Protein and gene for degrading dexamethasone and application of protein and gene

Abstract

The invention discloses a protein and a gene for degrading dexamethasone and application of the protein and the gene, and belongs to the technical field of biology. The HSD-like protein is found in a bacterial strain with a dexamethasone degradation function, the HSD-like protein is further proved to have the dexamethasone degradation function in vitro, a new theory can be provided for a steroid hormone metabolism action mechanism, an important reference is further provided for defining a metabolic pathway of steroid hormones in vivo and in vitro, and the HSD-like protein has a good application prospect. The method has important scientific research and application values. The amino acid sequence of the HSD-like protein provided by the invention is as shown in SEQ ID NO.1, and the protein can be used for degrading dexamethasone; the protein or the coding gene thereof can be used as a detection index to evaluate the sensitivity and drug resistance of patients with allergic rhinitis, allergic asthma and the like to hormone treatment, and is helpful for providing reference for clinical disease diagnosis and treatment and prognosis evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the degradation of steroid hormones, specifically to a protein and gene that degrades dexamethasone and their applications. Background Technology

[0002] Intranasal corticosteroid therapy is currently the most effective treatment for allergic rhinitis (AR); however, more than one-third of AR patients develop glucocorticoid resistance. This resistance in AR patients is related to abnormal hormone metabolism mechanisms. However, the metabolic pathways of glucocorticoids in vivo and in vitro are not yet fully understood, and the key metabolic enzymes or genes involved remain unclear. Therefore, in-depth research into the metabolic mechanisms of glucocorticoids is of great significance.

[0003] Chinese patent CN120230680A, entitled "A Steroid Hormone Degrading Bacterium and Its Screening Method and Application," discloses a steroid hormone-degrading bacterium, *Klebsiella pneumoniae* (KP2022), isolated from the nasal cavity of a patient with allergic rhinitis and steroid resistance, which possesses the ability to degrade dexamethasone. Steroid resistance in AR patients may be related to steroid hormone-degrading bacteria in vivo. Research on the genes responsible for dexamethasone degradation by in vivo steroid hormone-degrading bacteria can provide new theories for the metabolic mechanisms of steroid hormones and further provide important references for clarifying the metabolic pathways of steroid hormones in vivo and in vitro, possessing significant scientific research application value. In hormone-related diseases such as allergic rhinitis and allergic asthma, hormone sensitivity and resistance are crucial. Clarifying the expression of dexamethasone-degrading genes and the specific steroid hormone metabolic pathways in patients with these diseases can provide important references for disease diagnosis, treatment, and prognosis. Summary of the Invention

[0004] This invention discovered an HSD-like enzyme protein (HSD-like) in the bacterial strain *Klebsiella pneumoniae* (KP2022), which has the function of dexamethasone degradation. Further in vitro testing confirmed that this HSD-like enzyme protein has dexamethasone-degrading function. Based on this, the purpose of this invention is to provide a protein and gene for dexamethasone degradation and their applications.

[0005] The objective of this invention is achieved through the following technical solution: A protein that degrades dexamethasone, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] MTCPFIKETISDQIIMKILVTGGAGFIGSAVVRHIIENTLDEVRVMDCLTYAGNLESLAPVAGSERYSFSQTDITDAAAVAAQFSEFRPDIVMHLAAESHVDRSIDGPAAFIQTNVIGTFTLLEAARHYWSGLGEEQMQAFRFHHISTDEVYGDLHGTDDLFTEETPYAPSSPYSASKAGSDHLVRA WNRTYGLPVVVTNCSNNYGPYHFPEKLIPLTILNALAGKPLPVYGNGEQIRDWLYVEDHARALYKVATEGKSGETYNIGGHNERKNIDVVRTICAILDKVVAQKPGNISQFADLITFVTDRPGHDLRYAIDAAKIQRDLGWVPQETFESGIEKTVHWYLNNQTWWQRVLDGSYAGERLGLNN (SEQID NO.1).

[0007] A gene encoding the protein that degrades dexamethasone, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008]

[0009] A biomaterial expressing the protein that degrades dexamethasone, comprising any one of (1) to (4) below: (1) The nucleic acid molecule encoding the protein; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant expression vector containing the nucleic acid molecule described in (1), or a recombinant expression vector containing the expression cassette described in (2); (4) A host cell containing the nucleic acid molecule described in (1), or a host cell containing the expression cassette described in (2), or a host cell containing the recombinant expression vector described in (3).

[0010] In some embodiments, the expression vector is preferably pET-28a, and the host cell is preferably Escherichia coli.

[0011] The application of the aforementioned proteins, genes, or biological materials in the degradation of dexamethasone. This application includes research on the degradation mechanism of steroid hormones, such as using a carrier containing the protein to degrade dexamethasone in vitro, measuring the degradation products, exploring the dexamethasone degradation pathway, and thereby investigating the steroid hormone degradation mechanism.

[0012] A method for degrading dexamethasone involves adding the protein, a culture containing the protein, or cells expressing the protein to a body of water containing dexamethasone, thereby utilizing the protein to degrade the dexamethasone.

[0013] The aforementioned proteins or genes are used as detection indicators in the preparation of reagents for assessing the sensitivity and resistance of patients with allergic rhinitis, allergic asthma, etc., to hormone therapy. These reagents are used to detect the expression levels of the aforementioned proteins or genes. By detecting the expression of these proteins in patients with allergic rhinitis, allergic asthma, etc., the sensitivity and resistance to hormone therapy can be assessed, thus providing a reference for clinical diagnosis, treatment, and prognosis evaluation.

[0014] This invention has the following advantages and effects compared to existing technologies: It discovers an HSD-like enzyme protein in steroid hormone-degrading bacteria that functions to degrade dexamethasone. This invention provides a new theory for the metabolic mechanism of steroid hormones and further provides important reference for clarifying the metabolic pathways of steroid hormones in vivo and in vitro, possessing significant scientific research application value. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the construction of recombinant E. coli-HSD expressing HSD-like protein.

[0016] Figure 2This study investigated the expression of the HSD-like gene in *E. coli*-HSD. Using primers for HSD-like gene amplification, *E. coli* DNA was amplified by PCR. In the DNA gel after nucleic acid electrophoresis, compared to the empty vector without the HSD-like gene, a bright band amplified by the HSD-like primers was observed in the *E. coli*-HSD DNA, confirming successful transformation of *E. coli*-HSD.

[0017] Figure 3 This study describes the detection of HSD-like protein expression in E. coli-HSD. Total E. coli-HSD protein induced by IPTG was subjected to electrophoresis. Overexpressed HSD-like protein bands were observed in the protein gel after soaking in Coomassie Brilliant Blue solution.

[0018] Figure 4 The degradation of dexamethasone was achieved by heterologous HSD-like E. coli-HSD. LC-MS / MS analysis showed that the concentration of dexamethasone gradually decreased with increasing co-culture time between HSD-like E. coli and dexamethasone. Detailed Implementation

[0019] This invention identifies an HSD-like protein and its encoding gene from the dexamethasone-degrading strain K. pneumoniae KP2022 (CN120230680A), and performs heterologous expression of the protein, confirming its ability to degrade dexamethasone in vitro. The amino acid sequence of the HSD-like protein is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0020] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0021] Example 1: Construction of recombinant E. coli-HSD expressing HSD-like protein A gene fragment encoding an HSD-like protein was inserted into the pET-28a plasmid, which contained fi, ori, and ori (replicons), KanR (kanamycin resistance gene), and lacl (lactose operon). This plasmid was then introduced into BL21(DE3) recombinant *E. coli* to stably express the HSD-like protein, thus constructing a recombinant *E. coli* strain E. coli-HSD capable of expressing the HSD-like protein. Figure 1 ).

[0022] Example 2: HSD-like gene expression in E. coli-HSD DNA was extracted from *E. coli*-HSD using a bacterial genomic DNA extraction kit, and PCR amplification was performed using HSD-like gene primers (forward primer: 5'-CCGGAGAAACTGATCCCGCT-3', reverse primer: 5'-CACCTTGTCGAGAATGGCGC-3'). The amplification conditions were as follows: first stage: 95℃ pre-denaturation for 3 min; second stage: 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 60 s; second stage: 30 cycles; third stage: 72℃ complete extension for 5 min. The amplified products were then identified by 2% agarose gel electrophoresis to determine whether HSD-like expression was heterologous in recombinant *E. coli*. Results are shown below. Figure 2 .

[0023] Example 3: Expression of HSD-like proteins in Escherichia coli-HSD E. coli-HSD was added to LB medium and cultured with shaking at 37℃ and 180 rpm, while 30 μg / mL kanamycin was added to prevent contamination. When the absorbance at OD600 reached 0.5-1.0, 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce overexpression of HSD-like in E. coli-HSD. The bacterial culture was collected, centrifuged at 12000 rpm for 10 min at 4℃, and the supernatant was discarded to obtain bacterial pellet. The bacteria were lysed on ice for 30 min using RIPA strong lysis buffer and protease inhibitor, followed by ultrasonic lysis at 30% intensity for 30 min. 5× SDS-PAGE protein loading buffer was added and heated at 100℃ for 10 min. Finally, the heterologous expression of HSD-like was detected by 10% SDS-PAGE electrophoresis and Coomassie Brilliant Blue solution. The results are shown in the figure. Figure 3 .

[0024] Example 4: Degradation of dexamethasone by E. coli-HSD The cells were divided into E. coli-HSD and E. coli-Empty groups. E. coli expressing HSD-like protein (E. coli-HSD) and E. coli expressing the empty vector (E. coli-Empty) were added to 30 mL of LB medium and cultured with shaking at 37℃ and 180 rpm. 30 μg / mL kanamycin was added to prevent contamination. When the absorbance at OD600 reached 0.5-1.0, 0.5 mM IPTG was added to induce overexpression of HSD-like protein in E. coli-HSD, and 0.5 g / L (1.274 mmol / L) dexamethasone was added for continued co-culture. Degradation solution was collected at six time points (0 h, 8 h, 16 h, 24 h, 48 h, and 72 h) over 3 days, with 500 μL collected each time. The degradation of dexamethasone was then detected by LC-MS / MS. Results ( Figure 4 The results showed that the dexamethasone concentration in the E. coli-HSD group gradually decreased with increasing co-culture time, while no significant change was observed in the E. coli-Empty group.

[0025] The above embodiments are only used to help illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A protein that degrades dexamethasone, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the protein of claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

2.

3. A biomaterial expressing the protein of claim 1, characterized in that: Including any one of (1) to (4) below: (1) The nucleic acid molecule encoding the protein; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant expression vector containing the nucleic acid molecule described in (1), or a recombinant expression vector containing the expression cassette described in (2); (4) A host cell containing the nucleic acid molecule described in (1), or a host cell containing the expression cassette described in (2), or a host cell containing the recombinant expression vector described in (3).

4. The biomaterial according to claim 3, characterized in that: The expression vector is pET-28a.

5. The biomaterial according to claim 3, characterized in that: The host cell is Escherichia coli.

6. The use of the protein of claim 1, the gene of claim 2, or the biomaterial of claim 3 in the degradation of dexamethasone.

7. The application in the degradation of dexamethasone according to claim 6, characterized in that: This includes research on the degradation mechanisms of steroid hormones.

8. A method for degrading dexamethasone, characterized in that: The method involves adding the protein of claim 1 or a culture of a calcium-containing protein or cells expressing the protein to a body of water containing dexamethasone, and using the protein to degrade the dexamethasone.

9. The use of the protein of claim 1 or the gene of claim 2 as a detection indicator in the preparation of reagents for assessing the sensitivity and resistance of patients with allergic rhinitis or allergic asthma to hormone therapy.

10. The application according to claim 9, characterized in that: The reagents described are for detecting the expression levels of the protein or gene.

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