WO1999049035A2

WO1999049035A2 - Insecticidal compounds from segestria florentina

Info

Publication number: WO1999049035A2
Application number: PCT/GB1999/000907
Authority: WO
Inventors: John David Windass; Andrew Nicholas Blake; Evgeny Vasilievich Grishin; Elene Daniilovna Nosyreva; Sergei Aleksandrovich Koslov; Alexei Valerievich Lipkin
Original assignee: Zeneca Limited
Priority date: 1998-03-26
Filing date: 1999-03-23
Publication date: 1999-09-30
Also published as: AU3156399A; WO1999049035A3

Abstract

New insecticidal agents which comprise a polypeptide having a partial amino acid sequence: X?1ECMX2DX3TVCYIX4¿N (SEQ ID NO 3) or partial amino acid sequence: CENCWQYCDR (SEQ ID NO 4) where X?1, X2, X3 and X4¿ are variable amino acids; and a molecular weight of less than 6kDa, are described. Agents of this type may be obtainable from Segestria florentina or a fragment, variant or derivative thereof. Sequences encoding these toxins may be incorporated into insect viruses such as baculoviruses, or into recombinant plants in order to exert their toxic effect.

Description

INSECTICIDAL COMPOUNDS

The present invention relates to novel polypeptides such as proteins, which are useful as insecticidal agents, to nucleotide sequences encoding them as well as to insecticidal formulations or vectors such as insect virus vectors such as baculoviruses. or incorporating these.

Insecticidal agents have previously been isolated from insect species such as arthropods like scorpions or spiders. One such toxin has been obtained from the cellar spider Segestria florentina (Sagdiev et al., (1987) Bioorganischeskaya Khimiya, 13, 1013-1018). This toxin comprises the amino acid sequence

MRQDMVDESVCYITDNNCNGGKCLRSKACHADPWEL (SEQ ID NO 1)

These agents may be used as insecticides either by administration of the compounds per se, or by incorporating DNA which encodes the toxin into a suitable vector, for example a baculovirus (WO 92/16637) or other insect specific virus.

A further insecticidal agent isolated from Segestria spp. Is described in US Patent No. 5,674,846. The toxin here comprises a 31 amino acid sequence as follows:

KEXKPDGEQXGITNHNDXXNAXVXPNGPFMR (SEQ ID NO 2)

The applicants have now discovered a novel class of insecticidal compounds.

According to the present invention there is provided an insecticidal agent which comprises a polypeptide having a partial amino acid sequence:

X'ECMX²DX³TVCYIX⁴N (SEQ ID NO 3)

or partial amino acid sequence:

CENCWQYCDR (SEQ ID NO 4) where X', X², X³ and X⁴ are variable amino acids; said polypeptide having insecticidal properties and a molecular weight of less than 6kDa.

Suitably the polypeptide of the invention is a polypeptide obtainable from Segestria florentina or a fragment, variant or derivative thereof.

In a preferred embodiment, the polypeptides of the invention have the partial sequence SEQ ID NO 3 as defined above.

In particular in this structure, X¹ is selected from K or A, preferably K, X² is selected from A, V OR T, X³ is selected from E or G and X⁴ is H or I. The polypeptides of partial sequence (SEQ ID NO 3) are suitably up to 46 amino acids in length. In particular, they comprise one or more of the following partial sequences:

AECMVDETVCYIIN NNGTK (SEQ ID NO 5)

KECMTDGTVCYIINNNDDT (SEQ ID NO 6)

KECMADETVCYIINNNNDDG (SEQ ID NO 7)

It is believed that these polypeptides form part of a group or family of insecticidal agents and the invention includes members of this family. Preferably, the polypeptide comprises a partial SEQ ID No 7 above

Members of the family include the following sequences as well as insecticidal sequences which are at least 70%, preferably at least 80% and more preferably at least 90% homologous to any one of these structures.

KECMADETVCYIHNHNNCCGSCLCLNGPYARPWEMLVGNCKCGPKE

(SEQ ID NO 8)

KECMVDGTVCYIHNHNDCCGSCLCLNGPIARPWEMMVGNCKCGPKA (SEQ ID NO 9) KECMVDGTVCYIHNHNDCCGSCLCPNGPLARPWEMLVGNCKCGPKA

(SEQ ID NO 10)

KECMTDETVCYIHNHNDCCGSCLCLNGPIARPWEMMVGNCKCGPKA (SEQ ID NO 11)

KECMADGTVCYIHNHNDCCGSCLCPNGPLARPWEMLVGNCKCGPKA

(SEQ ID NO 12)

KECMTDGTVCYIHNHNDCCGSCLCSNGPIARPWEMMVGNCMCGPKA

(SEQ ID NO 13) As used herein, the term "homologous" is used to denote sequences which when aligned have similar (identical or conservatively replaced) amino acids in like positions or regions, where identical or conservatively replaced amino acids are those which do not alter the activity or function of the protein as compared to the starting protein. For example, two amino acid sequences which are "85% homologous" to each other have at least 85% similar (identical or conservatively replaced amino residues) in a like position when aligned optimally allowing for up to 3 gaps, with the proviso that in respect of the gaps a total of not more than 15 amino acid resides is affected. The degree of homology or similarity may be determined using methods well known in the art (see, for example, Wilbur, W.J. and Lipman, D.J. "Rapid Similarity Searches of Nucleic Acid and Protein Data Banks." Proceedings of the National Academy of Sciences USA 80, 726-730 (1983) and Myers E.and Miller W. "Optimal Alignments in Linear Space". Comput. Appl. Biosci. 4, 11-17(1988)). One programme which may be used in determining the degree of homology or similarity is the MegAlign Lipman-Pearson one pair method (using default parameters) which can be obtained from DNAstar Inc, 1228, Selfpark Street, Madison, Wisconsin, 53715, USA as part of the Lasergene system.

Alternatively, the polypeptide of the invention has a partial amino acid sequence SEQ ID NO 4 as defined above. Particular examples of the extended partial sequences of the invention as obtainable from Segestria florentina are given in Figure 4 hereinafter. Preferred examples of the agents of the invention include the following:

GARRCENCWQYCDRICRDKGKPRSTCKGFIIEWCECFD (SEQ ID NO 14)

or

YEPXXCENCWQYCDRYCKDSEKKPYSTCKGFIITWCECSDKPIPK (SEQ ID NO 15)

where X is an unknown amino acid residue.

Toxins having structures corresponding to each of the above SEQ ID NOS have been isolated from Segestria florentina venom and/or found to be encoded by S. florentina venom gland derived mRNA. These form a preferred aspect of the invention. In general the toxin of partial structure SEQ ID NO 4 will have a molecular weight of less than 6kDa and will be of from 38 to 43 amino acids in length.

The term "fragments" used herein refers to truncated fragments or deletion mutants of these toxins, which retain insecticidal properties.

Variants of the toxins are those in which one or more amino acids in the sequence have been replaced or deleted. As would be understood, some changes in amino acid sequence are possible without elimination of the activity of the polypeptide whilst specific changes may modify insecticidal activity. The replacement may be by way of "conservative substitution" where an amino acid is replaced with an amino acid of broadly similar properties, or there may be some non-conservative substitutions. In general more conservative substitutions will be feasible than non-conservative substitution. In general however, the variants will be at least 60% homologous, suitably at least 70% homologous and more preferably at least 90% homologous to the native toxin.

Variants may be isolated, for example from natural sources, by screening DNA libraries such as cDNA or genomic libraries, with a nucleotide sequence which encodes a sequence according to the invention or a probe or primer based thereon. These sequence may hybridise to any sequence which encodes a variant of the peptide of the invention. Such hybridisation occurs at, or between, low and high stringency conditions but preferably at high stringency conditions. In general terms, low stringency conditions can be defined as 3 x SCC at about ambient temperature to about 65°C, and high stringency conditions as 0.1 x SSC at about 65°C. SSC is the name of a buffer of 0.15M NaCl, 0.015M trisodium citrate. 3 x SSC is three time as strong as 1 x SSC and so on.

The term "derivative" relates to toxins which have been modified for example by chemical or biological methods.

The invention further provides a nucleotide sequence which encodes an agent as defined above. Such sequences may be used, for example in the production or application of the agents of the invention.

For instance, vectors incorporating nucleotide sequences which encode agents of the invention under the control of suitable expression regulation agents such as promoters, enhancers, signal sequences etc. may be introduced into an expression system, such as eukaryotic or prokaryotic cells, in particular prokaryotic cells such as E. coli. Culture of the transformed cells results in the production of the agents of the invention in significant quantities. These can then be recovered from the culture by conventional protein biochemical procedures. Agents produced in this way may then be incorporated into pesticidal formulations, for example in combination with agriculturally acceptable carriers, and applied to the insects or to an environment in which insects are found. In a preferred embodiment however, a nucleotide sequence which encodes an agent of the invention is incorporated either into a virus which infects insects, preferably selectively, such as baculovirus, so that the agent of the invention is expressed in insect cells which are infected with the virus.

Recombinant viruses of this type may be used in insect control. Alternatively, sequences which encode the agents of the invention may be incorporated into plants so that they are expressed in plant cells. In this way, they provide protective effect against insects feeding on the plants.

Vectors and expression hosts as defined above, together with methods of insect control utilising these form further aspects of the invention. The agents of the invention are preferably secreted from a cell in which they are produced. This is particularly true where they are applied in the form of a virus vector such as a baculovirus vector, as this allows the toxin to produce a significant effect on the insect as a whole by dissemination from the site of synthesis via bodily fluids (e.g. the haemolymph). Therefore, in a preferred embodiment, they may further include a signal peptide or a prepeptide amino acid sequence. Suitably signal peptide or prepeptide sequences are capable of mediating protein secretion from insect or plant cells. The selection of a suitable signal peptide sequence will depend upon the particular application of the agent of the invention and the host cell where it is being produced. A baculovirus signal sequence may be particularly useful, for example the secretory signal sequence of the gp67 protein. Other suitable signal sequences would be understood in the art, and many are listed in WO 92/16637, the content of which are incorporated herein by way of reference.

The invention will now be particularly described by way of example with reference to the accompanying drawings in which:

Figure 1 shows the results of size exclusion chromatography and SDS PAGE data for crude Segestria florentina venom; Figure 2 shows the results of reverse phase HPLC on the active fraction illustrated in Figure i;

Figure 3 shows the results of further reverse phase HPLC of fraction f5.2 illustrated in Figure 2; and

Figure 4 shows various partial amino acid sequences of toxins, as determined by different methods as described hereinafter.

Example 1

Separation and purification of toxins from Segestria florentina venom

Approximately 300 glands from Segestria florentina spiders from Central Asia were removed, extracted in Milli Q purified water, lyophilized and stored at -70°C.

Size-exclusion chromatography was selected for the first separation of the venom as well as for defining the molecular weight range of insecto toxins. The lyophilized venom was therefore dissolved in a running buffer (50mM Tris, 150mM NaCl, pH 7.0) and an aliquot of this solution was tested by Lowry assay. This indicated that crude venom contains more than 50% protein by dry weight. The solution was then separated using High-Performance Liquid Chromatography (HPLC) using a Gold System solvent module (Beckman) and a Waters 991 photodiode array detector. A TSK 4000SW column(10μM, 7.5 x 600mm Beckman)was first calibrated using gel filtration molecular weight markers (Serva Feinbiochemica - aldolase -147.00kDa, BSA - 66.00kDa, carbonic anhydrase -29.00kDa, cytochrome C - 12.40kDa, dinitrophenyl-L-Ala - 0.26kDa). lmg of crude venom was loaded onto the column in a volume of 0.1ml running buffer and eluted with a flow rate of 0.5ml/min by 50mM Tris buffer (pH 7.0) containing 150mM NaCl. The results are shown in Figure 1. Five well separated fractions were collected on a FRAC 100 collector (Pharmacia). 1/100 part of each fraction was then analysed by gel electrophoresis on a 12% acrylamide gel as described by Laemmli. Gels were stained with Coomassie R250 blue dye. Three different standard protein mixtures were used as markers. These were HMW - high molecular weight, LMW- low molecular weight and PS - peptide markers.

Each fraction was then tested for insectotoxin activity by injection of samples of 1 or 2 μl dissolved in water into 3^rd or 4^th instar H. virescens larvae of about lOOmg in weight. Each sample was tested on 5 larvae and toxic effects were monitored for up to 2 days. Similar buffers or saline were used as negative controls. These tests revealed that only fraction f5 in Figure 1 had insectotoxic effects. Its molecular weight was estimated to be less than 12kDa according to size-exclusion chromatography, a result which was confirmed by SDS PAGE data (Figure 1 ).

It appears therefore that Segestria florentina venom contains about 30% of low molecular weight proteins including species which are toxic to insects.

The active size fraction from this bioassay was loaded onto a DeltaPak reverse phase C₄ lOOA 3.9 xl 50mm column (Waters) for further purification. This was eluted at a flow rate of 0.7 ml/min. Solvents used were A 0.1 % trifluoroacetic acid, and B 0.1 % trifluoroacetic acid in acetonitrile. The separation was performed by applying a linear acetonitrile gradient to the column. This was created by controlled addition of Solvent B to Solvent A. Absorbance at 214 and 280nm was monitored.

Results are shown in Figure 2 hereinafter. Most of the polypeptides were eluted by 30-40% acetonitrile. Individual fractions were tested again for insectotoxic effects as described below. Fractions labeled f5.2, f5.5. f5.6 and f5.7 in Figure 2 were found to have toxic effects. Of these, f5.5, f5.6 and f5.7 were almost pure whilst f5.2 was a mixture of two related proteins.

The f5.2 fraction was therefore subjected to a final purification on the same column as that described above but by changing buffers from trifluoroacetic acid to sodium phosphate buffer (pH 6.0): Solvent A: (lOmM NaPi), Solvent B: (lOmM NaPi in 60% acetonitrile). A linear gradient of acetonitrile from 0 to 50%o solvent B over 100 minutes was established. Results are shown in Figure 3.

Each of the two separated polypeptides f5.2a and f5.2b obtained in this way had insectotoxin activity. The purity of the polypeptides was checked by capillary electrophoresis. Example 2

N-terminal Amino acid sequence determination of f5.2 toxins

In order to determine the N-terminal amino acid sequence of the f5.2a and f5.2b toxins, lyophilised samples (5μg) of each were reduced by dissolving a 500 fold molar excess of dithiothreitol (compared to toxin) in 6M guanidine chloride, 0.3M Tris buffer (pH 8.0), 2mM EDTA before overnight incubation under nitrogen at 25 °C. The thiol groups of the cysteine residues were then modified by alkylation with 4-vinylpyridine for 10 minutes at room temperature. After desalting on a DeltaPak column, the N-terminal sequences were determined on a gas-phase automatic sequencer Applied Biosystems model 470 A using an Edman degradation method. Phenylthiohydantoin amino acids were detected by an on-line HPLC module Applied Biosystems 120A. The partial sequence of f5.2a and f5.2b toxin was obtained and this is shown in Figure 4 and also in the sequence listings as SEQ ID Nos 16 and 17. Example 3 Isolation and sequencing of cDNA encoding f5.2

Venom glands of the spider Segestria florentina were removed, immediately frozen in liquid nitrogen and stored (-70°C). A sample of approximately 70 frozen venom glands (2.5g) was homogenized in liquid nitrogen. Total RNA was isolated from the frozen spider venom glands by the method of Feramisco (J. Biol. Chem. (1982) 257, 11024-11031). The first strand cDNA for amplification by the polymerase chain reaction (PCR) was synthesised by using total RNA and the RLdT oligonucleotide (Table 1) designed to act as a reverse transcription primer and to provide a unique 3' sequence to act as a recognition site during subsequent PCR mediated amplification. For this synthesis, 5μg of total RNA was reverse transcribed in 20μl for 60 minutes at 42°C (Promega). The structure of the oligonucleotide primers is presented in Table 1 below.

Table 1 Structure of oligonucleotide primers

Name Composition

RLdT |5 ' -GA-GAA-TTC-GGA-TCC-CTG-CAG- AAG-CTT-TTT-TTT-TTT-TTT- TTT-3' (SEQ ID NO 18)

RL 5 '-GA-GAA-TTC-GGA-TCC-CTG-CAG- AAG-CTT-3' (SEQ ID NO 19)

S4 5'-AAA-GA(G/A)-TG(T/C)-ATG-(G/A)CI-GA(T/C)-G(G/A)I- AC(G/A/T/C)-GT-3 ' (SEQ ID NO 20)

S7 5'-AAA-GA(G/A)-TG(T/C)-ATG-G(T/C)I-GA(T/C)-G(G/A)I- AC(G/A/T/C)-GT-3 ' (SEQ ID NO 21 )

SI 5 ' -GGT-GCI-( A C)G(G/A/T/C)-( A/C)GI-TG(T/C)-GA(G/A)- AA(T/C)-TG- 3' (SEQ ID NO 22)

S9 5 ' -TG(T/C)-GA(G/A)- AA(T/C)-TG(T/C)-TGG-C A(G/A)TA(T/C)-TG-3 ' (SEQ ID NO 23)

Rapid amplification of the 3'-cDNA ends (3'RACE- Frohman et al.,1988 (PNAS) 85:

8998) by PCR was then undertaken using the RLdT specific oligonucleotide primer RL and one of a range of degenerate oligonucleotide primer also shown in Table 1. These primers were designed in the following way: SI primer corresponded to the region between Gly, to Asn₇ residues in f5.2a; S9 primer corresponded to the region between Cys₅ to Cys_I2 residues in f5.2a and also to the region between Cys₆ to Cys₁₃ in the f5.2b, which were similar for both toxins.

PCR was performed in 25μl containing 1 x buffer (lOmM Tris-HCl, pH 9.0; 50mM KC1; 0.1% Triton X-100) for Taq DNA polymerase with 2mM MgCl₂, 0.05μg of the first strand cDNA, 0.2mM each dNTP, 1.5 units of Taq DNA polymerase, 5pmol RL oligonucleotide primer and 50pMol specific oligonucleotide primer. Samples were overlaid with mineral oil and subjected to 25 cycles, in a Perkin- Elmer thermocycler as follows: denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec. As a result of this PCR strategy, products of the expected sizes (approximately 250bp) were obtained. These PCR products were purified using the Wizard DNA Clean-up Purification System (Promega) and filled in with dNTPs and ATP using Klenow fragment and T4 polynucleotide kinase. The products were ligated into Smal digested pBlueScript SK vector (Stratagene). E. coli XL-1 Blue was used for plasmid propagation. The recombinant clones were analyzed using standard techniques and sequenced by the dideoxynucleotide chain- terminator method (Sanger et al., 1977) in the presence of [³³P]dATP by T7 DNA polymerase sequenase version 2.0 (Amersham).

At least ten separate clones for each PCR were isolated and sequenced on both forward (5'-3') and reverse (3 '-5') strands to confirm the cDNA sequence. It was found that the 3 '-untranslated region of these sequences contains a consensus polyadenylation signal (AATAAA) approximately 20 bp upstream from the polyadenylated tail.

The nucleotide sequences obtained using this method are set out in the sequence listing hereinafter. Specifically, the coding sequences corresponding the particular peptides are set out in Table 2.

Table 2

By this means, a full length amino acid sequence for f5.2a toxin and a partial amino acid sequence for f5.2b toxin were deduced. The close relationships between partial protein sequences determined directly using the Edman degradation method and sequences deduced following RACE-PCR of the cognate mRNAs are illustrated in Figure 4. In this Figure, the sequences correspond to those shown in the sequence listing provided in Table 3 Table 3

There are no differences between the partial sequences obtained using the two methods in respect of the f5.2b protein. Translation of the cDNAs shows that the sequence which encoded f5.2b toxin did not vary between the tested clones.

By contrast, the sequence encoded f5.2a toxins showed some variation with the sequence determined by Edman degradation and four of the closest cDNA encoded sequences s9-23, sl-2, s9-24 and s9-22 are shown in Figure 4.

Sequences 9-23 correlates with that obtained by N-terminal Edman degradation except for the Arg₂₃ residue. It is possible that incorrect sequence information was obtained by the Edman method as a result of the low yield of Arg residues on the gas-phase sequencer. It is probable that the other three sequences encode homologous sequences, for example as a result of polymorphism or allelic variation. Concentration of the toxins encoded by these genes in Segestria florentina venom are probably low. It is deduced therefore that the sequences of f5.2a and f5.2b are SEQ ID NOS 14 and 15 respectively as set out above.

This analysis revealed that both toxins are basic proteins with the same quantity of cysteine residues, probably forming 3 intramolecular disulphide bonds (predicted) and one additional SH-group. They contain a lot of positively charged amino acids. Homology between the amino acid sequences of the two f5.2 toxins was found to be around 15%. Two regions between Cys₅-Asp₁₈ and Lys₂₁-Asp₃₈ are well conserved (using the f5.2a sequence as a basis for the numbering) as is evident from Figure 4. There are differences in the N- terminal region, in particular in the Lys_I9-Gly₂₀ and in the extended C-terminus of the f5.2b toxin. Example 4

N-Terminal Amino Acid Sequencing of F5.5. F5.6 and F5.7 toxins

As with toxins f5.2a and f5.2b, to obtain N-terminal amino acid sequence of the f5.5, f5.6 & f5.7 toxins, lyophilised 5μg samples of each were reduced by dissolving with a 500 fold molar excess of dithiothreitol in 6M guanidine chloride, 0.3M Tris (pH8.0), 2mM EDTA before overnight incubation under nitrogen at 25°C. The thiol groups of the cysteine residues were then modified by alkylation with 4-vinylpyridine for 10 minutes at room temperature. After desalting on a Deltapak column, the N-terminal amino acid sequences were then determined on a gas-phase automatic sequencer using the Edman degradation method (Applied Biosystems model 470A). The sequences obtained were SEQ ID NOS 5, 6 and 7 respectively. Example 5

Isolation and Sequencing of cDNAs Encoding Members of the F5.5/F5.6/F5.7 Toxin Family This was undertaken essentially as described in Example 3 except that, after preparation of first strand cDNA using the RLdT primer, selective PCR amplification of cDNAs encoding members of the f5.5/f5.6/f5.7 toxin family was achieved by use of primer pairs RL/S4 and RL/S7 (see Table 1), the degenerate oligonucleotide primers S4 and S7 being designed on the basis of the sequence of the first 9 amino acids of f5.5, f5.6 and f5.7 as determined in Example 4. Amplification of PCR species of the anticipated size was then followed by cloning into pBlueScript SK and sequence analysis. Individual cDNA nucleotide sequences encoding members of the f5.5/f5.6/f5.7 toxin family are SEQ ID NOs 34, 35, 36, 37, 38 and 39 shown in the sequence listings hereinafter. These sequences encode SEQ ID NOS 8-13 given above. Example 6 Biological Activity

Toxic effect of Segestria florentina venom and of fractions prepared as described in Example 1 were assessed by intravenous injections of the various agents into adult mice. In this way, crude spider venom(4mg/kg weight) and fractions: f5.2 (1.1 mg/kg weight); f5.6 (1.5mg/kg weight); f5.7 (1.25 mg/kg weight) showed no toxic effects. However, after injection of crude venom (1 Oμg per larvae (0.1 μg/mg)), all tested insects had total flaccid paralysis after 15 minutes. After size-exclusion chromatography (Example 1 above), fraction f5 injected at a concentration of 4μg per larvae produced partial paralysis of all insects within 2 hours and 2 out of 5 deaths in one day. Fraction f5.2 (Example 1 above) at a concentration of 2.8μg per larvae resulted in partial paralysis of 3 out of 5 insects during the first 15 minutes and 2 out of 5 larvae died during day 2.

Fractions f5.2a, f5.2b, f5.5, f5.6 and f5.7 were tested in a similar manner and the results obtained are summarised in Table 4 below.

Table 4

In the above table, '"" indicates minutes, "h" is hours and "DAT" is Day(s) after test; "rp" is rigid paralysis, "fp" is flaccid paralysis, "sp" is semi-paralysed and "uc" is uncoordinated. Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention.

Claims

1. An insecticidal agent which comprises a polypeptide having a partial amino acid sequence:

X'ECMX²DX³TVCYIX⁴N (SEQ ID NO 3)

or partial amino acid sequence:

CENCWQYCDR (SEQ ID NO 4)

where X¹, X², X³ and X⁴ are variable amino acids; said polypeptide having insecticidal properties and a molecular weight of less than 6kDa.

2. An insecticidal agent according to claim 1 which is obtainable from Segestria florentina or a fragment, variant or derivative thereof.

3. An insecticidal agent according to any one of claim 1 or claim 2 which comprises a polypeptide having the partial sequence of SEQ ID NO 3 as defined in claim 1.

4. An insecticidal agent according to claim 3 wherein X¹ is selected from K or A, X² is selected from A, V or T, X³ is selected from E or G and X⁴ is H or I.

5. An insecticidal agent according to claim 3 or claim 4 which comprises a polypeptide of up to 46 amino acids in length.

6. An insecticidal agent according to any one of the preceding claims which comprises a polypeptide of partial sequence:

AECMVDETVCYIINNNNGTK (SEQ ID NO 5),

KECMTDGTVCYIINNNDDT (SEQ ID NO 6) or

KECMADETVCYIINNNNDDG (SEQ ID NO 7)

7. An insecticidal agent according to claim 6 which comprises a polypeptide of partial SEQ ID No 7.

8. An insecticidal agent according to claim 1 which comprises a sequence

KECMADETVCYIHNHNNCCGSCLCLNGPYARPWEMLVGNCKCGPKE

(SEQ ID NO 8)

KECMVDGTVCYIHNHNDCCGSCLCLNGPIARPWEMMVGNCKCGPKA

(SEQ ID NO 9) KECMVDGTVCYIHNHNDCCGSCLCPNGPLARPWEMLVGNCKCGPKA

(SEQ ID NO 10)

KECMTDETVCYIHNHNDCCGSCLCLNGPIARPWEMMVGNCKCGPKA (SEQ

ID NO 11)

KECMADGTVCYIHNHNDCCGSCLCPNGPLARPWEMLVGNCKCGPKA (SEQ ID NO 12) or

KECMTDGTVCYIHNHNDCCGSCLCSNGPIARPWEMMVGNCMCGPKA (SEQ

ID NO 13) or a sequence which is at least 70% homologous to any of these.

9. An insecticidal agent according to claim 1 or claim 2 which comprises a polypeptide of partial amino acid sequence SEQ ID NO 4.

10. An insecticidal agent according to claim 9 which comprises

GARRCENC WQ YCDRICRDKGKPRSTCKGFIIE WCECFD (SEQ ID NO 14)

or a variant or fragment thereof.

11. An insecticidal agent according to claim 9 which comprises

YEPXXCENCWQYCDRYCKDSEKKPYSTCKGFIITWCECSDKPIPK (SEQ ID NO 15)

where X is an unknown amino acid residue.

12. An insecticidal agent according to any one of the preceding claims which further comprises a signal sequence.

13. A nucleotide sequence which encodes an agent according to any one of the preceding claims.

14. A nucleotide sequence according to claim 13 which comprises any one of SEQ ID NOS 24, 26, 28, 30, or 32 as defined herein.

15. A nucleotide sequence according to claim 14 which comprises any one of SEQ ID NOS 34, 35, 36, 37, 38 or 39 as defined herein.

16. A vector incorporating a nucleotide sequence according to any one of claims 13 to 15.

17. A cell which has been transformed using a vector according to claim 16.

18. A cell according to claim 17 which comprises a plant cell.

19. A transgenic plant which comprises cells according to claim 18.

20. A recombinant virus which includes a nucleotide sequence according to any one of claims 13 to 15.

21. A recombinant virus according to claim 20 which comprises a recombinant baculovirus.

22. A method for killing or controlling insect pests which method comprises applying to the insect or to the environment thereof, an agent according to any one of claims 1 to 13 or a virus according to claim 20 or claim 21.