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HB-NC4 recombinant protein and preparation method and application thereof

A technology of HB-NC4 and recombinant protein, which is applied in the field of HB-NC4 recombinant protein and its preparation, can solve the problems that have not existed yet, and achieve the effect of long residence time and strong targeting

Pending Publication Date: 2021-06-25
SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Structural-improving drugs refer to those drugs that can improve the joint structure with appropriate imaging methods. So far, there is no real structural-improving drug.

Method used

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  • HB-NC4 recombinant protein and preparation method and application thereof
  • HB-NC4 recombinant protein and preparation method and application thereof
  • HB-NC4 recombinant protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0118] The preparation method of HB-NC4 recombinant protein of the present invention, the steps are as follows:

[0119] (1) Construction of fusion gene: prepare the HB-NC4 fusion gene containing the target gene encoding NC4 and the target gene encoding HB, and amplify;

[0120] (2) Double digestion of the empty plasmid pET28a: use XhoⅠ and BamHI to perform double digestion of the Escherichia coli expression plasmid pET28a, and recover the fragments;

[0121] (3) Ligation of the fusion gene and the plasmid: Add 31.92ng of the recovered product from PCR and 107.38ng of the recovered product from the digestion of the empty plasmid into the reaction system of the ClonExpress@Ⅱ kit, react at 37°C for 30min, and the ligated product is used for the next step of transformation ;

[0122] (4) Transformation of recombinant plasmids: add 4 μL of cooled reaction solution to 100 μL of LDH5α competent cells and mix well, bathe in ice for 30 minutes, heat shock at 42°C for 90 seconds, bath...

experiment example 1

[0129] Preparation of HB-NC4 fusion protein:

[0130] 1. The construction of fusion gene: adopt the method for PCR to obtain fusion gene with NC4 as template, and reclaim.

[0131] 2. Double digestion of the empty plasmid pET28a: XhoⅠ and BamHI were used to perform double digestion of the Escherichia coli expression plasmid pET28a, and the fragments were recovered.

[0132] 3. Ligation of the fusion gene and the plasmid: Add 31.92ng of the recovered product from PCR and 107.38ng of the recovered product from the digestion of the empty plasmid into the reaction system of the ClonExpress@Ⅱ kit, react at 37°C for 30min, and the ligated product is used for the next step of transformation.

[0133] 4. Transformation of recombinant plasmids: Add 4 μL of cooled reaction solution to 100 μL of LDH5α competent cells and mix well, ice-bath for 30 minutes, heat shock at 42°C for 90 seconds, ice-water bath for 2 minutes, add 900 μL of LB liquid medium, and incubate at 37°C Fully recover f...

experiment example 2

[0140] Determination of anti-complement activity of fusion protein:

[0141] Investigate the anti-complement activation activity of HB-NC4: with NC4 as a control (here, the preparation method of the protein as a control is the same as that of HB-NC4), and investigate the anti-complement activation activity of purified HB-NC4 by hemolysis method. The specific implementation steps are as follows :

[0142] First test the protein samples NC4 and HB-NC4 to inhibit the activity of the alternative complement pathway, take the rabbit red blood cell suspension, and use Mg 2+ - Wash with EGTA buffer three times until the supernatant is transparent and colorless. Reuse Mg 2+ - EGTA buffer dilutes the rabbit erythrocyte suspension to a certain concentration. The method for determining the release concentration is as follows: take 10ul of rabbit erythrocyte suspension, add 90ul of deionized water, measure the absorbance at 405nm, repeat the above operation until the absorbance is adjus...

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Abstract

The invention provides HB-NC4 recombinant protein and a preparation method and application thereof, and belongs to the technical field of biological medicines, the recombinant protein is formed by recombination of an N-terminal non-collagen structural domain 4 and an HB heparin binding structural domain of NC4 human collagen IX. The amino acid residue sequence of the recombinant protein is as shown in SEQ ID NO. 1. A nucleotide sequence for coding the recombinant protein is shown as SEQ ID NO.3 or a sequence with genetic code degeneracy. In the nucleotide sequence for coding the recombinant protein, the nucleotide sequence for coding the heparin binding domain is as shown in SEQ ID NO.4, and the nucleotide sequence for coding the N-terminal non-collagen domain 4 of the human collagen IX is as shown in SEQ ID NO.5 or has a sequence with genetic code degeneracy. The invention also discloses the preparation method of the HB-NC4 fusion protein. The HB-NC4 fusion protein retains the complement inhibition activity of the N-terminal domain 4 of collagen IX, can directly target cartilage and retain the complement inhibition activity, and the targeting property and retention time of the fusion protein are greatly improved.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a HB-NC4 recombinant protein and its preparation method and application. Background technique [0002] Osteoarthritis OA refers to joint diseases caused by various factors causing articular cartilage fibrosis, chapped, ulcers, and loss. The etiology is still unclear, and its occurrence is related to age, obesity, inflammation, trauma and genetic factors. OA tends to occur in joints with heavy load and many activities, such as knees, spine (cervical and lumbar), hips, ankles, hands and other joints, and is a common disease worldwide. [0003] The purpose of OA treatment is to relieve symptoms, delay joint structural changes, maintain joint function, and improve quality of life. The traditional treatment of OA is basically three aspects, i.e. non-drug treatment, drug treatment and surgical treatment. Internationally, all drugs for the treatment of OA are divided into two cat...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70A61K38/18A61K38/39A61P19/02A61P29/00
CPCC07K14/78C07K14/485C12N15/70A61K38/39A61K38/1808A61P19/02A61P29/00C07K2319/21
Inventor 李妍汪亚娅王福文李连牟艳玲
Owner SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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