GAD-IA2 modified fusion protein as well as construction method and application thereof

Abstract

The invention provides a GAD-IA2 modified fusion protein as well as a construction method and application thereof. The amino acid sequence of the GAD-IA2 modified fusion protein is as shown in SEQ ID No.5. GAD65 is modified, a part of N-terminal amino acid sequence 1-101 of GAD67 is used for replacing an amino acid sequence with relatively strong hydrophobicity at the N terminal of GAD65, the whole-gene synthesis is carried out on the whole-gene synthesis and an IA2 sequence, a recombinant gene is expressed in mammalian cells, and the fusion protein of GAD and IA2 can be prepared only through one-time expression, and the recombinant protein is easy to purify and has a good application prospect. The method can save time and reduce cost; when the protein is applied to T1DM detection, the detection sensitivity and specificity can be improved, missing detection is reduced, and the requirements of clinical reagent performance and patient cost reduction are better met.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a GAD-IA2 modified fusion protein, a construction method thereof and an application thereof. Background Art

[0002] Type 1 diabetes mellitus (T1DM) is caused by factors such as autoimmune deficiency or heredity, resulting in the depletion of pancreatic islet β cells in the body. Characterized by absolute insulin deficiency, its pathogenesis involves complex interactions between β cells and the innate immune system and the adaptive immune system. T1DM has a young onset age, a sudden onset, obvious symptoms such as polyphagia, polyuria, polydipsia and weight loss, a high blood sugar level, and many patients have ketoacidosis as the first symptom. The cellular immunity and humoral immunity of the patients are both abnormal.

[0003] Islet autoantibodies are markers of immune destruction of pancreatic islet β cells and are key indicators for diagnosing autoimmune T1DM. They are used for the differentiation between type 1 diabetes and type 2 diabetes, mainly including insulin antibody (IAA), islet cell antibody (ICA), protein tyrosine phosphatase antibody (IA-2A), zinc transporter 8 and glutamate decarboxylase antibody (GADA), etc. Among them, insulin treatment often causes patients to produce insulin antibody (IA), and the currently commonly used detection methods cannot distinguish IA from IAA. Therefore, the application of IAA in diabetes typing is limited to patients who have not used insulin or have used insulin for less than 2 weeks.

[0004] Among the currently known islet autoantibodies, the sensitivities of each index vary greatly. Among them, GADA has the highest sensitivity and specificity and is also a commonly used index in the laboratory, but its sensitivity is only about 80%. Summary of the Invention

[0005] The present invention aims to provide a GAD-IA2 modified fusion protein, a construction method thereof and an application thereof. When this protein is applied to a detection reagent, it can improve the sensitivity and specificity of the reagent and reduce missed detections.

[0006] The technical solution adopted by the present invention to achieve the above object is as follows:

[0007] The present invention provides a GAD-IA2 modified fusion protein, and its amino acid sequence is as shown in SEQ ID No.5.

[0008] The present invention also provides a gene encoding the above-mentioned GAD-IA2 modified fusion protein, and the nucleotide sequence of the gene is as shown in SEQ ID No.4.

[0009] The present invention also provides a T1DM detection reagent, which includes the above-mentioned GAD-IA2 modified fusion protein.

[0010] The present invention also provides a method for constructing a GAD-IA2 modified fusion protein, including:

[0011] S1, synthesizing the full gene sequence of the target gene: sequentially ligating the nucleotide sequences of the required GAD67 (aa 1-101), GAD65 (aa 95-585), and IA2 (aa 604-979) protein segments to obtain the target gene;

[0012] S2, constructing a recombinant plasmid: constructing the target gene obtained in step S1 into an expression vector to obtain a recombinant plasmid;

[0013] S3, expressing the fusion protein: expressing the recombinant plasmid obtained in step S2 in mammals to obtain the GAD-IA2 modified fusion protein.

[0014] Preferably, step S1 includes: based on the nucleotide sequences of different protein segments, directly splicing the GAD67 and GAD65 nucleotides without adding a linker gene, removing the terminator at the backend of GAD65, adding a Linker sequence between GAD65 and IA2, adding a eukaryotic KOZAK sequence before the target gene, adding a restriction endonuclease site Bam I at the upstream, adding an EcoR I restriction site at the downstream, and adding a 6*His tag sequence at the C-terminus. The sorting of each sequence is: restriction site Bam I - eukaryotic KOZAK sequence - GAD67 - GAD65 - Linker - IA2 - 6*His - restriction site EcoR I.

[0015] More preferably, the Linker sequence adopts a flexible peptide gene sequence (GGGGS) of 5 amino acid residues.

[0016] Preferably, the amino acid sequence of the GAD67 protein segment is as shown in SEQ ID No.1, the amino acid sequence of the GAD65 protein segment is as shown in SEQ ID No.2, and the amino acid sequence of the IA2 protein segment is as shown in SEQ ID No.3.

[0017] The present invention also provides the application of the above-mentioned GAD-IA2 modified fusion protein in the preparation of a T1DM detection reagent.

[0018] The present invention modifies GAD65, replaces the relatively hydrophobic amino acid sequence at the N-terminus of GAD65 with a part of the N-terminal amino acid sequence 1-101 of GAD67, and performs whole gene synthesis with the IA2 sequence. The recombinant gene is expressed in mammalian cells, and only one expression is required to prepare the fusion protein of GAD and IA2. This recombinant protein is easy to purify. The present invention can save time and reduce costs; applying this protein to T1DM detection can improve the sensitivity and specificity of detection, reduce missed detections, and better meet the clinical requirements for reagent performance and reducing patient costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the identification result of the recombinant plasmid construction in the embodiment of the present invention.

[0020] Figure 2 This is the electrophoresis result of the fusion protein in the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] To more clearly illustrate the present invention, the present invention will be further described in detail below in conjunction with embodiments and drawings. Those skilled in the art should understand that the specific content described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0022] Embodiment

[0023] I. Synthesis of the full gene sequence of the target gene

[0024] The nucleotide sequences of the required GAD67, GAD65, and IA2 protein segments are sequentially ligated to obtain the target gene. The terminator is removed from the back end of GAD65, a Linker sequence GGGGS is added between GAD65 and IA2, a eukaryotic KOZAK sequence is added in front of the target gene, a restriction endonuclease site Bam I is added upstream, an EcoR I restriction site is added downstream, and a 6*His tag sequence is added at the C-terminus, and the full gene is synthesized. The order of each sequence is: restriction endonuclease site Bam I - eukaryotic KOZAK sequence - GAD67 - GAD65 - Linker - IA2 - 6*His - restriction endonuclease site EcoR I.

[0025] The GAD67 protein sequence (Gene ID: 24379) is selected as follows:

[0026] MASSTPSSSATSSNAGADPNTTNLRPTTYDTWCGVAHGCTRKLGLKICGFLQRTNSL EEKSRLVSAFKERQSSKNLLSCENSDRDARFRRTETDFSNLFARDLLPAKNGEE(SEQ IDNo.1);

[0027] The GAD65 protein sequence (Gene ID: 2572) is selected as follows:

[0028] DLLPACDGERPTLAFLQDVMNILLQYVVKSFDRSTKVIDFHYPNELLQEYNWELADQPQNLEEILMHCQTTLKYAIKTGHPRYFNQLSTGLDMVGLAADWLTSTANTNMFTYEIAPVFVLLEYVTLKKMREIIGWPGGSGDGIFSPGGAISNMYAMMIARFKMFPEVKEKGMAALPRLIAFTSEHSHFSLKKGAAALGIGTDSVILIKCDERGKMIPSDLERRILEAKQKGFVPFLVSATAGTTVYGAFDPLLAVADICKKYKIWMHVDAAWGGGLLMSRKHKWKLSGVERANSVTWNPHKMMGVPLQCSALLVREEGLMQNCNQMHASYLFQQDKHYDLSYDTGDKALQCGRHVDVFKLWLMWRAKGTTGFEAHVDKCLELAEYLYNIIKNREGYEMVFDGKPQHTNVCFWYIPPSLRTLEDNEERMSRLSKVAPVIKARMMEYGTTMVSYQPLGDKVNFFRMVISNPAATHQDIDFLIEEIERLGQDL(SEQ ID No.2);

[0029] The IA2 protein sequence (Gene ID: 5798) is selected as follows:

[0030] ARQQDKERLAALGPEGAHGDTTFEYQDLCRQHMATKSLFNRAEGPPEPSRVSSVSSQFSDAAQASPSSHSSTPSWCEEPAQANMDISTGHMILAYMEDHLRNRDRLAKEWQALCAYQAEPNTCATAQGEGNIKKNRHPDFLPYDHARIKLKVESSPSRSDYINASPIIEHDPRMPAYIATQGPLSHTIADFWQMVWESGCTVIVMLTPLVEDGVKQCDRYWPDEGASLYHVYEVNLVSEHIWCEDFLVRSFYLKNVQTQETRTLTQFHFLSWPAEGTPASTRPLLDFRRKVNKCYRGRSCPIIVHCSDGAGRTGTYILIDMVLNRMAKGVKEIDIAATLEHVRDQRPGLVRSKDQFEFALTAVAEEVNAILKALPQ(SEQ ID No.3).

[0031] The full gene sequence of the target gene is shown in SEQ ID No.4.

[0032] II. Construction and Identification of Recombinant Plasmid

[0033] The full gene sequence obtained in the first step and the plasmid vector pcDNA3.1 were digested with Bam I and EcoR I, and the digested products were recovered using a kit and then ligated with T4 ligase.

[0034] The ligation product was transformed into Escherichia coli competent cells DH5α, with the volume not exceeding 10% of the competent cells. Among them, 100 μL of competent cells and 10 μL of ligation product were taken, mixed well by pipetting, placed on ice for 30 min, then subjected to a water bath at 42 °C for heat shock for 90 s, quickly transferred to a water bath, and placed for 2 min. 500 μL of LB medium was added to each tube, and the bacteria were gently incubated on a shaker at 37 °C for 1 h to allow the bacteria to recover. Then, centrifuged at 4000 rpm for 3 min, 500 μL of supernatant was removed, and 100 μL of medium was retained in the centrifuge tube.

[0035] 30 μL of the medium was taken and evenly spread on a resistant plate, and the plate was inverted and cultured overnight at 37 °C to obtain transformants; single colonies were picked and inoculated into 4 mL of LB resistant liquid medium and cultured overnight at 37 °C. The bacterial solution was taken for sequencing identification, and the result was consistent with the target gene; the plasmid was extracted using a plasmid extraction kit.

[0036] The identification result is as Figure 1 shown.

[0037] III. Expression and Identification of Fusion Protein

[0038] The recombinant plasmid obtained in the second step was transformed into DH10 Bac competent cells, with the volume not exceeding 10% of the competent cells. For every 100 μL of competent cells, 5 μL of recombinant plasmid was taken, mixed well by pipetting, placed on ice for 30 min, then subjected to a water bath at 42 °C for heat shock for 60 s, quickly transferred to a water bath, and placed for 2 min. 500 μL of LB medium was added to each tube, and the bacteria were gently incubated on a shaker at 37 °C for 4 h to allow the bacteria to recover;

[0039] 30 μL of the above bacterial solution and 30 μL of negative control were diluted 1:10 and 1:100 times, evenly spread on a resistant LB plate, and the plate was inverted and placed in a constant temperature incubator at 37 °C. Blue-white colonies would appear after about 48 h; colonies were picked and inoculated into 5 mL of resistant LB liquid medium and cultured overnight at 37 °C; the plasmid was extracted and subjected to PCR identification, showing that the recombinant target gene was correct;

[0040] Mammalian cell transfection: 24 h before transfection, 3*10 5Seed HEK293T cells at [X] cells / mL into a 6-well plate, with 2 mL of serum-free medium per well. Culture the cells in a 5% CO₂ incubator at 37°C. Mix Lipofectamine 2000, opti-MEM medium, and plasmid in a certain ratio. After incubating the mixture for 5 - 20 minutes, add it dropwise to the cells in the 6-well plate and place it in a 5% CO₂ incubator at 37°C for 48 - 72 hours. Then replace the medium with DMEM containing G418. After 3 - 5 days, transfer the cell clones to a 24-well plate and screen for monoclonal cells that highly express the protein. TM Mix the medium, plasmid in a certain ratio, incubate the mixture for 5 - 20 min, then add it dropwise to the cells in the 6-well plate, place it in a 5% CO₂ incubator, and culture at 37°C for 48 - 72 h. Replace it with DMEM medium containing G418. After 3 - 5 days, transfer the cell clones to a 24-well plate and screen for monoclonal cells that highly express the protein.

[0041] IV. Purification of the fusion protein

[0042] Cultivate the screened cells for large-scale expression; centrifuge at 2500 rpm for 3 - 5 minutes, collect the cells, wash the cells twice with 1×PBS buffer, and collect the cells; add 5 - 10 times the cell volume of lysis buffer (25 mM Tris-HCl, 300 mM NaCl, pH 8.0, 1% NP-40), incubate on ice for 30 minutes, centrifuge at 15000 rpm for 20 - 30 minutes, and collect the supernatant.

[0043] Incubate the supernatant with Ni-NTA RESIN pre-equilibrated with 5 column volumes at 2 - 8°C with rotation for 16 - 20 hours; discard the supernatant and transfer the packing material to a chromatography column.

[0044] Wash with loading buffer (25 mM Tris-HCl, 300 mM NaCl, pH 8.0) for 10 column volumes.

[0045] Wash with washing buffer 1 (25 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) for 10 column volumes; wash with washing buffer 2 (25 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH 8.0) for 5 column volumes; collect the protein, and store it at -80°C after replacing the buffer.

[0046] Detect the purity and concentration of the protein by SDS-PAGE electrophoresis. The electrophoresis results are shown in Figure 2 .

[0047] The amino acid sequence of the fusion protein is shown in SEQ ID No.5.

[0048] V. Application of the fusion protein to the detection of clinical samples

[0049] Link the obtained fusion protein to carboxyl magnetic beads.

[0050] The reaction was carried out by indirect immunoassay. M was a fusion protein solution containing 0.5 μg / mL. R1 was a solution of biotinylated GAD and IA2 protein complex. R2 was a solution of alkaline phosphatase-labeled streptavidin. The substrate was AMPPD.

[0051] 115 clinically diagnosed type 1 diabetes patient samples and 130 apparently healthy samples were selected for determination.

[0052] The reaction process of the chemiluminescence analyzer: Add 50 μL of the sample, 50 μL of reagent M, and 50 μL of reagent R1, react for 10 min, and wash 4 times; then add 100 μL of reagent R2 and react for 10 min, wash 4 times; add 200 μL of the substrate and react for 5 min, and then detect.

[0053] The test results are as follows: The sensitivity of the kit of the present invention is 93.9%, and the specificity is 100%; when using a commercially available single-item kit for detection, the sensitivity is 80.9% and 21.7%. The comparison results show that the sensitivity of this kit is higher than that of the kits for single detection items.

[0054]

[0055] Obviously, the above embodiments of the present invention are merely examples for more clearly illustrating the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. Any obvious changes or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A GAD-IA2 modified fusion protein, whose amino acid sequence is shown in SEQ ID No.

5.

2. A gene encoding the GAD-IA2 modified fusion protein according to claim 1, and the nucleotide sequence of said gene is shown in SEQ ID No.

4.

3. A T1DM detection reagent, comprising the GAD-IA2 modified fusion protein according to claim 1.

4. A method for constructing a GAD-IA2 modified fusion protein, comprising: S1, synthesizing the full gene sequence of the target gene: sequentially connecting the nucleotide sequences of the required GAD67, GAD65, and IA2 protein segments to obtain the target gene. For GAD67, select the aa 1-101 protein segment; for GAD65, select the aa 95-585 protein segment; for IA2, select the aa 604-979 protein segment; S2, constructing a recombinant plasmid: constructing the target gene obtained in step S1 into an expression vector to obtain a recombinant plasmid; S3, expressing the fusion protein: expressing the recombinant plasmid obtained in step S2 in mammals to obtain the GAD-IA2 modified fusion protein.

5. The construction method according to claim 4, characterized in that, Step S1 includes: based on the nucleotide sequences of different protein segments, directly splicing GAD67 and GAD65 without adding a linker gene, removing the terminator at the back end of GAD65, adding a Linker sequence between GAD65 and IA2, adding a eukaryotic KOZAK sequence in front of the target gene, adding a restriction endonuclease site Bam I upstream, adding an EcoR I cleavage site downstream, and adding a 6*His tag sequence at the C-terminus. The order of each sequence is: restriction endonuclease site Bam I - eukaryotic KOZAK sequence -GAD67-GAD65-Linker-IA2-6*His - restriction endonuclease site EcoR I.

6. The construction method according to claim 5, characterized in that, The Linker sequence uses a flexible peptide gene sequence GGGGS of 5 amino acid residues.

7. The construction method according to any one of claims 4 to 6, characterized in that The amino acid sequence of the GAD67 protein segment is shown in SEQ ID No. 1, the amino acid sequence of the GAD65 protein segment is shown in SEQ ID No. 2, and the amino acid sequence of the IA2 protein segment is shown in SEQ ID No.

3.

8. Use of the GAD-IA2 modified fusion protein according to claim 1 in the preparation of a T1DM detection reagent.

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