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Broad-spectrum chemokine receptor antagonistic polypeptide and application thereof

A chemokine receptor, antagonistic technology, applied in the direction of peptides, anti-inflammatory agents, antiviral agents, etc., can solve the problems of high toxicity, drug resistance of cytomegalovirus, and non-availability of anti-cytomegalovirus treatment for pregnant women. , to reduce death and inhibit replication

Inactive Publication Date: 2013-06-12
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Maribavir developed by Viro Pharm is in Phase III clinical trials, there have been reports of cytomegalovirus resistance
At the same time, due to the high toxicity of the above drugs, none of them can be used for anti-CMV treatment in pregnant women.

Method used

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  • Broad-spectrum chemokine receptor antagonistic polypeptide and application thereof
  • Broad-spectrum chemokine receptor antagonistic polypeptide and application thereof
  • Broad-spectrum chemokine receptor antagonistic polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Screening of polypeptide H9

[0028] 1. Construction of phage peptide library

[0029]According to the full-length genes of 25 human C, CC, CXC and CX3C chemokines on GenBank (Table 3) as targets, primers were designed with Premier 5.0, and DNA fragments encoding human chemokines were synthesized , PCR primers and sequencing primers, simultaneously synthesizing 5' and 3' PCR amplification primers and sequencing primers for amplifying 12-peptide random DNA fragments. This work was handed over to CORPEP, and the synthetic peptide library displayed 25 human chemokines on the surface of the phage, and this peptide library was named R.Ch-12.

[0030] Table 1 Chemokines required for constructing 4 classes of 25 chemokine peptide libraries

[0031]

[0032] 2. Screening R.Ch-12 phage peptide library with polypeptide H22 as target molecule

[0033] 2.1 Screening of H22-binding peptides

[0034] Coat the ELISA plate with 100ng H22, coat the negative control well...

Embodiment 2

[0043] Example 2 Bioinformatics Analysis of Polypeptide H9

[0044] A positive clone polypeptide leader was screened by phage display technology. The sequence consisted of 7 amino acids LNAHCAL. Using the sequence analysis tool ProtParam in the SWISS-PROT protein database, the sequence was analyzed by bioinformatics data and amino acid modifications were finally selected. The sequence VLNAHCALH was used as the target synthesis. The sequence stability, acidity and alkalinity, and in vitro expression timeliness all meet the experimental standards, and the analysis results are as follows:

[0045] Amino acid sequence VLNAHCALH

[0046] Number of amino acids: 9

[0047] Molecular weight: 977.1

[0048] Theoretical pI: 6.88

[0049] Amino acid composition:

[0050] Ala(A) 2 22.2% Gly(G) 0 0.0%

[0051] Arg(R) 0 0.0% His(H) 2 22.2%

[0052] Asn(N) 1 11.1% Ile(I) 0 0.0%

[0053] Asp(D) 0 0.0% Leu(L) 2 22.2%

[0054] Cys(C) 1 11.1% Lys(K) 0 0.0%

[0055] Gln(Q) 0 0.0% Met(M)...

Embodiment 3U

[0095] Construction and Identification of Example 3US28 Recombinant Plasmid cDNA Expression Vector

[0096] The virus liquid harvested after the AD169 strain of HCMV infected the host cells for 72 hours was boiled at 100° C. for 10 minutes, centrifuged at 12,000 rpm for 5 minutes, and then separated to obtain the template of the virus amplification gene. According to the US28 full-length sequence (serial number AY174271) provided by the US NCBI website, primers were designed using primer 5.0 software. The US28 gene was amplified by PCR. After purification and recovery, the product was double-digested with HindIII and Xba I respectively with the pcDNA3.1 plasmid. After the double-digestion product was recovered and purified by gel again after electrophoresis, the ligation reaction of the fragment and the carrier was carried out. .

[0097] Inoculate a ring of glycerin-preserved DH5α bacteria into a 3ml LB test tube, culture in a constant temperature shaking incubator at 37°C f...

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Abstract

The invention discloses a broad-spectrum chemokine receptor antagonistic polypeptide and an application thereof. The amino acid sequence of the broad-spectrum chemokine receptor antagonistic polypeptide is VLNAHCALH. The broad-spectrum chemokine receptor antagonistic polypeptide in the invention can be well combined with US28 receptors on cell membranes, lowers the increased calcium ion concentration caused by combination of physiological chemokines and receptors, has an effect of blocking receptor signal transduction, inhibits human cytomegalovirus AD169 and HIV-1 induced negativecolony fromformation in vitro, restrains virus from reproducing in cells, reduces death of cells infected by virus, and can be used as an antagonist for broad spectrum inflammation and virus chemokine and applied in relative work for researching virus prevention and treatment.

Description

technical field [0001] The invention relates to the field of antiviral polypeptides, in particular to a broad-spectrum chemokine receptor antagonistic polypeptide and its application. Background technique [0002] In the past 20 years, with the gradual increase of the immunocompromised population, human cytomegalovirus (HCMV) infection and the severe diseases caused by it have been increasing (Soderberg-Naucler C.HCMV microinfections in inflammatory diseases and cancer[J] .Journal of Clinical Virology, 2008, 41 (3): 218-223), however, there is no ideal treatment drug and means for HCMV infection so far. At present, five drugs are approved by the FDA for the treatment of human cytomegalovirus: Ganciclovir (Ganciclovir, GCV), Valganciclovir (Valganciclovir), Cidofovir (Cidofovir, CDV), foscarnet sodium (Foscarnet sodium, FOS ), and the first antisense nucleic acid drug, Formivirsen. Although these drugs have significant curative effects, they also produce toxic and side effe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06A61K38/08A61P31/00A61P31/12A61P31/20A61P31/18A61P35/04A61P29/00A61P9/10A61P37/06A61P37/08
Inventor 孙含笑莫雪梅张光李秀英
Owner JINAN UNIVERSITY
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