Chemokine binding activity of viral TNF receptors and related proteins

A chemokine and virus technology, which is applied in the field of chemokine binding activity of viral TNF receptors and related proteins, can solve the problems that are not clear, TNF binding is not an essential function, etc.

Inactive Publication Date: 2007-09-26
安东尼奥·阿尔卡米佩特霍
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This domain is not essential for TNF binding and its function has not been elucidated

Method used

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  • Chemokine binding activity of viral TNF receptors and related proteins
  • Chemokine binding activity of viral TNF receptors and related proteins
  • Chemokine binding activity of viral TNF receptors and related proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Materials and methods

[0051] 1.1-pox virus

[0052] CPV, EV and VV proliferate in vitro by infecting confluent monolayers of Bsc-I cells.

[0053] 1.2-Cloning of Camelpox Virus CrmB and Production of VaV CrmB

[0054] Using oligonucleotides CMLV 264 Eco (5'-GCGGAATTCATGAAGTCCGTATTATACTCG) and CMLV 264 Xho (5'-GCGCTCGAGTAAAAAGTGGGTGGGTTTGG) and purified CMLVDNA as a template to amplify the ORF 264 (corresponding to the CrmB gene) of the camelpox virus strain CMS by PCR . The PCR product was cloned into EcoRI / XhoI digested pBacl (Novagen) to generate plasmid pRA1. The lack of mutations in the amplified gene was confirmed by DNA sequencing. According to the manufacturer's instructions, the "QuikChange Multi Site-Directed Mutagenesis Kit (Strategene)" was used to obtain DNA corresponding to VaV (strain Bangladesh 1975; ORF 188) through multiple site-directed mutagenesis of plasmid pRA1. The introduced mutations are shown in Figure 6. After several consecutive round...

Embodiment 2

[0071] Example 2: Results

[0072] 2.1-vTNFR CrmD-binding chemokine encoded by EV and CPV

[0073] In order to find a new virus secreted protein that binds to chemokines, we performed cross-linking experiments with 1251-CXCL8 and identified a new secreted vCKBP encoded by poxvirus EV (Figure 3a). This activity is lacking in VV samples, and has a larger molecular size than the 35kDa vCKBP encoded by VV and other poxviruses. vCKPB is a kind of cross-linking CXCL8 but does not block its organism due to its low affinity for CXCL8. Active protein (Alcami et al., 1998, Graham et al., 1997, Lalani et al., 1998). Unexpectedly, we found that the vTNFR CrmD encoded by EV (known to bind TNF) has additional CXCL8 binding properties (Figure 3b). Using the truncated CrmD protein expressed in the VV expression system, we have confirmed that the three N-terminal CRDs of CrmD are necessary to block TNF activity (Figures 4a, 4b, and 4c), while CTD is not for TNF binding. It is necessary, but CTD gi...

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Abstract

Chemokine binding activity of viral TNF receptors and related proteins. The invention relates to a C-terminal domain (CTD) of viral tumour necrosis factor receptors (vTNFRs) CrmB or CrmD or CTD homologues (CTD1, CTD2 and CTD3) from poxvirus and their functional homologues, including derivatives, and fragments, for use in binding chemokines and their analogues and / or to enhance the immunomodulatory properties of TNFRs or in bloking binding of chemokines to their corresponding cell surface receptors and / or to modulate chemokine biological activity.

Description

Technical field [0001] The present invention relates to the C-terminal domain (CTD) of the tumor necrosis factor receptor (TNFR), which is encoded by a poxvirus and called cytokine response modifiers B and D (CrmB and CrmD), and its homologs, derivatives or The application of fragments to regulate the activity of chemokines and improve the immunomodulatory properties of TNFR. The present invention also relates to fusion polypeptides, pharmaceutical compositions and test kits containing the protein of the present invention. Background technique [0002] Poxviruses are complex DNA viruses that encode up to 200 genes. Smallpox virus (VaV) is the cause of smallpox. Smallpox is one of the most devastating human diseases. It was eradicated due to the use of vaccinia virus (VV) as a smallpox vaccine in the WHO global eradication campaign. Cowpox virus (CPV) is related to VV and is considered to be an aggressive virus that causes occasional infections in many mammals. Ectromelia virus (E...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/065A61K38/16G01N33/50C12N15/39C12N15/62
CPCC07K14/70578G01N2333/70578C12N2710/14143C07K2319/32C07K14/005C12N2710/24122A61P29/00A61P31/12A61P33/00A61P35/00A61P37/06A61P43/00C07K14/065G01N33/566G01N33/6863
Inventor 安东尼奥·阿尔卡米佩特霍阿里·阿莱霍赫贝格玛丽亚贝戈尼亚·鲁伊斯-阿尔盖洛因·霍马加里达·萨赖瓦维森特·P·史密斯
Owner 安东尼奥·阿尔卡米佩特霍
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