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Recombinant fusobacterium necrophorum leukotoxin vaccine and preparation thereof

a technology of fusobacterium necrophorum and leukotoxin, which is applied in the direction of bacteria peptides, antibody medical ingredients, peptide sources, etc., can solve the problems of ineffective protection, and increased risk of liver abscess in sheep and goats, so as to induce protective immunity and significant protection

Inactive Publication Date: 2009-05-07
KANSAS STATE UNIV RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0018]Expression of a polypeptide encoded by the 3.5 kb from the 5′ end of the lktA caused immediate cessation of the growth and lysis of E. coli host cells suggesting that regions of leukotoxin could be toxic to E. coli. Of course, the objective was to create overlapping gene truncations extending over the entire lktA ORF so that the resulting polypeptide products are small and relatively stable on expression, but are large enough to be immunogenic. Also, the effectiveness of various recombinant truncated leukotoxin polypeptides alone or in combinations as immunogens and evaluated protective immunity against challenge with F. necrophorum in mice was investigated. The use of mice as an experimental model for F. necrophorum infection in cattle is well established (Abe et al., 1976; Conion et al., 1977; Smith et al., 1989; Garcia and McKay, 1978; Emery and Vaughan, 1986). Extension of the patterns of immunity and infection to cattle has shown that mice can be a valuable model to evaluate the immunogenicity and protection provided by various F. necrophorum fractions (Garcia et al., 1975; Garcia and McKay, 1978). Studies have also indicated that strains of F. necrophorum that are pathogenic in domestic animals, frequently are pathogenic in mice suggesting necrobacillosis as a disease is similar among these species of animals (Smith and Thornton, 1993).
[0020]Truncated recombinant polypeptides were purified by nickel affinity chromatography, and injected into rabbits to raise polyclonal antisera. Antibodies raised against two of the five polypeptides (BSBSE and GAS) neutralized the toxicity of F. necrophorum leukotoxin against bovine neutrophils. The effectiveness of the purified truncated polypeptides to induce a protective immunity was determined by injecting the polypeptides, individually or in mixtures, homogenized with Ribi adjuvant in mice, followed by experimental challenge with F. necrophorum. Two polypeptides (BSBSE and SH) induced significant protection in mice against F. necrophorum infection and the extent of protection was greater than the full-length native leukotoxin or inactivated culture supernatant. The study provided further credence to the importance of leukotoxin as the major virulence factor of F. necrophorum and the protein carries a domain (s) or epitope (s) that induces protective immunity against experimental infection.

Problems solved by technology

Liver abscesses in feed lot cattle are a serious economic problem, causing condemnation of over 3 million livers and an estimated loss of $15 million annually in the United States.
To a lesser extent, liver abscesses in sheep and goats are also an economic problem.
The results of such attempts have varied from ineffectual to significant protection.
Antimicrobial compounds reduce the incidence of liver abscesses but do not eliminate the problem (Nagaraja et al., 1998).

Method used

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  • Recombinant fusobacterium necrophorum leukotoxin vaccine and preparation thereof
  • Recombinant fusobacterium necrophorum leukotoxin vaccine and preparation thereof
  • Recombinant fusobacterium necrophorum leukotoxin vaccine and preparation thereof

Examples

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example 1

Cloning of the Leukotoxin Encoding F. necrophorum Gene

[0042]Chromosomal DNA, extracted from Fusobacterium necrophorum subsp. necrophorum, strain A25 (Hull et al., 1981, Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect. Immun. 33:933-938.), was partially digested with the restriction endonuclease Sau3AI, and size-fractionated by sucrose gradient centrifugation (Baxter-Gabbard, 1972, A simple method for the large scale preparation of sucrose gradients. FEBS. Lett. 20117-119). The 10-12 kb DNA fragments were ligated in BamHI-digested, dephosphorylated λZAP Express vector, packaged into lambda phage head and tail protein components (Stratagene, La Jolla, Calif.), and recombinant phages were infected into Escherichia coli XL1-Blue MRF′ and plated onto agar plates. Plaque lifts were performed (with polyclonal antiserum raised in rabbits against affinity purified leukotoxin) using...

example 2

Preparation of Polyclonal Antileukotoxin Antiserum

[0050]Leukotoxin from F. necrophorum subsp.necrophorum strain A25 was purified using an immunoaffinity column containing antileukotoxin monoclonal antibody, F7B10 (Tan, Z. L., T. G. Nagaraja, M. M. Chengappa, J. J. Staats. 1994. Purification and quantification of Fusobacterium necrophorum leukotoxin using monoclonal antibodies. Vet. Microbiol. 42:121-133.). Affinity-purified native leukotoxin (0.5 mg) in 100 μl of PBS was homogenized with an equal volume of Freund's complete adjuvant and injected intramuscularly in rabbits. A booster dose was given on day 21 with 0.5 mg of native toxin in 100 μl of PBS homogenized with an equal volume of Freund's incomplete adjuvant. Serum samples were collected on day 42. Naturally occurring rabbit antibodies that react to E. coli proteins were removed from the antisera as follows. Cell pellets of E. coli XL1-Blue MRF′ host cells grown overnight in Luria broth were sonicated in PBS and centrifuged t...

example 3

Construction of Truncated Forms of the Leukotoxin

[0074]A 3.5 kb sequence from the 5′ end of lktA gene was amplified by PCR and cloned in-frame in the expression vector pQE 30 (Qiagen Corporation). Induced expression of this truncated version of the leukotoxin protein with IPTG resulted in the immediate cessation of growth and caused lysis of the host E. coli cells. In order to obtain better expression of recombinant protein, smaller truncations of the leukotoxin gene were constructed. Polymerase chain reaction using thermostable polymerase with proof reading ability (EXTaq; Takara Corp.) was used to amplify five overlapping regions of the leukotoxin gene. The forward primers were designed to contain a SacI site, and the reverse primers had a XmaI site. F. necrophorum A25 chromosomal DNA was used as the template, and the amplified products were digested with restriction enzymes SacI and XmaI, and cloned in-frame in the His-tag expression vector pQE 30. Five truncated leukotoxin prote...

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Abstract

The F. necrophorum gene expressing leukotoxin was sequenced and cloned. The leukotoxin open reading frame (lktA) is part of a multi-gene operon containing 9,726 bp, and encoding a protein containing 3,241 amino acids with an overall molecular weight of 335,956 daltons. The protein encoded by the gene was truncated into five polypeptides having overlapping regions by truncating the full length gene into five different sections and amplifying, expressing, and recovering the protein encoded by each of these sections. Additionally, a region upstream of the gene was sequenced and the polypeptide encoded by that nucleotide sequence was purified and isolated. These polypeptides along with the full length protein are then tested to determine their immunogenicity and protective immunity in comparison to the efficacy of immunization conferred by inactivated native leukotoxin in F. necrophorum culture supernatant.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional application of application Ser. No. 09 / 841,786 filed Apr. 24, 2001 which was a continuation-in-part application to application Ser. No. 09 / 558,257, Filed Apr. 25, 2000. The content and teachings of each of these applications is hereby incorporated by reference herein.SEQUENCE LISTING[0002]A printed Sequence Listing accompanies this application, and also has been submitted with identical contents in the form of a computer-readable ASCII file on a floppy diskette with application Ser. No. 09 / 558,257, filed Apr. 25, 2000. Use of this previously filed CRF sequence listing is requested.BACKGROUND OF THE INVENTION[0003]1. Field of the Invention[0004]The present invention is concerned with methods of cloning and expressing the leukotoxin gene from Fusobacterium necrophorum (F. necrophorum), sequencing and characterizing the leukotoxin protein expressed by this gene, truncating the gene into a series of nucleotide...

Claims

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Application Information

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IPC IPC(8): A61K39/114C12N15/11C12N15/00A61K39/00C07K14/195
CPCC07K14/195A61K39/00
Inventor NAGARAJA, TIRUVOOR G.STEWART, GEORGE C.NARAYANAN, SANJEEV K.CHENGAPPA, MUCKATIRA M.
Owner KANSAS STATE UNIV RES FOUND