Classifications

• • 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
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/555—Interferons [IFN]
  • C07K14/56—IFN-alpha

Definitions

• This invention relates to a human . interferon- ⁇ and to the production thereof.
• the invention relates to a complete nucleotide sequence of a human interferon- ⁇ gene, and recombinant DNA molecules comprising this nucleotide sequence, as well as processes utilising said recombinant DNA molecules for producing the human interferon- ⁇ .
• the human interferons are a group of proteins possessing potent antiviral, antiproliferative and immune response modulating activities (1) .
• the potential therapeutic value of the interferons together with their limited availability from natural sources, considerable effort has been directed towards the cloning and expression of interferon genes.
• Three distinct types of interferon, ⁇ , ⁇ and ⁇ , have been described, based on differences in antigenicity and biological characteristics of the molecules (for review, 2) .
• nucleotide sequence of this aspect of the invention may be obtained from natural, synthetic or semi-synthetic sources; furthermore, the nucleotide sequence may be a naturally-occurring sequence, or may be related by mutation, including single or multiple base substitutions, deletions insertions and inversions, to such a naturally-occurring sequence, provided always that the DNA molecule comprising such a sequence is capable of being expressed as the desired amino acid sequence.
• the nucleotide sequence may have expression control sequences positioned adjacent to it, such control sequences being derived either from homologous or heterologous sources.
• the nucleotide sequence of IFN- ⁇ Ml according to this invention is further characterised in having a restriction map substantially as shown in Figure lb.
• IFN- ⁇ 4a 153 of 189 amino acids
• ⁇ 4b a complete amino acid sequence for another IFN- ⁇ denoted ⁇ 4b.
• the amino acid sequences were derived from unpublished nucleotide sequences of clones isolated from the same human gene library as that used in the present report (12) .
• IFN- ⁇ 4a and IFN- ⁇ 4b are considered to be allelic as they have similar flanking DNA sequences and, on currently published data, have only two amino acid differences (3) .
• an Alul fragment containing the coding region of the IFN- ⁇ Ml gene has been inserted into the Hindi site of the phage Ml3 mp 11, resulting in a fusion of the IFN- ⁇ Ml gene and the ⁇ -galactosidase gene.
• E. coli infected with the recombinant M13 phage carrying the fused gene has been cultured and extracts have shown antiviral activity in cytopathic effect inhibition assays. This antiviral activity was completely neutralised by IFN- ⁇ antibodies.
• Figure la shows the restriction map of the ⁇ Ml PstI fragment containing the IFN- ⁇ Ml gene. Restriction sites are indicated by the symbols ⁇ Q, Pstl ? - ⁇ , EcoRI; f r Hindlll. The hatched area indicates the IFN- ⁇ Ml coding region. The direction of transcription is from left to right.
• Figure lb shows the restriction map of the ⁇ Ml Rsal fragment containing the IFN- ⁇ Ml gene and the strategy for sequencing the IFN- ⁇ Ml gene. Restriction sites are indicated by the symbols: ⁇ >, Rsal; ⁇ *, Bspl; ⁇ r, Alul; , Sau3AI; ⁇ , EcoRII; ⁇ , Hindlll. Arrowed segments below the map indicate the extent and direction of nucleotide sequence data obtained from M13 subclones. The asterisks indicate the Bspl and EcoRII sites which are present in IFN- ⁇ Ml but absent from both IFN- ⁇ 4a and IFN- ⁇ 4b (see below) .
• Figure 2 shows the nucleotide and predicted amino acid sequence of IFN- ⁇ Ml.
• the initiation codon for pre-interferon, the codon for the N-terminal amino acid of the mature interferon and the termination codon are underlined.
• the putative 'TATA' box is underlined twice-.
• Figure 3 shows the nucleotide sequence of the M13 recombinant phage M13- ⁇ Ml-Bl in the region of the fusion between the ⁇ -galactosidase gene of Ml3mpll and the IFN- ⁇ Ml gene.
• Hybridization was carried out using the synthetic oligonucleotide probes (Table 1) . Restriction maps were constructed using standard methods, including-
• a PstI fragment of the- ⁇ Ml DNA to which the oligonucleotide probes hybridized was subcloned into p ⁇ C9 by standard methods. Following amplification in E. coli the purified PstI fragment was digested with selected restriction enzymes and the resulting fragments were separated by electrophoresis on an agarose gel. The fragments were transferred to nitrocellulose paper, hybridized to specific oligonucleotide probes and a restriction map of the PstI fragment was derived (Fig.la) .
• the gene was expressed in E. coli.
• An Alul-fragment of 669 bp (Fig.lb) was cloned into the Hindi site of Ml3mpll and clones with the correct orientation of the Alul fragment were selected by hybridization with oligonucleotides 4 and 5 (Table 1) .
• Ml3- ⁇ Ml-Bl contained the ⁇ -galactosidase promoter and the N-terminal 15 nu ⁇ leotides of the ⁇ -galactosidase gene coding region, 20 nucleotides of the M13mpll polylinker sequence, and 25 nu ⁇ leotides of the IFN- ⁇ Ml leader sequence followed by the mature IFN- ⁇ Ml coding region (Fig.3) .
• the 23 amino acid interferon leader would be replaced by a 19 amino acid leader (11 residues of which are non-interferon) , assuming the N-terminal methionine is removed from the ⁇ -galactosidase N-terminus.
• the interferon activity in both the culture supernatant and in the cell pellet extracts' was completely neutralized by both a polyclonal antiserum against human IFN- ⁇ (Cantell) and a monoclonal anti-human IFN- ⁇ antibody. It may be noted that the level of interferon expression obtained was lower than that previously reported with a M13 vector (20) .
• Factors which may account for this difference include the intrinsic specific activities of the particular interferons, the specific activity of the product of the particular fused gene constructed here

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  • 1984-12-20 EP EP19850900024 patent/EP0165942A1/de not_active Withdrawn
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