Recombinant human TSG6-IFN alpha fusion protein, preparation method thereof and application of recombinant human TSG6-IFN alpha fusion protein as antiviral drug

A TSG6-IFN, TSG-6-technology, applied in antiviral agents, recombinant DNA technology, animal/human proteins, etc., can solve the problems of unfavorable clinical treatment, short half-life, and limited scope of clinical application

Active Publication Date: 2020-10-27
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] According to clinical research and practice, interferon-α needs to be effective in the early stage of viral infection or preventive medication, which greatly limits the scope of clinical application
Moreover, the half-life in humans and animals is only 3h to 5h, and the half-life is relatively short, requiring multiple injections to maintain the drug effect; in addition to being unfavorable for clinical treatment, it also increases the pain and economic burden of patients

Method used

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  • Recombinant human TSG6-IFN alpha fusion protein, preparation method thereof and application of recombinant human TSG6-IFN alpha fusion protein as antiviral drug
  • Recombinant human TSG6-IFN alpha fusion protein, preparation method thereof and application of recombinant human TSG6-IFN alpha fusion protein as antiviral drug
  • Recombinant human TSG6-IFN alpha fusion protein, preparation method thereof and application of recombinant human TSG6-IFN alpha fusion protein as antiviral drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] A preparation method of recombinant human TSG-6 and porcine IFN-α fusion protein (rhTSG6-PoIFNα fusion protein), including the following steps:

[0062] (1) Codon optimization of the recombinant human TSG-6 gene shown in SEQUENCE LISTING 400 by Escherichia coli codon preference to obtain the optimized recombinant human TSG shown in SEQUENCE LISTING 400 -6 gene, the amino acid sequence of recombinant human TSG-6 encoded by it is shown in SEQUENCE LISTING 400;

[0063] Codon optimization of the recombinant porcine IFN-α gene shown in SEQUENCE LISTING 400 by Escherichia coli codon preference to obtain the optimized recombinant porcine IFN-α gene shown in SEQUENCE LISTING 400 , The amino acid sequence of the encoded recombinant porcine IFN-α is shown in SEQUENCE LISTING 400 ;

[0064] Use a connecting peptide gene fragment shown in SEQUENCE LISTING 400 to combine the optimized recombinant porcine IFN-α gene shown in SEQUENCE LISTING 400 with the optimized recombination shown in S...

Embodiment 2

[0079] Determination of antiviral activity titer of rhTSG6-PoIFNα fusion protein standard

[0080] a. Experimental materials:

[0081] Recombinant human interferon alpha (rhIFN-α) standard: its titer is 15000IU / mL, as a known positive control, purchased from Beijing China Institute of Food and Drug Identification, interferon alpha national standard, batch number: 97 / 04 , Hereinafter referred to as standardized interferon;

[0082] RhTSG6-PoIFNα fusion protein product: prepared in Example 1, as the test product;

[0083] Cell line: bovine kidney cell (MDBK), purchased from ATCC;

[0084] Virus strain: The attack virus is Vesicular Stomatitis Virus (VSV), a gift from the Institute of Clinical Virology of Anhui Medical University.

[0085] b. Experimental method

[0086] Operate under aseptic conditions, take 1 rhTSG6-PoIFNα fusion protein standard and add 1mL water for injection to dissolve it, and then dilute it in a reagent bottle with DMEM cell nutrient solution containing 10% newborn c...

Embodiment 3

[0108] Determination of the half-life of rhTSG6-PoIFNα in rabbit plasma

[0109] 3.1. The half-life of rhTSG6-PoIFNα in rabbit plasma was determined. 40μg / kg recombinant porcine interferon-α (i.e. rPoIFN-α) was used as a positive control, rhTSG6-PoIFNα was injected intramuscularly into rabbits at a dose of 40μg / kg, and then blood samples (1ml) were collected at different times. Specifically: 1h before administration, 0.25h, 1h, 3h, 4h, 8h, 10h, 12h, 24h, 48h, 72h, 96h, 120h, 144h and 168h after administration, blood was collected from the ear vein of the rabbit. After the blood sample was collected, it was transferred into an EP tube containing EDTA and centrifuged together, and the supernatant was stored in a refrigerator at -80°C.

[0110] 3.2. Use the "cytopathic inhibition method" in the 2015 edition of "Chinese Pharmacopoeia" to determine the retention time of rhTSG6-PoIFNα in rabbit blood. The abscissa is the plasma collected at each time point in the rabbit, and the ordina...

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Abstract

The invention discloses a recombinant human TSG6-IFN alpha fusion protein, a preparation method thereof and application of the recombinant human TSG6-IFN alpha fusion protein as an antiviral drug. Thepreparation method comprises the following steps: after optimizing existing human TSG-6, porcine IFN-alpha or human IFN-alpha genes, connecting the porcine IFN-alpha or human IFN-alpha genes with thehuman TSG-6 genes through a Link connecting peptide gene fragment, cloning to a pET-32a expression vector, transforming into escherichia coli, carrying out inducible expression, and washing, denaturing and renaturing the expressed bacteria; and after separating and purifying, adding a freeze-drying protective agent, and carrying out freeze drying so as to prepare the rhTSG6-PoIFN alpha fusion protein and an rhTSG6-hIFN alpha fusion protein. The VSV virus resistance titer of the rhTSG6-PoIFN alpha fusion protein is up to (1.60+/-0.45)*10<7>IU/mL; and the VSV virus resistance titer of the rhTSG6-hIFN alpha fusion protein is up to (4.50+/-1.32)*10<6>IU/mL. By virtue of the method, efficient expression and batch preparation of non-naturally existing characteristic fusion proteins are achieved; and the half-life period of the recombinant human TSG6-IFN alpha fusion protein in a body is up to 60-72 hours, the antiviral effect is relatively good, and the recombinant human TSG6-IFN alpha fusion protein can be developed and applied as a novel antiviral drug.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant human TSG6-IFNα fusion protein, a preparation method thereof, and its application as an antiviral drug. Background technique [0002] Coronavirus disease 2019 (COVID-19), which started in China at the end of 2019 and followed the global outbreak, is caused by a new type of coronavirus infection. The clinical symptoms are fever, fatigue, and dry cough. Some patients may be affected by a "cytokine storm". "The rapid development from mild to severe, manifested as acute respiratory distress syndrome, metabolic acidosis and sepsis, and even multiple organ failure leading to death. Severely ill patients with acute viral major infectious diseases such as the Middle East Respiratory Syndrome (MERS) that broke out in 2012 and SARS that broke out in 2003 also have similar clinical features and pathological processes. Due to the lack of specific therapeutic drugs in the cli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N1/21A61K38/21A61K47/64A61P31/12C12R1/19
CPCA61K38/00A61P31/12C07K14/465C07K14/47C07K14/56C07K2319/00C12N15/70C12N2800/22
Inventor 王明丽夏兵兵吴博何志远蒋敏之周炜许智勇赵俊张俊玲余莉
Owner ANHUI MEDICAL UNIV
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