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CN-121975758-A - Histamine oxidase protein and application thereof

CN121975758ACN 121975758 ACN121975758 ACN 121975758ACN-121975758-A

Abstract

The invention discloses a histamine oxidase protein and application thereof, and the amino acid sequence of the histamine oxidase protein is shown as SEQ ID NO. 1. The histamine oxidase protein PAO can be used for preparing medicines for treating allergic rhinitis, and can be used for reducing the aggregation of local histamine of nasal mucosa by the local degradation of the histamine of the nasal mucosa, improving symptoms of the allergic rhinitis such as sneeze and the like and relieving nose type 2 inflammatory reaction, so that the treatment effect of the allergic rhinitis is achieved, and a brand new view angle and target point are provided for the treatment of the allergic rhinitis.

Inventors

  • LIU ZHENG
  • HU DANQING
  • WANG HAI

Assignees

  • 华中科技大学同济医学院附属同济医院

Dates

Publication Date
20260505
Application Date
20260402

Claims (10)

  1. 1. A histamine oxidase protein has an amino acid sequence shown in SEQ ID NO. 1.
  2. 2. A gene tynA for coding the histamine oxidase protein PAO as claimed in claim 1, wherein the nucleotide sequence of the tynA gene is shown in SEQ ID NO. 2.
  3. 3. A recombinant expression vector pET28a-tynA is characterized in that the recombinant expression vector pET28a-tynA is obtained by inserting the tynA gene of claim 2 into a pET28a vector.
  4. 4. A tynA recombinant engineering bacterium E.coli-tynA is characterized in that the tynA recombinant engineering bacterium E.coli-tynA is an engineering bacterium containing the recombinant expression vector pET28a-tynA of claim 3.
  5. 5. The recombinant engineering bacterium according to claim 4, wherein the engineering bacterium is Escherichia coli BL21-DE3.
  6. 6. A preparation method of histamine oxidase protein PAO is characterized by comprising the following steps: 1) Transferring tynA recombinant engineering bacteria E.coli-tynA described in claim 4 or 5 into LB liquid medium for culturing, and adding IPTG for induction when OD value is 0.6-0.8; 2) After the induction is completed, centrifuging the bacterial liquid to obtain precipitate, cleaning, re-suspending in a cracking liquid, cracking on ice, and performing ultrasonic crushing; 3) Collecting the crushed bacterial liquid, centrifuging and collecting the supernatant; 4) Purifying the supernatant to obtain histamine oxidase protein.
  7. 7. The method according to claim 6, wherein in the step 1), the induction concentration is 0.5 mM, the induction temperature is 25 ℃, and the induction time is 16 hours; In the step 4), the purification method is His tag affinity chromatography purification.
  8. 8. Use of a histamine-oxidase protein PAO according to claim 1 or a histamine-oxidase protein PAO prepared by the method according to claim 6 for the manufacture of a medicament for the treatment of allergic rhinitis.
  9. 9. A medicament for treating allergic rhinitis, which is characterized by comprising the histamine-oxidase protein PAO of claim 1 or the histamine-oxidase protein PAO prepared by the method of claim 6.
  10. 10. The drug for treating allergic rhinitis according to claim 9, wherein the drug for treating allergic rhinitis comprises histamine oxidase protein PAO, and wherein the concentration of histamine oxidase protein PAO is 1 mg/mL.

Description

Histamine oxidase protein and application thereof Technical Field The invention relates to the technical field of biological medicines, in particular to histamine oxidase protein and application thereof. Background Allergic Rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa caused by allergen exposure of sensitized individuals and is characterized by symptoms such as nasal obstruction, runny nose, sneezing and itching. Worldwide, AR affects 10% -40% of the population, often coexists with asthma, severely affects the quality of life of patients, and brings about a heavy socioeconomic burden. The pathogenesis of AR involves genetic susceptibility and environmental factors that together promote an inflammatory response based on type 2 helper T cells (T H 2), characterized by upregulation of key cytokines such as Interleukins (IL) -4, IL-5 and IL-13, and pronounced eosinophil infiltration in the nasal mucosa. Histamine is a central mediator in allergic reactions and is released mainly by degranulation of mast cells by immunoglobulin (Ig) E-dependence (e.g. allergen cross-linked IgE-bound fcer 1 receptor) or non-IgE-dependence (e.g. complement activation or neuropeptide stimulation) and acts through four G-protein coupled receptors (H1R-H4R). Among these, H1R signals mediate AR key symptoms such as vasodilation, runny nose and immune cell recruitment. Although H1R antagonists (antihistamines) remain the first line of treatment for AR, there are individual differences in the response to treatment, and many patients have incomplete relief from symptoms, highlighting the need for more targeted strategies. Histamine is synthesized by L-Histidine Decarboxylase (HDC) and is degraded primarily by two enzymes, diamine oxidase (DAO, EC.1.4.3.22) and histamine N-methyltransferase (HNMT, EC.2.1.1.8). DAO is responsible for the clearance of extracellular histamine as a secreted protein, whereas HNMT is a cytoplasmic protein that can methylate intracellular histamine, converting it to imidazole-4-acetaldehyde or 1-methyl histamine, respectively. These metabolites are further oxidized by aldehyde dehydrogenase (ALD) to produce imidazole-4-acetic acid or 1-methylimidazole-4-acetic acid. In the nasal mucosa, HNMT appears to be the primary catabolic enzyme. Notably, increased HDC mRNA expression in nasal tissues of AR patients and decreased HNMT mRNA expression suggested that there was a local imbalance in histamine metabolism. However, there is no report on whether a patient with allergic rhinitis can be treated by targeting histamine degrading enzymes. Disclosure of Invention The invention aims to solve the treatment problem of respiratory tract inflammatory diseases and provides a histamine oxidase protein and application thereof, and the invention discovers that the histamine oxidase protein (PRIMARY AMINE oxidase, PAO) can degrade local histamine of nasal mucosa for the first time, thereby reducing aggregation of the local histamine of the nasal mucosa, improving symptoms of allergic rhinitis such as sneeze and the like and relieving inflammatory reaction of nose T H, and further has a treatment effect on the allergic rhinitis. In order to achieve the above purpose, the technical scheme of the invention is as follows: The invention provides a histamine oxidase protein, the amino acid sequence of which is shown as SEQ ID NO. 1. The invention also provides a gene tynA for coding the histamine oxidase protein PAO, and the nucleotide sequence of the tynA gene is shown as SEQ ID NO. 2. The invention also provides a recombinant expression vector pET28a-tynA, wherein the recombinant expression vector pET28a-tynA is obtained by inserting the tynA gene into the pET28a vector. The invention also provides tynA recombinant engineering bacteria E.coli-tynA, wherein the tynA recombinant engineering bacteria E.coli-tynA are engineering bacteria containing the recombinant expression vector pET28 a-tynA. Further, the engineering bacteria are escherichia coli BL21-DE3. The invention also provides a preparation method of the histamine oxidase protein PAO, which comprises the following steps: 1) Transferring the tynA recombinant engineering bacteria E.coli-tynA into an LB liquid culture medium for culture, and adding IPTG for induction when the OD value is 0.6-0.8; 2) After the induction is completed, centrifuging the bacterial liquid to obtain precipitate, cleaning, re-suspending in a cracking liquid, cracking on ice, and performing ultrasonic crushing; 3) Collecting the crushed bacterial liquid, centrifuging and collecting the supernatant; 4) Purifying the supernatant to obtain histamine oxidase protein. Further, in the step 1), the induction concentration is 0.5 mM, the induction temperature is 25 ℃, and the induction time is 16 hours; In the step 4), the purification method is His tag affinity chromatography purification. The invention also provides application of the histamine oxidase protein PAO or the histamine oxidase