WO1997047649A1 - Neurotoxine de la tique d'anderson - Google Patents

Neurotoxine de la tique d'anderson Download PDF

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Publication number
WO1997047649A1
WO1997047649A1 PCT/AU1997/000366 AU9700366W WO9747649A1 WO 1997047649 A1 WO1997047649 A1 WO 1997047649A1 AU 9700366 W AU9700366 W AU 9700366W WO 9747649 A1 WO9747649 A1 WO 9747649A1
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tick
sequence
amino acid
polypeptide
acid sequence
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PCT/AU1997/000366
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English (en)
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Kevin William Broady
Michael Joseph Thurn
Slavica Masina
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Insearch Limited
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Priority to AU30192/97A priority Critical patent/AU720801B2/en
Publication of WO1997047649A1 publication Critical patent/WO1997047649A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43527—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from ticks
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to a paralysis tick neurotoxin and to polynucleotides encoding the neurotoxin.
  • the present invention further relates to compositions for use in raising an immune response in animals against tick neurotoxin, antibodies against the paralysis tick neurotoxin and methods of obtaining a protective effect against tick paralysis in mammals. Background of invention
  • the Australian paralysis tick Ixodes holocyclus is present along portions of the eastern coastal strip of Australia from north Queensland down to the Lakes entrance in Victoria (Roberts, 1970). Envenomation by the tick results in severe toxicosis for thousands of Australian domestic pets and livestock each year.
  • the toxicoses caused by Ixodes holocyclus are characterised by rapidly ascending flaccid paralysis. Other symptoms include loss of appetite, loss of coordination, excessive vomiting, respiratory distress and often death in the absence of speedy antitoxin treatment (Stone et al., 1989). Human cases of tick paralysis have been known to occur. Although deaths are now rare, hypersensitivity reactions are still common (Stone et al., 1983; Dorey and Broady, 1995).
  • the paralysis caused by Ixodes holocyclus is due to the presence of a neurotoxin in the salivary gland of the tick (Ross 1926, 1935; Stone et al., 1983).
  • Recent studies by Thurn et al (1992) resulted in the isolation of a neurotoxin from I. holocyclus which was shown to bind to rat brain synaptosomes (pinched off nerve terminals) in a temperature dependent manner. Proteins which had absorbed to the synaptosomes were resolved under reducing conditions by tricine-SDS (sodium dodecyl sulphate)-? AGE (polyacrylamide gel electrophoresis) and visualised by autoradiography.
  • This technique successfully identified 3 neurotoxins which had an apparent molecular weight of 5kDa and a pi of 4.5.
  • the neurotoxins were designated HT-I, HT-II and HT-III.
  • the second approach involves administration of an antiserum.
  • the present inventors have now purified the neurotoxin HT-1 of Ixodes holocyclus and determined the corresponding gene sequence.
  • the present invention provides an isolated polynucleotide which hybridises under stringent conditions to the polynucleotide sequence set out in Figure 12.
  • the polynucleotide comprises at least 10 nucleotides, more preferably at least 18 nucleotides and more preferably at least 25 nucleotides.
  • the isolated polynucleotide has a sequence substantially as shown in Figure 12 or natural variants or functional equivalents thereof.
  • naturally variants of the polypeptide sequence shown in Figure 12 we mean variants of the sequence which occur naturally and which encode the HT-1 neurotoxin.
  • functional equivalents of the polypeptide sequence shown in Figure 12 we mean sequences which encode polypeptides which differ from the polypeptide sequence shown in Figure 13 by way of substitutions, additions or deletions where such differences do not eliminate the biological activity of the polypeptide.
  • the isolated polynucleotide sequence encodes a polypeptide comprising an amino acid sequence corresponding to amino acids 23 to 72 as shown in Figure 13.
  • sequences which hybridise to the sequence shown in Figure 12 hybridise under stringent conditions.
  • stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0/1% NaDodSO 4 at 50°C; (2) employ during hybridisation a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 nM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10%
  • the present invention provides an isolated polypeptide having a sequence substantially as shown in Figure 13 or a biologically active fragment thereof.
  • biologically active fragment we mean a fragment which retains at least one of the activities of native HT-1 neurotoxin which activities include (i) the ability to cause a toxic effect in animals; and (ii) ability to mimic the binding of native HT-1 neurotoxin to at least one antibody or ligand molecule.
  • the polypeptides of the present invention may be used in controlled amounts as relaxants. For example, the polypeptides may be administered to subjects undergoing surgery where muscle relaxation is desirable.
  • the present invention provides a chimeric peptide comprising a first amino acid sequence substantially as shown in Figure 13 or a biologically active fragment thereof fused to a second amino acid sequence.
  • the first amino acid sequence corresponds to amino acids 23 to 72 of Figure 13.
  • the second amino acid sequence facilitates presentation of the tick neurotoxin sequence for the purpose of raising an immune response against the tick neurotoxin.
  • the second amino acid sequence may be any sequence which is suitable for this function.
  • the second amino acid sequence comprises a secretion signal.
  • the second amino acid sequence may be the MalE secretion signal. It will be appreciated that use of a secretion signal as the second amino acid sequence also facilitates purification of a recombinantly expressed chimeric peptide according to this aspect of the present invention.
  • the invention provides a composition for use in raising an immune response in animals against tick neurotoxin, the composition including a carrier and a polypeptide according to the second or third aspects of the present invention.
  • the present invention also extends to a composition for use in raising an immune response in animals against tick neurotoxin, the composition including a polynucleotide encoding a polypeptide according to the second or third aspects of the present invention.
  • polypeptides and fragments of the present invention without deleteriously affecting the biological activity of the polypeptides or fragments.
  • This may be achieved by various changes, such as sulfation, phosphorylation, nitration and halogenation; or by amino acid insertions, deletions and substitutions, either conservative or non- conservative (eg. D-amino acids, desamino acids) in the peptide sequence where such changes so not substantially alter the overall biological activity of the peptide.
  • Preferred substitutions are those which are conservative, i.e., wherein a residue is replaced by another of the same general type.
  • amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar. Furthermore, three of the encoded amino acids are aromatic. It is generally preferred that encoded peptides differing from the determined polypeptide contain substituted codons for amino acids which are from the same group as that of the amino acid replaced.
  • the basic amino acids Lys, Arg, and His are interchangeable; the acidic amino acids Asp and Glu are interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gin, and Asn are interchangeable; the nonpolar aliphatic amino acids Gly, Ala, Val, He, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely related and Val, He and Leu are more closely related), and the aromatic amino acids Phe, Trp and Tyr are interchangeable.
  • amino acids which are not naturally encoded by DNA may also be made.
  • alternative residues include the omega amino acids of the formula NH 2 (CH 2 ) n COOH wherein n is 2-6. These are neutral, nonpolar amino acids, as are sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine.
  • Phenylglycine may substitute for Trp, Tyr or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic.
  • Proline may be substituted with hydroxyproline and retain the conformation conferring properties.
  • the compostion further includes a suitable adjuvant.
  • Preferred adjuvants include DEAE Dextran/mineral oil, Alhydrogel, Auspharm adjuvant and Algammulin.
  • the present invention provides an antibody which binds to a polypeptide of the second aspect of the present invention.
  • antibody should be construed as covering any specific binding substance having a binding domain with the required specificity.
  • the term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide including an immunoglobulin binding domain, whether natural or synthetic. Chimeric molecules including an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.
  • the present invention provides a method of obtaining a protective effect against tick paralysis in an animal which method includes inoculating the animal with a polypeptide according to the second aspect of the invention.
  • the tick is Ixodes Holocyclus.
  • the animal is selected from a cow, horse, goat, cat and dog.
  • the word "comprise” or variations thereof will be understood to imply the inclusion of a stated element or integer or group of stated elements or integers but not the exclusion of any other element of integer of group of integers.
  • Figure 1 Elution profile of fractions obtained after reverse- phase C4 HPLC chromatography of the toxic fraction obtained from DEAE Affi-Gel Blue chromatography.
  • a 4.6x150mm C4 (Vydac) reverse-phase column was equilibrated with 0.1% aqueous TFA at a flow rate of lml/min.
  • the injection volume was 0.8ml and the column was eluted with a gradient of 0.8%TFA/80% acetonitrile.
  • FIG. 2 Elution profile obtained from the HIC of the toxic fraction obtained by DEAE Affi-Gel Blue chromatography.
  • HIC was carried out on a 0.5x10cm Alkyl Sepharose (Pharmacia) column. The column was equilibrated at a flow rate of 0.5 ml/mi n with 2.5M ammonium sulphate, lOOmM ammonium acetate, pH 6.8 (buffer A). A 2.0ml aliqout of sample was injected and the column allowed to equilibrate before a gradient of 100% buffer A to 100% lOOmM ammonium acetate, pH 6.8 (buffer B) was initiated. The eluate was monitored at 280nm and 1ml fractions collected by a Isco model 400 fraction collector. The fractions constituting each peak were pooled, concentrated and the buffer-exchanged by ultrafiltration.
  • FIG. 3 Separation of peak 1 (HIC) by Reverse-phase C4 HPLC.
  • the toxic fraction isolated by HIC was chromatographed on a 4.6x150mm C4 (Vydac) reverse phase column.
  • the column was equilibrated with 0.1% aqueous TFA at a flow rate of 0.25ml/min.
  • the injection volume was 0.8ml and the column was eluted with a gradient of 0.8%TFA/80% acetonitrile. Protein peaks were collected manually in Eppendorf tubes and analysed by Tricine-SDS-PAGE.
  • FIG 4 Separation of peak 1 ( Figure 3) by reverse-phase C8 HPLC.
  • Peak 1 isolated by reverse-phase C4 HPLC was chromatographed on a 4.6x300mm C8 (Brownlee) reverse phase column. The column was equilibrated with 0.1% aqueous TFA at a flow rate of l.Oml/min. 1.0ml of sample was injected and a gradient of 0.8%TF A/80% acetonitrile executed. Protein peaks were collected manually in Eppendorf tubes and analysed by Tricine-SDS-PAGE.
  • Figure 5 Separation of peak 2 ( Figure 3) by reverse-phase C8
  • Peak 2 isolated by reverse-phase C4 HPLC was chromatographed on a 4.6x300mm C8 (Brownlee) reverse phase column. The column was equilibrated with 0.1% aqueous TFA at a flow rate of l.Oml/min. 1.0ml of sample was injected and a gradient of 0.8%TFA/80% acetonitrile executed. Protein peaks were collected manually in Eppendorf tubes and analysed by Tricine-SDS-PAGE.
  • Figure 6 Derivation of Pi, P2 and P3 degenerate primers.
  • the putative tick amino acid sequence is given (top of page in bold). For all primers the amino acid sequence is shown in bold and the 4 base sites (N) were designated as inosine (I).
  • Figure A is the 23 bp antisense primer Pi The 4 base site at the 3' end of the sense strand was omitted.
  • Figure B is the 29 bp antisense primer P2. The A at the 3' end of the antisense strand was omitted and an EcoR I site was added to the 5' end.
  • Figure C is the 23 bp sense primer P3.
  • Figure 7 Possible tick toxin peptide orientations, (i): original putative tick peptide orientation, (ii); original putative tick peptide orientation with unknown (Xn) number of amino acids at junction spanning two adjoining peptides. (iii): reverse putative tick toxin orientation, with sequence coding for primers P4 (N-terminal) and P5 (C-terminal) in bold. (iv): reverse putative tick toxin orientation with Xn amino acids at junction spanning adjoining peptides.
  • Figure 8 Derivation of the degenerate primers P4 and P5.
  • the amino acid sequences were based on the sequence in Figure 3.8 (iii) and are shown in bold.
  • the 4 base sites (N) were designated inosine (I).
  • A the 24 bp antisense primer P4.
  • B the 23 bp sense primer P5.
  • the [CT] at the 3' end was omitted.
  • Figure 9 Sequence data obtained for the open reading frame from the PCR product amplified with primers P4 and P5.
  • Peptide B (5') and peptide A (3') from tryptic peptide information are underlined. The intervening amino acid sequence is not underlined.
  • Figure 10 Derivation of the non-degenerate primers Si and S2.
  • Amino acid sequences are shown in bold and were based on the sequence data in Figure 3.12.
  • A The 22 bp sense primer Si which was designed to extend toward the 3' end of the gene. The final A at the 3' end and the final C at the 5' end of the sense strand were omitted.
  • B The 22 bp antisense S2 primer which was designed to extend toward the 5' extremity of the toxin transcript. The final two G's at the 5' end of the antisense strand were omitted.
  • Figure 11 Sequence data obtained for 3' RACE product. The amino acids encoded by the open reading frame of the tick toxin are shown. Homologous data to previous peptide sequence information is underlined.
  • Stop codons TGA, TAA are indicated (*), followed by the 3' untranslated region including polyadenylation signal (AATAAA) and poly A tail.
  • Figure 12 The gene sequence encoding neurotoxin HT-1 from Ixodes holocyclus.
  • Figure 13 The deduced amino acid sequence for the unprocessed HT neutotoxin from Ixodes holocyclus.
  • Figure 14 Western blot of expressed products detected with antibodies to MBP.
  • Lanes 1 and 2 uninduced whole culture
  • Lanes 3 and 4 induced whole culture, 2 hrs
  • Lanes 5 and 6 induced whole culture, 4 hrs
  • Lanes 7 and 8 periplasmic extract, 2 hrs
  • Figure 15 Western blot of expressed product detected with dog anti-tick serum. Lane 1: uninduced whole culture
  • Lane 2 induced periplasmic extract
  • Lane 3 MBP standard
  • Figure 16 Dot Blots of Mouse Immune Sera against Whole Tick Extract and Periplasm Extract
  • Figure 17 Western blots of periplasmic extract.
  • Lanes 1,3,5 Serum from mice immunised with periplasmic extract with adjuvant
  • Lanes 7,9 Serum from mice immunised with periplasmic extract without adjuvant
  • Lane 11 Normal mouse serum
  • Lane 15 dog anti-tick serum
  • Lane 18 Western blots of crude whole tick extracts.
  • Lane 1 Serum from mouse immunised with periplasmic extract and adjuvant
  • the crude tick extract prepared from 275 female adult engorged ticks, was fractionated by ammonium sulphate precipitation. After a preliminary precipitation with 60% saturated ammonium sulphate, the resulting supernatant was adjusted to 70% saturation and the precipitate collected by centrifugation. The bulk of the neurotoxicity was recovered in the 70% precipitate and this fraction was in turn chromatographed on heparin Sepharose to principally remove the major host blood protein, haemoglobin and its derivatives. The unbound or sample flow through was collected and rechromatographed on a column of DEAE-Affi Gel Blue.
  • Neurotoxicity was associated with fraction obtained after eluting with 0.1M ammonium acetate, This fraction was used as the starting material for further purification and manipulation. It represents a 7.9 fold increase in purity of Holocyclus toxin and a recovery of 36%.
  • Degenerate primers were designed based on the partial sequence described above. The primers were degenerate as they take into account the codon usage for all of the amino acids.
  • the primers Pi and P2 are nested primers ( Figure 6) which were designed so that they could be used in 5' RACE PCR.
  • the Pi primer is 23 bp in length and is complementary to the 3' end of the sequence. It was designed to reverse transcribe a first strand cDNA copy of tick toxin mRNA.
  • the P2 primer is complementary to the proposed junction spanning the two adjoining peptides. This primer of 29 bp was designed so that it could prime towards the 5' end of the gene and be used in a PCR with an oligodeoxyribonucleotide anchor.
  • the P2 primer was used rather than Pi to maximise target specificity.
  • the anchor primer is complementary to the anchor sequence ligated onto the 3' end of the cDNA.
  • the P2 primer like the anchor primer encoded an EcoR I adaptor for efficient cloning into the EcoR I multiple cloning site in the pCR-Script vector.
  • an alternative (and more successful) method of ligation was chosen incorporating a blunt end ligation into the Srf site of pCR-Script in the presence of a Srf restriction enzyme.
  • the P2 and anchor primers amplified a DNA fragment of 150 bp in size.
  • the 150 bp product was purified and ligated into the pCR-Script sequencing vector .
  • the ligation mix was then used to transform electrocompetent DH5a bacterial cells.
  • RNA from engorged ticks was the starting material for all mRNA isolation procedures.
  • the total RNA was prepared according to the method of Okayama et al. (1987).
  • the PolyA tract mRNA isolation kit (Promega) was used to isolate mRNA from total tick RNA.
  • DNA CLONING METHODS Synthesis of cDNA from mRNA was carried out using a reverse transcriptase method (Goodman and MacDonald, 1974), with a Promega cDNA synthesis kit.
  • HT-I Ixodes holocyclus
  • 5'- AmpliFinder RACE kit from Clontech.
  • the 5' and 3' terminal sequences of the HT-I gene were investigated using the Marathon cDNA Amplification kit (Clontech).
  • Northern hybridisation was used to determine the size and presence of specific mRNA molecules in total or mRNA preparations. The procedure was performed as described by Sacchi et al. (1986), using a formaldehyde/agarose denaturing system. USE OF AN OLIGOdT TEMPLATE
  • First strand cDNA was synthesised from the tick mRNA using an oligodT primer. Subsequent to the formation of oligodT first strand cDNA template, additional amplifications were performed by PCR using Pi and P3 primers. Ubiquitin primers were used as a positive amplification control.
  • the P3 primer is a sense primer of 23 bp ( Figure 6) that spans the region of the adjoining tryptic peptides.
  • the ubiquitin primers were chosen as they amplify a well conserved 76 amino acid polypeptide that appears to be expressed by all eukaryotic cells. Several PCR products were obtained following PCR using ubiquitin primers, as was anticipated (result not shown).
  • Figure 7 (i) depicts the putative Ixodes holocyclus neurotoxin sequence, where peptide A is followed by B and where there is an undefined number of residues at the N- terminally blocked end. Equally likely, Figure 7 (ii) shows that the junction spanning the two adjoining peptides may also contain an undefined number of residues that were not detected during sequencing as there was uncertainty as to how many cleavage sites were present in the whole toxin peptide.
  • the primers P4 and P5 were used in a PCR with oligodT cDNA template. The resulting amplification produced a product of approximately 120 bp. This product of 120 bp was subsequently eluted from the gel and purified. Purification was analysed by electrophoresing an aliquot of the amplified product on a 10% polyacrylamide gel. A single pure band was obtained with a concentration of approximately 10-15ng /3 ⁇ L of eluted DNA, as determined by comparison with PhiX174/Hae III known standards. The purified product was ligated into the vector pBluescript. The ligation mix was used to transform into electrocompetent DH5a cells.
  • Recombinant colonies were grown overnight and plasmid DNA was purified by alkaline lysis.
  • An aliquot of the recombinant vector DNA was digested with Not I and Xho I restriction enzymes, each of which have restriction sites flanking the multiple cloning site EcoR V in the pBluescript vector.
  • the restriction digests were analysed for the presence of the 120 bp insert on a 10% polyacrylamide gel. One clone was obtained which had an insert of appropriate size (120 bp).
  • Recombinant vector containing the 120 bp insert was gel purified and subsequently sequenced by manual sequencing. The resulting DNA and translated amino acid sequence are shown in Figure 9.
  • the primers Si and S2 were non- degenerate primers designed to anneal to complementary strands on the same region of the putative tick toxin gene ( Figure 10).
  • the Si primer is a 22 bp sense primer designed to prime toward the 3' end of the gene. It was used in conjunction with an adaptor primer and with adaptor ligated cDNA as template.
  • the product obtained by PCR was approximately 300 bp in size.
  • the S2 primer is an antisense primer (22 bp) that was designed to prime toward the unknown 5' end of the gene.
  • the S2 primer was used in a PCR with the adaptor primer as for 3' RACE.
  • the 5' RACE product was approximately 280 bp.
  • the individual RACE products were purified, ligated into pBluescript sequencing vector and transformed into electrocompetent DH5a cells and recombinant clones purified by alkaline lysis. Plasmid DNA was digested with Bam HI and Xho I restriction enzymes and vectors containing insert of correct size were purified and sequenced.
  • the consensus sequence for the sense strand of the 3' RACE product is shown in Figure 11. Within the sequence overlap of the P4/P5 amplified product there were 4/18 differences at the amino acid level. The region of the C-terminal end coding for the toxin is approximately 19 amino acids in length. This region is clearly defined by the presence of the two stop codons (TGA and TAA) at the end of the open reading frame. Downstream of the stop codon is a non-coding region (—160 bp, that would not be translated during toxin processing). This region includes a polyadenyalation sequence AAUAAA which is upstream from the polyA tail. All these characteristics indicate that a functional eukaryotic mRNA has been amplified. 5' RACE SEQUENCED PRODUCT
  • 5' RACE PCR with the above antisense primer gave a product of — 280 bp which was sequenced to reveal the 5' sequence of the gene including the start codon and the 5' untranslated sequence.
  • a non- degenerate PCR primer was designed to the 5' untranslated region and used in 3' RACE PCR.
  • the product (—400 bp was sequnced and shown to include the start codon, stop codons, polyA tail and regions corresponding to the peptides sequenced directly from the protein HT-1.
  • the gene sequence for the Australian paralysis tick Ixodes holocyclus neurotoxin HT-1 is shown in Figure 12.
  • the deduced amino acid sequence of the unprocessed HT-1 is shown in Figure 13. DEDUCTION OFHT-1 TOXIN SEQUENCE FOR EXPRESSION
  • the length of the signal peptide sequence was predicted based on the available database prediction programs for signal sequence cleavage sites (PSORT) and from the published methodologies of von Heijne (von Heijne 1996). From these predictions the N-terminal residue of the mature toxin protein correlated to that of a serine residue (residue 23 figure 13). To ensure that the entire toxin sequence would be translated when cloned into an expression vector it was decide to extend the beginning of the expressed toxin seqeunce to a glutamic acid residue (residue 19 in figure 13).
  • the expression sequence for HT-1 was amplified by PCR using the gene specific primers shown below incorporating restriction endonuclease sites for cloning into the expression vector system pMAL (New England Biolabs protein fusion and purification system).
  • Antisense primer E2 (38 mer):
  • the PCR product was successfully ligated into the expression vector pMAL-p2 (the expression vector pMAL-P2 contains a MalE secretion signal which directs the secretion of maltose binding protein (MBP) which results in the expression of an MBP fusion protein) using a NEB ligation kit as per manufacturers instructions.
  • the sequence of the cloned product was then confirmed by preparing the DNA for analysis by cycle sequencing based on the LI-COR automated Sequitherm cycle sequencing protocol, using reagents and equipment under standard conditions as per manufacturers instructions.
  • EXPRESSION OF THE TOXIN SEQUENCE HT-1 The cloned fusion protein was electroporated into E.coli host TBl cells using a Bio-Rad Gene Pulser under standard conditions as outlined by the manufacturer. The expression of the fusion protein essentially was carried out as outlined in the NEB instruction manual. Briefly the conditions involved inoculating an overnight culture of cells containing the fusion plasmid. The cells were grown at 37°C to optimal density. The cell culture was then induced using IPTG and further incubated for 4 hours. The cells were harvested by centrifugation and periplasmic extraction performed as described by the manufacturer.
  • Samples of periplasmic extract, supernatants and whole culture from the expression experiment were analysed via immunological detection by Western blot essentially as described by Sambrook et al 1989.
  • the sample proteins were electrophoresed in a 12% SDS-PAGE under reducing conditions (1M DTT) using standard buffers and conditions.
  • the immobilised proteins were then assembled in a Western blot apparatus.
  • a 0.45uM nitrocellulose membrane (BioRad) and Whatmann 3MM filter paper were used to transfer the proteins.
  • the antibodies used to detect the proteins were appropriate dilutions of anti-MBP (NEB) and anti-rabbit alkaline phosphatase (AP) (Promega) and in Figure 15, anti-tick dog commercial antiserum (Australian Veterinary Serum Laboratories) and anti- dog lgG AP(Sigma).
  • Figure 14 depicts expressed protein samples being detected by anti- MBP showing that the major protein being detected is that of the fusion protein at 48 kDa.
  • the identity of the fusion protein and that of MBP (at 42kDa) was confirmed by N-terminal sequencing of the first 10 residues (carried out by Dr. D. Shaw, ANU).
  • the yield of expression culture as analysed on SDS-PAGE against standard proteins is estimated as between 5- 10 mg per litre of culture.
  • Figure 15 depicts periplasm extract being detected by the polyclonal dog antisera.
  • the dog antiserum is shown to be specifically reacting with the fusion protein at 48kDa in lane 2 but not reacting with standard MBP in lane 3.
  • the faint higher molecular weight band in lane 2 is likely to be the fusion protein containing the MalE secretion signal from the difference in size.
  • the semi-purified material obtained from the C8 column was further purified by rechromatographing the semi-purified fractions on a C18 reverse phase column (VYDAC) with the same buffers and flow conditions. Purified material was collected in a single peak/fraction being identified as the fusion protein by SDS-PAGE analysis as described above.
  • mice Seven week old female Balb/c mice were immunised I. P. with expressed periplasmic extract (lOug protein/lOOul) in the presence (4 mice) or absence (4 mice) of complete Freunds adjuvant (Pierce). Fourteen days following the primary injection a secondary boost was given of periplasm preparation as described above. Three weeks (28 days) following the primary injection a third boost using a 2ug/100ul semi-purified preparation of periplasm extract was given as the immunogen as described above. On the same day the mice were bled by cardiac puncture and 200ul of blood was collected from each mouse. The blood was allowed to clot overnight at 4°C. The clotted blood was then spun down using a bench top centrifuge (1500 rpm) and sera removed. The sera and blood were then stored separately at - 20°C.
  • the anti mouse sera are shown to react with periplasm extract antigen and with the native tick extract. Sera immunised with adjuvant react more strongly at various dilutions than that from mice immunised without adjuvant.
  • the anti-MBP reacts strongly with the periplasm extract antigen as expected.
  • the normal mouse sera did not react with either antigens as expected. This experiment provides preliminary evidence indicating that the fusion protein can induce a specific immune response to both the carrier protein [MBP] and the recombinant tick toxin.
  • a Western blot was performed to confirm that the immune response detected by dot blot experiments was directed against both the carrier protein [MBP] and the recombinant tick toxin.
  • Protein antigens were electrophoresed on a 15% SDS-PAGE.
  • the antigens were crude native tick extract and expressed periplasm extract containing the fusion protein.
  • the gels were blotted onto nitrocellulose and Western blot performed under standard conditions.
  • the primary antibodies used to detect the antigens were appropriate dilutions of the three mouse sera from animals immunised with adjuvant; two mice sera immunised without adjuvant; normal mouse sera and commercial polyclonal dog antisera.
  • the secondary antibodies used were as described for the dot blot experiment with the addition of anti-dog IgG-AP for detection of dog antiserum.
  • Figure 17 depicts the Western blot experiment detecting proteins in the periplasmic extract.
  • the mouse immune sera and the anti-MBP and dog immune serum all detect the fusion protein in the periplasmic extract
  • the normal mouse negative control does not detect any antigen as expected
  • Figure 18 depicts the Western blot detecting proteins in the crude tick homogenate. Both the mouse immune sera and the dog immune serum detect a low molecular weight band corresponding to the native tick toxin [— 5kDa]. This protein is not detected by the anti-MBP or the normal mouse serum. Some non-specific binding of anti-MBP to the crude tick extract is evident but not at the size of the tick toxin. The experiment indicates that the fusion protein is able to elict antibodies which react speicifically with the recombinant tick toxin.
  • mice appeared to be normal for the 1/16-1/32 dilutions.
  • the initial protection assay therefore used a 1/4 dilution of crude tick homogenate to produce toxin induced paralysis.
  • the following experimental groups were set up where each animal [5 day old Q/S neonatal mice] with lOOul of the relevant mixture:
  • Test a Tick homogenate + mouse sera [immunised with adjuvant]
  • Test b Tick homogenate + mouse sera
  • mice were left overnight (16 hours) on a 37°C heating pad.
  • results from this preliminary protection assay indicates that the immune mouse sera raised against the crude expressed periplasmic extract containing recombinant fusion protein was able to produce partial protection against the native tick toxin. Complete protection may require more antibody than was available in the small amounts of immune mouse serum available for this experiment.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Insects & Arthropods (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
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  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un polynucléotide isolé codant la neurotoxine HT-1 de la tique d'Anderson Isodes holocyclus. La présente invention se rapporte également à un polypeptide isolé correspondant à la neurotoxine HT-1, à des anticorps se fixant à la neurotoxine HT-1 et à des compositions de vaccins utilisés dans le but de provoquer une réaction immunitaire contre la neurotoxine HT-1 chez des animaux.
PCT/AU1997/000366 1996-06-11 1997-06-11 Neurotoxine de la tique d'anderson WO1997047649A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU30192/97A AU720801B2 (en) 1996-06-11 1997-06-11 Paralysis tick neurotoxin

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPO0395A AUPO039596A0 (en) 1996-06-11 1996-06-11 Paralysis tick neurotoxin
AUPO0395 1996-06-11

Publications (1)

Publication Number Publication Date
WO1997047649A1 true WO1997047649A1 (fr) 1997-12-18

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ID=3794713

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1997/000366 WO1997047649A1 (fr) 1996-06-11 1997-06-11 Neurotoxine de la tique d'anderson

Country Status (3)

Country Link
AU (1) AUPO039596A0 (fr)
WO (1) WO1997047649A1 (fr)
ZA (1) ZA975161B (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2344591A (en) * 1998-08-24 2000-06-14 Bioline Limited A chimaeric DNA polymerase
WO2014018724A1 (fr) 2012-07-27 2014-01-30 Zoetis Llc Compositions de toxine de tique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ALLERGY, 50 (Supplement), Abstract P-0922, COREY C.N. et al., "Characterisation of the Major 35 kDa Allergen of the Australian Paralysis Tick", p. 389. *
TOXICON, Vol. 30, (1992), THURN M.J. et al., "Identification of the Neurotoxin from the Australian Paralysis Tick Ixodes Holocyclus"; & BIOSIS, AN 92:490545. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2344591A (en) * 1998-08-24 2000-06-14 Bioline Limited A chimaeric DNA polymerase
GB2344591B (en) * 1998-08-24 2003-11-12 Bioline Ltd Thermostable DNA polymerase
WO2014018724A1 (fr) 2012-07-27 2014-01-30 Zoetis Llc Compositions de toxine de tique
AU2017261603B2 (en) * 2012-07-27 2019-07-18 Zoetis Services Llc Tick toxin compositions
AU2019204773B2 (en) * 2012-07-27 2021-02-18 Zoetis Services Llc Tick toxin compositions

Also Published As

Publication number Publication date
ZA975161B (en) 1998-03-03
AUPO039596A0 (en) 1996-07-04

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