Recombinant Twist1 protein, preparation method and application of recombinant Twist1 protein in keloid

Abstract

The invention provides a recombinant Twist1 protein, a preparation method and application of the recombinant Twist1 protein in keloid. The recombinant Twist1 protein has the advantages of strong specificity, high affinity and high immune titer, has extremely high affinity with the Twist1 protein antibody, and has the advantages of high sensitivity and high specificity when being used for detecting the Twist1 protein antibody. Reference basis is provided for clinical diagnosis and prevention and control of keloid, and the method also plays an important role in prevention of keloid.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a recombinant Twist1 protein, a preparation method and application thereof in keloid. Background Art

[0002] Keloid is a pathological scar that is formed due to excessive proliferation of fibroblasts and excessive deposition of extracellular matrix after skin injury, which causes scar tissue to extend beyond the original injury range and continue to grow. Recent studies have shown that Twist1 protein plays an important role in the pathogenesis of keloid. Twist1 is a transcription factor that can regulate a variety of cell biological processes, such as cell proliferation, migration, differentiation, and the synthesis and remodeling of extracellular matrix. In keloid tissue, the expression level of Twist1 protein is often significantly increased, which promotes the abnormal activation of fibroblasts by activating a series of downstream signaling pathways, thereby promoting the formation and development of keloid. Therefore, Twist1 protein is expected to become a key target for the diagnosis and treatment of keloid, and the detection of its related indicators is also of great clinical significance.

[0003] However, the Twist1 protein itself is unstable and easy to decompose, which further increases the difficulty of conducting effective research and application around it. When using the existing Twist1 protein as an immunogen to prepare antibodies, the antibody titer is generally not ideal, and it is impossible to accurately identify and bind to the Twist1 protein in keloid tissue, and it is difficult to effectively neutralize and block its in vivo and in vitro activities, which does not help to build an efficient diagnosis and immunotherapy system to prevent and treat keloids. This is mainly because the traditional way of obtaining Twist1 protein (mostly derived from prokaryotic or eukaryotic conventional expression systems) has the problem of low expression levels, which is difficult to meet the needs of a large number of experiments and subsequent applications. Prokaryotic expression often faces protein folding errors and the formation of inclusion bodies, and the subsequent renaturation is cumbersome and the yield is not high; although the protein folding of eukaryotic expression is relatively correct, the expression level is restricted by many factors, the cost is high, and it takes a long time. It is difficult to prepare a sufficient amount of natural conformation Twist1 protein on a large scale for in-depth research and treatment development. Conventional methods for purifying Twist1 protein do not completely remove impurities, and co-purification with other impurity proteins occurs frequently, resulting in poor protein purity. The interaction between impurity molecules and Twist1 protein will also destroy its active site conformation and shield the antigenic epitope. When it is used in immune response to prepare antibodies, immune cell recognition is hindered, and the efficiency of stimulating immune response is low. The titer of the produced antibodies is naturally difficult to increase, limiting its application in precise targeted intervention of keloids.

[0004] At present, the clinical diagnosis of keloids mainly depends on the patient's medical history, clinical manifestations and histopathological examination. Although medical history and clinical manifestations can provide preliminary diagnostic clues, they lack specificity and it is difficult to accurately distinguish keloids from other types of scars. Although histopathological examination is the gold standard for diagnosis, it is an invasive examination and requires obtaining the patient's lesion tissue, which not only causes additional pain to the patient, but also may cause complications such as infection and bleeding. In addition, the accuracy of histopathological examination results is also affected by multiple factors such as sampling site and sample processing. Moreover, these traditional diagnostic methods are difficult to make an accurate diagnosis in the early stages of keloid onset, which is not conducive to timely and effective intervention measures. Therefore, it is urgent to develop a non-invasive, highly specific and sensitive method for keloid diagnosis.

[0005] Antigen-antibody reaction has high specificity and sensitivity. Detection techniques based on this principle, such as enzyme-linked immunosorbent assay (ELISA), have been widely used in the diagnosis of various diseases. By detecting the antibody level of a specific protein, the expression of the protein in the body and the body's immune response to related diseases can be indirectly reflected. For Twist1 protein, detecting its antibody level can provide new ideas and methods for the diagnosis of keloids. Summary of the invention

[0006] One of the purposes of the present invention is to provide a recombinant Twist1 protein, the amino acid sequence of the recombinant Twist1 protein is shown as SEQ ID No.1; the nucleotide sequence of the recombinant Twist1 protein is shown as SEQ ID No.2.

[0007] The second object of the present invention is to provide a method for preparing a recombinant Twist1 protein, comprising the following steps: S1 clones the coding gene shown in SEQ ID No. 2 into a vector to construct a recombinant expression plasmid.

[0008] S2 transforms the obtained recombinant expression plasmid into competent cells, and then obtains the recombinant antigen protein through culture and purification.

[0009] Preferably, the vector is a pET28a vector, and the competent cell is Escherichia coli.

[0010] The third object of the present invention is to provide a use of a recombinant Twist1 protein in a detection kit for preparing Twist1 protein antibodies.

[0011] A fourth object of the present invention is to provide an application of a recombinant Twist1 protein in the preparation of a keloid vaccine.

[0012] Beneficial effects of the present invention: The recombinant Twist1 protein provided by the present invention has high expression, better purification, high immune titer, can effectively stimulate the body to cause a protective immune response, and has the advantages of strong specificity and high affinity. It has no serological cross reaction with Twist1 protein antibody negative serum, has extremely high affinity with Twist1 protein antibody, and the recombinant Twist1 protein antibody also has extremely high affinity with Twist1 protein. It is further applied to ELISA detection kits, and the detection conclusion can be quickly obtained through the ELISA detection results. The conclusion can detect keloids, provide a reference basis for the clinical diagnosis and prevention and control of keloids, and also play an important role in the prevention of keloids. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the predicted three-dimensional structure of the recombinant antigen protein; Figure 2 The results of protein monoclonal induced expression SDS-PAGE, M: Marker; Lanes 1-5: recombinant Twist1 protein samples expressed with pET32a, pBAD / His A, pGEX-4T-1, pMAL-p2X and pET28a as expression vectors, respectively; Figure 3 The SDS-PAGE results of the purified samples, where M: Marker; Lane 1: Liuchuan; Lanes 2-4: Twist1 protein supernatant purified samples; Lanes 5-7: recombinant Twist1 protein inclusion body (precipitate) purified samples; Lanes 8-10: recombinant Twist1 protein supernatant purified samples; Figure 4 The SDS-PAGE results of the final protein product, where M: Marker; Lane 1: the final product of recombinant Twist1 protein purification; Figure 5 is a curve showing the change of absorbance value (OD) with antibody dilution in Example 2; Figure 6 is a curve showing the change of absorbance value (OD) with antibody dilution in Example 3; Figure 7 This is a curve showing the change of absorbance value (OD) with antibody dilution in Example 4. DETAILED DESCRIPTION

[0014] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0015] The technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0016] Example 1 Preparation of recombinant Twist1 protein Step 1: Connect the target gene shown in SEQ ID No. 2 to a prokaryotic expression vector. The prokaryotic expression vector selected is pET32a, pBAD / His A, pGEX-4T-1, pMAL-p2X and pET28a to obtain a recombinant expression plasmid containing the target gene fragment.

[0017] Step 2: Transfer the recombinant expression plasmid containing the target gene fragment into BL21 (DE3) competent cells, pick a single colony, culture the bacteria to OD600 to 0.4-0.6, add 1mmol / L IPTG and induce at 37℃ for 12h before collecting the bacteria.

[0018] Step 3: Perform SDS-PAGE detection on the collected bacteria. Refer to the molecular cloning experiment guide to prepare the reaction liquid. The stacking gel concentration is 4%, the separation gel concentration is 12%, the stacking gel is electrophoresed at a constant current of 10mA, and the separation gel is electrophoresed at a constant current of 25mA. After electrophoresis, Coomassie Brilliant Blue staining is performed to verify the expression of the recombinant protein. The results are as follows: Figure 2 As shown, the recombinant expression vector was efficiently expressed, and the expression level of the recombinant Twist1 protein using pET28a as the expression vector was the highest.

[0019] Step 4: Amplify the expression Obtain recombinant antigen protein suitable for ELISA experiment. The specific steps are: 10ml of overnight culture of expression bacteria is inoculated into 1L LB medium (containing 100μg / ml ampicillin), and cultured at 37℃ for 2-3 hours with shaking. When the OD value reaches 0.6, add IPTG to a final concentration of 0.5mM, continue shaking and inducing culture at 37℃ for 4 hours, and then harvest the bacteria by centrifugation for use.

[0020] Step 5: Purification The collected bacterial sludge was resuspended in 40 ml 20 mM PB + 150 mM Nacl, pH 8.0; ultrasonically disrupted in an ice bath at 420 W for 3 seconds, with an interval of 5 seconds, for 15 minutes; centrifuged at 12000 rpm, 4°C for 20 minutes, and the supernatant and precipitate were collected for later use; Among them, the supernatant was filtered through a 0.22um filter membrane and then purified by Ni column affinity enrichment.

[0021] Equilibration buffer: "20mM PB+150mM NaCl, pH8.0"; Washing buffer: 20mM PB+150mM NaCl+5mM imidazole, pH 8.0 20mM PB+150mM NaCl+20mM imidazole, pH 8.0 20mM PB+150mM NaCl+50mM imidazole, pH 8.0 20mM PB+150mM NaCl+80mM imidazole, pH8.0; Elution buffer: 20 mM PB + 150 mM NaCl + 300 mM imidazole, pH 8.0; After flow-through and purification of samples, loading buffer was added and the samples were boiled at 100°C and analyzed by SDS-PAGE.

[0022] Inclusion body (precipitate) purification: (1) Wash the precipitate once with 20 mM PB + 150 mM NaCl, pH 8.0 buffer; (2) Wash once with 20 mM PB + 150 mM NaCl, pH 8.0 buffer plus 0.3% Triton. (3) Gradient elution 20mM PB+150mM NaCl+1M Urea, pH8.0 20mM PB+150mM NaCl+2MUrea, pH8.0 20mM PB+150mM NaCl+3M Urea, pH8.0 20mMPB+150mMNaCl+4MUrea, pH8.0; (4) Dissolve the precipitate in 15 ml of 20 mM PB + 150 mM NaCl + 8 M Urea, pH 8.0; (5) Centrifuge, precipitate and re-dissolve the supernatant, filter it through a 0.22 μm filter membrane, and then perform Ni column affinity enrichment and purification.

[0023] Equilibration buffer: "20mM PB+150mM NaCl+8M Urea, pH8.0" Washing buffer: 20mM PB+150mM NaCl+8M Urea+5mM imidazole, pH 8.0 20mM PB+150mM NaCl+8M Urea+20mM imidazole, pH 8.0 20mM PB+150mM NaCl+8M Urea+50mM imidazole, pH 8.0 20mM PB+150mM NaCl+8M Urea+80mM imidazole, pH 8.0 Elution buffer: 20mM PB+150mM NaCl+8M Urea+300mM imidazole, pH 8.0 After flow-through and purification of the sample, the sample was boiled at 100°C after adding loading buffer and analyzed by SDS-PAGE. Figure 3 The SDS-PAGE results of the final protein product are shown in Figure 4 shown.

[0024] The amino acid sequence of the recombinant antigen protein is shown in SEQ ID No: 1, specifically MMQDVSSSPVSPADDSLSNSEEEPDRQQPPSGKRGGRKRRSRRSAGGGAGPGGAAGGGVGGGDEPGPAQGKRGKSAGCGGGGGAGGGGGSSSGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKHHHHHHSVWRMEGAHHHHHH.

[0025] Example 2 Immunoassay of recombinant Twist1 protein Material: (1) Antigen: recombinant Twist1 protein and traditional Twist1 protein; (2) Experimental animals: Healthy female BALB / c mice of similar age and weight (e.g., 6-8 weeks old, weighing 20-25 g) were selected, with at least 6 mice in each group. They were housed in a standard SPF animal room and allowed to acclimate to the environment for 1 week before the experiment. The mice were randomly divided into a recombinant Twist1 protein immunization group, a traditional Twist1 protein immunization group, and a blank control group.

[0026] (3) Serum: serum from the recombinant Twist1 protein immunization group, serum from the traditional Twist1 protein immunization group, and Twist1 mouse negative serum (blank control group).

[0027] Recombinant Twist1 protein immunization group: The recombinant Twist1 protein was fully emulsified with Freund's complete adjuvant in a volume ratio of 1:1, and injected subcutaneously into mice at multiple points for the first immunization, with a dose of 100 μg protein per mouse per injection; 2 weeks later, the recombinant Twist1 protein was emulsified with Freund's incomplete adjuvant (same volume ratio) for a second immunization, with the same dose as the first immunization; 2 weeks later, the third immunization booster was performed using the same method and dose.

[0028] Traditional Twist1 protein immunization group: The operation is the same as the recombinant Twist1 protein immunization group, except that the immunogen is replaced with the traditionally prepared Twist1 protein, and the dosage, adjuvant type and ratio, immunization time interval, etc. remain the same.

[0029] Blank control group: injected with equal volumes of physiological saline and adjuvant emulsion, and the immunization procedure was synchronized.

[0030] 10-14 days after the last immunization, the blood of the mice was collected by orbital blood sampling. After standing at room temperature for 2 hours, the blood was centrifuged at 4°C and 3000 rpm for 15 minutes. The serum was collected and stored in aliquots at -80°C for later use.

[0031] (4) Antibody: Sheep anti-mouse-HRP Buffer preparation: (1) Antigen dilution buffer: 0.01 M PBS, pH 7.2.

[0032] (2) Elution buffer: 0.01 M PBS, pH 7.2, containing 0.05% Tween 20.

[0033] (3) Blocking solution: 0.01 M PBS, pH 7.2, containing 5% fetal bovine serum.

[0034] (4) Stop solution: 2 M sulfuric acid.

[0035] 96-well plate preparation: On the first day, dilute the original (traditional Twist1 protein) to 1 μg / ml with antigen dilution buffer, then add 100 μL of antigen (traditional Twist1 protein) to each well, seal and incubate at 4°C overnight. On the second day, discard the supernatant. Add 200 μL of blocking buffer to each well and incubate at 37°C for 30 minutes for later use. Discard the supernatant before use.

[0036] ELISA titration (1) According to the 10-fold gradient dilution method, the serum from the recombinant Twist1 protein immunization group, the serum from the traditional Twist1 protein immunization group, and the Twist1 mouse negative serum were diluted 10 6 times, prepare 10 2 Up to 10 6 Five dilutions of samples were prepared. 100 μL of the samples were added to appropriate wells, sealed, and incubated at 37°C for 40 min.

[0037] (2) Discard the supernatant and wash five times with wash buffer.

[0038] (3) Add 100 μL of HRP-labeled goat anti-mouse secondary antibody (1:10,000 dilution) to each well and incubate at 37°C for 30 min.

[0039] (4) Discard the supernatant and wash five times with wash buffer.

[0040] (5) Add 100 μL of TMB (mixture of Solution A and Solution B) one-step substrate reagent to each well and incubate at room temperature in a dark room for 5 min with gentle shaking.

[0041] (6) Add 50 μL of stop solution to each well and read the sample immediately at 450 nm.

[0042] (7) Principles for calculating serum positive titer: At the same dilution factor, a sample OD450 value ≥ 2.1 times the negative control (Twist1 mouse negative serum) is considered positive, and the corresponding serum dilution factor is used as the antibody titer. If the negative control absorbance value is < 0.05, it is calculated as 0.05. The ELISA test results are shown in Table 1.

[0043] Table 1 Serum titer

[0044] From the data in the table, we can see that the titer of the recombinant Twist1 protein immunization group is 10 6 The traditional Twist1 protein immunization group titer is 10 5 .

[0045] Example 3 ELASA experiment with rabbit serum as the source to verify the sensitivity and specificity of recombinant Twist1 protein Material: (1) Antigen: recombinant Twist1 protein and Twist1 protein.

[0046] (2) Serum: Twist1 rabbit tetraimmune serum and Twist1 rabbit negative serum (3) Antibody: goat anti-rabbit-HRP Buffer preparation: (1) Antigen dilution buffer: 0.01 M PBS, pH 7.2.

[0047] (2) Elution buffer: 0.01 M PBS, pH 7.2, containing 0.05% Tween 20.

[0048] (3) Blocking solution: 0.01 M PBS, pH 7.2, containing 5% fetal bovine serum.

[0049] (4) Stop solution: 2 M sulfuric acid.

[0050] Experimental preparation: The ELISA plate was divided into recombinant Twist1 protein group, Twist1 protein and blank control group.

[0051] (1) On the first day, dilute the recombinant Twist1 protein and Twist1 protein to 1 μg / ml with antigen dilution buffer, and add 100 μL of antigen to each well of each group. After sealing, incubate at 4°C overnight. On the second day, discard the supernatant. Add 200 μL of blocking buffer to each well and incubate at 37°C for 30 minutes for use. Discard the supernatant before use. Only antigen dilution buffer was added to the wells of the blank control group, and no antigen was coated.

[0052] (2) This step divides the samples into five groups, namely, recombinant Twist1 protein-positive group, recombinant Twist1 protein-negative group, Twist1 protein-positive group, Twist1 protein-negative group and blank group.

[0053] According to the 10-fold serial dilution method, Twist1 rabbit tetraimmune serum and Twist1 rabbit negative serum were diluted 10 6 times, prepare 10 2 Up to 10 6 Five dilutions of samples were prepared for use. 100 μL of five dilutions of Twist1 rabbit tetra-rat serum and Twist1 rabbit negative serum samples were added to the recombinant Twist1 protein and Twist1 protein wells, respectively, and incubated at 37°C for 40 min after sealing. The blank control group was also added with samples of the same dilution.

[0054] Among them, the recombinant Twist1 protein-positive group was a group in which five dilutions of Twist1 rabbit tetrasera were added to the recombinant Twist1 protein wells, and then sealed and incubated at 37°C for 40 minutes.

[0055] The recombinant Twist1 protein-negative group was composed of five dilutions of Twist1 rabbit negative serum samples added to the recombinant Twist1 protein wells, which were sealed and incubated at 37°C for 40 minutes.

[0056] The Twist1 protein-positive group consisted of four rabbit serums of Twist1 at five dilutions added to the Twist1 protein wells, which were sealed and incubated at 37°C for 40 min.

[0057] The Twist1 protein-negative group consisted of five dilutions of Twist1 rabbit negative serum samples added to the Twist1 protein wells, which were sealed and incubated at 37°C for 40 min.

[0058] (3) Discard the supernatant and wash five times with wash buffer.

[0059] (4) Add 100 μL of HRP-labeled goat anti-rabbit secondary antibody (1:10,000 dilution) to each well and incubate at 37°C for 30 min. The same secondary antibody was also added to the blank control group.

[0060] (4) Discard the supernatant and wash five times with wash buffer.

[0061] (5) Add 100 μL of TMB (mixture of Solution A and Solution B) one-step substrate reagent to each well and incubate at room temperature in a dark room for 5 min with gentle shaking.

[0062] (6) Add 50 μL of stop solution to each well and use a microplate reader to quickly read the absorbance (OD) value of each well at a wavelength of 450 nm. The results are shown in Table 2.

[0063] Table 2 OD values ​​of each group at different dilutions in the ELASA experiment with rabbit serum

[0064] Sensitivity analysis: Draw five groups of curves showing the change of absorbance value (OD) with antibody dilution, such as Figure 5 shown.

[0065] Comparing the curves of the absorbance value (OD) of the recombinant Twist1 protein-positive group and the Twist1 protein-positive group as a function of antibody dilution, the curve of the recombinant Twist1 protein-positive group is above the Twist1 protein-positive group. It can be seen that the sensitivity of the recombinant Twist1 protein is higher than that of the Twist1 protein.

[0066] The absorbance value of the highest dilution group in the recombinant Twist1 protein-positive group was 0.18, which was still significantly higher than the absorbance value of 0.02 in the highest dilution group in the recombinant Twist1 protein-negative group. It can be seen that the recombinant Twist1 protein is more sensitive in detecting Twist1 protein antibodies.

[0067] Specificity analysis: The absorbance value of the recombinant Twist1 protein-negative group was similar to that of the blank group, which means that no nonspecific color reaction would occur. The absorbance of the Twist1 protein-negative group was slightly higher than that of the blank group and the recombinant Twist1 protein-negative group, indicating that the recombinant Twist1 protein has higher specificity because it is less likely to produce nonspecific binding.

[0068] Example 4 ELASA experiments using mouse serum as the source validate the sensitivity and specificity of recombinant Twist1 protein In this example, the serum used was Twist1 mouse tetraimmune serum and Twist1 mouse negative serum; the antibody used was goat anti-mouse-HRP.

[0069] The remaining experimental steps and parameters were consistent with those in Example 2. The absorbance (OD) values ​​of each group are shown in Table 3.

[0070] Table 3 OD values ​​of each group at different dilutions in the ELASA experiment with serum from mice

[0071] Sensitivity analysis: Draw five groups of curves showing the change of absorbance value (OD) with antibody dilution, such as Figure 6 shown.

[0072] Comparing the curves of the absorbance value (OD) of the recombinant Twist1 protein-positive group and the Twist1 protein-positive group as a function of antibody dilution, the curve of the recombinant Twist1 protein-positive group is above the Twist1 protein-positive group. It can be seen that the sensitivity of the recombinant Twist1 protein is higher than that of the Twist1 protein.

[0073] The absorbance value of the highest dilution group of the recombinant Twist1 protein-positive group was 0.21, which was still significantly higher than the absorbance value of 0.022 of the highest dilution group of the recombinant Twist1 protein-negative group. It can be seen that the recombinant Twist1 protein is more sensitive in detecting Twist1 protein antibodies.

[0074] Specificity analysis: The absorbance value of the recombinant Twist1 protein-negative group was similar to that of the blank group, which means that no nonspecific color reaction would occur. The absorbance of the Twist1 protein-negative group was slightly higher than that of the blank group and the recombinant Twist1 protein-negative group, indicating that the recombinant Twist1 protein has higher specificity because it is less likely to produce nonspecific binding.

[0075] Example 5 ELASA experiment using sheep serum to verify the sensitivity and specificity of recombinant Twist1 protein In this example, the serum used was Twist1 sheep tetraimmune serum and Twist1 sheep negative serum; the antibody used was rabbit anti-sheep-HRP.

[0076] The remaining experimental steps and parameters were consistent with those in Example 2. The absorbance (OD) values ​​of each group are shown in Table 4.

[0077] Table 4 OD values ​​of each group at different dilutions in the ELASA experiment with sheep serum

[0078] Sensitivity analysis: Draw five groups of curves showing the change of absorbance value (OD) with antibody dilution, such as Figure 7 shown.

[0079] Comparing the curves of the absorbance value (OD) of the recombinant Twist1 protein-positive group and the Twist1 protein-positive group as a function of antibody dilution, the curve of the recombinant Twist1 protein-positive group is above the Twist1 protein-positive group. It can be seen that the sensitivity of the recombinant Twist1 protein is higher than that of the Twist1 protein.

[0080] The absorbance value of the highest dilution group in the recombinant Twist1 protein-positive group was 0.25, which was still significantly higher than the absorbance value of 0.02 in the highest dilution group in the recombinant Twist1 protein-negative group. It can be seen that the recombinant Twist1 protein is more sensitive in detecting Twist1 protein antibodies.

[0081] Specificity analysis: The absorbance value of the recombinant Twist1 protein-negative group was similar to that of the blank group, which means that no nonspecific color reaction would occur. The absorbance of the Twist1 protein-negative group was slightly higher than that of the blank group and the recombinant Twist1 protein-negative group, indicating that the recombinant Twist1 protein has higher specificity because it is less likely to produce nonspecific binding.

Claims

1. A recombinant Twist1 protein, characterized in that: The amino acid sequence of the recombinant Twist1 protein is shown in SEQ ID No.1; the nucleotide sequence of the recombinant Twist1 protein is shown in SEQ ID No.

2.

2. A method for preparing a recombinant Twist1 protein, characterized in that: The following steps are involved: S1 clone the coding gene shown in SEQ ID No.2 into a vector to construct a recombinant expression plasmid; S2 transforms the obtained recombinant expression plasmid into competent cells, and then obtains the recombinant antigen protein through culture and purification.

3. The method for preparing the recombinant Twist1 protein according to claim 2, characterized in that: The vector is a pET28a vector, and the competent cell is Escherichia coli.

4. Use of the recombinant Twist1 protein according to claim 1 in preparing a detection kit for Twist1 protein antibodies.

5. Use of the recombinant Twist1 protein according to claim 1 in the preparation of a keloid vaccine.

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