidance of penetrating device under can make arthropodan prey paralysis.Little, move slowly or the immobilized arthropods has adapted to a kind of like this strategy, promptly utilize the neurotoxin composition of the venom of extremely low concentration that its prey is benumbed rapidly.These compositions or neurotoxin pass through the function to the effective competition interference insect nervous tissue in special receptor site.Many this neurotoxins are polypeptide.Based on its host specificity and binding mode, these toxin are divided into dissimilar (20).For example, the neural phallotoxins from multiple scorpion is divided into the arthropodan type of infection and infects mammiferous type.

There is the special toxin of several arthropodss to be accredited as insect selectivity peptide.For example, the Buthinae scorpion is expressed in two types different insect selectivity neurotoxin of biological effect on the target insect.In blow fly, somatotype be excitotoxin toxin by inducedmotion neurone tip get excited repeatedly cause immediately, rapidly with reversible shrinkability paralysis (21-23).These toxin are about 70 amino acid whose single chain polypeptides, and by four disulfide bond crosslinkings.The reason of hormesis is the enhancing that the pathway closure of activation action potential, sodium conduction and current potential dependence slows down.AaIT is a kind of toxin (24) for preparing from the venom of scorpion Androctonus australis, and it is first active insect toxins of this excitement of isolating demonstration from these organisms.

Second type insect selectivity neurotoxin is the inhibition toxin, comprises BjIT2 (25), LqqIT2 (26) and LqhIT2 (27).These toxin are 60 to 65 amino acid whose polypeptide, and their primary amino acid sequence is different from excitotoxin.These toxin-induced slowly, progressive paralysis and insect muscle lax fully.This activity is the result (26,28) that action potential excites obstruction, and owing to the inhibition of activation sodium conduction and the depolarize of axolemma.

The method and the strategy that are used to prepare the recombinant baculovirus of expression of heterologous genes are (4,5,29) well-known in the art.These gene expression methods can prepare mammalian proteins economically in the carrier for expression of eukaryon system, produce the protein of having finished suitable tertiary structure and having formed active necessary suitable disulfide linkage as a rule.

Usually, to import the baculovirus viral genome be to be undertaken by virus genom DNA and the homologous recombination that contains between suitable " transfer vector " of foreign gene interested to heterologous gene.These carriers generally be can be in host bacterium plasmid DBA self-replicating, that flexible genetic manipulation can be provided.Baculovirus transfer vector also contains the hereditary box that comprises a virus genomic zone, and this hereditary box is modified to have following feature (listing by 5 ' to 3 ' direction): the viral DNA that 1) contains the 5 ' zone in dispensable gene group zone; 2) viral promotors; 3) one or more codings are convenient to the dna sequence dna of the restriction site of allogeneic dna sequence insertion; 4) transcription termination sequence; With 5) contain dispensable gene group zone 3 ' zone viral DNA.An interested heterologous gene is inserted the restriction enzyme site in viral promotors downstream in the transfer vector.The box that is produced contains a mosaic gene.Wherein heterologous gene is in transcribing under the control of the viral promotors that exists on the transfer vector and transcription termination sequence.And the flank of this mosaic gene is a viral DNA sequence of being convenient to carry out at virus genomic nonessential region homologous recombination.Recombinant virus can support the insect cell of virus replication to prepare by virus genom DNA and recombinant transfer vector cotransfection.Between the homologous sequence that exists in flank viral DNA sequence that exists on the transfer vector and the virus genom DNA homologous recombination takes place, and cause mosaic gene to insert a virus genomic zone, the basic function that the insertion in this zone can viral interference.Infective recombinant virus particle is made up of the recombination group DNA that is called as rhabdovirus expression vector that is wrapped up by one deck protein coat.

In a preferred embodiment, the virus genomic nonessential zone that exists on the transfer vector comprises the viral DNA zone of being responsible for synthetic polyhedrin.The transfer vector that more preferably between the flanking sequence that participates in homologous recombination, contains complete polyhedron gene.After recombinating, will cause the positive phenotype of polyhedrin to be recovered from the genomic dna of the synthetic defective virus of polyhedrin (because of defectiveness in the genome copy of polyhedron gene).This strategy is convenient to the evaluation and the selection of recombinant virus.

In another embodiment, the baculovirus genomic dna can directly be modified by introducing a single restriction enzyme recognition sequence in virus genomic nonessential zone.Can constitute one contain will with the heterologous gene of expression of recombinant virus and operably connect with it can instruct the mosaic gene of this gene in the adjusting sequence of the expressed in insect cells of baculovirus infection, and it is directly inserted virus genomic single restriction enzyme site.This strategy has been eliminated and has been made up the necessary of transfer vector and to the dependence of the homologous recombination that is used to prepare recombinant virus.This method is introduced by (30) such as Ernst, and sees WO94/28114 (31).

Be suitable for transmitting by the best toxin gene of the recombination rhabdovirus expression vector needs of the insect-specific neurotoxin of genetic coding and express the efficient that obtains maximum.Those skilled in the art can use multiple strategy to design and prepare recombinant baculovirus, and wherein toxin gene is expressed the suitable time cause in course of infection and produced the toxin of the functional form of q.s, and are used in the insect host and combine with target cell.

The best mass-produced key of reorganization NPVs is to select suitable system to come regulatory gene to express.A kind of optimum system choosing like this uses the inhibition/operon element that can suppress the active genetic transcription of coded insect-killing.Existing several inhibition systems that can influence genetic expression are introduced, and comprise different induction type eukaryotic promoters (32-33).

In addition, the regulation and control of genetic expression can be finished by using colibacillary lac inhibition/operon inducible system in the eukaryotic cell.Existing three kinds of approach have obtained introduction: (ⅰ) initial (34-38) that transcribes by suitably placing the promotor site to stop the lac operon; (ⅱ) by transcribe (39) of lac inhibition/operon complex in the mediation of extended peroid blocking-up RNA polymerase II; (ⅲ) activate the promotor corresponding to fusion rotein, fusion rotein is formed (40-41) by the activating area of the virion protein 16 (VP16) of lac arrestin and hsv.But because the activation of lac inductor isopropyl-(IPTG) is slow and invalid, this system only produces medium adjusting (42).

The present invention relates in particular to tsiklomitsin (tet) operon-inhibition system constructing and is used for the system that regulatory gene is expressed.The tsiklomitsin inhibition is to have the DNA of high affinity conjugated protein with the tet operon sequence.The tet inhibition of Tn10 coding comes regulatory gene to express (43,44) by combination and promotor eclipsed operon site.Tsiklomitsin or its analogue stop inhibition to combine with its manipulation sequence, thereby make RNA polymerase combine and mediate transcribing of mRNA with promoter sequence.

Tet operon-inhibition system has been successfully used to controlling gene expression effectively in plant.For example, Gatz etc. can make constitutive promoter (cauliflower mosaic virus 35S promoter with this system in transgenic plant; CaMV 35S) activity reduces by 500 times (45).Specifically, they have prepared the every cell of a kind of energy synthetic 1 * 10 ⁶Tet suppresses the rotaring gene tobacco plant of molecule.Three tet operons are introduced near the TATA box of CaMV35S promotor.Genetic expression is suppressed substantially under the normal physiological environment.But, add tsiklomitsin and stoped inhibition to combine with handling the site, cause disinthibiting fully of CaMV35S promotor.

In the time of more recently, tet operon-inhibition system is used to controlling gene expression (46) in the protozoon parasite trypanosoma bocagei.The transgenosis trypanosome of expressing intestinal bacteria tsiklomitsin arrestin shows the expression of inductor (tsiklomitsin) dependence of integrating in the karyomit(e) that is in the reporter gene under the promotor control that has the tet operon.The expression of reporter gene can be subjected to the control corresponding to four magnitudes of tsiklomitsin concentration, and this has substantially exceeded other based on the shown transcriptional control of the system of eucaryon inhibition.

The present invention's control is by the expression of the insecticidal proteins of recombinant virus genomes coding.Experimental evidence shows, with respect to the PIBs that insect produced that handles with wild-type virus, uses the PIBs that insect produced that infects through the genetically engineered reorganization NPVs that expresses a kind of insecticidal proteins to reduce about (see figure 1) more than 10 times.Specific insecticidal proteins can have negative impact (as cell toxicant) maybe can induce insect host paralysis and dead to insect cell.These effects can cause daughter of virus to reduce and/or the virus structure instability.The expression that suppresses insecticidal proteins can make insect cell can support the propagation of recombinant virus to produce the output that approaches wild-type virus.

The present invention regulates the expression of toxin gene by several method.A kind of method is used the trans-activation molecule (being also referred to as " tsiklomitsin trans-activator " here) under the tsiklomitsin control to control and is instructed the toxin gene expression promoter.This feature that can the strict expression system of regulating is at Gossen﹠Bujard, and U.S. Patent number 5,464 is introduced in 758, is hereby incorporated by (47,48), and is shown in Fig. 2.The controlling elements that is used for generegulation is a fusion rotein, and this fusion rotein is made up of the activating area fusion of the virion protein 16 of tet arrestin and hsv (tTA).

The present invention also comprises the structure of the proteic transgenic insect of a kind of inhibition of composing type ground expression (or suppressing sample).When the reorganization NPV that contains mosaic gene (by the tet operon, promotor and the toxin structure genomic constitution that operably connect) duplicated in the transgenic insect host, arrestin combined and suppresses the expression of toxin with operon.For making up the condition regulator control system of this genetic transcription, carry out basic genetic manipulation of two steps: ⅰ) tet operon fragment is inserted the upstream of toxin gene, the zone of preferred insertion and transcription initiation site and/or enhanser region overlapping; And ⅱ) eukaryotic genome is introduced on inhibition stable gene ground, be preferably the genome of insect or insect cell line.

Inhibition can place the expression that comes to drive inhibition under a constitutive promoter (being Actin muscle) or the bacilliform virus promoter (being IE1, p10 or hybrid promoter) when the recombinant virus infection host.When using a kind of bacilliform virus promoter such as IE1, p10 or polyhedrin, inhibition is only expressed when virus infection.This design can provide giving full expression to of arrestin, because virus can provide virus replication necessary rerecording device.Tet handles the site and is placed near virus genomic transcription initiation site and/or the enhanser zone, blocks transcribing of heterologous protein (being preferably a kind of insecticidal proteins) that driven by viral promotors.

This strategy need be used for foreign gene is inserted the efficient carrier of insect genes group.Dissimilar removable nucleic acid entities has carried out identifying (49) in different insects.Displaceable element is inserted dna sequence dna can cause the genetic expression that changes or interrupt, they are accredited as mutant.At present, use has been finished the efficient conversion (50) of insect based on the conversion carrier system of P-element in Drosophila.But these conversion carriers can not be used for the conversion (51) of other insect.What therefore, needs were other can be used for the removable genetic elements that other insect comprises the efficient conversion of lepidopterous insects.

In addition, other method for transformation concentrates on other transfer system.Wherein a kind of such technology is " a parent microinjection ", comprises DNA is injected in the conceived female ovum or ovary.This technology has been successfully used to plasmid dna sequence is imported predatory mite Metaseiulusoccidenalis (52) and Amblyseius finlandicus (53).Recently, existing report electricity transforms among the embryo who successfully plasmid DNA is shifted insect into (keith Hughes, USDA, Fargo, ND, person-to-person communication).

A kind of method for preparing transgenic insect is to use heterologous gene is imported the very useful swivel base increment element (TE) of lepidopterous insects genome.These genetic elements are convenient to make up to express and are regulated proteic transgenic insect, and regulate the expression that albumen is controlled shaft-like virogene.This TE be in the arthropods genome, extensively exist and in lepidopterous insects sailor's element of omnipresence (mariner element) (55) almost.Sailor's element can use the degenerated primer of being derived by two conservative regions of sailer transposase enzyme open reading frame to identify and prepare (55).Recently, from predatory mite Metaseiulus occidentalis, be separated to a sailer transposase element.Use two reverse primers, sailor's element (Moc1) of total length is checked order, find the long 1284bp of this sequence, comprise the inverted terminal repeat sequence of 28bp.But Moc1 sailor's element contains and comprises the mass mutation that moves frame, and the transposase activity can not detect.Find that based on these Moc1 is considered to the transposable element of a non-activity.But sailor's element of non-activity is an attractive instrument for import foreign DNA by homologous recombination, because it has accumulated a large amount of sudden changes, comprises deletion, insertion and terminator codon.It will be favourable that foreign DNA is inserted such sailor site, because transposase gene no longer includes activity, it is stable that such foreign DNA will keep.Tet suppressor gene and contain a segmental selective marker of sailor (as cyclic diolefine resistant gene-Rdl; (56,57)) the sailor site of inserting lepidopterous insects takes place by homologous recombination.Conversion is carried out with the electric method for transformation of microinjection technology of introducing previously or new development.

Embodiment

The present invention is further illustrated in the following embodiments.Be to be understood that these embodiment only are illustrative, the present invention is not limited to use the introduction among the embodiment.According to discussion and the following examples above, those skilled in the art can determine, and under the prerequisite that does not deviate from its spirit and scope, the present invention be carried out various changes and modifies to make it to be applicable to different application and environment.All these are modified all in desired claim scope.

Embodiment 1

The adjusting that insecticidal proteins is expressed also can be finished by tTA is incorporated into viral genome self with the manipulation site.In this method, the controlling elements of the tetracyclin resistance operon of encoding among the colibacillary Tn10 is inserted into the expression of regulating insecticidal proteins in the AcNPV genome.TTA is placed under the control of immediate early promoter IE1, IE1 expression alien gene immediately after NPV infects.Baculovirus transfer vector pAcP+IE1TV3 (is provided by Dr.DonaldJarvis, Texas Agricultural Experiment Station, Texas A﹠MUniversity, College Station, TX) be used to this structure, it is a U.S. Patent number 5,162, the derivative of the baculovirus early promoter transfer vector of introducing in 222.At first, tTA with enzyme EcoR I and BamH I by plasmid pUHD15-1 (available from Dr.RobertHorlick, DuPont-Merck Pharmaceutical Co Wilmington, downcut and be inserted in DE) plasmid Bluescript SK with EcoRI and the digestion of BamH I ± ^(Stratagene, Menasha, WI) in.After the connection, plasmid DNA is transformed into bacillus coli DH 5 alpha (Gibco/BRL; Gaithersburg, MD) in, the existence of inserting son confirms with restriction analysis.

Confirmed after the insertion of tTA, with a SV40 polyadenylation fragment (SEQ ID NO:22) (by Gary Chun, E.I.duPont de Nemours and Company provides) that has a BamH I restriction site at 5 ' and 3 ' end be inserted into Bluescript SK ± ^In the BamH I site in tTA downstream and then.With the HpaI site (polyA) of inside and Bluescript SK ± ^Multiple clone site 3 ' end in the Xho I site found confirm that this inserts segmental correct direction.Contain the segmental novel plasmid of tTA and polyA and put down, and add a Bgl II restriction site by integrating a BglII joint (SEQ ID NO:1) with EcoR1 digestion and with Klenow polysaccharase benefit.After the connection, plasmid DNA is converted in the bacillus coli DH 5 alpha.Then the Bluescript SK that from then on modifies with restriction enzyme Bgl II and Not I ± ^Downcut the fragment that contains tTA and SV40 in the plasmid.This fragment is connected among the transfer vector pAcP+IE1TV3 that cuts with restriction enzyme Bgl II and Not I.This construction cause encoding the composite structure gene of tTA and the downstream that the polyadenylation fragment is inserted into baculovirus IE1 promotor and hr5 enhanser zone.After the connection, plasmid DNA is converted into bacillus coli DH 5 alpha, the transformant of the plasmid that contains a single asymmetric site that is positioned at tTA gene 3 ' end is carried in screening in the transformant that is produced.Novel plasmid is named as TV3tTa (Fig. 3).

The next step that makes up regulation system for recombinant baculovirus insecticides comprises a minimal promoter sequence and tetracycline operator sequence is integrated in the viral genome.Use restriction enzyme Xho I and BamH I from plasmid pF43h (SEQ ID NO:2; Available from Dr.R.Horlick, Dupont-Merck Pharmaceutical Co.) downcuts the minimal promoter (hGH) that merges with placed in-line tetracycline operator sequence (7 tet operons).With this fragment cloning to earlier with the Bluescript SK of Xho I and the digestion of BamH I ± ^In.After the connection, plasmid DNA is converted into bacillus coli DH 5 alpha, positive colony is identified with restriction enzyme analysis.Then this plasmid is digested (in 3 ' end multiple clone site) with restriction enzyme Xho I, flat with Klenow polysaccharase benefit, connection again under the condition that has Not I joint (SEQ ID NO:3).After the connection, plasmid DNA is converted into bacillus coli DH 5 alpha, resulting transformant screens with the digestion of restriction enzyme Not I.Behind 2% agarose gel electrophoresis, find to have an appointment the existence of the segmental existence of 450bp with regard to the prove out construction.This plasmid is named as ptetophGH.

At last, the structure gene of a kind of insect selectivity neurotoxin LqhIT2 of coding is placed the BamH I cloning site in minimal promoter downstream.For preparation LqhIT2 gene, designed ten kinds of oligonucleotide (Fig. 4 is made up of SEQ ID NO:4-13), and synthetic with the phosphoramidite chemical method of standard.According to the diagram among Fig. 5, the method that adopts manufacturer to recommend, use Gibco/BRL (Gaithersburg, MD) kinases with these oligonucleotide phosphorylations, annealing, and connect with the Gibco/BRL ligase enzyme.Use Perkin-Elmer CetusAmpliTaq then ^(Norwalk, CT), according to method and the hereinafter modification of introduction that manufacturer is recommended, (PCR's Polymerase) increases to junction fragment by polymerase chain reaction.Oligonucleotide Lq1 (SEQ ID NO:4) is as forward primer, and oligonucleotide Lq10 (SEQ ID NO:13) is as reverse primer.List the introduction of these methods below in detail.

Carry out ten independent phosphorylation reactions (each oligonucleotide carries out a reaction) altogether.In the 1.5mL micro-centrifuge tube, insert every kind of oligonucleotide (SEQ ID NO:4-13) of 250pmol.(Gibco/BRL, 10 units/uL) and the water of sufficient volume make the reaction cumulative volume reach 50uL to add the kinases of 1mM ATP, 6uL of 10 * kinase buffer liquid, 1uL of 5uL in every pipe.With 10 arms 37 ℃ of incubations 1 hour.Behind the incubation, the oligonucleotide (25pmol) of every kind of phosphorylation of 5uL is placed an independent micro-centrifuge tube, then pipe is placed 95 ° drying heater.Closing upper heater then makes it be cooled to room temperature so that the oligonucleotide annealing of phosphorylation.(Gibco/BRL, 5 units/uL) and 3uL deionized water place independent micro-centrifuge tube together with the ligase enzyme of 50uL phosphorylation, annealed oligonucleotide mixture and 5 of 15uL * be connected 10mM ATP, the 4uL of damping fluid, 3uL.This pipe 37 ℃ of incubations 30 minutes, is spent the night at room temperature storage subsequently.

Containing annealing increases with PCR with the nucleic acid fragment of the oligonucleotide that is connected.Carry out three PCR reactions with the dilution template DNA of difference (oligonucleotide that contains phosphorylation, annealing and be connected).Following reaction mixture is used for the PCR reaction:

61.5uL deionized water

10 * PCR damping fluid of 10uL (Perkin-Elmer Cetus)

Every kind of dATP, the dCTP of 2uL, dGTP, dTTP (each 200uM)

0.5uL AmpliTaq ^Polymerase (2.5 units/100uL)

Template DNA with deionized water be diluted to 1: 100,1: 1,000 and 1: 10,000 (v/v).Every reaction mixture that adds 80uL in three 0.5mL micro-centrifuge tubes.The oligonucleotide Lq1 (SEQ ID NO:4) and 5uL (100pmol) the oligonucleotide Lq10 (SEQ ID NO:13) (being used separately as forward and inverse PCR primer) that in every pipe, add 5uL (100pmol).In every pipe, add the suitably template of dilution of 10uL.Use a Perkin-Elmer DNA Thermocycler ^Carry out the PCR reaction by following amplification method:

Step 1 (1 circulation): 96 ℃ 3 minutes

75 ℃ 3 minutes

Step 2 (25 circulations): 95 ℃ 30 seconds

75 ℃ 2 minutes

Step 3 (1 circulation): 95 ℃ 30 seconds

75 ℃ 5 minutes

Product electrophoresis on 2% sepharose that amplification is obtained is analyzed.The amplified fragments of about 300 base pairs sees every group reaction.

Pcr amplification after LqhIT2 gene and the flanking region, separate this 300bp fragment by 2% sepharose, and use SpinBind ^(ME) method of recommending according to manufacturer is carried out purifying to the DNA recovery system for FMC, Rockland.Isolating fragment contains the synthetic oligonucleotide of LqhIT2 gene and signal sequence with generation with BamH I digestion the 5 ' end and 3 ' of viscosity is held (Fig. 6, SEQ ID NO:14).Molecule clone technology with standard is inserted into pTZ-18R plasmid (PHarmacia, Piscataway, BamH I cloning site NJ) with the fragment that digests then.After the transformed into escherichia coli DH5 α MCR, select isolating clone and preparation plasmid DNA.The forward of 8 positive colonies and commodity in useization and oppositely pTZ-18R primer order-checking have been identified.Find that a clone (No.16) contains the correct sequence of coding synthetic gene and signal sequence.The plasmid that is obtained (pTZ-18RLq) contains two BamH I restriction sites: a site is near 5 ' end of toxin gene, and another site is terminator codon and then.

Method according to standard prepares the pTZ-18RLq plasmid DNA, and contains the insertion fragment of 300 base pairs of LqhIT2 gene and signal sequence with release with the digestion of BamH I.Separate from the carrier sequence by this fragment of swimmer that powers at 1.2% sepharose, and use SpinBind ^DNA recovery system purifying.With the molecule clone technology of standard isolating fragment is inserted into the BamH I cloning site of ptetophGH then.After being converted into bacillus coli DH 5 alpha MCR, select isolating clone and preparation plasmid DNA.

Then this plasmid is digested with restriction enzyme Not I, discharge the fragment of 750bp, separate this fragment by 2.0% sepharose, and use SpinBind ^DNA recovery system purifying.This fragment contains tet operon site, minimum hGH promotor and LqhIT2 gene/leader sequence.The fragment that to separate and digest with the molecule clone technology of standard is inserted into the Not I cloning site of novel baculovirus vector TV3tTa (above) then.After being converted into bacillus coli DH 5 alpha MCR, select isolating clone and with restriction enzyme analysis technical Analysis plasmid DNA.Be separated to and bred the segmental clone of the several Not of containing I, and the preparation plasmid DNA is used for cotransfection.New transfer vector is called the TV3tTaTo-LqhIT2 (see figure 7).

Spodoptera frugiperda cell (sf-9) is at the ExCell that adds 3.0% foetal calf serum ^(JRH Biosciences, Lenexa breed in KS) 401 substratum.At the AcNPV of the TV3tTaTo-LqhIT2 of every part of 50uL (500ng) and linearizing polyhedrin feminine gender (2.5ug, Baculogold ^Viral DNA, Pharmigen, San Diego, CA) the middle Lipofectin that adds ^(50ug, 0.1mg/mL, Gibco/BRL).Sf-9 cell (about 50% individual layer) viral DNA/transfer vector solution cotransfection.Transfection by collecting supernatant liquor in the cotransfection experiments, and separated recombinant virus with the plaque purification process of standard after 5 days, wherein only selected polyhedrin male plaque (6).

The isolating plaque of picking is suspended in the ExCell that 500uL adds 2.5% foetal calf serum ^In the substratum.Sf-9 cell (50% individual layer) in the 35mM culture dish infects and collects supernatant liquor after 5 days with 100uL viral suspension incubation.Carry out the extensive propagation of recombinant virus with this supernatant liquor inoculation medium.

The expression of the LqhIT2 of recombinant baculovirus AcTV3tTaTo-LqhIT2 coding confirms with immunoblotting assay and biological assay.Sf-9 cell (50mL) infects with wild-type AcNPV (negative control), AcLqhIT2 (positive control) or AcTV3tTaTo-LqhIT2.Under the situation that does not have tsiklomitsin or analogue, heterologous protein can be expressed in the cell that AcTV3tTaTo-LqhIT2 infects effectively.But when having tsiklomitsin or analogue, the expression of toxin is suppressed.Then the tsiklomitsin of different concns is added and add experiment in the substratum and determine that substrate effectively controls the ability of heterologous protein expression.In metainfective several pitch times, collect infected cells, cell suspension is centrifugal to remove growth medium.Outwell remaining substratum, use Branson Sonifier ^(450 type) is being provided with the cell that 2 processing cracking in 30 seconds keeps.In refrigerated microcentrifuge (4 ℃) 15,000rpm removed cell residue in centrifugal 10 minutes then.

(Pierce, Rockford IL) carry out quantitatively according to the explanation of manufacturer protein concentration in the cells infected ultrasonic disruption thing with BCA ProteinAssay.Concentration known production standard curve according to bSA.Sample and standard substance are 37 ℃ of incubations 30 minutes, with the absorption value at spectrophotometer measurement 562 places.Determine the protein concentration of each sample with linear regression analysis.

Quantitatively the single protein of planting in the sample separates with protein electrophoresis.Sample is diluted to 3-4mg/mL with deionized water.Every kind of sample of 25uL is added to electrophoresis sample buffer (3.8mL deionized water, 1.0mL 0.5M Tris-HCl, the pH6.8 of 75uL, 0.8mL glycerine, 10% 1.6mL (w/v) SDS, 0.4mL beta-mercaptoethanol, 0.4mL 0.5% tetrabromophenol sulfonphthalein).The molecular weight standard thing is used in the 100uL sample buffer dilution biotinylated molecular weight of albumen standard substance of 1uL (Gibco) and is prepared.Sample and standard substance were 95 ℃ of heating 2 minutes.Sample on the sample (20uL/ hole) is in 15%Mini-Protean II Tris-HCl ReadyGel ^(Bio-Rad, Melville, NY).In mounted running gel device, add electrophoretic buffer (1.0g SDS is dissolved in the 1L deionized water for 3g Tris alkali, 14.4g glycine), sample about 1.5 hours with the 40mA electrophoresis.

After the electrophoresis, isolating protein is transferred to nitrocellulose filter with western blotting technique from running gel.Trace paper (3MM; Schleicher and Schuell, Keene, NH) and nitrocellulose filter (BA-S NC; Schleicher and Schuell) cut to the gel size, and in Western blot transfering buffering liquid (11.6g Tris alkali, pH8.3,5.8g glycine, 0.74g SDS, 400mL methyl alcohol, 1.6L deionized water), soak.Trace paper, nitrocellulose filter and gel are arranged in the following order: three trace paper, gel, nitrocellulose filter and three trace paper.This " centre-fills " placed transfer device, make gel towards negative electrode and nitrocellulose filter towards anode.In transfer device, protein is shifted with the 60mA energising.After the transfer, take transfer device apart, at the air drying nitrocellulose filter.

Follow these steps to the protein that shifts is carried out immunoblotting assay, all steps are all at room temperature carried out on rotary shaker.Be not transferred on the nitrocellulose filter site that albumen occupies be dissolved in TTBS (20mM Tris, 500mM NaCl, pH7.5,0.05%Tween-20) 3% gelatin (w/w) insulation in was sealed in 30 minutes.Remove the sealing damping fluid, filter membrane rinsing 5 minutes in 100mL TTBS.By in 10mL TTBS, add the special rabbit polyclonal antibody of 10uL rabbit Antibody Preparation LqhIT2 (available from Dr Bruce Hammock, University of California, Davis, CA).This anti-solution was added on the nitrocellulose filter of sealing and incubation 1 hour.After the incubation, change 100mL TTBS washing nitrocellulose filter 3 times, each washing continues 10 minutes.Two anti-by in 20mL TBSS, add the peroxidase-conjugated goat anti-rabbit igg of 10uL (Sigma, St.Louis, MO) and the streptavidin of 10uL peroxidase labelling be prepared.Nitrocellulose filter is solution incubation 1 hour together therewith.After the incubation, change 3 times 100 mL TTBS washing nitrocellulose filters, each washing continues 10 minutes.On nitrocellulose filter, add detection reagent (ECL Detection Kit; Amersham, Arling Heights, IL) and incubation 60 seconds.With detection reagent venting from the filter membrane, and cover filter membrane with Saran Wrap.(Kodak X-OMAT AR, Rochester NY) go up exposure 2-10 second to detect the signal on the nitrocellulose filter at the X-ray sheet with the filter membrane handled well.Can provide near 7, the separating clone of the positive immunoblotting reaction of 000Mr is considered to the toxin gene positive.

The condition of introducing above of carrying out western blotting method is to obtain with natural LqhIT2 toxin, and wherein the limit of Jian Ceing is determined as the 15ng toxin.In the experiment of usability transfect cell ultrasonic disruption thing, LqhIT2 toxin by recombinant baculovirus AcTV3tTaTo-LqhIT2 coding is not detected, express in this explanation cells infected with specimen in the amount of the toxin that exists be lower than the limit of detection of this experiment.

Embodiment 2

Make up a kind of tetR of containing gene and AcMNPV IE-1 gene activation district and met novel trans-activator genetic heterozygosis of reading the fusion of frame ground.The tetR Gene Partial of tTA is carried out pcr amplification with following Oligonucleolide primers:

TETR1?5’-ATGTCTAGATTAGATAAAAG-3’ (SEQ?ID?NO：15)

TETR2?5’AGATCTGGACCCACTTTACATTTAAG-3’(SEQ?ID?NO：16)

The dna profiling of this reaction is plasmid TV3tTa.Following reaction mixture is used for this PCR reaction:

The aseptic deionized water of 33uL

5 * Taq damping fluid D (InVitrogen) of 10uL

10 * dNTP stock solution of 5uL (every kind of dNTP of 2.5mM)

The oligonucleotide TETR1 of 1uL (SEQ ID NO:15) (100pmol)

The oligonucleotide TETR2 of 1uL (SEQ ID NO:16) (100pmol)

The plasmid DNA TV3tTa of 1uL (10ng)

0.2uL AmpliTaq Polymerase (5 units/uL) carry out the PCR reaction by following amplification method:

Step 1 (1 circulation): 95 ℃ 1 minute

72 ℃ 4 minutes

Step 2 (30 circulations): 95 ℃ 30 seconds

45 ℃ 30 seconds

72 ℃ 30 seconds

Step 3 (1 circulation): 95 ℃ 30 seconds

45 ℃ 30 seconds

75 ℃ 5 minutes

The product of 622 base pairs that obtained (SEQ ID NO:17) comprise tTA with respect to the tetR gene of translation initiation codon (ATG)+1 to+616 base pairs.An extra Bgl II restriction site is integrated into the 5 ' end of oligonucleotide TETR2, after being used for the IE-1 active region is inserted into this site.

Two Oligonucleolide primers,

IE1A1

5 '-CGGGATCCATGACGCAAATTAATTTTAACG-3 ' (SEQ ID:18) and

IE1A2

5’-CCAGATCTTTAACCTTGTGAATTGTCCAAGTATTC-3’(SEQ?ID：19)

Be used for by the 454 base pair fragments (SEQ ID NO:20) of plasmid pIE1H/C (58) amplification corresponding to preceding 145 amino-acid residues of IE-1 (IE1A).The existing report in this zone is represented the active region (59) of IE-1.Tail end at IE1A in amplification procedure has added a BamH I or a Bgl II site (being incorporated into the 5 ' end of IE1A1 and IE1A2 respectively), inserts IE1A (seeing below) so that tetR is met reading frame ground.The condition of pcr amplification IE-1 active region is identical with the condition of above introducing that is used for the tetR gene.

Use TA clone's test kit (Invitrogen) with two pcr amplification product tetR and IE1A directly be cloned into plasmid vector PCR II (Invitrogen, San Diego, CA) in, produce plasmid ptetR and pIE1A respectively.The potential clone who digests several ptetR with BamHI and Bgl II is to identify that wherein the upstream flank of tetR is that BamH I and downstream flank are the clone of Bgl II.The two fidelity of tetR and IE1A confirms with sequential analysis.Be inserted into the Bgl II with BamH I and the Bgl II digestion pIE1A and the 454bp fragment that will contain IE1A and partly digest and make up final heterozygosis trans-activator gene among the linearizing ptetR.With the digestion of BamH I and Bgl II is that the wherein correct clone of IE1A and the equidirectional insertion of tetR is identified on the basis.Only contain tetR and IE1A and meet the fragment that the clone who reads the insertion of frame ground just produces one 1080 base pair.The plasmid ptetRIE1A that is produced contain by tetR gene and IE1A meet the heterozygous genes reading the fusion of frame ground and form (SEQ ID NO:21, Fig. 8).Joint between tetR and the IE1A confirms with sequential analysis.The BamHI fragment cloning that will contain SV40 polyadenylation sequence (SEQ ID NO:22) again arrives the and then Bgl II site in tetRIE1A gene downstream.

Replace the VP16 active region except using from the zone similarity of IE1, another modification that this system is done comprises with AcMNPV p35 minimal promoter replacement human growth hormone (hGH) minimal promoter.This promotor contains early stage and late transcription element simultaneously, but mainly is an early promoter (60).Two complementary oligonucleotide (200pmol) P35PR01 (SEQ IDNO:23) and P35PR02 (SEQ ID NO:24) are by being cooled to room temperature gradually 100 ℃ of heating 5 minutes and through 1 hour, annealing in 10 * connection damping fluid (New England Biolabs Inc.).What the annealing product that is obtained was represented the p35 gene is-8 to-57bp p35 minimal promoter (Fig. 9) with respect to translation initiation codon.Oligonucleotide is designed to, and the terminal and Sst I coupling in the back of annealing mutually could in fact produce a Sst I site again but have only 3 ' of promotor to hold after being cloned into Sst I site.The p35 minimal promoter is cloned into the Sst I site of plasmid ptetophGH, causes the hGH promotor to be replaced, and the p35 minimal promoter is placed under the transcriptional regulatory of tetop sequence by the p35 promotor.The p35 promotor is confirmed by digestion of Sst I and sequential analysis with respect to the correct orientation of tetop sequence.The plasmid called after ptetopp35 that is produced.

Similarly, with top identical condition under, made up the p35 promotor of a modification with another group oligonucleotide P35MPR01 (SEQ ID NO:25) and the mutual annealing of P35MPR02 (SEQ ID NO:26), be integrated into the p35 promotor (Figure 10) of having eliminated TTAAG RNA initiation site in late period in the promoter sequence thereby produce a wherein single sequence change.Removing the late period initiation site can place transcribing under the control of tetRIE1A fully with LqhIT2 or other expression of heterologous genes, and the minimizing system at later period of infection by initiation site transcriptional start and the leak that produces in late period.The p35 minimal promoter of modifying is cloned into the Sst I site of ptetophGH, produces plasmid ptetopp35m.Ptetopp35 and ptetopp35m contain single BamH I and Not I site, are used for inserting DNA in the downstream of p35/p35m minimal promoter.

Method with the digestion of BamH I is separated LqhIT2 gene and leader sequence from pTV3tTaTo-LqhIT2, and the 300 base pair fragment subclones that will contain LqhIT2 gene and leader sequence are to the single site of Bgl II that ptetopp35 and ptetopp35m all have, and plasmid ptetopp35Lq and the ptetopp35mLq that produced this moment contain the and then LqhIT2 gene/leader sequence in p35/p35m promotor downstream.The clone who contains the LqhIT2 directed correct with respect to the p35/p35m promotor identifies with the digestion of Sst I.Make the tetopp35/35mLq box complete by inserting a BamH I fragment (SEQ ID:22) that contains 204 base pairs of SV40 polyadenylation signal sequence.The Hpa I site of an inside is used to identify that the polyA signal sequence has the clone of correct direction with respect to LqhIT.The plasmid ptetopp35/35mLqA of Chan Shenging is used for providing the tetop that is inserted into last transfer vector box at last.

Design is used for that tetRIE1A and ptetopp35/p35mLqA are incorporated into the genomic transfer vector of AcMNTV and makes up through two steps.At first, and transfer vector pAcP+IE1TV3 (provide by Dr.Donald Jarvis, Texas A﹠M University, CollegeStation TX) makes the carrier linearizing with Bgl II and Not I (in multiple clone site) digestion.Will be from 1.3 kilobase of ptetRIE1A right BamH I/Not I fragment directed cloning makes the trans-activator gene place transcribing under the control of hr5/ie-1 promotor in these sites of pAcP+IE1TV3.The second, tetopp35LqA or tetopp35mLqA box be inserted at tetRIE1A be inserted in the single site of Not I that regenerates in the process of pAcP+IE1TV3.This step finishes with the following step: with Not I digested vector, use the big fragment of archaeal dna polymerase (Klenow) that 5 ' protruding terminus is mended the life end of showing no increases in output.Similarly, ptetopp35/35mLqA uses the T4 archaeal dna polymerase to equal endization with Xho I and the digestion of Sst I, the 0.8Kbp fragment that contains the tetopp35/p35mLqA box.Then this fragment is inserted into the Not I site of flush end.The clone that selection contains with respect to each box of two kinds of directions of tetRIE1A gene prepares recombinant virus to determine the influence of direction contratoxin genetic expression.According to toxin gene and trans-activator is in the same way or oppositely, and the clone is by called after Lq+ or Lq-(Figure 11) respectively.The result has made up four different transferring plasmids (pTV3lq+, pTV3lq-, pTV3Mlq+ and pTV3Mlq-).

In addition, also can make up transferring plasmid, make the tetop-hGH-Lq box among the tetopp35/p35mLqA box replacement pTV3TaTo-LqhIT2, like this, this moment, last box was in transcribe (Figure 12) under the control of original tetR-VP16 (tTA) trans-activation system.These plasmids are by making up with making up the same way as of introducing among pTV3Lq+ and the pTV3Lq-, and finally are used for the effect of trans-activator aspect the ability of being reduced by tsiklomitsin that relatively change.Specifically, after carrier pTV3tTaTo-LqhIT2 equalled endization with Not I digestion (discharging the tetop-hGH-LqhIT2 box) and with Klenow, the Xho I/Sst II fragment (seeing above) of 0.8kbp being equalled endization was cloned among the carrier pTV3tTaTo-LqhIT2 by two kinds of directions.The four kinds of transferring plasmids that produce contain tetopp35LqA or the tetopp35mLqA with respect to two kinds of directions of tTA.These plasmids are named as ptTAlq+, ptTAlq-, ptTAMlq+ and ptTAMlq-.

(GIBCO/BRL, Gaithersburg is MD) with special transferring plasmid and the 0.5ug BaculoGold of 1ug to use lipofectin ^TM(CA) cotransfection advances about 1.0 * 10 to viral DNA for Pharmingen, San Diego ⁶The Sf-9 cell prepares recombinant virus.Collect the transfection supernatant in 5-7 days after the transfection, and be used for the plaque purification process of standard.Select 5 to 6 different separation plaques from the test of first run plaque, each plaque repurity once infects about 1.0 * 10 in 35mm tissue culture ware ⁶The preliminary amplicon virus of Sf-9 cell.With resulting viral stock at 75cm ²Preparation work stock in the tissue culture flasks.Zhi Bei 8 kinds of different recombinant viruses see table 1 in this way.

Table 1. recombinant virus is expressed LqhIT2 virus characteristic 1.vTV3lq+ and is contained tet inhibition with operon/identical 5 '-3 ' direction of p35/lq box under different trans-activators

-HR5 trans-activator 2.vTV3lq-except opposite identical 3.vTV3Mlq+ with operon/p35/lq direction with (1) except the modification p35 promotor that late period, expression was removed identical 4.vTV3Mlq-with (1) except with lq box direction on the contrary identical 5.vtTAlq+ with (3) to contain tet inhibition-VP16 trans-activator (identical with the lq box

5 '-3 ' direction) 6.vtTAlq-except opposite identical 7.vtTAMlq+ with lq box direction with (5) except the p35 promotor of modification identical 8.vtTAMlq-with (5) except identical with (7) on the contrary with lq box direction

The activity in vivo of the segregant of selecting from several these recombinant viruses is estimated in the feeding study of use Heliothis virescens larva in 3 age, to determine in infection, whether having active toxin to express, if have, whether the existence of tsiklomitsin or analogue (as doxycycline or duomycin) can increase viral LT with respect to untreated contrast ₅₀In preliminary study, several different processing have been carried out estimating to determine whether microbiotic can effectively be absorbed by insect midgut, if can not, whether the composition in the recipe influences absorption, and by whether promoting to absorb directly for insect injection of antibiotics or the local microbiotic that uses.When existing or not having 0.3% doxycycline (" Dc ", Sigma, Chemical Co Cat.No.D9891), the food filler that the vTV3Mlq+ of the Heliothis virescens larva feeding usefulness 2000PIBs that gave for 3 ages infects 24 hours.After 24 hours, one larva is transferred to not to be had in the contaminated foodstuff accordingly to infecting back 40 hours.Larva is handled in the following manner.

A: maintain on the foodstuff (0% or 0.3%Dc)

B: 0.3% the Dc aqueous solution of injection 4uL

C: maintain 2% (w/v) agar+or-0.3%Dc on; Or

D: use with the Dc acetone soln of 4uL0.3% is local

In handling A, B and D, larva is shifting back after the processing on their corresponding foodstuffs.Write down the paralysis symptom of insect different periods after infection.The result of this test is summarized in table 2, and this result provides evidence to show the LT of the virus of surveying ₅₀Be subjected to the influence of Dc, and this result depends on the approach that Dc is imported into insect.

Table 2. doxycycline infects the LT of Heliothis virescens larva to vTV3Mlq+ ₅₀Influence

Handle LT ₅₀A ¹Maintain 84-92 on 0% the foodstuff

Injection w/0.3%Dc 92-108

Maintain agar w/o Dc 84-92

The local 0.3%Dc 92-108B that uses ²Maintain on 0.3% the foodstuff 92

Injection w/0.3%Dc 116-135

Maintain agar+0.3%Dc 135

The local 0.3%Dc 108-116 that uses

¹The insect that on the 0%Dc foodstuff, infects and keep

²On the 0.3%Dc foodstuff, infect and infected by vTV3Mlq+ with the insect of keeping and on agar, injected or insect that feeding Dc keeps gets slowly than the contrast insect death that does not contact with Dc.This influence is especially strong when agar is handled, its LT ₅₀Increased 45-60%.

The activity in vivo of selected vtTAlq+, vtTAMlq-and vTV3Mlq+ virus is also measured with foodstuff medicament biological test.Transfer to untreated foodstuff the animal that will eat up foodstuff fully and (contain 0.3% (w/v) tsiklomitsin (Sigma, Cat.No.T3258), 0.3% (w/v) doxycycline (Sigma, Cat.No.D9891) or the soyflour of 1% (w/v) duomycin (Bioserve)/Fructus Hordei Germinatus insect substratum (Bioserv, Cat.No.9967)) before on, use 2000PIBs (to represent about 20 * LT for the Heliothis virescens larva feeding in 3 ages from vtTAlq+, vtTAMlq-, vTV3Mlq+, CG201 or AcMNPV (C6) ₅₀) the foodstuff medicament (50uL volume) that infects 24 hours.Temperature at foodstuff is lower than the foodstuff that after 50 ℃ microbiotic is added thawing.The antibiotic amount that selection is used for foodstuff makes the microbiotic amount of contact maximum but insect is not had harmful effect (being dysplasia).With insect 28 ℃ of cultivations, and be recorded in the poisoning/virus infection symptom that infects the back different time, and determine LT for every kind of virus of every kind of antibiotic treatment with VISTAT/TIME-for Analysis of Time Response Data program (Boyce ThompsonInstitute at Cornell University, 1990) ₅₀(table 3).CG201 is a kind of recombinant virus of expressing LqhIT2 and not having tsiklomitsin trans-activator system under the control of hr5/ie-1 promotor.

The LT of the reorganization trans-activation virus of the different tetracycline analogues of table 3. contact ₅₀Virus treated ¹LT ₅₀(h) vtTAlq+3 is untreated 78.4

Duomycin 95.0

Tsiklomitsin 97.4

Doxycycline 88.5vtTAlq+4 is untreated 88.2

Duomycin 99.9

Tsiklomitsin 96.9

Doxycycline 105.4vtTAlq+6 is untreated 66.6

Duomycin 78.3

Tsiklomitsin 87.4

Doxycycline 91.6vtTAMlq-2 is untreated 89.5

Duomycin 113.7

Tsiklomitsin 117.2

Doxycycline 113.8vTV3Mlq+1 is untreated 77.3

Duomycin 85.3

Tsiklomitsin 79.3

Doxycycline 81.9vTV3lq+5 is untreated 76.9

Duomycin 82.3

Tsiklomitsin 80.0

Doxycycline 75.9CG201 is untreated 52.6

Duomycin 59.6

Tsiklomitsin 58.4

Doxycycline 62.7AcMNPV (C6) is untreated 97.2

Duomycin 107.8

Tsiklomitsin 104.7

Doxycycline 97.5

¹Antibiotic treatment concentration all except that duomycin (1%) are 0.3%

In general, antibiotic treatment has increased all viral LT ₅₀But the influence of vtTAlq+6 and vtTAMlq-2 is obviously compared CG201 with these microbiotic and AcMNPV is big.Specifically, these two kinds of viruses show with respect to untreated contrast insect when having tsiklomitsin or doxycycline can increase by 30 to 40% kill and wound the time.This difference is 2 to 3 times of observed difference in the insect that infects of CG201.Other strain isolated of vtTAlq+ shows with respect to CG201 L ₅₀Be worth less increase.The strain difference of this same recombinant virus may be that each strain causes at the JND of expressing on trans-activator and the toxin, and is people's viewed normal phenomenons when using the rhabdovirus expression vector system.Therefore we have detected several different plaques to every kind of recon.The poorest recon of performance is to contain the recon that the trans-activator tetRIE1A (vTV3Mlq+) that is in modification transcribes the LqhIT2 under the control.The LT of these recons ₅₀AcMNPV with respect to wild-type significantly descends, though this shows that tetRIE1A can express by trans-activation LqhIT2, is expressed in when having microbiotic not to be suppressed.This may be that the tetR of trans-activator has partly produced the conformational change of not anticipating, thereby has damaged the interactional ability of trans-activator and tsiklomitsin or analogue because when the VP16 active region is replaced by the IE1 active region.

Summary is got up, selected AcMNPV recon is expressed LqhIT2 under the control of the trans-activator of a sensitive tetracycline biological test results shows, when infect can interact with trans-activator and a kind of microbiotic of attenuatings/delay insect selectivity toxin expression in the presence of when carrying out, the LT of recombinant virus ₅₀Can be prolonged greatly.

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P Moss, B Proceedings of the National Academy of Sciences (1989) 86, pp 2549-2533 (38) Deutschle, U Pepperkok, R Wang, F Giordano, T.J McAllister.W.T Ansorge, W Bujard, H Proceedings of the National Academy of Sciences (1989) 86, pp 5400-5405 (39) Deutschle, U Hipskind, R.A Bujard, H science (1990) 248, pp 480-483 (40) Labow, M.A Baim, S.B Shenk, T Levine, A.J molecule and cytobiology (1990) 10, pp 3343-3356 (41) Baim, S.B Labow, M.A Levine, A.J Shenk, T Proceedings of the National Academy of Sciences (1991) 88, pp 5072-5076 (42) Wyborski﹠Short, nucleic acids research 19, pp 4647-4653 (43) Bertrand, K.P Postle, K Wray, L.V Reznikoff, W.S gene (1983) 23, pp 149-156 (44) Hillen, W Schollmeier, K Gatz, C molecular biology assorted (1984) 172, pp 185-201 (45) Gatz, C Kaiser, A Wendenburg, R molecule and General Genetics (1991) 227, pp 229-237 (46) Wirtz, E Clayton C science (1995) 268,1179-1182 (47) Gossen, M Bujard, H Proceedings of the National Academy of Sciences (1992) 89, pp 5547-5551, (48) W.O.Pub.No.94/29442 (49) M é vel-Ninio, M Mariol, M.-C Gans, M (1989) (50) Rubin and Spradling (1983) (51) Handler et al. 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(A) title: E.I.DUPONT DE NEMOURS AND COMPANY

(B) street: 1007 MARKET STREET

(C) city: WILMINGTON

(D) state: DELAWARE

(E) country: U.S.A.

(F) postcode: 19898

(G) phone: 302-892-8112

(H) fax: 302-773-0164

(I) telegram: 6717325 (ⅱ) denomination of invention: the production of recombinant baculovirus (ⅲ) sequence number: 26 (ⅳ) computer-reader form

(A) media type: 3.50 inches floppy disks

(B) computer: IBM PC compatibility

(C) operating system: MICROSOFT WINDOWS 3.1

(D) the existing request for data of software: MICROSOFT WORD 6.0A (ⅴ):

(A) application number:

(B) applying date:

(C) classification number: (ⅵ) request for data formerly:

(A) application number: 60/009,120

(B) applying date: December 22 nineteen ninety-five (ⅶ) proxy/business quarters's information:

(A) name: FLOYD, LINDA AXAMETHY

(B) login numbering: 33,692

(C) information of reference/document number: BA-9078-A (2) SEQ ID NO:1: (ⅰ) sequence signature:

(A) length: 8 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:1:CAGATCTG (2) SEQ ID NO:2: (ⅰ) sequence signature:

(A) length: 407 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ⅱ ) ：DNA ( ) ( ⅹⅰ ) ：SEQ ID NO：2：CTCGAGTTTA CCACTCCCTA TCAGTGATAG AGAAAAGTGA AAGTCGAGTT TACCACTCCC 60TATCAGTGAT AGAGAAAAGT GAAAGTCGAG TTTACCACTC CCTATCAGTG ATAGAGAAAA 120GTGAAAGTCG AGTTTACCAC TCCCTATCAG TGATAGAGAA AAGTGAAAGT CGAGTTTACC 180ACTCCCTATC AGTGATAGAG AAAAGTGAAA GTCGAGTTTA CCACTCCCTA TCAGTGATAG 240AGAAAAGTGA AAGTCGAGTT TACCACTCCC TATCAGTGAT AGAGAAAAGT GAAAGTCGAG 300CTCAACAGTG GGAGAGAAGG GGCCAGGGTA TAAAAAGGGC CCACAAGAGA CCAGCTCAAG 360GATTCCAAGG CCGCGGCCCC GAATTCGAGC TCGGTACCCG GGGATCC 407 ( 2 ) SEQ ID NO：3： ( ⅰ ) ：

(A) length: 10 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:3:AGCGGCCGCT (2) SEQ ID NO:4: (ⅰ) sequence signature:

(A) length: 75 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:4:ACGATGAATT CGGATCCTAT GAAGATCCTC CTTGCTATTG CCCTTATGCT TAGCACCGTG 60ATGTGGGTGA GCACC 75 (2) SEQ ID NO:5: (ⅰ) sequence signature:

(A) length: 51 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:5:GACGGCTACA TCAAACGCCG CGACGGCTGC AAAGTGGCCT GCCTTATCGG C 51 (2) SEQ ID NO:6: (ⅰ) sequence signature:

(A) length: 51 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:6:AACGAGGGCT GCGACAAAGA GTGCAAAGCC TACGGCGGCA GCTACGGCTA C 51 (2) SEQ ID NO:7: (ⅰ) sequence signature:

(A) length: 51 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:7:TGCTGGACCT GGGGCCTCGC ATGCTGGTGC GAGGGCCTCC CCGACGACAA A 51 (2) SEQ ID NO:8: (ⅰ) sequence signature:

(A) length: 48 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:8:ACCTGGAAAA GCGAGACCAA CACCTGCGGC TAAGGATCCT CTAGAGTC 48 (2) SEQ ID NO:9: (ⅰ) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:9:CACCCACATC ACGGTGCTAA GCATAAGGGC AATAGCAAGG AGGATCTTCA TAGGATCCGA 60ATTCATCGT 69 (2) SEQ ID NO:10: (ⅰ) sequence signature:

(A) length: 51 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:10:AAGGCAGGCC ACTT TGCAGC CGTCGCGGCG TTTGATGTAG CCGTCGGTGC T 51 (2) SEQ ID NO:11: (ⅰ) sequence signature:

(A) length: 51 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:11:GTAGCTGCCG CCGTAGGCTT TGCACTCTTT GTCGCAGCCC TCGTTGCCGA T 51 (2) SEQ ID NO:12: (ⅰ) sequence signature:

(A) length: 51 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:12:GTCGGGGAGG CCCTCGCACC AGCATGCGAG GCCCCAGGTC CAGCAGTAGC G 51 (2) SEQ ID NO:13: (ⅰ) sequence signature:

(A) length: 54 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:13:GACTCTAGAG GATCCTTAGC CGCAGGTGTT GGTCTCGCTT TTCCAGGTTT TGTC 54 (2) SEQ ID NO:14: (ⅰ) sequence signature:

(A) length: 243 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:14:ATGAAGATCC TCCTTGCTAT TGCCCTTATG CTTAGCACCG TGATGTGGGT GAGCACCGAC 60GGCTACATCA AACGCCGCGA CGGCTGCAAA GTGGCCTGCC TTATCGGCAA CGAGGGCTGC 120GACAAAGAGT GCAAGGCCTA CGGCGGCAGC TACGGCTACT GCTGGACCTG GGGCCTCGCA 180TGCTGGTGCG AGGGCCTCCC CGACGACAAA ACCTGGAAAA GCGAAACCAA CACCTGCGGC 240TAA 243 (2) SEQ ID NO:15: (ⅰ) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:15:ATGTCTAGAT TAGATAAAAG 20 (2) SEQ ID NO:16: (ⅰ) sequence signature:

(A) length: 26 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:16:AGATCTGGAC CCACTTTACA TTTAAG 26 (2) SEQ ID NO:17: (ⅰ) sequence signature:

(A) length: 623 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ⅱ ) ：DNA ( ) ( ⅹⅰ ) ：SEQ ID NO：17：ATGTCTAGAT TAGATAAAAG TGATTAACAG CGCATTAGAG CTGCTTAATG AGGTCGGAAT 60CGAAGGTTTA ACAACCCGTA AACTCGCCCA GAAGCTAGGT GTAGAGCAGC CTACATTGTA 120TTGGCATGTA AAAAATAAGC GGGCTTTGCT CGACGCCTTA GCCATTGAGA TGTTAGATAG 180GCACCATACT CACTTTTGCC CTTTAGAAGG GGAAAGCTGG CAAGATTTTT TACGTAATAA 240CGCTAAAAGT TTTAGATGTG CTTTACTAAG TCATCGCGAT GGAGCAAAAG TACATTTAGG 300TACACGGCCT ACAGAAAAAC AGTATGAAAC TCTCGAAAAT CAATTAGCCT TTTTATGCCA 360ACAAGGTTTT TCACTAGAGA ATGCATTATA TGCACTCAGC GCTGTGGGGC ATTTTACTTT 420AGGTTGCGTA TTGGAAGATC AAGAGCATCA AGTCGCTAAA GAAGAAAGGG AAACACCTAC 480TACTGATAGT ATGCCGCCAT TATTACGACA AGCTATCGAA TTATTTGATC ACCAAGGTGC 540AGAGCCAGCC TTCTTATTCG GCCTTGAATT GATCATATGC GGATTAGAAA AACAACTTAA 600GATGTGAAAG TGGGTCCAGA TCT 623 ( 2 ) SEQ ID NO：18： ( ⅰ ) ：

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:18:CGGGATCCAT GACGCAAATT AATTTTAACG 30 (2) SEQ ID NO:19: (ⅰ) sequence signature:

(A) length: 35 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:19:CCAGATCTTT AACCTTGTGA ATTGTCCAAG TATTC 35 (2) SEQ ID NO:20: (ⅰ) sequence signature:

(A) length: 455 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ⅱ ) ：DNA ( ) ( ⅹⅰ ) ：SEQ ID NO：2O：CGGGATCCAT GACGCAAATT AATTTTAACG CGTCGTACAC CAGCGCTTCG ACGCCGTCCC 60GAGCGTCGTT CGACAACAGC TATTCAGAGT TTTGTGATAA ACAACCCAAC GACTATTTAA 120GTTATTATAA CCATCCCACC CCGGATGGAG CCGACACGGT GATATCTGAC AGCGAGACTG 180CGGCACGTTC AAACTTTTTG GCAAGCGTCA ACTCGTTAAC TGATAATGAT TTAGTGGAAT 240GTTTGCTCAA GACCACTGAT AATCTCGAAG AAGCAGTTAG TTCTGCTTAT TATTCGGAAT 300CCCTTGAGCA GCCTGTTGTG GAGCAACCAT CGCCCAGTTC TGCTTATCAT GCGGAATCTT 360TTGAGCATTC TGCTGGTGTG AACCAACCAT CGGCAACTGG AACTAAACGG AAGCTGGACG 420AATACTTGGA CAATTCACAA GGTTAAAGAT ACTGG 455 ( 2 ) SEQ ID NO：21： ( ⅰ ) ：

(A) length: 354 amino acid

(B) type: amino acid

(C) chain: strand

( D ) ： ( ⅱ ) ： ( ⅹⅰ ) ：SEQ ID NO：21：Met Ser Arg Leu Asp Lys Ser Lys Val Ile ASn Ser Ala Leu Glu Leu1 5 10 15Leu ASn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His 50 55 60Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg65 70 75 80Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125Aan Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr145 150 155 160Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser Arg 195 200 205Ser Met Thr Gln Ile Asn Phe Asn Ala Ser Tyr Thr Ser Ala Ser Thr 210 215 220Pro Ser Arg Ala Ser Phe Asp Asn Ser Tyr Ser Glu Phe Cys Asp Lys225 230 235 240Gln Pro Asn Asp Tyr Leu Ser Tyr Tyr Asn His Pro Thr Pro Asp Gly 245 250 255Ala Asp Thr Val Ile Ser Asp Ser Glu Thr Ala Ala Arg Ser Asn Phe 260 265 270Leu Ala Ser Val Asn Ser Leu Thr Asp Asn Asp Leu Val Glu Cys Leu 275 280 285Leu Lys Thr Thr Asp Asn Leu Glu Glu Ala Val Ser Ser Ala Tyr Tyr 290 295 300Ser Glu Ser Leu Glu Gln Pro Val Val Glu Gln Pro Ser Pro Ser Ser305 310 315 320Ala Tyr His Ala Glu Ser Phe Glu His Ser Ala Gly Val Asn Gln Pro 325 330 335Ser Ala Thr Gly Thr Lys Arg Lys Leu Asp Glu Tyr Leu Asp Asn Ser 340 345 350Gln Gly ( 2 ) SEQ ID NO：22： ( ⅰ ) ：

(A) length: 205 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:22:GGATCCAGAC CATGATAAGA TACATTGATG AGTTTGGACA AACCACAACT AGAATGCAGT 60GAAAAAAATG CTTTATTTGT GAAATTTGTG ATGCTATTGC TTTATTTGTA ACCATTATAA 120GCTGCAATAA ACAAGTTAAC AACAACAATT GCATTCATTT TATGTTTCAG GTTCATTTTT 180AGGTGTGGGA GGTTTTTTCG GATCC 205 (2) SEQ ID NO:23: (ⅰ) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:23:CTATAAATAT TCAACGTTGC TTGTATTAAG TGAGCATTTG AGCTTTACCA AGGATCCGCG 60GCCGCAGCT 69 (2) SEQ ID NO:24: (ⅰ) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:24:GCGGCCGCGG ATCCTTGGTA AAGCTCAAAT GCTCACTTAA TACAAGCAAC GTTGAATATT 60TATAGAGCT 69 (2) SEQ ID NO:25: (ⅰ) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: the information of SEQ ID NO:25:CTATAAATAT TCAACGTTGC TTGTATTCAG TGAGCATTTG AGCTTTACCA AGGATCCGCG 60GCCGCAGCT 69 (2) SEQ ID NO:26: (ⅰ) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: line style (ⅱ) molecule type: DNA (genome) (ⅹ ⅰ) sequence statement: SEQ ID NO:26:GCGGCCGCGG ATCCTTGGTA AAGCTCAAAT GCTCACTGAA TACAAGCAAC GTTGAATATT 60TATAGAGCT 69

Claims

1. the method expressed by the insecticidal proteins of the mosaic gene coding that exists in the recombinant baculovirus genome of a control comprises:

(b) make up a recombination rhabdovirus expression vector, this expression vector contains second mosaic gene, this mosaic gene contains the 3rd nucleic acid fragment of second promotor of encoding, second promotor influenced by the adjusting albumen of second nucleic acid fragment coding, and the 3rd nucleic acid fragment operably is connected on the 4th nucleic acid fragment of a kind of insecticidal proteins of coding;

(c) recombination rhabdovirus expression vector that makes up in the step (b) is stably imported in the recombinant insect cell that makes up in the step (a); And

(d) under the environment of supporting shaft-like virus replication, keep the recombinant insect cell for preparing in the step (c); Wherein the proteic expression of adjusting of second nucleic acid fragment coding influences the expression of insecticidal proteins.

2. the method expressed by the insecticidal proteins of the mosaic gene coding that exists in the recombinant baculovirus genome of a control comprises:

(a) make up a recombination rhabdovirus expression vector that contains following ingredients, (1) first mosaic gene, this mosaic gene contains first nucleic acid fragment of first promotor of encoding, and first nucleic acid fragment operably is connected in one of coding can be influenced on proteic second nucleic acid fragment of adjusting of genetic expression; (2) second mosaic genes, this mosaic gene contain the 3rd nucleic acid fragment of second promotor of encoding, and second promotor regulated albumen to be influenced, and the 3rd nucleic acid fragment operably is connected on the 4th nucleic acid fragment of a kind of insecticidal proteins of coding;

(b) recombination rhabdovirus expression vector with step (a) stably imports in a kind of insect cell; And

(c) under the environment of supporting shaft-like virus replication, keep the insect cell for preparing in the step (b); Wherein regulating proteic expression influences the expression of insecticidal proteins.

3. method of producing the pesticidal recombinant baculovirus comprises:

(b) make up a recombination rhabdovirus expression vector, this expression vector contains second mosaic gene, this mosaic gene contains the 3rd nucleic acid fragment of second promotor of encoding, second promotor influenced by the adjusting albumen of second nucleic acid encoding, and the 3rd nucleic acid fragment operably is connected on the 4th nucleic acid fragment of a kind of insecticidal proteins of coding;

(c) recombination rhabdovirus expression vector with step (b) imports in the recombinant insect cell of step (a);

(d) under the environment of supporting shaft-like virus replication, in an external cell culture or in insect body complete, that live, keep the recombinant insect cell for preparing in the step (c); Wherein the proteic expression of adjusting of second nucleic acid fragment coding influences the expression of insecticidal proteins; And

(e) collect progeny virus.

4. method of producing the pesticidal recombinant baculovirus comprises:

(a) make up a recombination rhabdovirus expression vector that contains following ingredients, (1) first mosaic gene, this mosaic gene contains first nucleic acid fragment of first promotor of encoding, and first nucleic acid fragment operably is connected in one of coding can be influenced on proteic second nucleic acid fragment of adjusting of genetic expression; (2) second mosaic genes, this mosaic gene contains the 3rd nucleic acid fragment of second promotor of encoding, second promotor influenced by the adjusting albumen of second nucleic acid fragment coding, and the 3rd nucleic acid fragment operably is connected on the 4th nucleic acid fragment of a kind of insecticidal proteins of coding;

(b) recombination rhabdovirus expression vector with step (a) imports in a kind of insect cell; And

(c) under the environment of supporting shaft-like virus replication, in an external cell culture or in insect body complete, that live, keep the insect cell for preparing in the step (b); Wherein the proteic expression of adjusting of second nucleic acid fragment coding influences the expression of the insecticidal proteins of the 4th nucleic acid fragment coding; And

(d) collect progeny virus.

5. the method for claim 4, wherein in step (a):

(ⅰ) first nucleic acid fragment that operably is connected with proteic second nucleic acid fragment of coding and regulating is a tsiklomitsin trans-activator;

(ⅱ) second promotor of the 3rd nucleic acid fragment coding contains one or more tsiklomitsins manipulation sites that operably are connected in a minimal promoter sequence;

(ⅲ) insecticidal proteins of the 4th nucleic acid fragment coding is a kind of insect selectivity neurotoxin; And wherein, the environment of the shaft-like virus replication of support comprises the existence of tsiklomitsin or a kind of tetracycline analogue of effective dose in step (c), make the tsiklomitsin trans-activator not handle the site and combine, operably be connected in the sequence-directed genetic expression of minimal promoter that tsiklomitsin is handled the site thereby make it to induce with the tsiklomitsin that comprises the 3rd nucleic acid fragment.

6. the recombinant insect cell that contains mosaic gene, mosaic gene comprises first nucleic acid fragment of first promotor of encoding, first nucleic acid fragment operably is connected in second nucleic acid fragment, and second nucleic acid fragment coding can influence the adjusting albumen of the genetic expression of being instructed by second promotor.

7. the transgenic insect that contains one or more recombinant insect cells, contain mosaic gene in the recombinant insect cell, mosaic gene contains first nucleic acid fragment of first promotor of encoding, first nucleic acid fragment operably is connected on second nucleic acid fragment, and second nucleic acid fragment coding can influence the adjusting albumen of the genetic expression of being instructed by second promotor.

8. the transgenic insect of the recombinant insect cell of claim 6 or claim 7, wherein regulating albumen is the tsiklomitsin trans-activator.

9. the recombination rhabdovirus expression vector that contains mosaic gene, first nucleic acid fragment that contains the promotor of encoding in the mosaic gene, the adjusting albumen that this promotor is expressed by the recombinant insect cell of claim 6 influences, and first nucleic acid fragment operably is connected on second nucleic acid fragment of encoding insecticidal proteins.

10. the recombination rhabdovirus expression vector of claim 9, wherein first nucleic acid fragment contains one or more tsiklomitsins that operably are connected in a minimal promoter and handles sites.