Method for processing tif1 gamma protein, storage reagent and application

Abstract

The application provides a TIF1 gamma protein processing method, a storage reagent and application, and relates to the technical field of biology. The TIF1 gamma protein processing method comprises contacting TIF1 gamma protein with Zn 2+ , and the like. 2+ The TIF1 gamma protein processing method can improve the thermal stability of TIF1 gamma and the detection effect of anti-TIF1 gamma antibody, and is beneficial to the detection and diagnosis of dermatomyositis in clinic.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for treating TIF1γ protein, a storage reagent, and its application. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] TIF1γ (Transcriptional Intermediary Factor 1γ) is a protein with multiple biological functions. Also known as TRIM33 (Tripartite motif-containing 33), it belongs to the E3 ubiquitin ligase family and functions as an E3 ubiquitin ligase, transcription cofactor, and non-canonical TGF-β signaling molecule. TIF1γ possesses a ring-box-coiled-coil (RBCC) domain. TIF1γ plays a role in various biological processes, including transcriptional elongation, cell differentiation, embryonic development, mitosis, and DNA repair. It plays a crucial role in the homeostasis and function of various immune cell types, particularly in the myeloid lineage, where it inhibits the excessive proliferation of granulocyte-macrophage precursors (GMPs) and granulocytes, and supports the proper differentiation and activation of monocytes and macrophages. TIF1γ also plays an important role in the differentiation and homeostasis of dendritic cells (DCs). The absence of TIF1γ significantly affects the homeostasis of DCs, resulting in a significant reduction in the number of pDCs and cDC subsets in the spleen, thymus, and lymph nodes.

[0004] Dermatomyositis (DM) is an idiopathic autoimmune inflammatory disease characterized by myopathy with a distinctive skin rash. Dermatomyositis may be accompanied by interstitial lung disease, cardiac involvement, esophageal involvement, nutritional calcification, and potential malignancy.

[0005] Studies have found that serum anti-TIF1-γ antibody is a sensitive and specific serological marker for dermatomyositis complicated with tumors. It provides an effective means for screening dermatomyositis complicated with tumors. For patients with dermatomyositis, anti-TIF1γ antibody can be detected at the same time as diagnosis, which can be used for early diagnosis of tumors and is of great significance for improving prognosis.

[0006] Currently, the poor thermal stability of the TIF1γ antigen results in low signal values ​​and sensitivity in the detection of TIF1γ autoantibodies, making it unsuitable for effective detection.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The purpose of this invention is to provide Zn 2+ Applications in the preparation of TIF1γ storage reagents and TIF1γ storage reagents obtained based on this concept, to improve the thermal stability of TIF1γ in the reagents and the detection capability of TIF1γ against target antibodies when TIF1γ is used as an antigen.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] In a first aspect, a method for treating TIF1γ protein is provided, the method comprising reacting TIF1γ protein with Zn 2+ touch.

[0011] In an optional embodiment, the TIF1γ protein and the Zn 2+ The concentration ratio is TIF1γ protein: Zn 2+ =1 mg / mL: 0.5~2 mM, for example, but not limited to 1 mg / mL: 0.5 mM, 1 mg / mL: 1 mM, 1 mg / mL: 1.5 mM, 1 mg / mL: 2 mM, TIF1γ protein: Zn 2+ The preferred concentration ratio is TIF1γ protein:Zn 2+ =1 mg / mL:1~2 mM.

[0012] In an optional implementation, the Zn 2+ It is provided by one or more salts selected from halides, sulfates, nitrates, carbonates, phosphates, oxalates, acetates, and citrates.

[0013] In an optional implementation, the Zn 2+ Provided by ZnCl2.

[0014] In an optional embodiment, the TIF1γ protein is reacted with Zn in an environment containing surfactant, buffer, and NaCl at a pH of 7.0–8.5. 2+ touch.

[0015] In an optional embodiment, the concentration of NaCl is 150 mM to 1 M, for example, but not limited to 150, 200, 300, 400, 500, 600, 700, 800 or 900 mM or 1 M.

[0016] In an optional embodiment, the surfactant includes Tween series surfactants, preferably including Tween 20, wherein the volume percentage of Tween 20 is 0.075% to 0.1%, for example, but not limited to 0.075%, 0.08%, 0.09% or 0.1%.

[0017] In optional embodiments, the buffer solution includes Tris buffer or Hepes buffer, and the concentration is preferably 20-50 mM, for example, but not limited to 20, 25, 30, 35, 40, 45 or 50 mM.

[0018] In an optional embodiment, the TIF1γ protein is reacted with Zn in an environment containing the following components. 2+ Contact: 20-50 mM Tris buffer or Hepes buffer, 150 mM-1 M NaCl, 0.075-0.1 v / v% Tween 20 and 1-2 mM ZnCl2, pH 7.0-8.5.

[0019] In an optional embodiment, the amino acid sequence of the TIF1γ protein is shown in SEQ ID NO.1.

[0020] Secondly, a TIF1γ protein storage reagent is also provided, which includes Zn 2+ The ingredients include surfactants, buffer solutions, and NaCl, with a pH of 7.0–8.5.

[0021] In an optional embodiment, the concentration of NaCl is 150 mM to 1 M, for example, but not limited to 150, 200, 300, 400, 500, 600, 700, 800 or 900 mM or 1 M.

[0022] In an optional embodiment, the surfactant includes Tween series surfactants, preferably including Tween 20.

[0023] In an optional embodiment, the TIF1γ storage reagent includes Tween20 with a volume percentage of 0.075% to 0.1%, such as, but not limited to, 0.075%, 0.08%, 0.09%, or 0.1%.

[0024] In an optional implementation, the buffer solution includes Tris buffer or Hepes buffer.

[0025] In an optional embodiment, the concentration of the Tris buffer or Hepes buffer is 20-50 mM, for example, but not limited to 20, 25, 30, 35, 40, 45 or 50 mM.

[0026] In an optional embodiment, the TIF1γ storage reagent comprises 20-50 mM Tris buffer or Hepes buffer, 150 mM-1 M NaCl, 0.075-0.1 v / v% Tween 20 and 1-2 mM ZnCl2, with a pH of 7.0-8.5.

[0027] In an optional embodiment, the TIF1γ storage reagent further includes an eluent.

[0028] In an optional embodiment, the eluent includes desulfurized biotin.

[0029] Thirdly, a TIF1γ protein solution is also provided, the TIF1γ protein solution comprising the TIF1γ protein storage reagent and TIF1γ protein described in the second aspect, wherein the TIF1γ protein and the Zn 2+ The concentration ratio is TIF1γ protein: Zn 2+ =1 mg / mL:0.5~2 mM, for example, but not limited to 1 mg / mL:0.5 mM, 1 mg / mL:1 mM, 1 mg / mL:1.5 mM, 1 mg / mL:2 mM, TIF1γ protein:Zn 2+ The preferred concentration ratio is TIF1γ protein:Zn 2+ =1 mg / mL:1~2 mM.

[0030] In an optional embodiment, the amino acid sequence of the TIF1γ protein is shown in SEQ ID NO.1.

[0031] In an optional embodiment, the TIF1γ protein solution comprises 20-50 mM Hepes buffer, 1 M NaCl, 0.1 v / v% Tween 20, 0.5-2 mg / mL TIF1γ protein, and 1-2 mM ZnCl2, with a pH of 7.0-8.5.

[0032] Fourthly, a method for preparing a TIF1γ antibody detection reagent is also provided, the method comprising contacting the TIF1γ protein solution described in the third aspect with a solid-phase support, thereby immobilizing the TIF1γ protein therein onto the solid-phase support.

[0033] Alternatively, it may involve mixing a stock solution containing TIF1γ protein, a zinc salt, and a solid support, thereby immobilizing the TIF1γ protein onto the solid support.

[0034] Alternatively, after immobilizing the TIF1γ protein on the solid support, Zn can be added. 2+ Contact with coupling material.

[0035] TIF1γ protein and Zn 2+ The concentration ratio of TIF1γ protein to Zn is 1:1. 2+ =1 mg / mL: 0.5~2 mM.

[0036] In optional embodiments, the solid-phase support includes, but is not limited to, microtubes, columns, microparticles, nitrocellulose membranes, or side-flow devices; more specifically, it can be, but is not limited to, enzyme-labeled wells, immunochromatographic test strips, or magnetic beads. In optional embodiments, the magnetic beads include barcoded magnetic beads (BMB).

[0037] Fifthly, a TIF1γ antibody detection reagent prepared by the method described in the fourth aspect is also provided.

[0038] Sixthly, the application of the TIF1γ protein storage reagent described in the second aspect, or the TIF1γ protein solution described in the third aspect, or the TIF1γ antibody detection reagent described in the fifth aspect, in any of the following:

[0039] (I) Preparation of TIF1γ antibody detection kit;

[0040] (II) Non-diagnostic and therapeutic target detection of TIF1γ antibodies;

[0041] (III) Prepare diagnostic reagents or kits for dermatomyositis.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention discovers that using a certain concentration of Zn 2+ Treatment of TIF1γ protein (TIF1γ protein and Zn) 2+ The optimal concentration ratio (1 mg / mL: 0.5~2 mM) significantly improves the detection effect of TIF1γ antibody, specifically by enhancing the signal-to-noise ratio and detection sensitivity (sensitivity can reach 70%). Compared with the untreated Zn... 2+ The processed antigen showed a 33% improvement in signal-to-noise ratio and a 27% improvement in sensitivity. Furthermore, this invention also discovered that after Zn... 2+ The treatment significantly improved the thermostability of TIF1γ protein, which is crucial for reagent preservation and enhancing detection effectiveness. This invention enables more effective detection of TIF1γ antibodies in serum, and is of great significance for the auxiliary diagnosis of dermatomyositis in clinical practice. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Example 1

[0046] The antigen preparation process in this embodiment is as follows:

[0047] Step 1: Insert the nucleic acid fragment containing the full-length TIF1γ (protein sequence as shown in SEQ ID NO.1) into the pcDNA3.1+ vector to obtain the expression plasmid.

[0048] Step 2: Transfect the recombinant expression plasmid from Step 1 into HEK293F cells, and culture them in suspension at 37°C and 5% CO2 for 6 days. Collect the cell pellet, add lysis buffer to resuspend the cells, and then sonicate them.

[0049] Step 3: After lysing, the cell lysate is centrifuged, the supernatant is collected, filtered, and then purified by affinity using Streptactin-Beads. Elution is then performed using elution buffer containing 2.5 mM dethiobiotin.

[0050] Different eluent formulations are set in step 3 as shown in Table 1.

[0051] Table 1 (Eluent) Stock Solution Formulation

[0052]

[0053] Add 2.5 mM desulfurized biotin during elution, and after elution, concentrate the target protein to 1 mg / mL.

[0054] The TIF1γ antigen from the above seven stock solutions was coated onto BMB using the same method, with a coating ratio of 25 μg / 50K. Using phycoerythrin-labeled mouse anti-human IgG secondary antibody working solution, the detection efficacy of antigens stored in different stock solutions was tested using a fully automated multiplex immunoassay analyzer developed by Livzon Pharmaceutical Group Co., Ltd. (Zhuhai Livzon Reagent Co., Ltd.) to optimize the detection of serum samples (the serum sample tray included 40 anti-TIF1γ positive dermatomyositis serum samples and 20 anti-TIF1γ negative serum samples).

[0055] The signal-to-noise ratio, sensitivity, and specificity of the detection were statistically analyzed. The signal-to-noise ratio was calculated as the ratio of the mean signal value of positive samples to the mean signal value of negative samples; sensitivity was calculated as the ratio of the number of positive samples detected to the number of positive samples; and specificity was calculated as the ratio of the number of negative samples detected to the number of negative samples. The results are shown in Table 2.

[0056] Table 2. Detection results of TIF1γ antigen against TIF1γ-positive dermatomyositis in different storage solutions.

[0057]

[0058] As shown in Table 2, the effects of different buffer systems on the detection results of TIF1γ antigen storage solutions are not significantly different.

[0059] Example 2

[0060] The TIF1γ protein solution prepared using the elution / storage solution of formulation ① in Example 1 was divided into 5 groups and different final concentrations of ZnCl2 (0 mM / 0.5 mM / 1 mM / 2 mM / 10 mM) were added. Then, BMB was coated on each group in the same way to further test the effect of each coating on recognizing the TIF1γ antibody.

[0061] The detection method was the same as in Example 1. The test samples were optimized serum disc samples (including 40 anti-TIF1γ positive dermatomyositis serum samples and 20 negative serum samples). The results are shown in Table 3 below.

[0062] Table 3 Different Zn 2+ Detection results of TIF1γ antigen against TIF1γ-positive dermatomyositis after concentration treatment

[0063]

[0064] As shown in Table 3, an appropriate concentration of Zn was added to the TIF1γ antigen solution. 2+ (0.5~2 mM) can significantly improve the signal-to-noise ratio and sensitivity without changing the detection specificity. The final concentration is 1~2 mM Zn. 2+ The optimal signal-to-noise ratio is 4.4, and the sensitivity is 70%, which is better than the result without Zn. 2+ Sensitivity increased by 15%; however, excessive Zn 2+ On the contrary, it significantly reduces performance, possibly due to excessive Zn. 2+ This is related to protein precipitation.

[0065] Example 3

[0066] Thermal stability test

[0067] Take the 3TIF1γ protein solution from Example 2 above (with 1 mM Zn added). 2+ ) and Group 1 protein solution (without added Zn) 2+ The sample was subjected to accelerated thermal treatment (37°C, 1 week). After treatment, BMB was coated using the same method to prepare a coating material coated with TIF1γ antigen protein at a ratio of 25 μg / 50K. The effect of the coating material on optimizing serum plate detection was tested according to the method in Example 1, and the results are shown in Table 4 below.

[0068] Table 4. Results of TIF1γ antigen detection against TIF1γ-positive dermatomyositis after thermal acceleration.

[0069]

[0070] As shown in Table 3, the signal-to-noise ratio and sensitivity of the TIF1γ antigen reagent in Example 7 are superior to those in Comparative Example 1, indicating that the addition of Zn to the stock solution... 2+ It can significantly improve the thermal stability of TIF1γ antigen.

[0071] Comparative Example 1

[0072] Comparative Example 1 provides a Ro52 antigen solution containing 1 mg / mL Ro52 antigen (target antigen of dermatomyositis) and a stock solution. The stock solution contains 50 mM Tris, 1 M NaCl, 2.5 mM dethiobiotin, 0.5 mM ZnCl2, 0.10% v / v Tween 20, and pH 8.0.

[0073] Comparative Examples 2-4

[0074] Comparative Examples 2-4 each provided a Ro52 antigen solution, differing from Comparative Example 1 only in the presence of Zn in the storage solution. 2+ The final concentrations were 1 mM, 10 mM and 0 mM (containing no Zn), respectively. 2+ ).

[0075] Comparative Example 5

[0076] Comparative Example 5 provides a Mi-2 antigen solution containing 1 mg / mL Mi-2 antigen (target antigen for dermatomyositis) and a stock solution. The stock solution contains 50 mM Tris, 1 M NaCl, 2.5 mM dethiobiotin, 0.5 mM ZnCl2, 0.10% v / v Tween 20, and pH 8.0.

[0077] Comparative Examples 6-8

[0078] Comparative Examples 6-8 each provided a Mi-2 antigen reagent, differing from Comparative Example 5 only in the presence of Zn in the storage solution. 2+ The final concentrations were 1 mM, 10 mM and 0 mM (containing no Zn), respectively. 2+ ).

[0079] Comparative Example 9

[0080] Solutions containing Ro52 antigen (Comparative Examples 1-4) were coated with BMB using the same method (coating ratio of 25 μg / 50 K), and the detection effect of each coating on the Ro52 optimized serum disc (containing 35 positive samples and 5 negative samples) was tested. The results are shown in Table 5 below.

[0081] Table 5 Different Zn 2+ Detection results of Ro52 antigen concentration-treated against Ro52-positive dermatomyositis

[0082]

[0083] As shown in Table 5, adding Zn to the Ro52 antigen storage solution... 2+ The detection performance (signal-to-noise ratio, sensitivity, specificity) of anti-Ro52 antibodies was not improved, and the excessive Zn... 2+ It will also lead to a significant decrease in its detection signal-to-noise ratio and sensitivity.

[0084] Comparative Example 10

[0085] Solutions containing Mi-2 antigen from comparative examples 5 to 8 were coated with BMB using the same method (coating ratio of 25 μg / 50K), and the detection effect of each coating on the Mi-2 optimized serum disc (35 positive samples and 5 negative samples) was tested. The results are shown in Table 6 below.

[0086] Table 6 Different Zn 2+ Detection results of Mi-2 antigen against Mi-2 positive dermatomyositis after concentration treatment

[0087]

[0088] As shown in Table 6, adding Zn to the Mi-2 antigen storage solution... 2+ It did not improve the performance (signal-to-noise ratio, sensitivity, specificity) of detecting anti-Mi-2 antibodies, and the excessive Zn... 2+ It will also lead to a significant decrease in its detection signal-to-noise ratio and sensitivity.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a TIF1γ antibody detection reagent, characterized in that, This includes contacting a TIF1γ protein solution with a solid support to immobilize the TIF1γ protein onto the solid support; The TIF1γ protein solution includes TIF1γ protein and Zn. 2+ Surfactants, buffer solutions, and NaCl; pH 7.0–8.5; The TIF1γ protein and the Zn 2+ The concentration ratio is TIF1γ protein: Zn 2+ =1 mg / mL: 0.5~2 mM.

2. A method for preparing a TIF1γ antibody detection reagent, characterized in that, This includes the stock solution containing TIF1γ protein, Zn 2+ Mix with a solid support to immobilize the TIF1γ protein onto the solid support; In this process, TIF1γ protein was reacted with Zn in an environment containing surfactant, buffer, and NaCl at a pH of 7.0–8.

5. 2+ Contact, the TIF1γ protein and the Zn 2+ The concentration ratio is TIF1γ protein: Zn 2+ =1 mg / mL: 0.5~2 mM.

3. A method for preparing a TIF1γ antibody detection reagent, characterized in that, This includes immobilizing the TIF1γ protein on the solid support and then adding Zn. 2+ Contact with coupling material; In this process, TIF1γ protein was reacted with Zn in an environment containing surfactant, buffer, and NaCl at a pH of 7.0–8.

5. 2+ Contact, the TIF1γ protein and the Zn 2+ The concentration ratio is TIF1γ protein: Zn 2+ =1 mg / mL: 0.5~2 mM.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The amino acid sequence of the TIF1γ protein is shown in SEQ ID NO.

1.

5. The preparation method according to any one of claims 1 to 3, characterized in that, The TIF1γ protein: Zn 2+ =1 mg / mL: 1~2 mM.

6. The preparation method according to any one of claims 1 to 3, characterized in that, The concentration of NaCl is 150 mM to 1 M.

7. The preparation method according to any one of claims 1 to 3, characterized in that, The surfactants include Tween series surfactants.

8. The preparation method according to claim 7, characterized in that, The surfactant includes Tween20.

9. The preparation method according to any one of claims 1 to 3, characterized in that, The buffer solution includes Tris buffer or Hepes buffer.

10. The preparation method according to claim 9, characterized in that, The concentration of the Tris buffer or Hepes buffer is 20-50 mM.

11. The preparation method according to any one of claims 1 to 3, characterized in that, The TIF1γ protein solution comprises 20–50 mM Hepes buffer, 1 M NaCl, 0.1 v / v% Tween 20, 1 mg / mL TIF1γ protein, and 0.5–2 mM ZnCl2, with a pH of 7.0–8.

5.

12. The TIF1γ antibody detection reagent prepared by the method according to any one of claims 1 to 11.

13. The use of the TIF1γ antibody detection reagent according to claim 12 in any of the following: (I) Preparation of TIF1γ antibody detection kit; (II) Non-diagnostic and therapeutic target detection of TIF1γ antibodies; (III) Prepare diagnostic reagents or kits for dermatomyositis.

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