Tumor necrosis factor-gamma

A nucleic acid molecule and sequence technology, applied in antitumor drugs, fungi, DNA/RNA fragments, etc., can solve problems such as unclear regulation of blood vessels

Inactive Publication Date: 2001-11-14
HUMAN GENOME SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the complex mechanisms regulating angiogenesis are still not well understood, it is becoming clear that the process begins and ends as a result of a balance of positive and negative factors

Method used

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Experimental program
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Effect test

Embodiment 1

[0391] Embodiment 1, bacterial expression and purification of TNF-γ

[0392] The DNA sequence encoding the full-length TNF-γ ORF, ATCC Accession No. 75927 was first amplified with PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the TNF-γ protein. Additional nucleotides corresponding to TNF-γ were added to the 5' and 3' sequences, respectively. The 5' oligonucleotide primer is shown as SEQ ID NO: 3, the sequence is 5'-GCGCGGATCCACCATGAGACGCTTTTTAAGCAAAGTC-3', which contains a -BamHI restriction enzyme site followed by the TNF-γ code starting from the initial methionine codon The first 24 nucleotides of the sequence. The 3' sequence is 5'-CGCGTCTAGACTATAGTAAGAAGGCTCCAAAGAAGG-3' (SEQ ID NO: 14), which contains an XbaI site and a sequence of 22 nucleotides complementary to TNF-γ. This restriction enzyme site corresponds to the restriction enzyme site in the bacterial expression vector pQE-9 (Qiagen), which was then digested with BamHI and XbaI. The ampli...

Embodiment 2

[0394] Embodiment 2: Clone and express TNF-γ with the baculovirus expression system

[0395] The DNA sequence (ATCC NO.75927) encoding the full-length TNF-γ protein is amplified with PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene, and the 5' primer sequence is 5'-GCGCGGATCCACCATGAGACGCTTTTTAAGCAAAGTC-3' (SEQ ID NO: 15), which contains a -BamHI restriction enzyme site (in bold) followed by 24 nucleotides of the TNF-γ gene. The 3' primer sequence is 5'-CGCGTCTAGACTATAGTAAGAAGGCTCCAAAGAAGG-3' (SEQ ID NO: 16), which contains a restriction endonuclease XbaI site and 22 nucleotides complementary to the 3' untranslated sequence of the TNF-γ gene, amplified Sequences were isolated from 1% agarose gels using a commercially available kit ("Geneclean", BIO 101 Inc., La Jolla, CA). This fragment was then digested with endonucleases BamHI and XbaI and purified on a 1% agarose gel. This fragment is called F2.

[0396] Utilize the baculovirus expression s...

Embodiment 3

[0404] Embodiment 3. Expression of recombinant TNF-γ in COS cells

[0405]The expression of plasmid TNF-γ-HA was derived from a vector pcDNA I / Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E coli origin of replication, 4) CMV promoter sub, followed by a linker region, a SV40 intron and a polyadenylation site. A DNA fragment encoding the entire TNF-γ precursor and a hemagglutinin antigen (HA) tag fused to the 3' end of its framework was cloned into the polylinker region of the vector. Expression of the recombinant protein is thus under the control of the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). Fusion of the HA tag to the target protein allows easy detection of the recombinant protein with antibodies recognizing HA epitopes.

[0406] The mechanism of plasmid construction is...

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Abstract

The invention discloses human TNF-γ-α and TNF-γ-β polypeptides, DNA (RNA) encoding these polypeptides and methods for producing these polypeptides through recombinant technology. The present invention also discloses methods of using these polypeptides to inhibit cell growth such as tumor or cancer, promote wound healing, provide resistance to infection, induce inflammatory activity and stimulate growth of certain cell types to treat diseases such as restenosis. The invention also relates to a diagnostic method for detecting mutations in TNF-γ-α and TNF-γ-β nucleic acid sequences or overexpression of TNF-γ-α and TNF-γ-β polypeptides. The present invention also discloses antagonists against these polypeptides and their application as therapeutic agents for treating cachexia, septic shock, cerebral malaria, inflammation, arthritis and transplant rejection.

Description

field of invention [0001] The present invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, uses of such polynucleotides and polypeptides, and production of such polynucleotides and polypeptides. Specifically, the polypeptide of the present invention has been identified as a new member of the tumor necrosis factor family, hereinafter referred to as "TNF-γ-α". The present invention also relates to the protein encoded by a splice variant of the gene encoding TNF-γ-α, hereinafter referred to as "TNF-γ-β". The invention also relates to inhibiting the function of these polypeptides. Background of the invention [0002] Human tumor necrosis factor-a (TNF-a) and b (TNF-b or lymphotoxin) are related members of a large class of polypeptide mediators, including interferons, interleukins and growth factors, collectively referred to as cell Factor (Beutler, B. and Cerami, A., Annu. Rev. Immunol., 7:625-655 (1989)). [0003] Tumor necrosi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K31/7088A61K38/00A61K39/395A61K39/40A61K39/42A61K48/00A61P29/00A61P35/00C07H21/04C07K14/525C07K16/24C12N1/15C12N1/19C12N1/21C12N5/00C12N5/02C12N5/10C12N15/00C12N15/28C12N15/63C12N15/70C12N15/74C12P21/00C12P21/02C12P21/06
CPCY10S930/14A61K48/00A61K38/00C07K14/525A61P29/00A61P35/00Y02A50/30C12N15/11
Inventor 余国良倪健克雷格·A·罗森张筠
Owner HUMAN GENOME SCI INC
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