WO1992011363A1 - Recombinant molecules useful for producing insecticidal microbes - Google Patents

Abstract

Insecticidal microbes with enhanced insecticidal activity are produced by the introduction into the microbial genome of a genetic sequence coding for an insect selective toxin. Also disclosed are methods for enhancing the insecticidal activity of microbes and methods of producing insect selective toxin.

Description

Recombinant molecules useful for producing insecticidal microbes

The present invention relates to insecticidal microbes with enhanced insecticidal activity. More particularly, the present invention relates to insecticidal microbes in which a genetic sequence coding for an insect selective toxin has been introduced into the genome of the microbe.

Venom is defined as a mixture of substances which are produced in specialized glandular tissues in the body of venomous animal. The venom is introduced into the body of its prey or opponent by the aid of a stinging-piercing apparatus in order to paralyze and/or kill it. Scorpions contain in their venom a number of proteins, or neurotoxins, which are toxic and act on the nervous system. The individual neurotoxins differ in their potency on various species of animals.

The venoms derived from scorpions belonging to the Buthinae subfamily have three main groups of polypeptide neurotoxins which modify axonal sodium conductance. One group of neurotoxins are the a-toxins, which specifically affect mammals through an extreme prolongation of the action potentials due to a slowing or blockage of the sodium channel in activation (Catterall, 1984; Rochat et al., 1979). The second group of neurotoxins are the depressant insect selective toxins which induce a progressively developing flaccid paralysis of insects by the blockage of action potentials substantially due to the suppression of sodium current (Lester et al., 1982; Zlotkin et al., 1985). The third group of neurotoxins are the excitatory insect selective toxins which cause an immediate (knock down) spastic paralysis of insects by the induction of repetitive firing in their motor nerves due to an increase of the sodium peak current and the voltage dependent slowing of its inactivation (Walther et al., 1976; Pelhate and Zlotkin, 1981).

The scorpion venom derived insect toxins were detected and their isolation was monitored by the typical responses of Sarcophaga blowfly larva which develop an immediate and transient contraction paralysis when injected with the excitatory toxins and progressively developing flaccidity for the depressant toxins (Zlotkin et al., 1971b; Lester et al., 1982). In spite of the opposite symptomatology induced by the depressant and excitatory insect toxins, both affect exclusively sodium conductance and share the same binding site in the insect's neuronal membranes (Zlotkin et al., 1985; Gordon et al., 1984).

Insect-selective toxins have also been identified in venoms from a number of other arthropods (Zlotkin, 1985). The venoms of braconid wasps are highly toxic to lepidopterous larvae. The venom of the braconid Bracon hebetor causes a flaccid paralysis in lepidopterous larvae by inducing presynaptic interruption of the excitatory glutaminergic transmission at the insect neuromuscular junction (Piek et al., 1982). The venoms of solitary wasps are toxic to a large number of insects and spiders from different orders (Rathmeyer, 1962). An example of these venoms is the venom of Philanthus triangulum which induces in insects a flaccid paralysis substantially due to presynaptic blockage of neuromuscular transmission; this venom affects both excitatory and inhibitory transmission (May and Piek, 1979). The venom of the black widow spider, Latrodectus mactans, contains components which are neurotoxic to insects, but not to mammals, and other components with the opposite selectivity (Fritz et al., 1980; Ornberg et al., 1976).

Polyhedrosis viruses have been identified as potentially useful to express foreign genes in cells. US 4,745,051 discloses a method for producing a recombinant baculovirus expression vector capable of expression of a selected gene in a host insect cell utilizing baculoviruses from Autographa californica, Trichoplusia ni, Rachiplusia ou and Galleria mellonella. Bishop (1988) relates to genetic engineering of baculoviruses for improved pesticidal activity. At page 3, Bishop discusses the possibility of including foreign genes in a nuclear polyhedrosis virus of the alfalfa looper moth Autographa californica.

US 4,870,023 relates to recombinant Autographa californica and Heliothis zea baculoviruses which encode fusion polyhedrin proteins capable of forming occlusion bodies containing foreign peptides. At column 40, it is suggested that genes coding for neurotoxins may be expressed in order to increase the insecticidal activity of baculoviruses.

EP 222,412, discloses methods of producing insulin-like growth factor I (IGF-1) using recombinant Bombyx mori nuclear polyhedrosis viruses. Maeda (1989b) describes gene transfer vectors of the Bornbyx mori nuclear polyhedrosis virus, and their use for expression of foreign genes in insect cells. Adachi et al. (1989) relates to the cDNA structure of the bombyxin protein. EP 225,777, relates to recombinant viruses containing DNA sements of two different nuclear polyhedroses viruses from different host insects. The recombinant virus is disclosed by EP 225,777 to be effective in destroying or controlling both species of host insects. Maeda (1989c) discloses that genetically engineered insect viruses containing a recombinant gene encoding the diuretic hormone of the tobacco hornworm resulted in increased insecticidal activity of the baculovirus.

Miller et al. (1983) contains the suggestion that recombinant DNA technology could be used to enhance the toxicity of a virus, for example by introducing an insect-specific toxin gene into the genome of Autographa californica. Until the present invention, however, attempts to express genes coding for insect-selective neurotoxins and thereby increase the insecticidal activity of recombinant baculoviruses have been unsuccessful. Carbonell et al. (1988) attempted to use insect-specific scorpion neurotoxins to improve the effectiveness of baculovirus pesticides. Carbonell et al. cloned the gene encoding insectotoxin-1 of the scorpion Buthus eupeus in E. coli, and attempted to express the gene in Autographa californica nuclear polyhedrosis virus. However, Carbonell et al. reported that biological activity of the toxin was not observed, and no paralytic activity was detected. Carbonell and Miller (1987) further report that insect baculoviruses did not express genes in infected mammalian cell lines. Dee et al. (1990) report the expression of insecticidal neurotoxin Aalt under the control of the Moloney murine sarcoma virus in cultured mouse cells. However, Dee et al. report that they were unsuccessful in expressing the neurotoxin in E. coli.

Thus, the identification of a foreign gene effective for insect control is crucial for the construction of a baculovirus insecticide. Although certain toxins, an insect diuretic hormone and a juvenile hormone esterase have shown insecticidal and physiological effects on infected insects when the proteins or baculoviruses carrying the corresponding genes were applied, recombinant viruses did not show a strong increase in potency when compared to control virus (Maeda, 1989a; Hammock et al., 1990).

In the present invention, the disadvantages of the prior art in being unsuccessful in expressing scorpion neurotoxins in baculoviruses have largely been overcome.

Accordingly, it is one object of the present invention to provide baculoviruses with enhanced insecticidal activity.

It is another object of the present invention to provide expression systems to produce insect-selective scorpion neurotoxins. The present invention is directed to the production of insecticidal microbes which possess a genetically enhanced toxicity to insects through the introduction of genes which will induce the production of insect-selective toxins, normally found in scorpions, by the microbes. This invention further relates to methods of enhancing the toxicity of the insecticidal microbes comprising incorporating into the genome of the microbe a recombinant DNA molecule comprising a genetic sequence coding for a scorpion toxin selective for insects.

In order to use the insect-selective toxins of the present invention to enhance the toxicity of insecticidal microbes, a genetic sequence coding for the insect-selective toxin is introduced into the genome of the microbe. For example, the toxicity of the Bornbyx mori nuclear polyhedrosis virus may be enhanced by introducing a recombinant DNA molecule coding for an insect toxin. The coding genetic sequence may be operably linked to a signal sequence in order to aid the toxicity. The signal sequence may be isolated from an insect protein, for example, the signal sequence of the Bornbyx mori protein bombyxin (Adachi et al., 1989).

In a preferred embodiment of the present invention, the recombinant baculovirus comprises a genetic coding sequence which codes for the production of the scorpion insect toxin AaIT. The toxin AaIT from the venom of the scorpion A. australis is a single polypeptide chain of 70 amino acids cross-linked by four disulfide bonds (Darbon et al., 1982). Although scorpion venom contains various types of toxins, AaIT has toxicity only towards insects and is reported to be non-toxic to isopods and mammals (Zlotkin et al., 1971a). Electrophysical and toxin binding studies using insects, crustaceans, and arachnids have demonstrated that AaIT exclusively affects insect nervous systems (Walther et al., 1976; Teitelbaum et al., 1979; Gordon et al., 1984).

In one embodiment of the present invention, a synthetic gene encoding AaIT behind a secretion signal sequence from the silkworm neuropeptide bombyxin (Adachi et al., 1989) is constructed and inserted into a transfer vector of Bornbyx mori nuclear polyhedrosis virus (BmNPV), pBK273, after the strong polyhedrin gene promoter. Three μg of the resulting recombinant plasmid pBmAaIT and two μg of BmNPV DNA are cotransfected into BmN cells. A recombinant virus, BmAaIT, lacking polyhedra production, is isolated by a plaque assay from the cotransfected culture supernatant by the method described in Maeda et al. (1985); Maeda (1989b). When BmN cells are separately infected with

BmAaIT, BmDH5, carrying the diuretic hormone gene3, and a control BmM14 with a deletion in the polyhedrin gene, all infections show the same cytopathic effects. BmM14 was constructed by homologous recombination of BmNPV and a plasmid containing a deletion (between 70 and 166 nucleotides from the translational initiation site) in the polyhedrin coding area generated by Bal31 digestion, as described in Maeda et al. (1985). The deletion in the BmM14 polyhedrin gene caused production of a major polypeptide similar to polyhedra, but uncrystalized.

The physiological and insecticidal effects of BmAaIT were examined in the silkworm, B. mori, by the method described in Maeda et al. (1985) and Maeda (1989). When second instar larvae are injected with 10⁵ p.f.u. (plaque forming units) of virus, they show dramatic changes in behavior at about 40 hours post injection. The larvae show continuous rotations of the head, dorsal arching, and shaking of the body. All larvae stop feeding between 45 to 55 hours post injection and do not move, although they are still able to respond to prodding. All larvae die by 60 hours post injection. This is approximately a 40 % increase in the speed of insect killing compared to the control BmM14 virus. As reported in Maeda (1989c), infection with BmDH5 virus causes about a 20 % increase in the speed of killing, but the larvae infected with BmDH5 do not show any apparent changes in behavior.

To confirm the production of active AaIT peptide in infected silkworm larvae, hemolymph at 55 hours post-injection is injected into larvae of the blow fly, Sarcophaga falculata. Symptoms following injection occurred immediately and are similar to those induced by AaIT purified from venom. However, hemolymph from larvae infected with control BmM14 virus produces no acute symptoms upon injection. A rough estimate based upon this biological assay indicates that the hemolymph contained 5 μg/ml of toxin. The expression of the AaIT gene in infected 5th instar larvae is also confirmed by immunoblot of hemolymph samples taken at various times after infection. An immunoreactive band which co-migrated with authentic AaIT is detected in the 48 hour and 55 hour samples showing that the expressed material and the toxin purified form venom have similar specific activity. The amount of material in the 55 hour sample is about 5 μg/ml. The estimated molecular weight of the toxin is identical with that of the native toxin, indicating the correct cleavage of the heterologous signal sequence in insect larvae and secretion of the peptide toxin. A recombinant BmNPV construct without the signal sequence does not secrete the peptide efficiently, demonstrating the necessity of the signal sequence for secretion. The DNA fragment containing the AaIT coding sequence was preceded by the sequence GAGCTCGAATTCATG containing a SacI site and methionine for the transcriptional start signal without any insertion of nucleotides. This AaIT gene without a signal sequence was transferred into pBE283. The resultant plasmid was cotransfected with BmNPV T3 DNA into BmN cells and a recombinant BmNPV was isolated as described in Maeda (1989b). Larvae infected with this virus show paralytic symptoms at 60 hours p.i., but the time to death is similar to that caused by the control virus.

To determine whether or not the effects of the BmAaIT virus are caused directly by the expressed scorpion toxin, native scorpion toxin is purified by HPLC from commercially obtained venom (Sigma, Latoxan) and injected into second instar silkworm larvae (0.6 μl per 5 to 10 mg larva). Doses over 300 ng cause death in most larvae within 24 hours with onset of symptoms at 30 min post injection. Doses of 12 to 60 ng cause effects similar to infection with BmAaIT virus in more than half of the larvae within 24 hours; however, these larvae recover within 48 hours. Low doses (less than 6 ng) have no apparent effects. The abnormal behavior of larvae injected with the native toxin is the same as that observed following infection with BmAaIT. The sensitivity of the silkworm to AaIT is comparable to that of the cutworm, Spodoptera littoralis, for which de Dianous et al. (1987) reported an LD₅₀ of 130 ng/10 mg body weight These data indicate that the BmNPV-silkworm system is useful for screening toxin genes for pest control. This system may be useful as a new approach in characterizing neurotoxin molecules and their effects in in vivo systems.

Baculoviruses have long been attractive biological agents for insect control, however, one limitation to their use has been the slow speed of killing which allows the pest insects to continue to damage crops. The present invention demonstrates that the incorporation into the virus' genome of a genetic sequence coding for the production of an insect specific scorpion toxin, such as the AaIT toxin, can significantly increase a baculovirus' insecticidal activity in a manner consistent with sodium channel blocking caused by chemical insecticide. The AaIT toxin is also highly insect specific with doses of 1 mg/mouse reported to produce no effects in mice (de Dianous et al., 1987).

The present invention also includes methods of producing insect-selective toxins utilizing expression vector systems. An expression vector is prepared, which expression vector comprises a recombinant DNA molecule which contains a genetic sequence coding for an insect-selective protein neurotoxin. The expression vector is introduced into the genome of an expression system host. Suitable hosts include bacteria such as E. coli, and yeast, including the strain Saccharomyces cerevisiae. Other suitable expression system hosts in clude insect cells grown in culture. These insect cells may be infected with a baculovirus. Alternatively, the baculovirus may be used to infect the cells of a living insect, and the insect cells used as the expression system host. The expression system host is then allowed to produce an expression supernatant. The insect-selective toxin may then be isolated from the expression supernatant.

Figures

Fig. 1 shows the restriction map of pBK283, the Bombyx mori baculovirus transfer vector (cf. Maeda, 1989b).

Fig.2 shows the restriction map of pCIB4223, which is comprised of pBK283, the bombyxin signal sequence and the gene coding for the production of the toxin AaIT.

Fig. 3 shows the restriction map of pCIB4222, the expression vector for ompA

leader- AaIT fusion protein.

I. Insect Selective Toxins Derived from Venom

The amino acid sequences of various insect toxins (AaIT from Androctonus australis, LqqIT2 from Leiurus quinquestriatus quinquestriatus, BJIT2 from Buthotus judaicus, LqhIT2 from Leiurus quinquestriatus hebraeus, SmpIT2 from Scorpio maurus palmatus and LqhP35 from Leiurus quinquestriatus hebraeus) is published in EP 374,753 (page 4, lines 38 to 54 and page 5, line 2).

II. Genetic Engineering of Insect Selective Toxins

This invention further comprises the genetic sequences coding for the insect selective toxins, expression vehicles containing the genetic sequence, hosts transformed therewith, the toxin produced by such transformed host expression, and uses for the toxin.

The sequence of amino acid residues is designated herein either through the use of their commonly employed single-letter designations. A listing of these one-letter and the three-letter designations may be found in textbooks such as Lehninger (1975). When the amino acid sequence is listed horizontally, the amino terminus is intended to be on the left end whereas the carboxy terminus is intended to be at the right end. Using the amino acid sequence information, the DNA sequences capable of encoding them are examined in order to clone the gene encoding the toxin. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson et al., 1977).

Although occasionally an amino acid sequence may be encoded by only a single oligonucleotide, frequently the amino acid sequence may be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of this set contain oligonucleotides which are capable of encoding the peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the nucleotide sequence of the gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.

Using the genetic code (Watson et al., 1977), one or more different oligonucleotides can be identified, each of which would be capable of encoding the toxin peptides. The probability that a particular oligonucleotide will, in fact, constitute the actual toxin encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Such "codon usage rules" are disclosed by Lathe (1985). Using the "codon usage rules" of Lathe, a single oligonucleotide, or a set of

oligonucleotides, that contains a theoretical "most probable" nucleotide sequence capable of encoding the toxin peptide sequences is identified.

The oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence capable of encoding the toxin gene fragments is used to identify the sequence of a complementary oligoncleotide or set of oligonucleotides which is capable of hybridizing to the "most probable" sequence, or set of sequences. An oligonucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the toxin gene (Maniatis et al., 1982).

Thus, in summary, the actual identification of toxin peptide sequences permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide. By constructing an oligonucleotide complementary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides), one obtains a DNA molecule (or set of DNA molecules), capable of functioning as a probe to identify and isolate the toxin gene.

The cloning and use of the various toxins described above will hereinafter be described generally as "a toxin." It should be understood that any of the above-detailed toxins may be used as described in any of the methods according to this invention. The process for genetically engineering the toxin according to the invention is facilitated through the cloning of genetic sequences which are capable of encoding the toxin and through the expression of such genetic sequences. As used herein, the term "genetic sequences" is intended to refer to a nucleic acid molecule (preferably DNA). Genetic sequences which are capable of encoding the toxin may be derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof.

Genomic DNA may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with the 5' promoter region of the toxin gene sequences. To the extent that a host cell can recognize the transcriptional regulatory and translational initiation signals associated with the expression of the protein, then the region 5' may be retained and employed for transcriptional and translational initiation regulation.

For cDNA, the cDNA may be cloned and the resulting clone screened with an appropriate probe for cDNA coding for the desired sequences. Once the desired clone has been isolated, the cDNA may be manipulated in substantially the same manner as the genomic DNA. However, with cDNA there will be no introns or intervening sequences. For this reason, a cDNA molecule which encodes the toxin is the preferred genetic sequence of the present invention.

Genomic DNA or cDNA may be obtained in several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from a cell which produces the toxin and used to produce cDNA by means well known in the art. Such suitable DNA preparations are enzymatically cleaved, or randomly sheared, and ligated into recombinant vectors to form a gene library. Such vectors can then be screened with the above-described oligonucleotide probes in order to identify a toxin encoding sequence. A suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the toxin gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) identified using the above-described procedure, is synthesized, and hybridized by means well-known in the art, against a DNA or, more preferably, a cDNA preparation derived from cells which are capable of expressing the toxin gene. The source of DNA or cDNA used will preferably have been enriched for toxin sequences. Such enrichment can most easily be obtained from cDNA obtained by extracting RNA from cells which produce high levels of the toxin gene. Techniques of nucleic acid hybridization are disclosed by Maniatis et al. (1982), and by Hames and Higgins (1985).

To facilitate the detection of the desired toxin encoding sequence, the above-described DNA probe may be labeled with a detectable group. Such detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of immunoassays and in general most any label useful in such methods can be applied to the present invention. Particularly useful are enzymatically active groups, such as enzymes (Wisdom, 1976), enzyme substrates (GB 1,548,741), co- enzymes (US 4,230,797 and US 4,238,565) and enzyme inhibitors (US 4,134,792);

fluorescers (Soini and Hemmila, 1979); chromophores; luminescers such as chemiluminescers and bioluminescers (Gorus and Schram, 1979); specifically bindable ligands; proximal interacting pairs; and radioisotopes such as ³H, ³⁵S, ³²P, ¹²⁵I and ¹⁴C. Such labels and labeling pairs are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their reactive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors).

For example, a cofactor-labeled probe can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme. For example, one can use an enzyme which acts upon a substrate to generate a product with a measurable physical property. Examples of the latter include, but are not limited to, β-galactosidase, alkaline

phosphatase and peroxidase.

General procedures for hybridization are disclosed, for example, in Maniatis et al. (1982), and in Hames and Higgins (1985). Those members of the above-described gene library which are found to be capable of such hybridizations are then analyzed to determine the extent and nature of the toxin encoding sequences which they contain. In an alternative way of cloning the toxin gene, a library of expression vectors is prepared by cloning DNA or, more preferably cDNA, from a cell capable of expressing toxin into an expression vector. The library is then screened for members capable of expressing a protein which binds to anti-toxin antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as the toxin or fragments of the toxin.

The cloned toxin encoding sequence, obtained through the methods described above, may be operably linked to an expression vector, and introduced into bacterial or eukaryotic cells to produce toxin, or a functional derivative thereof. Techniques for such manipulations are disclosed by Maniatis et al. (1982) and are well-known in the art.

The above-described methods are, therefore, capable of identifying genetic sequences which are capable of encoding the toxin or fragments thereof. In order to further characterize such genetic sequences, it is desirable to express the toxins which these sequences encode, and confirm that they possess characteristics of toxin peptides. Such characteristics may include the ability to specifically bind anti-toxin antibody, the ability to elicit the production of antibody which are capable of binding to the toxin, the ability to provide a toxin function to a recipient cell, etc.

An alternative way of obtaining a genetic sequence which is capable of encoding the toxin is to prepare it by oligonucleotide synthesis. This method is especially feasible for proteins, such as the toxins of this invention, which have less than 100 amino acids. The genetic code is used to determine an oligonucleotide sequence which is capable of encoding the amino acid sequence.

In a preferred embodiment, this oligonucleotide sequence is predicted using the codon frequency appropriate for the organism in which the gene is to be expressed. Such codon frequencies for some organisms are available as part of the sequence analysis computer programs of the University of Wisconsin Genetics Computer Group. Codons frequencies for other organisms may be calculated with the aid of the same computer package using data in the available sequence data banks. In some cases, alternative codons may be selected to facilitate synthesis and/or provide convenient restriction sites. Translational stop and start signals are added at the appropriate points and sequences to create convenient cloning sites are added to the ends. The above nucleotide sequence constitutes the "coding strand". The sequence of the "complementary strand" is predicted using the computer programs mentioned above.

A series of oligonucleotides ranging from 20 to 50 bases is synthesized in order to provide a series of overlapping fragments which when annealed and ligated will produce both strands of the gene. These fragments are then annealed and ligated together using techniques well-known to those skilled in the art (Maniatis et al., 1982). The resulting DNA fragment with the predicted size is isolated by electrophoresis and ligated into a suitable cloning vector for amplification and further manipulation. This synthetic gene may be handled using the techniques described above for genes isolated from genomic and/or cDNA. III. Secretion Signal Sequences

Another aspect of the present invention comprises the use of polypeptide sequences which are known to direct proteins to which they are operably linked into the cellular secretory pathway, or the genetic sequences encoding such polypeptide sequences, for the purpose of enhancing the effectiveness of toxins such as those described in Section I when expressed in either transgenic plants or in recombinant microbes, or to facilitate the recovery of active toxins from expression systems such as E. coli, yeast, and virus-infected cells in culture. It is expected that secretion signal sequences from a wide variety of sources would be competent to carry out the function intended, because it is well-documented that the specificity of the signal recognition and processing apparatus in prokaryotic and eukaryotic cells is low (von Heijne, 1985). For the various purposes, however, it is likely that particular sources of secretion signal sequences will tend to be more useful than others.

For the purpose of producing recombinant baculoviruses which are effective in controlling insects, or which can be used in culture to infect cells and produce active toxins, the preferred secretion signal sequence is encoded by a nucleotide sequence, and when translated by the cellular translational apparatus, consists of any natural or artificial sequence of amino acids which can be demonstrated to promote secretion of an operably linked amino acid sequence, especially that set of amino acid sequences which are made up of the following four components: (1) a region at the amino terminal end which contains one or more basic amino acids, (2) a central region which is composed largely of hydrophobic amino acids, (3) a region at the carboxyl end which contains a larger number of polar amino acids than the central region, and (4) a site appropriate for recognition and cleavage by the signal peptidase enzyme (von Heijne, 1986).

More preferred are secretion signal sequences derived from proteins of bacteria, yeast, fungi, or higher eukaryotes, including both animals and plants (Watson, 1984). More preferred are secretion signal sequences from proteins of insect origin, for example those of cecropin B from Hyalophora cecropia (van Hofsten et al., 1985), and the eclosion hormone from Manduca sexta (Horodyski et al., 1989). Also preferred are the secretion signal sequences naturally associated with scorpion toxins, which can be determined by the analysis of mRNA, cDNA, or genomic DNA as described in Section I. More preferred is the natural secretion signal sequence of AaIT (Bougis et al., 1989). Also preferred are signal sequences from those higher eukaryotes which have been the source of genes whose translated products are effectively secreted by recombinant baculovirus-infected cells, for example, mammals and plants. More preferred are the signal sequences encoded in genes whose translated products are effectively secreted by recombinant baculovirus-infected cells, for example, human colony stimulating factor I (Luckow and Summers, 1989), human α-interferon (Id.), human β-interferon (Id.), human interleukin-2 (Id.), French bean (Phaseolus vulgaris) phaseolin (Id.) and mouse interleukin-3 (Miyajima et al., 1987). Especially preferred are secretion signal sequences from proteins of Bombyx mori, for example, those of storage proteins 1 (Sakurai et al., 1988) and 2 (Fujϋ et al., 1989). Most preferred is the secretion signal sequence of bombyxin (Adachi et al., 1989), which is composed of the following amino acid sequence:

MKILLAIALMLSTVMWVST

For the purpose of producing recombinant bacteria, especially E. coli, which are capable of producing and secreting active scorpion toxins, the most preferred secretion signal sequence is that of the ompA gene (Ghrayeb et al., 1984).

For the purpose of producing recombinant yeast, especially Saccharomyces cerevisiae, which are capable of producing and secreting active scorpion toxins, preferred secretion signal sequences are those which contain the 4 components detailed above. More preferred arc secretion signal sequences derived from scorpion toxins, especially AaIT. More preferred are the secretion signal sequences derived from the PHO5 (Smith et al., 1985), and SUC2 (Carlson et al., 1983) gene sequences of S. cerevisiae. Most preferred is the 85-amino acid sequence which comprises the prepro-peptide of the S. cerevisiae mating factor alpha (Bitter et al., 1987):

MRFPSIFTAV LFAASSALAA PVNTTTEDET AQIPAEAVIG YLDLEGDFDV AVLPFSNSIN NGLLFINTTI ASIAAKEEGV QLDKR

IV. Antibodies to Neurotoxins

Another aspect of this invention are antibodies to these neurotoxins. In the following description, reference will be made to various methodologies well-known to those skilled in the art of immunology. Standard reference works setting forth the general principles of immunology include the work of Klein (1982); Kennett et al. (1980); Campbell (1984); and Eisen (1980).

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term

"epitope" is meant to refer to that portion of a hapten which can be recognized and bound by an antibody. An antigen may have one or more than one epitope. An "antigen" is capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

The term "antibody" (Ab) or "monoclonal antibody" (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab')2 fragments) which are capable of binding an antigen. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., 1983).

The antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing the neurotoxin or a fragment thereof, can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding the neurotoxin.

In a preferred method, a neurotoxin fragment is prepared and purified to render it substantially free of natural contaminants. In another preferred method, a neurotoxin fragment is synthesized, according to means known in the art. Either the purified fragment or the synthesized fragment or a combination of purified natural fragment or synthesized fragment may be introduced into an animal in order to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present invention are monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., 1975; Kohler et al., 1976a and b; Hammerling et al., (1981)). In general, such procedures involve immunizing an animal with neurotoxin antigen. The splenocytes of such animals are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the American Type Culture Collection, Rockville, Maryland. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then cloned by limiting dilution as described by Wands and Zurawski (1981). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the neurotoxin antigen.

Through application of the above-described methods, additional cell lines capable of producing antibodies which recognize epitopes of the desired insect selective toxin can be obtained.

V. Expression of the Insect Selective Toxin and Its Functional Derivatives

The toxin encoding sequences, obtained through the methods described above, may be operably linked to an expression vector, and introduced into prokaryotic or eukaroytic cells in order to produce the toxin or its functional derivatives. The present invention pertains both to the intact toxin and to the functional derivatives of this toxin. A "functional derivative" of the toxin is a compound which possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of the toxin. The term "functional derivative" is intended to include the "fragments," "variants," "analogues." or "chemical derivatives" of a molecule. A "fragment" of a molecule such as the toxin is meant to refer to any polypeptide subset of the molecule. A "variant" of a molecule such as the toxin is meant to refer to a molecule substantially similar in structure and function to either the entire molecule, or to a fragment thereof. A molecule is said to be "substantially similar" to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity. Thus, provided that two molecules possess a simlar activity, they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequence of amino acid residues is not identical. An "analog" of a molecule such as the toxin is meant to refer to a molecule substantially similar in function to either the entire molecule or to a fragment thereof. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule.

Such moieties may improve the molecule's solubility, absorption, biological half-life, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art.

A DNA sequence encoding the toxin or its functional derivatives may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis et al. (1982) and are well-known in the art.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of the toxin synthesis. Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.

If desired, the non-coding region 3' to the gene sequence coding for the toxin may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3'-region naturally contiguous to the DNA sequence coding for the toxin, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.

Two DNA sequences (such as a promoter region sequence and the toxin encoding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the toxin gene sequence, or (3) interfere with the ability of the toxin gene sequence to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.

Thus, to express the toxin transcriptional and translational signals recognized by an appropriate host are necessary.

The present invention encompasses the expression of the toxin protein (or a functional derivative thereof) in either prokaryotic or eukaryotic cells. Preferred prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc. The most preferred prokaryotic host is E. coli. Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli W3110 (F-, lambda-, prototrophic (ATCC 27325)), and other enterobacterium such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species.

Under such conditions, the toxin will not be glycosylated. The procaryotic host must be compatible with the replicon and control sequences in the expression plasmid.

To express the toxin (or a functional derivative thereof) in a prokaryotic cell (such as, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.), it is necessary to operably link the toxin encoding sequence to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage 1, the bla promoter of the β-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pPR325, etc. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage 1 (PL and PR), the trp, recA, lacZ, lad, and gal promoters of E. coli, the α-amylase (Ulmanen et al., 1985) and the s-28-specific promoters of B. subtilis (Gilman et al., 1984), the promoters of the bacteriophages of Bacillus (Gryczan, 1982), and Streptomyces promoters (Ward et al., 1986). Prokaryotic promoters are reviewed by Glick and Whitney (1987); Cenatiempo (1986); and Gottesman (1984).

Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream of the gene-encoding sequence. Such ribosome binding sites are disclosed, for example, by Gold et al. (1981)).

Preferred eukaryotic hosts include yeast, fungi, insect cells, mammalian cells either in vivo or in tissue culture. Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or the myeloma P3x63Sg8, and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as HMR 332 that may provide better capacities for correct post-translational processing.

For a mammalian host, several possible vector systems are available for the expression of the toxin. A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, etc., may be employed.

Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., metabolite.

Yeast provides substantial advantages in that it can also carry out post-translational peptide modifications. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which can be utilized for production of the desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., prepeptides). Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast are grown in mediums rich in glucose can be utilized. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

Another preferred host is insect cells, for example, the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, 1988). Alternatively, baculovirus vectors can be engineered to express large amounts of the toxin in insect cells (Jasny, 1987; Miller et al., 1986). In a preferred embodiment, expression of the toxin in baculovirus vectors is enhanced by the presence of a signal sequence coding for a secretory peptide.

As discussed above, expression of the toxin in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer and Walling, 1982); the TK promoter of Herpes virus (McKnight, 1982); the SV40 early promoter (Benoist and Chambon, 1981); the yeast ga14 gene promoter (Johnston and Hopper, 1982; Silver et al., 1984).

As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the toxin (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as the toxin encoding DNA sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the toxin encoding sequence).

The toxin encoding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the toxin may occur through the transient expression of the introduced sequence. Alter natively, permanent expression may occur through the integration of the introduced sequence into the host chromosome.

In one embodiment, a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama and Berg (1983).

In a preferred embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColE1, pSC101, pACYC 184, xVX.

Such plasmids are, for example, disclosed by Maniatis et al. (1982). Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan (1982). Suitable Streptomyces plasmids include pIJ101 (Kendall and Cohen, 1987), and Streptomyces bacteriophages such as x2C31 (Chateret al., (1986). Pseudomonas plasmids are reviewed by John and Twitty (1986) and Isaki (1978)).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein et al., 1982; Broach, 1981; Broach, 1982; Bollon and Stauver, 1980; Maniatis, 1980).

Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the DNA constructs) may be introduced into an appropriate host cell by any of a variety of suitable means: transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the toxin, or in the production of a fragment of this toxin. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).

The expressed protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.

VI. Use of the Insect-Selective Toxin to Improve Insecticidal Microbes

In one embodiment of the present invention, the insect selective toxin alone or in combination with any of the enhancing compounds mentioned above is used to enhance the toxicity of insecticidal microbes. The microbes useful in the present invention include baculoviruses, fungi and bacteria. Several baculoviruses including those that infect cotton bollworm, Heliothis virescens, Douglas fir tussock moth, Orgia pseudotsugata, gypsy moth, Lymantria dispar, alfalfa looper, Autographica californica, European pine fly, Neodiiprion sertifer and codling moth, Laspeyresia pomonella have been registered as pesticides. The preferred baculoviruses for the present invention include the Bombyx mori nuclear polyhedrosis virus (BmNPV) and the Autographica californica nuclear polyhedrosis virus (AcNPV). Introduction of an insect-selective toxin into the genome of such a baculovirus can significantly enhance the potency of such pesticides. Methods for the introduction of foreign genes into the genome of baculoviruses are the subject of two patents (US 4,745,051 and JP 61-09288,-97). EP 309,368 discloses the production of a microbial insecticide effective against two species of insects by construction of a recombinant baculovirus containing DNA segments of two species of nuclear polyhedrosis (baculovirus) virus. Numerous fungi are capable of infecting insects. Introduction of the insect-selective toxin into the genome of such fungi could enhance the potency as pesticides. For example, Beauvaria bassania and Beauvaria brongniartii have a wide host range and have been suggested as candidates for microbial pesticides (Miller et al., 1983). Bacteria (other than Bacillus thuringiensis) that have been considered as insect control agents include Bacillus popilliae, B. lentimorbus and B. sphaericus. Their potential as pesticides can be enhanced by improving their potency through the incorporation of an insect-selective toxin into their genome.

Thus, one embodiment of the present invention comprises insecticidal microbes, especially baculoviruses, which exhibit enhanced toxicity to insects. The genome of the baculoviruses comprises a genetic sequence coding for an insect-selective protein neurotoxin isolated from venom, such as disclosed in part I above.

In another embodiment of the present invention, methods are provided for enhancing the toxicity of an insecticidal microbe. The methods of the present invention comprise a) isolating a recombinant DNA molecule comprising a genetic sequence coding for an insect-selective protein neurotoxin isolated from venom, and b) introducing the recombinant DNA molecule into the genome of the insecticidal microbe.

The recombinant DNA molecule comprises a genetic sequence for an insect-selective protein neurotoxin isolated from venom, such as scorpion venom. The preferred insectselective toxins of the present invention are those insect-selective toxins which, when the DNA coding sequence is inserted into the genome of a baculovirus, will result in a recombinant baculovirus having improved insecticidal properties. Preferred are the alpha, depressant and intermediate toxins. Most preferred are those insect-selective toxins having an amino acid sequence of from about 60 to about 70 amino acids in length. Especially preferred are the AaIT, LqhP35, LqhIT2 and LqqIT2 toxins. The most preferred insectselective neurotoxin is the AaIT toxin.

The recombinant DNA molecule comprises regulatory sequences to effect the expression of the coding sequence. These regulatory sequences preferably include promoter sequences, untranslated leader sequences, and a signal sequence to promote the secretion of the toxin protein, once expressed.

The present invention also includes methods of producing insect selective toxins comprising preparing an expression vector which codes for the production of an insectselective toxin, introducing the expression vector into the genome of an expression system host, and allowing the host to produce an expression supematant. The supernatant may be extracted from the host and the insect-selective toxin may be isolated from the supernatant. Preferred hosts include E. coli and yeast. Especially preferred as the host are insect cells, either cultured or in living insects, which have been infected with a baculovirus containing a DNA sequence coding for the production of the insect-selective toxin.

Having now generally described this invention, the same will be better understood by reference to specific examples, which are included herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

EXAMPLES

Deposits pCIB4223 has been deposited with the American Type Culture Collection in Rockville, Maryland on October 4, 1990 and has been designated with the accession number

ATCC 40906.

Example 1: Synthesis of Genes Encoding Insect-Selective Toxins

A. Purification of Insect-Selective Toxins

The purification of several insect toxins has already been described in several works by Zlotkin (Zlotkin, supra; Lester et al., 1982). An altemative method which allows purification with high recoveries from limited amounts of venom is to use high

performance liquid chromatography. This technique is exemplified in the purification of LqhIT2.

For purification of LqhIT2, lyophilized Leiurus quinquestriatus hebraeus venom (Sigma) is extracted three times using 0.5 ml water/20 mg venom. The water extracts are combined and subjected to ion-exchange chromatography on sulfoethylaspartamide HPLC column (Nest Group). The extract is applied to the column previously equilibrated in 5 mM KPO₄, pH 3.0 in 25 % acetonitrile and the column eluted with a gradient from 0 to 0.5 M KCI in the same buffer over 60 min. Individual fractions are desalted and further separated by reverse phase chromatography on a Vydac C-8 column equilibrated in 0.1 % trifluoroacetic acid and eluted with a 75 minute gradient from 0 to 70 % B (B = acetonitrile:isopropanol 1:1 in 0.1 % trifluoroacetic acid). Individual fractions are tested for toxicity to insects by injection into Sarcophaga or Heliothis larvae as described by Zlotkin et al. (1985).

B. Amino Acid Sequencing of Insect Toxins

The insect toxin is reduced by incubating samples in 6 M guanidine HCl, 1 M Tris HCl, pH 8.6, 10 mM EDTA, 20 mM dithiothreitol for 1 h at 37°C. 4-vinylpyridine (Sigma) is added to 50 mM and incubation continued at room temperature for 1 h. The modified protein is desalted on a Vydac C-8 column as described above. Peptides are produced by enzymatic digestion with trypsin, Lys-C, or Glu-C or by partial acid hydrolysis following standard procedures (Allen, 1981). Peptides are separated by reverse phase HPLC prior to sequencing. The amino acid sequences of the intact toxin and the individual peptides are determined by automatic Edman degradation using a Model 470A Protein Sequencer (Applied Biosystems, Foster City, CA) equipped with an on-line reverse phase HPLC for analysis of the phenylthiohydantoin derivatives of the amino acids and a Model 900 data analysis system. The sequence determined for the LqhIT2 toxin is:

DGYIKRRDGC KVACLIGNEG CDKECKAYGG S YGYCWTWGL ACWCEGLPDD KTWKSETNTC G.

Sequences of other insect toxins determined using the same techniques are provided in EP 374,753.

C. Synthesis of Gene Encoding Insect Toxin

Since the insect toxins are small proteins (<80 amino acids), a gene encoding a toxin may be constructed by DNA synthesis. The synthesis of a gene which encodes AaIT, the Androctonus australis insect toxin is described in EP 374,753.

Example 2: Preparation of antibodies to insect-selective toxin

A. Preparation of immunogen

Standard computer analyses (Hopp and Woods, 1983) are used to predict antigenic sites from the amino acid sequences of insect-selective toxins. Synthetic peptides are prepared corresponding to these regions. The peptides are coupled through an interval cysteine to an ovalbumin carrier using the reagent N-succinimidyl-3-(2-pyridyldithio)propionate (Pierce Chemical Co.) as described by Carlsson et al. (1978). The degree of conjugation is estimated by amino acid analysis of the conjugate.

B. Production of Antisera

Rabbits are immunized with 0.5 to 1.0 mg antigen emulsified in complete Freund's adjuvant and boosted monthly with antigen in incomplete Freund's adjuvant. Sera are titered by conventional ELIS A assays using the peptide conjugated to a heterologous carrier (typically bovine serum albumin). Positive sera are titered against the appropriate insect toxin.

C. Results

Typically dilutions of 1:10,000 allow detection of 1 to 10 ng of the homologous peptides. Dilutions of 1:300 allow detection of 3 to 10 ng of intact toxin protein. Table I shows results of immunization with several different toxin peptides.

Table I: Detection Limits in ELIS A Assay of Antisera Raised against Toxin

Peptides

Toxin Immunizing Peptide Detection of Peptide Detection

of Toxin AaIT N- terminal 1-16 3 ng at 1:10,000 3 ng at 1:300 AaIT C-terminal 52-70 3 ng at 1:10,000 3 ng at 1:300

LqhlT2 N-terminal 1-13 1 ng at 1:1,000

LqhIT2 C-terminal 46-61 1 ng at 1:10,000

BjIT2 N-terminal 1-13 0.3 ng at 1:3,000

BjIT2 C-terminal 46-60 10 ng at 1:3,000 1 ng at 1:300 Example 3: Enhancement of Insecticidal Activity of Baculoviruses by

Incorporation of Gene Coding for AaIT Toxin

A. Construction of pCIB4223 (Bombyxin signal sequence- AaIT in baculovirus transfer vector)

A DNA fragment containing the signal sequence of the Bombyx mori protein bombyxin (Adachi et al., 1989) is produced by annealing and extending the primers S V69A23 (TGTTGACACC CACATTACTG TTGACAACAT TAATGC) and S V70A23

(TAGAGCTCAT GAAGATACTC CTTGCTATTG CATTAA) using the Gene Amp kit (Perkin-Elmer Cetus) and standard conditions for PCR amplification.

The reaction product is a 65 bp fragment composed of the bombyxin signal sequence with the addition of a SacI restriction site at its 5' end.

A DNA fragment containing the AaIT gene is produced using the primers S V65A23 (AATCTAGAGG ATCCTAGTTG ATGATAGTAG TGTCGC) and S V68A23

(GTAATGTGGG TGTCAACAAA AAAAAACGGC TACGCT) to amplify by PCR a fragment from the AaIT clone described in Example 1. The reaction product is a 247 bp fragment with 18 bp of the bombyxin signal sequence attached in correct reading frame to the 5' end of the AaIT gene sequence. The AaIT sequence is modified to include a penultimate isoleucine codon that is not included in the original clone but is indicated in the protein sequence published in Darbon et al. (1982). This fragment has an XbaI restriction site at its 3' end.

The two products above are precisely fused using an overlapping PCR strategy that uses the bombyxin sequence contained in both to initiate the reaction, and then uses the two flanking primers, SV65A23 and SV70A23, to amplify the fused product (Higuchi et al., 1988). The final product is a 291 bp fragment containing the bombyxin signal sequence fused to the AaIT sequence and having SacI and XbaI restriction sites at its 5' and 3' ends, respectively. This product [Bs-AaIT] is cut with SacI and XbaI (NEB) and isolated by electrophoresis through 2.5 % NuSieve GTG agarose (FMC Bioproducts). The appropriate fragment is excised, melted at 65°C, and approximately 1 % is used directly in a standard ligation reaction with 50 ng of SacI/XbaI digested pBK283 (Fig. 1). 10 % of the ligation mix is transformed into E. coli strain HB101 and transformants are selected on L-broth containing 100 μg/ml ampicillin. Individual colonies are picked and screened by standard miniprep/restriction analysis and the correct clone is amplified using a large scale alkaline lysis plasmid prep procedure. Dideoxy sequencing is performed to verify the absence of PCR-introduced mutations. The correct clone is designated pCIB4223 (Fig. 2 and SEQ ID NO:1).

B. Preparation of recombinant baculovirus carrying the BS-AaIT gene.

The pCIB4223 transfer vector is used to prepare a recombinant Bombyx mori nuclear polyhedrosis virus carrying the Bs-AaIT gene as described by Maeda (1989b).

C. Infection of Bombyx mori larvae Bombyx mori larvae are infected with the recombinant virus. Infection of 2nd, 3rd, 4th or 5th instar larvae leads to death of all insects 48 to 55 hr after infection. The time to death of insects infected with virus lacking the AaIT gene is 5 days. Insects infected with the Bs-AaIT recombinant virus show abnormal behaviors with spastic contractions and arching starting about 40 to 50 hr after injection, then become immobile before death. This set of symptoms, which is consistent with a neurotoxic effect, is not observed with the control virus. Data from a typical experiment is shown in table II below.

Table II: Effect of Various Recombinant Baculoviruses on Third Instar

Bombyx mori Larvae

Virus Bs-AaTT^a M14^b Uninfected

Hours post

Infection Wt. Condition Wt. Condition Wt. Condition

0 25.9 Healthy 50.1 Healthy 23.4 Healthy

26.2 Healthy 25.1 Healthy 25.2 Healthy

40.1 Healthy 24.5 Healthy 25.2 Healthy

50.2 Healthy 45.5 Healthy 31.3 Healthy

32.4 Healthy 30.1 Healthy c Healthy Virus Bs-AaTT^a M14^b Uninfected

Hours post

Infection Wt. Condition Wt. Condition Wt. Condition

21 37.8 Healthy 95.6 Healthy 61.7 Healthy

59.5 Healthy 53.7 Healthy 33.9 Healthy

99.5 Healthy 49.8 Healthy 47.0 Healthy

84.2 Healthy 43.7 Healthy 33.0 Healthy

55.3 Healthy 47.5 Healthy 89.0 Healthy 4 13.1 alive 97.8 alive 55.8 alive

11.0 alive 54.2 alive 51.6 alive

73.0 alive 55.9 alive 41.2 alive

71.2 alive 50.8 alive 71.4 alive

48.2 alive 63.6 alive 100.5 alive 1 2 arching all healthy all healthy

1 off diet

2 restless 3 all arching all healthy all healthy

none feeding all feeding all feeding 5 all dead all alive all alive 6 2 dead all alive 9 3 dead all alive 5 all dead all alive a Recombinant virus carrying Bs-AaIT gene

b Recombinant virus carrying a gene for juvenile hormone esterase, used as control

c Not recorded D. Construction of an AcNPV recombinant baculovirus containing the gene encoding AaIT

In addition to Bombyx mori nuclear polyhedrosis virus (BmNPV), Autographica californica nuclear polyhedrosis virus (AcNPV) is used as a recombinant baculovirus with enhanced insecticidal activity according to the present invention. Several transfer vectors of AcNPV are available for expression of foreign genes in agricultural pest insects including Spodoptera exignia, Spodopterafungiporda, Heliothis zea, Heliothis armigen, Heliothis virescens, Trichoplusia ni and related insects (Miller, 1988). Some of these vectors can be used for insertion of foreign genes without disrupting the original polyhedrin gene. Recombinant AcNPVs produced by these transfer vectors will produce polyhedral inclusion bodies as well as foreign gene products. Recombinant viruses having polyhedral inclusion bodies are especially preferred for their ability to infect an insect orally, which is the natural mode of infection of insects in the field.

Among the transfer vectors which can be used for expression of insect toxin genes in AcNPV is pAcUW(B) (Weyer et al., 1990). The pAcUW(B) transfer vector contains the original polyhedrin gene with the original promoter and an insertion site (BglII) for expression of the foreign genes after the p10 promoter. The insect toxin gene is then inserted at the BglII site by ligation, and its orientation is checked by double digestion with restriction enzymes or by direct sequencing. For example, the AaIT toxin gene in the transfer vector pBK283, which has been designated pCIB4223, is digested with SacI and XbaI to excise the complete AaIT gene including a signal sequence of Bombyxin for secretion. The cleaved AaIT toxin gene is inserted into pTZ18R (Pharmacia), which is similar to a pUC plasmid, at the SacI and XbaI sites. The resultant plasmid is cleaved with SacI and a synthesized oligomer (5'-CGGATCCGATCG-3') is inserted. The insertion of this oligomer is confiremed by screening with BamHI for the fragment containing the AaIT gene; the correct plasmid has a BamHI site due to the inserted oligomer and the BamHI site between the stop codon of the AaIT gene and XbaI site used for construction. For the insertion of the AaIT gene, the pAcUW(B) is cleaved with BglII. Cleaved pAcUW (B) and the constructed plasmid are then ligated and transformed into E. coli, JM101. The correct plasmid containing the AaIT gene has an insertion of about 10 to 12 kb with one SacI site, but no BamHI or BglII sites. The direction of the inserted AaIT gene is confirmed by double digestion with SacI (5' end of the AaIT gene) and BamHI (within the coding sequence of the polyhedrin gene). A recombinant transfer vector derived from pAcUW(B) carrying the AaIT gene in the correct orientation has a fragment of about 500 bp while the plasmids with insertions in opposite direction have a fragment of about 800 bp.

A recombinant AcNPV is obtained by cotransfection of this recombinant transfer vector and viral DNA of AcNPV, which lacks production of the polyhedral protein (e.g., a virus with a deletion in the polyhedral gene coding sequence). The cotransfection is performed by the method described in Example 3a above. Recombinant viruses are screened by looking for viruses producing polyhedral inclusion bodies since the transfer vector pAcUW(B) has the polyhedrin gene with the original promoter. Confirmation of the insertion of the foreign gene is performed by Southern blot analysis. The recombinant virus is propagated in Sf cells and the occlusion bodies are collected and purified from the infected cells by centrifugation and washing with distilled water. Polyhedral inclusion bodies containing recombinant viruses carrying the AaIT gene are mixed in diet and larvae are allowed to feed. The effects of the recombinant baculovirus are measured in accordance with the data of Table π above. In all species tested, the speed of kill significantly increased.

E. Injection of Manduca sexta with recombinant AcNPv containing the gene for AaIT

When Manduca sexta larvae are infected with the recombinant AcNPV containing the AaIT gene, larvae typically show symptoms between 60 to 72 hours post-injection and are functionally dead in 4 to 5 days. No larvae injected with wild-type AcNPV are dead at this time. Control larvae injected with wild-type AcNPV die 7 to 8 days post-injection.

F. Oral Activity of recombinant AcNPV virus containing the gene for AaTT

Heliothis virescens larvae are fed 1000 to 5000 occlusion particles of the recombinant AcNPV containing the AaIT gene (AcNPV-AaIT) mixed in diet. Symptoms including spastic contractions and cessation of feeding appear in all larvae fed AcNPV-AaIT virus by 75 hours after feeding. The LT50 [time to death for 50 % of larvae] is 90 hours for larvae fed recombinant virus. At 90 hours, larvae fed wild-type control AcNPV virus are still healthy and feeding normally. This result demonstrates that the recombinant virus is orally active against Heliothis virescens. Example 4: Production of AaIT Protein by Expression of the BS-AaIT Gene in

Bombyx mori Cells in Tissue Culture

The recombinant baculovirus carrying the Bs-AaIT gene (described in Example 3) is used to infect Bombyx mori cells in culture. AaIT is secreted into the tissue culture medium. The amount of AaIT is measured by isolating the material by HPLC and detecting it with an immunological assay. These methods are described in Example 6. Medium collected 48 hrs post-infection contains about 370 ng AaIT/ml. Injection of this material into Sarcophaga falculata larvae demonstrates that this material has neurotoxic activity.

Example 5: Expression of AaIT in Yeast Strain Saccharomyces cerevisiae

A. Construction of pCIB4224

A DNA fragment encoding the AaIT gene is produced in a manner similar to that described for construction of pCIB4223 in Example 3. PCR amplification is used to obtain the AaIT coding sequence from the AaIT clone described in Example 1 by using the primers SV65A23 and SV71A23 (GAGAGCTCGA ATTCATGAAA

AAAAACGGCT ACGC). The reaction product is a 243 bp fragment encoding the AaIT gene with the penultimate isoleucine codon inserted, and having SacI and XbaI restriction sites at its 5' and 3' ends, respectively. This fragment is isolated and cloned into an appropriate vector, for example, the pBK283 vector as described for pCIB4223. The correct clone is designated pCIB4224.

B. Construction of expression vector pP[alpha]AaIT

An example of a method to construct an expression vector for the production of AaIT in yeast is the assembly of pP[alpha] AaIT. This requires the AaIT gene (which resides in its correct form in the baculovirus transfer vector pBK283+AaIT), and plasmids

pUC18/PH05-[alpha]FL and pJDB207/PHO5-RIT12. Plasmid pJDB207/PHO5-RIT12 has a LEU2 gene which allows for selection on leucine-deficient medium, but which is poorly transcribed because of its truncated promoter region. The AaIT gene is inserted into pJDB207/PHO5-RIT12 behind the PHO5-[alpha] promoter and leader sequence from pUC18/PHO5-[alpha]FL. High plasmid copy numbers are required to obtain complete complementation of the host's LEU-phenotype. The expression vector pP[alpha]AaIT is constructed in a two-step ligation process which is begun with the adaptation of the AaIT gene by the polymerase chain reaction (PCR). Two oligomers are used to prime the PCR:

1) 5'-CTG GAT AAA AGA AAA AAA AAC GGC TAC GCT- 3'

2) 3'-G TGA TGA TAG TAG TTG ATC GGA GCT CCC AGT-5'

Primer (1) adds the final four codons of the yeast alpha factor preproprotein to the amino end of the AaIT sequence, and primer (2) adds an Xho I restriction enzyme site just after the termination codon of the AaIT sequence. Primer (1) is phophorylated by treating it with T4 polynucleotidekinase in the presence of ATP, and the reaction is stopped by incubation at 65°C for 15 minutes. The oligomers and plasmid pBK283+AaIT are incubated with DNA polymerase from T. aquaticus in a series of denaturation, annealing, and polymerization steps as recommended by the manufacturer of the Gene Amp PCR kit, PerkinElmer/Cetus. The reaction is stopped by the addition of chloroform, the removal of the aqueous phase, and the precipitation of the product with ammonium acetate and ethyl alcohol.

A DNA fragment containing the repressible acid phosphatase (PHO5) promoter and the majority of the alpha factor prepro peptide is obtained from plasmid

pUC18/PHO5-alphaFL by digesting with restriction enzymes BamHI and PvuII, treating with alkaline phosphatase, and purifying the 800 bp fragment by agarose gel electrophoresis. This fragment is combined with at least a 10-fold molar excess of the phosphorylated PCR product described above and incubated overnight at room temperature with T4 DNA ligase and ATP. The ligation reaction is stopped by heating to 65°C for 15 minutes, and then the ligation products are diluted in an appropriate buffer and digested by restriction endonucleases BamHI and XhoI for 4.5 hours.

The products of this digestion are run on an agarose gel, and compared to the unreacted 800 bp PHO5 promoter-alpha factor prepro peptide DNA fragment. Formation of the correct ligation product results in the appearance in the digest mixture of a DNA fragment which migrates slightly slower than the unreacted 800 bp fragment. This new fragment is isolated from the gel. The vector fragment is prepared from plasmid pJDB207/PHO5-RIT12 by digesting it with restriction endonucleases BamHI and XhoI and purifying the large fragment by agarose gel electorphoresis. The vector fragment and the ligation product isolated above are incubated together with T4 DNA ligase at 15°C overnight, and the ligation mixture is used to transform E. coli strain DH5 [alpha].

Small scale plasmid preparations from several transformants are tested by restriction enzyme analysis and representative strains bearing plasmids with the correct gross structure are cultured on a larger scale to provide plasmid DNA for sequence analysis. Convenient primers for determining the sequence of the construct are:

3) 5'-ATTGCC AGC ATTGCTGCT-3'

4) 5'-GACTGG CGTTGTAATGAG-3'

Oligomer (3) primes synthesis in the alpha factor prepro sequence, 15 bp from the junction with the AaIT sequence. Oligomer (4) primes synthesis near the XhoI site. The information obtained from the use of these two primers covers the entire AaIT coding sequence as well as the junction with the alpha factor prepro peptide. The plasmid which contains the correct sequence is designated pP[alpha]AaIT.

C. Expression of AaIT in yeast

Plasmid pP[alpha]AaIT is transformed into either of two yeast strains, GRF18 [MAT alpha, his3-11, 3-15, leu2-3, 2-112] or HT246 [MAT a, leu2-3, 2-112, prb, cps] by the spheroplast method (Burgers and Percival, 1987). The regeneration agar which is used to plate the transformation mixture contains a suboptimal level of leucine, 1.5 mg/100 ml agar. After growth of the transformed yeast colonies (3 to 7 days) they are gridded on minimal medium plates lacking leucine. Growth of colonies on this medium is considered evidence of transformation.

To produce AaIT from yeast, the transformed yeast cells are initially cultured in high phosphate minimal medium with vigorous aeration at 25°C. Synthesis of AaIT is initiated by collecting the cells by centrifugation, washing them in sterile 0.8 % NaCl, resuspending them in low phosphate minimal medium (SC-3 medium), and continuing the incubation at 25°C. AaIT accumulation is monitored by performing ELISA assays or SDS polyacryl amide gel and Western blot analysis on conditioned culture fluids or on solubilized cell pellets.

Example 6: Isolation of AaIT from Supernatants of Expression Systems

A. Isolation of immunoreactive material

Immunoreactive material is partially purified from extracts of yeast and supernatant from cultured insect cells infected with Bombyx mori baculovirus (Maeda, 1989b) with the use of high performance liquid chromotography (HPLC). A Brownlee RP-300 reversed-phase column (2.1 × 100 mm, 7 μm pore size) is equilibrated in 0.1 % trifluoroacetic acid (TFA). Extracts are applied to the column and eluted by the following gradient of solvent A (0.1 % TFA in water) to solvent B (0.1 % TFA in 1:1 acetonitrile:isopropanol):

Time (min) % solvent B

0 0

5 0

40 50

45 90

50 90

60 0

Native AaIT purified from scorpion venom elutes from the column under these conditions at approximately 34 minutes. Therefore the eluent from extract injections is collected in three fractions: the first from 0 to 32 minutes, the second from 32 to 38 minutes, and the third from 38 to 70 minutes. After completion of the run, all three fraction volumes are reduced by use of a Savant Speed Vac Concentrator. HPLC fractions are stored frozen at -20°C.

B. Detection of immunoreactive material by enzyme-linked immunosorbant

assay (ELISA)

HPLC-fractionated extract samples are screened for immunoreactivity by typical ELISA. 50 μl of various dilutions of a sample are applied to an ethanol-washed 96-well vinyl microtiter plate (samples diluted in borate buffered saline). The plate is left overnight at 4°C to allow for adsorption of proteins contained in the sample to the plate. The next day the plate is washed three times with ELISA wash buffer to remove any unbound protein. The plate wells are then blocked by filling them with bovine serum albumin blocking buffer and soaking them for at least one hour. The plate is then rinsed three times with ELISA wash buffer.

50 μl of concentrated rabbit polyclonal anti- AaIT antibody (the preparation of which is described in Example 2) is added to each well at a concentration of 1:1000 in ELISA diluent. The plate is incubated at 37°C for one hour, then washed three times with ELISA wash buffer. 50 μl of a 1 :500 dilution of anti-rabbit antibody developed in goat (Sigma) is applied to each well, then incubated for one hour at 37°C. The plate is washed three times in ELISA wash buffer, then 50 μl of substrate solution (2.3 mM p-nitrophenyl phosphate in substrate buffer) is added to each well. The plate is incubated at room temperature for thirty minutes, allowing a colorimetric reaction to proceed. This reaction is quenched by the addition of 50 μl of 3 M sodium hydroxide to each well.

The absorbance of each well at 405 nm is measured by spectrophotometer, and compared to a standard curve of known concentrations of native AaIT purified from scorpion venom. Sample AaIT concentration is determined by computer comparison of the unknown's absorbance to that of the standard (Enzfitter or Molecular Dynamics). Typical results indicating the concentration of AaIT in expression supernatants are:

Sample AaTT (mg/l)

Baculovirus (24 hr) 0.01

Baculovirus (48 hr) 0.37

Yeast GRF-pPal (24 hr) 2.3

Yeast GRF-pPal (48 hr) 0.08

Yeast HT246-pPa AaIT (24 hr) 3.4

Example 7: Expression of AaIT in E. coli

The vector used for expression of AaIT in E. coli is the secretion vector pIN-III-ompA (Masui et al., 1983; Ghrayeb et al., 1984). Fusion of the AaIT gene to the ompA signal peptide allows its export into the E. coli periplasmic space, which facilitates purification and may provide a more favorable environment for the production of biologically active AaIT protein.

A. Construction of pCIB4222 (E. coli expression vector for AaIT)

A DNA fragment encoding the E. coli ompA signal sequence is produced by PCR amplification using the primers S V47A23 (GGAACTCTAG ATAA CGAGGG) and S V67A23 (GGCCTGCGCT ACGGTAGCGA), with pIN-III-ompA3 as a template. The reaction product is an 89 bp fragment encoding the ompA signal peptide, with an XbaI restriction site at its 5' end.

A DNA fragment encoding the AaIT gene is produced by PCR amplification using the primers S V65A23 and SV66A23 (ACCGTAGCGC AGGCCAAAAA AAACGGCTAC GC) with the AaIT clone described in Example 1 as a template. The reaction product is a 243 bp fragment with 15 bp of the ompA signal sequence fused to the 5' end of the AaIT gene. The AalT gene sequence includes the addition of the penultimate isoleucine codon and has a BamHI restriction site at its 3' end.

The two fragments above are fused by overlapping PCR using the flanking primers SV47A23 and SV65A23 to amplify the fusion. The reaction product is a 317 bp fragment composed of the ompA signal sequence attached to the AaIT gene, and having XbaI and BamHI restriction sites at its 5' and 3' ends, respectively. This fragment is ligated by standard methods into XbaI/BamHI digested pIN-III-ompA3, and transformed into E. coli strain HB101. The correct clone is identified, its sequence is verified, and the plasmid is designated pCIB4222 (Fig. 3). The pCIB4222 transformed cell line is designated

CGE1535.

B. Assay for expression of AaIT in E. coli

The CGE1535 cells are grown to log phase in L Broth and induced for 2 to 4 hours with 2 mM IPTG (Isopropyl-β-D- hiogalactopyranoside). The cells are harvested and periplasmic extracts are prepared using osmotic shock (Koshland and Botstein, 1980).

Samples are run on a 10 to 20 % Tricine peptide gel (Novel Experimental Technology), electroblotted onto a nitrocellulose membrane and assayed for AaIT expression using standard Western blotting techniques. The primary antibody is directed against the C-terminal residues 52-70 and is described in Example 2. This antibody is used at a dilution of 1:25,000; the secondary antibody is alkaline phosphatase conjugated Anti-Rabbit IgG (Sigma) used according to manufacturer's recommendation. Detection is by standard colorimetric assay for alkaline phosphatase. Quantitation is based on comparison with known amounts of purified AaIT. Immunoreactive bands corresponding to predicted molecular weights for both the unprocessed (~9.8 kD) and the correctly processed (~7.8 kD) AaIT protein are detected. Expression of the putative mature AaIT protein is estimated by comparison with authentic AaIT standards to be 200 to 400 μg/ml.

C. Assay for AaIT activity in E. coli extracts

The periplasmic extract from CGE1535 is tested in the Sarcophaga larvae injection assay for biologically activity of AaIT. To obtain samples sufficiently concentrated for the assay, the AaIT is precipitated from the periplasmic extracts using 80 % (NH₄)₂SO₄. Residual salt is removed by buffer exchange in a Centricon-3 microconcentrator

(Amicon). Western blot analysis estimates the concentration of AaIT in this sample to be 30 to 50 ng/μl. 100 % of Sarcophaga larvae injected with 10 μl [300 to 500 μg] of concentrated sample are killed. Larvae injected with a ten-fold dilution [30 to 50 μg] are not killed. Control larvae are similarly unaffected, but 100 % of larvae injected with 10 ng purified authentic AaIT are killed. Thus, only 2 to 3 % of the immunoreactive material is biologically active.

* * * * * * * * * * * *

While the present invention has been described with reference to specific embodiments thereof, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly, all such variations, modifications and embodiments are to be regarded as being within the spirit and scope of the present invention.

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Sequence listing

SEQ ID NO:1

SEQUENCE TYPE: Nucleotide with corresponding protein

SEQUENCELENGTH: 287 base pairs

GAG CTC ATG AAG ATA CTC CTT GCT ATT GCA TTA ATG TTG TCA 42

Met Lys Ile Leu Leu Ala Ile Ala Leu Met Leu Ser ACAGTA ATG TGG GTG TCA ACA AAA AAA AAC GGC TAC GCT GTT 84 Thr Val Met Trp Val Ser Thr Lys Lys Asn Gly Tyr Ala Val GAC TCT TCT GGC AAA GCT CCG GAA TGC CTG CTG TCT AAC TAC 126 Asp Ser Ser Gly Lys Ala Pro Glu Cys Leu Leu Ser Asn Tyr TGC AAC AAC CAG TGC ACT AAA GTT CAT TAC GCT GAC AAA GGC 168 Cys Asn Asn Gln Cys Thr Lys Val His Tyr Ala Asp Lys Gly TAC TGC TGC CTG CTG TCT TGC TAC TGC TTC GGC CTG AAC GAC 210 Tyr Cys Cys Leu Leu Ser Cys Tyr Cys Phe Gly Leu Asn Asp GAC AAA AAA GTT CTG GAA ATC TCT GAC ACT CGT AAA TCT TAC 252 Asp Lys Lys Val Leu Glu Ile Ser Asp Thr Arg Lys Ser Tyr TGC GAC ACT ACT ATC ATC AAC TAG GAT CCT CTA GA 287

Cys Asp Thr Thr Ile Ile Asn

Claims

What is claimed is:

1. An insecticidal microbe with enhanced toxicity to insects, the genome of said insecticidal microbe comprising a recombinant DNA molecule, said recombinant DNA molecule further comprising a genetic sequence coding for an insect-selective protein neurotoxin, wherein the insect-selective protein neurotoxin is selected from the group consisting of AaIT, buthoid scorpion toxins and chactoid scorpion toxins.

2. An insecticidal microbe of claim 1, wherein the insecticidal microbe is a baculovirus.

3. An insecticidal baculovirus of claim 2, wherein the baculovirus is a nuclear polyhedrosis virus.

4. An insecticidal baculovirus of claim 3, wherein the nuclear polyhedrosis virus is selected from the group consisting of the Bombyx mori nuclear polyhedrosis virus and the Autographica californica nuclear polyhedrosis virus.

5. An insecticidal baculovirus of claim 3, wherein the genetic sequence codes for an insect-selective protein neurotoxin selected from the group consisting of AaIT, LqhIT2, LqqIT2 and LqhP35.

6. An insecticidal baculovirus of claim 2 further comprising a gene secretion signal sequence operably linked in correct reading frame with a promoter.

7. An insecticidal baculovirus of claim 6 wherein the gene secretion signal sequence originates from a gene whose translated products are effectively secreted by recombinant baculovirus-infected cells.

8. An insecticidal baculovirus of claim 6 wherein the gene secretion signal sequence originates from a gene from the insect target of the baculovirus.

9. An insecticidal baculovirus of claim 4 further comprising a gene secretion signal sequence operably linked in correct reading frame with a promoter, wherein the gene secretion signal sequence is the secretion signal sequence of bombyxin.

10. An insecticidal microbe of claim 1 wherein the recombinant DNA molecule further comprises one or more additional genetic sequences operably linked on either side of the toxin coding region, wherein the additional genetic sequences are selected from the group consisting of promoters and terminators.

11. An insecticidal microbe of claim 10, wherein the additional genetic sequences include a promoter selected from the group consisting of the promoter originating from the toxin gene and promoters originating from a gene from the insecticidal microbe.

12. An insecticidal microbe of claim 11, wherein the additional genetic sequences include a polyhedrin promoter from the microbe.

13. An insecticidal microbe of claim 11, wherein the additional genetic sequences include the p10 promoter of AcNPV.

14. An insecticidal microbe of claim 1, wherein the recombinant microbe is Bornbyx mori nuclear polyhedrosis virus and the recombinant DNA molecule comprises a coding DNA sequence derived from pCIB4223.

15. An insecticidal microbe of claim 1, wherein the recombinant microbe is Autographica californica nuclear polyhedrosis virus and the recombinant DNA molecule comprises the p10 promoter of AcNPV.

16. A method of enhancing the toxicity of insecticidal microbes comprising: a) isolating a recombinant DNA molecule comprising a genetic sequence coding for an insect-selective protein neurotoxin, wherein the genetic sequence codes for an

insect-selective protein neurotoxin selected from the group consisting of AaIT, buthoid scorpion toxins and chactoid scorpion toxins; and b) introducing the recombinant DNA molecule into the genome of the insecticidal microbe.

17. A method according to claim 16, wherein the insecticidal microbe is a baculovirus.

18. A method according to claim 17 wherein the genetic sequence codes for an insect-selective protein neurotoxin selected from the group consisting of AaIT, LqhIT2, LqqIT2 andLqhP35.

19. A method according to claim 18, wherein the baculovirus is the Bombyx mori nuclear polyhedrosis virus.

20. A method according to claim 18, wherein the baculovirus is the Autographica californica nuclear polyhedrosis virus.

21. A method according to claim 16, wherein the recombinant DNA molecule comprises a signal sequence isolated from an insect protein operably linked to the coding genetic sequence.