Polypeptide combinative with PKR kinase structure field specificity and uses thereof

A kinase domain and specific technology, applied in peptides, material inspection products, biological tests, etc., can solve the problems of no PKR inhibitors and PKR polypeptide inhibitors reported

Inactive Publication Date: 2008-10-29
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the PKR kinase domain plays a role in diseases such as Alzheimer's d

Method used

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  • Polypeptide combinative with PKR kinase structure field specificity and uses thereof
  • Polypeptide combinative with PKR kinase structure field specificity and uses thereof
  • Polypeptide combinative with PKR kinase structure field specificity and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: recombinant PKR cat Preliminary study on the expression, purification and activity of

[0027] 1. Recombinant PKR cat Plasmid construction

[0028] Use the existing pET28a-PKR template, the following PCR primers and Pfu polymerase to PCR amplify PKR cat :

[0029] Forward primer: 5′-CATGCCATGGGCGACATGAAAGAAACAAAGTATACTG-3′

[0030] Reverse primer: 5′-GGCTCTCGAGACATGTGTGTCGTTCATTTTTCTC-3′

[0031] The amplified DNA fragment was digested with Nco I / Xho I and cloned into plasmid pET28a (Novagen). The recombinant plasmid thus constructed was named pET28a-PKR cat And introduced Escherichia coli BL21. Recombinant plasmid pET28a-PKR cat Allowed in mature PKR cat Recombinant PKR with an additional methionine residue at the N-terminus and an additional 6 histidines at the C-terminus cat expression.

[0032] 2. Recombinant PKR cat purification of

[0033] ①. Directly containing pET28a-PKR cat BL21 in 5 mL LB medium (50 μg / ml kanamycin), 37 ° C, 250 rpm...

Embodiment 2

[0055] Example 2: Obtaining recombinant phage that can specifically bind to PKRcat through five rounds of screening

[0056] 1. The first round of screening and elution:

[0057] ①. Use NaHCO 3 solution dilution PKR cat Protein to 100 μg / mL solution, requires NaHCO 3 The final concentration was 0.1M. Add 100 μL of this solution to one well of a 96-well plate, and coat overnight at 4°C.

[0058] ②. Add 150 μL of freshly prepared TBS (BTBS) solution containing 0.5% (w / v) BSA to the protein-coated wells in the previous step, and place on a rocking platform at room temperature for 1 hour.

[0059] ③. At the same time, the phage (10 11 pfu) was added to 100 μL BTBS, added to an empty well, and placed on a rocking platform at room temperature for 1 hour, the purpose was to absorb the phage that could bind non-specifically to the ELISA plate.

[0060] ④. Pour off the protein / BTBS solution and wash 6 times with 200 μL TBST (TBS containing 0.1% Tween, pH 7.4), taking care not to...

Embodiment 3

[0087] Embodiment 3: ELISA detection screened phage to PKRcat and control binding ability

[0088] ①. Use NaHCO 3 Solution Dilute protein to 100 μg / mL solution, call for NaHCO 3 The final concentration was 0.1M. Add 100 μL of this solution to two wells of a 96-well plate, and fix overnight at 4°C. At the same time, BSA was added to the other two wells as a control according to the same method.

[0089] ②. Add 100 μL of 3% BSA to each well to block the part of unbound protein, and place on a rocking platform at room temperature for 1 hour.

[0090] ③. According to the measured concentration of the phage eluted in the fifth round, dilute the phage with TBST, add 100 μL to each well, and the phage is about 10 9 -10 10 . Place on rocking platform at room temperature for 1 hour.

[0091] ④. Each well was washed 6 times with 200 μl TBST.

[0092] ⑤. HRP-anti-M13 antibody (Pharmacia Biotech Company) was diluted 1:5000 with TBST, added 100 μL to each well, and placed on a rock...

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Abstract

The invention relates to a polypeptide capable of specifically binding with the structural domain of PKR kinase and an application thereof. The invention relates to a 12-peptide sequence with amino acid sequence of Ser-Val-His-Leu-Tyr-His-Ser-Thr-Lys-Thr-Leu-Arg, which is capable of specifically binding with the PKR kinase structural domain (PKRcat) and inhibiting the activity of the PKR kinase. Therefore, the 12-peptide has practical value in research on the role of PKRcat in cell apoptosis and development of PKRcat inhibitors.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and biomedicine, and relates to the use of phage display technology to screen the PKR kinase domain (PKR cat ) specifically binding polypeptides and applications thereof. Background technique [0002] PKR is an interferon-induced double-stranded RNA-dependent protein kinase and a member of the eukaryotic translation initiation factor eIF2α kinase family. It is a serine-threonine kinase composed of 551 amino acids, and recent studies have also found that it has tyrosine kinase activity [Su, Q. et al. Tyrosine phosphorylation acts as a molecular switch to full-scale activation of the eIF2α RNA-dependent protein kinase. 2006. PNAS]. Structurally, it mainly includes two functional domains: the N-terminal and RNA-binding regulatory domain and the C-terminal catalytic domain. PKR plays a major role in the interferon-induced antiviral defense response and is considered to be the most characteri...

Claims

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Application Information

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IPC IPC(8): C07K7/08G01N33/68
Inventor 曹又佳张宏恺常云松杜明娟张翠竹
Owner NANKAI UNIV
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