Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Patent
1984-09-28
1988-06-14
Hazel, Blondel
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
424 85, 435 68, 435811, C07K 1300, C07K 1526, A61K 4502, C12P 2100
Patent
active
047512873
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates generally to genetic engineering and has particular reference to human leukocyte interferon N and a method for producing same in bacterial cells.
BACKGROUND ART
Interferons, and the leukocyte interferon inclusive, are essentially a class of inducible proteins in the vertebrates that are capable of exhibiting antiviral and antimicrobial activity, participating in regulation of immunological reactions of a cell and producing radioprotective and antitumorigenic effect. Human leukocyte interferons make up a multigene family incorporating not less than twelve members, which has been proved by an analysis of the structure of the chromosomal DNA and cDNA synthesized on the interferon mRNA, as well as by that of the aminoacid sequence of individual proteins from a heterogeneous mixture of human leukocyte interferons.
Known in the present state of the art are diverse human leukocyte interferons obtained by virtue of genetic engineering, e.g., interferon A produced by recombinan DNA in the cells of Escherichia coli (cf. Goeddel D. V. et al., Human leukocyte interferon produced by E. coli is biologically active, Nature, 1980, 287, 411-416), or human leukocyte interferon F (cf. Ovchinnikov Yu. A. et al., Direct expression of the gene of human leukocyte interferon F in the cells of E. coli, Transactions of the USSR Academy of Sciences, 1982, 265, 238-242 (in Russian), which is in effect a protein consisting of 166 aminoacids arranged in the following sequence: LHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDMEACVIQEVGVEETPLM NVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSKIFQERLRRKE
A-ala; C-cys; D-asp; E-glu; F-phe; G-gly; H-his; l-ile; K-lys; L-leu; M-met; N-asn; P-pro; Q-gln; R-arg; S-ser; T-thr; V-val; W-trp; Y-tyr.
To produce the aforesaid interferon F the summary messenger RNA is produced from human blood leukocytes induced by Newcastle disease virus using the guanidine-chloride-guanidine-thiocyanate method. The poly-A fraction of mRNA is produced by double purification on oligo-dT-cellulose. The synthesis of cDNA is carried out using a synthetic oligonucleotide complementary to the gene of interferon within the sphere of translation termination. The synthesis of the second chain is performed with the aid of the Klenov's fragment of DNA-polymerase I. The thus-produced double-chain DNA is inserted after reconstruction, in the vector plasmid pBR 322 under the control of a tryptophane promotor. It is with the aforesaid recombinant plasmid that the E coli cells are transformed.
The aforementioned human leukocyte interferons differ from each other both in the efficacy of an inhibitory effect they produce on the cytopathic action of the same virus, and in the potency against most various viruses.
DISCLOSURE OF THE INVENTION
The invention is aimed at the provision of a novel human leukocyte interferon featuring the specificity of an antiviral and antiproliferative effect.
The human leukocyte interferon N of the invention is an essentially novel one and has not so far been described in literature.
The object of the invention is attained due to the fact that, according to the invention, the interferon N of the invention is in effect a protein featuring the following sequence of aminoacids: HEMIQQTFNLFSTKDSSAAWDETLLDKFYIELFQQLNDLEACVTQEVGVEEIALMNE DSILAVRKYFQRITLYLMGKKYSPCAWEVVRAEIMRSFSFSTNLQKGLRRKD
The novel human leukocyte interferon N disclosed in this invention features the specificity of both antiviral and antiproliferative effect. Practical application of this novel kind of interferon will make it possible to extend the range of medicines for producing a selective effect on viruses and malignant neoplasms.
A method of producing the aforementioned human leukocyte interferon N, consisting in isolation of matrix poly(A)-mRNA from induced human leukocytes, synthesis of the interferon gene, insertion in the vector plasmid pBR 322 under the control of a tryptophane promotor, and transformation of the thus-produced recombinant DNA of the E-coli bacterial cells, wherein, according to t
REFERENCES:
patent: 4414150 (1983-11-01), Goeddel
Goeddel et al., Nature, vol. 290, pp. 20-26, 1981.
Nagata et al., J. Interferon Research, vol. 1, pp. 333-336, 1981.
Apsalon Uldis R.
Berzin Valdis M.
Dishler Andris V.
Duk Arnis E.
Feldmane Guna Y.
Hazel Blondel
Institut Bioorganicheskoi Khimii Imeni M.M. Shemyakina akademii
Institut Organicheskogo Sinteza Akademii Nauk Latviiskoi SSR
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