TIF1 gamma protein treatment method, storage reagent and application

Abstract

The invention provides a TIF1 gamma protein treatment method, a storage reagent and application, and relates to the technical field of biology. According to the TIF1 gamma protein treatment method, the TIF1 gamma protein is in contact with Zn < 2 + >, and the Zn < 2 + > can improve the TIF1 gamma thermal stability and the detection effect of the anti-TIF1 gamma antibody, so that clinical detection and diagnosis of dermatomyositis are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a TIF1γ protein processing method, a storage reagent and an application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] TIF1γ (Transcriptional Intermediary Factor 1γ) is a protein with multiple biological functions. TIF1γ is also called TRIM33 (Tripartite motif-containing 33). It belongs to the E3 ubiquitin ligase family and has the functions of E3 ubiquitin ligase, transcription co-factor and non-classical TGF-β signaling molecule. TIF1γ has a ring-box-coiled-coil (RBCC) domain. TIF1γ plays a role in a variety of biological processes, including transcription elongation, cell differentiation, embryonic development, mitosis and DNA repair. TIF1γ plays a key role in the homeostasis and function of multiple immune cell types, especially in the myeloid lineage, where it inhibits the overexpansion of granulocyte-macrophage precursors (GMPs) and granulocytes, and supports the correct differentiation and activation of monocytes and macrophages. TIF1γ also plays an important role in the differentiation and homeostasis of dendritic cells (DCs). The loss of TIF1γ significantly affects the homeostasis of DCs, resulting in a significant decrease 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 rash that may be associated with interstitial lung disease, cardiac involvement, esophageal involvement, nutritional calcifications, and potential malignancy.

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

[0006] Currently, the TIF1γ antigen has poor thermal stability, which results in low signal values ​​and sensitivity in the detection of TIF1γ autoantibodies, and cannot be well applied in detection.

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

[0008] The present invention aims to provide Zn 2+ The application in the preparation of TIF1γ storage reagent and the TIF1γ storage reagent obtained based on the conception are used to improve the thermal stability of TIF1γ in the reagent and the detection ability of target antibodies when TIF1γ is used as an antigen.

[0009] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

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

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

[0012] In an optional embodiment, the Zn 2+ Provided by one or more salts of halides, sulfates, nitrates, carbonates, phosphates, oxalates, acetates and citrates.

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

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

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

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

[0017] In an optional embodiment, the buffer comprises 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 mixed 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 ZnCl 2, pH 7.0-8.5.

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

[0020] In a second aspect, a TIF1γ protein storage reagent is also provided, wherein the TIF1γ protein storage reagent comprises Zn 2+ , surfactant, buffer and NaCl, pH 7.0-8.5.

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

[0022] In an optional embodiment, the surfactant includes a Tween series surfactant, preferably Tween20.

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

[0024] In an optional embodiment, the buffer comprises 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 includes 20-50 mM Tris buffer or Hepes buffer, 150 mM-1 M NaCl, 0.075-0.1 v / v% Tween20 and 1-2 mM ZnCl2, with a pH of 7.0-8.5.

[0027] In an optional embodiment, the TIF1γ storage reagent further comprises an eluting agent.

[0028] In an optional embodiment, the eluent comprises desthiobiotin.

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

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

[0031] In an optional embodiment, the TIF1γ protein solution includes 20-50 mM Hepes buffer, 1 M NaCl, 0.1 v / v% Tween20, 0.5-2 mg / mL TIF1γ protein and 1-2 mM ZnCl2, with a pH of 7.0-8.5.

[0032] In a fourth aspect, 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 carrier, so that the TIF1γ protein therein is fixed on the solid phase carrier.

[0033] Or, comprising mixing an original solution containing TIF1γ protein, a zinc salt and a solid phase carrier, so that the TIF1γ protein therein is fixed on the solid phase carrier;

[0034] Alternatively, after the TIF1γ protein is fixed on the solid phase carrier, Zn 2+ Contact with the conjugate.

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

[0036] In an optional embodiment, the solid phase carrier includes but is not limited to microtubes, columns, microparticles, nitrocellulose membranes or lateral flow devices, and more specifically, for example, but not limited to, enzyme-labeled wells, immunochromatographic test strips or magnetic beads. In an optional embodiment, the magnetic beads include barcoded magnetic beads (BMB for short).

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

[0038] In the sixth aspect, there is also provided the use 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) preparing a TIF1γ antibody detection kit;

[0040] (II) Testing for TIF1γ antibodies for non-diagnostic and therapeutic purposes;

[0041] (III) Preparing a diagnostic reagent or kit for dermatomyositis.

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

[0043] The present invention finds that using a certain concentration of Zn 2+ TIF1γ protein was treated with Zn 2+ The concentration ratio of Zn was 1 mg / mL: 0.5-2 mM (preferably 1 mg / mL: 0.5-2 mM), which can significantly improve the detection effect of TIF1γ antibody, specifically the improvement of signal-to-noise ratio and detection sensitivity (sensitivity can reach 70%); compared with the detection without Zn 2+ The signal-to-noise ratio of the treated antigen increased by 33% and the sensitivity increased by 27%. 2+ The thermal stability of TIF1γ protein after treatment is also significantly improved, which plays an important role in the preservation of reagents and improving the effectiveness of detection. The present invention can more effectively detect TIF1γ antibodies in serum, which is of great significance for the auxiliary diagnosis of dermatomyositis in clinical practice. DETAILED DESCRIPTION

[0044] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Example 1

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

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

[0048] Step 2: The recombinant expression plasmid in step 1 was transfected into HEK293F cells, and after suspension culture at 37° C., 5% CO 2 for 6 days, the cell pellet was collected, lysate was added for re-suspending, and then ultrasonically disrupted.

[0049] Step 3: After the lysis, the cell solution was centrifuged and the supernatant was filtered and affinity purified by Streptactin-Beads. The elution was then eluted by adding 2.5 mM desthiobiotin.

[0050] In step 3, different eluent recipes are set as shown in Table 1.

[0051] Table 1 (Eluent) Storage Solution Formula

[0052]

[0053] During elution, 2.5 mM desthiobiotin was added, and after elution, the target protein was concentrated to 1 mg / mL.

[0054] The TIF1γ antigens in the above 7 groups of storage solutions were coated on BMB in the same way, with a coating ratio of 25μg / 50K. In combination with the phycoerythrin-labeled mouse anti-human IgG secondary antibody working solution, the detection effect of antigens stored in different storage solutions on the optimized serum plate (the serum plate included 40 anti-TIF1γ positive dermatomyositis serum samples and 20 negative serum samples) was tested using the Livzon self-developed fully automatic multiple immunoassay analyzer (Zhuhai Livzon Reagent Co., Ltd.).

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

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

[0057] Storage solution Signal-to-Noise Ratio Sensitivity Specificity ① 3.3 55％ 95％ ② 3.0 55％ 95％ ③ 3.3 55％ 95％ ④ 3.2 55％ 95％ ⑤ 3.1 55％ 95％ ⑥ 3.3 55％ 95％ ⑦ 3.3 55％ 95％

[0058] As shown in Table 2, the effects of TIF1γ antigen storage solutions in different buffer systems on the detection results are similar.

[0059] Example 2

[0060] The TIF1γ protein solution prepared using the elution / storage solution of formula ① in Example 1 was divided into 5 groups, and different final concentrations of ZnCl2 (0mM / 0.5mM / 1mM / 2mM / 10mM) were added, and then coated with BMB respectively according to the same method, and the effect of each coated object in recognizing TIF1γ antibody was further tested.

[0061] The detection method was carried out in the same manner as in Example 1. The detection samples were optimized serum plate 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 after concentration treatment against TIF1γ positive dermatomyositis

[0063] Group <![CDATA[Add Zn 2+ concentration]]> Signal-to-Noise Ratio Sensitivity Specificity 1 none 3.3 55％ 95％ 2 0.5mM 4.1 65％ 95％ 3 1mM 4.4 70％ 95％ 4 2mM 4.4 70％ 95％ 5 10mM 1.3 10％ 100％

[0064] As shown in Table 3, an appropriate concentration of Zn was added to the TIF1γ antigen solution. 2+ (0.5~2mM), which can significantly improve the signal-to-noise ratio and sensitivity without changing the detection specificity. Among them, the final concentration is 1~2mM Zn 2+ The best, signal-to-noise ratio was 4.4, sensitivity was 70%, which was higher than that without Zn addition. 2+ Sensitivity increased by 15%; however, excess Zn 2+ On the contrary, the performance is significantly reduced, which may be related to the excessive Zn 2+ Caused protein precipitation.

[0065] Example 3

[0066] Thermal stability test

[0067] Take the TIF1γ protein solution of group 3 in Example 2 (added with 1 mM Zn 2+ ) and group 1 protein solution (without Zn added 2+ ) was subjected to heat acceleration treatment (37°C, 1 week). After treatment, BMB was coated in the same manner to prepare a coating material coated with TIF1γ antigen protein, and the coating ratio was 25μg / 50K. The coating material was tested for the detection effect of the optimized serum plate according to the method of Example 1, and the results are shown in Table 4 below.

[0068] Table 4 Detection results of TIF1γ antigen after heat acceleration against TIF1γ positive dermatomyositis

[0069] Group <![CDATA[Add Zn 2+ concentration]]> Signal-to-Noise Ratio Sensitivity Specificity 3 <![CDATA[1mM Zn 2+ ]]> 3.8 65％ 95％ 1 <![CDATA[Zn-free 2+ > 2.3 45％ 95％

[0070] As shown in Table 3, the signal-to-noise ratio and sensitivity of the TIF1γ antigen reagent of Example 7 are better than those of Comparative Example 1, indicating that the addition of Zn 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 (dermatomyositis target antigen) and a storage solution containing 50 mM Tris, 1 M NaCl, 2.5 mM desthiobiotin, 0.5 mM ZnCl2, 0.10% v / v Tween20, pH 8.0.

[0073] Comparative Examples 2 to 4

[0074] Comparative Examples 2 to 4 provide a Ro52 antigen solution, which differs from Comparative Example 1 only in that Zn in the storage solution is 2+ The final concentrations were 1mM, 10mM and 0mM (without Zn 2+ ).

[0075] Comparative Example 5

[0076] Comparative Example 5 provides a Mi-2 antigen solution containing 1 mg / mL Mi-2 antigen (dermatomyositis target antigen) and a storage solution containing 50 mM Tris, 1 M NaCl, 2.5 mM desthiobiotin, 0.5 mM ZnCl2, 0.10% v / v Tween20, pH 8.0.

[0077] Comparative Examples 6 to 8

[0078] Comparative Examples 6 to 8 provide a Mi-2 antigen reagent, which differs from Comparative Example 5 only in that the Zn in the storage solution 2+ The final concentrations were 1mM, 10mM and 0mM (without Zn 2+ ).

[0079] Comparative Example 9

[0080] The solutions containing Ro52 antigen (Comparative Examples 1 to 4) were coated with BMB (coating ratio of 25 μg / 50K) according to the same method, and the detection effect of each coating on the Ro52 optimized serum plate (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 after concentration treatment against Ro52 positive dermatomyositis

[0082] Group <![CDATA[Zn concentration in the stock solution 2+ > Signal-to-Noise Ratio Sensitivity Specificity Comparative Example 4 <![CDATA[Zn-free 2+ > 4.7 71％ 100％ Comparative Example 1 <![CDATA[0.5mM Zn 2+ ]]> 4.6 71％ 100％ Comparative Example 2 <![CDATA[1mM Zn 2+ ]]> 4.6 71％ 100％ Comparative Example 3 <![CDATA[10mM Zn 2+ ]]> 1.4 17％ 100％

[0083] As shown in Table 5, the addition of Zn 2+The performance of its detection of anti-Ro52 antibodies (signal-to-noise ratio, sensitivity, specificity) was not improved, and 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] The solutions containing Mi-2 antigen in Comparative Examples 5 to 8 were coated with BMB (coating ratio was 25 μg / 50K) according to the same method, and the detection effect of each coating on the Mi-2 optimized serum plate (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 Ro52 antigen after concentration treatment against Ro52 positive dermatomyositis

[0087] Group <![CDATA[Zn concentration in the stock solution 2+ > Signal-to-Noise Ratio Sensitivity Specificity Comparative Example 8 <![CDATA[Zn-free 2+ > 3.4 71％ 100％ Comparative Example 5 <![CDATA[0.5mM Zn 2+ ]]> 3.4 71％ 100％ Comparative Example 6 <![CDATA[1mM Zn 2+ ]]> 3.3 71％ 100％ Comparative Example 7 <![CDATA[10mM Zn 2+ ]]> 1.4 17％ 100％

[0088] As shown in Table 6, adding Zn to the Mi-2 antigen storage solution 2+ There was no effect on its performance in detecting anti-Mi-2 antibodies (signal-to-noise ratio, sensitivity, specificity), and 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 treating TIF1γ protein, characterized in that: TIF1γ protein and Zn 2+ touch.

2. The TIF1γ protein processing method according to claim 1, characterized in that: The TIF1γ protein and the Zn 2+ The concentration ratio of TIF1γ protein:Zn 2+ =1mg / mL: 0.5~2mM; Optionally, the amino acid sequence of the TIF1γ protein is as shown in SEQ ID NO.1; Optionally, the TIF1γ protein: Zn 2+ =1mg / mL: 1~2mM.

3. A TIF1γ protein storage reagent, characterized in that: Including Zn 2+ , surfactant, buffer and NaCl, pH 7.0-8.

5. Optionally, the concentration of NaCl is 150mM to 1M; Optionally, the surfactant includes a Tween series surfactant, preferably Tween 20; Optionally, the TIF1γ storage reagent comprises 0.075% to 0.1% by volume of Tween20; Optionally, the buffer comprises Tris buffer or Hepes buffer; Optionally, the concentration of the Tris buffer or Hepes buffer is 20-50 mM.

4. The TIF1γ protein storage reagent according to claim 3, characterized in that It comprises 20-50 mM Tris buffer or Hepes buffer, 150 mM-1 M NaCl, 0.075-0.1 v / v% Tween20 and 1-2 mM ZnCl2, and the pH value is 7.0-8.

5.

5. The TIF1γ protein storage reagent according to claim 3, characterized in that Also included is an eluent.

6. A TIF1γ protein solution, characterized in that: The invention comprises the TIF1γ protein storage reagent according to any one of claims 3 to 5 and the TIF1γ protein, wherein the TIF1γ protein and the Zn 2+ The concentration ratio of TIF1γ protein:Zn 2+ =1mg / mL: 0.5~2mM; Optionally, the amino acid sequence of the TIF1γ protein is as shown in SEQ ID NO.1; Optionally, the TIF1γ protein: Zn 2+ =1mg / mL: 1~2mM.

7. The TIF1γ protein solution according to claim 6, characterized in that The method 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 ZnCl 2, with a pH value of 7.0-8.

5.

8. A method for preparing a TIF1γ antibody detection reagent, characterized in that: The method comprises contacting the TIF1γ protein solution according to any one of claims 6 or 7 with a solid phase carrier, so that the TIF1γ protein therein is fixed on the solid phase carrier; or, comprising: adding a raw solution containing TIF1γ protein, Zn 2+ Mixing with a solid phase carrier so that the TIF1γ protein therein is fixed on the solid phase carrier; Alternatively, after the TIF1γ protein is fixed on the solid phase carrier, Zn 2+ Contact with the conjugate.

9. The TIF1γ antibody detection reagent prepared by the method of claim 8.

10. Use of the TIF1γ protein storage reagent according to any one of claims 3 to 5, or the TIF1γ protein solution according to any one of claims 6 or 7, or the TIF1γ antibody detection reagent according to claim 9 in any of the following: (I) preparing a TIF1γ antibody detection kit; (II) Testing for TIF1γ antibodies for non-diagnostic and therapeutic purposes; (III) Preparing a diagnostic reagent or kit for dermatomyositis.

Images