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Expression of lipoproteins

a technology of lipoproteins and lipids, applied in the field of gene engineering, can solve the problems of presenting problems such as the problem of pneumococcal disease control, applicants' difficulty in obtaining detectable expression of recombinant ospc, and antibiotic resistance, and achieve the effect of reducing the risk of pneumococcal resistan

Inactive Publication Date: 2005-08-04
HUEBNER ROBERT +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0086] In view of the difficulties associated with the use of adjuvants, it is thus an advantage of the present invention that the recombinant lipidated proteins are the most immunogenic forms, and are capable of eliciting immune responses both without any adjuvant and with alum.
[0087] The following examples illustrate but do not limit the scope of the invention disclosed in this specification. EXAMPLES

Problems solved by technology

However, the later systemic phases have proved to be more refractory to antibiotics.
Based on the findings regarding OspA, one might expect that lipidation of recombinant OspC would be useful to enhance its immunogenicity; but, as discussed below, the applicants experienced difficulties in obtaining detectable expression of recombinant OspC.
The increase in the frequency of multiple antibiotic resistance among pneumococci and the prohibitive cost of drug treatment in poor countries make the present prospect for control of pneumococcal disease problematical.
In some individuals, however, the organism carried in the nasopharynx can give rise to symptomatic sinusitis of middle ear infection.
If pneumococci are aspirated into the lung, especially with food particles or mucus, they can cause pneumonia.
Although pneumococcal meningitis is less common than other infections caused by these bacteria, it is particularly devastating; some 10% of patients die and greater than 50% of the remainder have life-long neurological sequelae.
The 23-valent vaccine is not effective in children less than 2 years of age because of their inability to make adequate responses to most polysaccharides.
However, immunogenicity and protection studies in mice have demonstrated-that the truncated recombinant form of PspA is not immunogenic in naive mice.
Many studies have suggested that urease, a complex of the products of the ureA and ureB genes, may be a protective antigen, However, until now it has not been known how to produce a sufficient-mucosal immune response to urease.
However, these adjuvants have deficiencies.
For instance, while cholera toxin B is not toxic in the sense of causing cholera, there is general unease about administering a toxin associated with a disease as harmful as cholera, especially if there is even the most remote chance of minor impurity.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of a Vector Containing a Gene Encoding the OspA Leader Sequence

[0088] Plasmid pBluescript KS+ (Stratagene) was digested with XbaI and BamHI and ligated with a 900 bp XbaI-BamHI DNA fragment containing the complete coding region of B. burgdorferi strain ACA1 ospA gene, to form a lipoprotein fusion vector pLF100. This procedure is shown schematically in FIG. 1.

[0089] The vector pLF100 has been deposited with the American Type Culture Collection at Rockville, Md. on Feb. 2, 1995 under Accession No. 69750. This deposit was made under the terms of the Budapest Treaty.

example 2

construction of a pET9a Expression Vector Containing a Hybrid ospA-ospC Gene

[0090] Specifically designed oligonucleotide primers were used in a polymerase chain reaction (PCR) to amplify the portion of the ospc gene downstream from the cysteine-encoding codon terminating the signal-peptide recognition-encoding sequence to the C-terminal end of the coding region from the Pko and B31 strains of B. burgdorferi.

[0091] The 5′-end primer had the nucleotide sequences respectively for the Pko and B31 strains:

5′-GGC GCG CAT GCA ATA ATT(Pko)(SEQ ID NO: 3)CAG GGA AAG G-3′5′-GGC GCG CAT GCA ATA ATT(B31)(SEQ ID NO: 4)CAG GGA AAG A-3′while the 3′-end primer had the nucleotidesequence:5′-CGC GGA TCC TTA AGG TTT(B31 &(SEQ ID NO: 5)TTT TGG-3′Pko)

[0092] The PCR amplification was effected in a DNA Thermal Cycler (Perkins-Elmer Cetus) for 25 cycles with denaturation for 30 secs at 94° C., annealing at 37° C. for 1 minute and extension at 72° C. for 1 minute. A final extension was effected at 72° C...

example 3

Expression and Purification of Lipidated OspC.

[0095] Plasmid pPko9a, prepared as described in Example 2, was used to transform E. coli strains BL21(DE3) (pLysS) and HMS174(DE3)(pLysS). The transformed E. coli was inoculated into LB media with 30 μg / ml kanamycin sulfate and 25 μg / ml of chloramphenicol at a rate of 12 ml of culture for every liter prepped. The culture was grown overnight in a flask shaker at 37° C.

[0096] The next morning, 10 ml of overnight culture medium was transferred to 1 L of LB media containing 30 μg / ml of kanamycin sulfate and the culture was grown in a flask shaker at about 37° C. to a level of OD600=0.6-1.0 (although growth up to OD600=1.5 can be effected), in approximately 3-5 hours.

[0097] To the culture medium was added isopropylthiogalactoside (IPTG) to a final concentration of 0.5 mM and the culture medium was grown for a further two hours at about 30° C. The cultures were harvested and samples analyzed on Coomassie stained SDS-PAGE gels (FIG. 5). The...

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Abstract

Heterologous lipidated proteins formed recombinantly are disclosed and claimed. The expression system can be E. coli. The heterologous lipidated protein has a leader sequence which does not naturally occur with the protein portion of the lipidated protein. The lipidated protein can have the Borrelia OspA leader sequence. The protein portion can be OspC, PspA, UreA, Ure B, or a fragment thereof. Methods and compositions for forming and employing the proteins are also disclosed and claimed.

Description

REFERENCE TO RELATED APPLICATIONS [0001] Reference, especially with respect to recombinant Borrelia proteins, is made to each of applications Ser. No. 07 / 973,338; filed Oct. 29, 1992; Ser. No. 08 / 373,455 (Rule 62 FWC of U.S. Ser. No. 07 / 973,338), filed Jan. 17, 1995, Ser. No. 07 / 888,765, filed May 27, 1992; Ser. No. 08 / 211,891; filed Oct. 16, 1992 (national phase of PCT / US92 / 08697); and Ser. No. 07 / 779,048, filed Oct. 18, 1991. [0002] Reference, especially with respect to structural genes of pneumococcal proteins, epitopic regions thereof, and administration of pneumococcal proteins, is made to each of applications Ser. Nos. 656,773, filed Feb. 15, 1991; Ser. No. 835,698, filed Feb. 12, 1992; Ser. No. 072,056, filed Jun. 3, 1993; Ser. No. 072,068, filed Jun. 3, 1993; Ser. No. 214,222, filed Mar. 17, 1994; Ser. No. 214,164, filed Mar. 17, 1994; Ser. No. 247,491, filed May 23, 1994; Ser. No. 048,896, filed Apr. 20, 1993; Ser. No. 246,636, filed May 20, 1994; ______ (continuation-in-pa...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00C07K14/195C07K14/205C07K14/315C12N15/62C12P21/02
CPCA61K38/00C07K14/195C07K14/205C12P21/02C07K2319/02C07K2319/40C12N15/625C07K14/315Y02A50/30
Inventor HUEBNER, ROBERTERDILE, LORNEWARAKOMSKI, DONALDBECKER, ROBERTGRAY, MARYANNPYLE, DEREK
Owner HUEBNER ROBERT
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