Truncated body of recombinant human glutamate decarboxylase 65 and application thereof

Abstract

The invention relates to the technical field of molecular biology, in particular to a truncated body of recombinant human glutamate decarboxylase 65 and application of the truncated body. According to the invention, a main immunogenic fragment of GAD65 is screened and retained, and a truncated body of the recombinant human glutamate decarboxylase 65 is constructed. The truncation provided by the invention can be efficiently expressed in insect cells, and a stable antigen can be obtained after an expression product is purified and prepared and can be used for glutamate decarboxylase antibody detection.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a truncated form of recombinant human glutamate decarboxylase 65 and an application thereof. Background Art

[0002] Glutamate decarboxylase (GAD) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that is widely present in organisms. It undergoes structural changes in an acidic environment and irreversibly catalyzes the α-decarboxylation of L-glutamate or glutamate to produce γ-aminobutyric acid (GABA).

[0003] In 1991, human pancreatic islet GAD65 was cloned and sequenced, and it was found that GAD has two isoforms, namely GAD65 and GAD67. The GAD65 gene is located on chromosome 10p11, while GAD67 is located on chromosome 2q31, confirming that GAD65 and GAD67 are derived from single isolated genes. GAD67 is mainly distributed in the cytoplasm of neurons, and the GABA synthesized by it does not play the role of a neurotransmitter; while GAD65 is mainly concentrated in the preganglionic nerve terminals, catalyzing the production of inhibitory neurotransmitter GABA. Only GAD65 is expressed in human pancreatic islets, and mainly in B cells. Therefore, GAD65 is closely related to human diabetes. In recent years, GAD antibodies have received more and more attention and have become a hot topic in diabetes research.

[0004] Both GAD isoforms form functional dimers. According to the primary structure sequence of the GAD gene, the GAD monomer consists of three parts: the C-terminal domain, the 5'-phosphate pyridoxal binding region, and the N-terminal domain. The N-terminal domain has two parallel α-helices, corresponding to the 5'-phosphate pyridoxal binding domain of itself and the other monomer; the 5'-phosphate pyridoxal binding region adopts a type 1 5'-phosphate pyridoxal domain containing three α-helices and a 2-layer antiparallel β-pleated sheet. The two catalytic active centers of the GAD dimer are located in the center of the 5'-phosphate pyridoxal binding domain, on the inner surface of the dimer. The active site of GAD67 is covered by an extended catalytic loop, while in GAD65, its catalytic loop is highly mobile, allowing the auxiliary factor 5'-phosphate pyridoxal to enter and exit, controlling the enzyme activity. The active sites of both isozymes have a lysine residue that contacts PLP (Lys405 in GAD67; Lys396 in GAD65) and are covered by a catalytic loop.

[0005] The highly sensitive GAD antibody test can screen out as many people at high risk of type 1 diabetes as possible from the normal population. Then, the ICA test can increase the specificity of the screening.

[0006] GAD65 is composed of 585 amino acids and has a molecular weight of 65kDa. The N-terminus of its sequence is hydrophobic, and the protein is unstable and difficult to express and purify. Summary of the invention

[0007] In view of this, the present invention provides a truncated form of recombinant human glutamate decarboxylase 65 and its application. The present invention provides a truncated form of recombinant human glutamate decarboxylase 65, a biological material, and its application in the preparation of a GAD65 antibody detection kit. The present invention screens and retains the main immunogenic fragments of GAD65 to construct the truncated form of recombinant human glutamate decarboxylase 65. The truncated form provided by the present invention can be efficiently expressed in insect cells, and the expression product is purified to obtain a stable antigen, which can be used for glutamate decarboxylase antibody detection.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a truncated form of recombinant human glutamate decarboxylase 65, comprising any of the following:

[0010] (I) a truncated sequence having the amino acid sequence as shown in SEQ ID NO: 2 with the 1st to (52-154) amino acids replaced or deleted; or

[0011] (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0012] (III) A sequence having a homology of 90% or more to the amino acid sequence shown in (I) or (II).

[0013] The present invention provides a truncated form of recombinant human glutamate decarboxylase 65, comprising any of the following:

[0014] (I) a truncated sequence having the amino acid sequence as shown in SEQ ID NO: 2 but lacking the 1st to (52nd to 154th) amino acids; or

[0015] (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0016] (III) A sequence having a homology of 90% or more to the amino acid sequence shown in (I) or (II).

[0017] In some specific embodiments of the present invention, the truncated body is connected to a tag for detection or purification.

[0018] In some specific embodiments of the present invention, the truncation comprises a truncated sequence having an amino acid sequence as shown in SEQ ID NO: 2 with the 1st to (71st to 154th) amino acids replaced or deleted.

[0019] In some specific embodiments of the present invention, the truncated form includes a truncated sequence having an amino acid sequence as shown in SEQ ID NO: 2 with amino acids 1 to 52, 1 to 71, 1 to 92, 1 to 135 or 1 to 154 replaced or deleted.

[0020] Preferably, the truncation comprises a truncated sequence having an amino acid sequence as shown in SEQ ID NO: 2 with amino acids 1 to 71 replaced or deleted.

[0021] In some specific embodiments of the present invention, the tag comprises a 10*his tag.

[0022] The present invention also provides a biomaterial comprising any of the following:

[0023] (i) a nucleic acid molecule encoding the truncation; or

[0024] (ii) an expression vector comprising the nucleic acid molecule described in (i); or

[0025] (iii) a virus comprising the expression vector described in (ii); or

[0026] (iv), transducing (iii) the virus, transforming or transfecting (ii) the expression vector, or a host cell having the nucleic acid molecule (i) integrated into its genome.

[0027] In some specific embodiments of the present invention, the nucleic acid molecule has a nucleotide sequence as shown in any of SEQ ID NOs: 3 to 7.

[0028] Preferably, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:4.

[0029] In some specific embodiments of the present invention, the expression vector comprises a pFastBac1 backbone vector.

[0030] In some embodiments of the invention, the virus comprises a baculovirus.

[0031] In some embodiments of the invention, the host comprises an insect cell.

[0032] In some specific embodiments of the present invention, the insect cells include sf9 or High Five cell lines.

[0033] The present invention also provides a culture product obtained after culturing the host cells in the biological material.

[0034] In some specific embodiments of the present invention, the method for preparing the truncated body comprises: culturing the host cell, breaking it, and extracting and purifying it to obtain the truncated body.

[0035] In some specific embodiments of the present invention, the disruption comprises disrupting the host cells by high-pressure homogenization; and the extraction and purification comprises extracting and purifying by Ni metal chelating chromatography and DEAE ion exchange chromatography.

[0036] The present invention also provides the use of any of the following items in the preparation of a GAD65 antibody detection kit:

[0037] (A), the truncated body; and / or

[0038] (B), the biological material; and / or

[0039] (C) the culture product.

[0040] The present invention also provides a GAD65 antibody detection kit, comprising any of the following items:

[0041] (A), the truncated body; and / or

[0042] (B), the biological material; and / or

[0043] (C) the culture product.

[0044] The present invention provides the following beneficial effects:

[0045] The full-length GAD65 protein is only expressed in trace amounts in insect cells. The present invention screens and retains the main immunogenic fragments of GAD65 to construct the truncated form of the recombinant human glutamate decarboxylase 65. The truncated form provided by the present invention can be efficiently expressed in insect cells, and the expression product is purified to obtain a stable antigen, which can be used for glutamate decarboxylase antibody detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0047] Figure 1 The figure shows the SDS-PAGE detection result of the human glutamate decarboxylase 65 recombinant protein implemented in the present invention. DETAILED DESCRIPTION

[0048] The present invention discloses a truncated form of recombinant human glutamate decarboxylase 65 and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0049] In view of this, the technical problem to be solved by the present invention is to provide a GAD65 recombinant protein efficiently expressed in insect cells and its application.

[0050] The present invention reduces the effects of unstable factors on the stable and efficient expression of proteins by modifying the GAD65 sequence. The glutamate decarboxylase 65 recombinant protein obtained by the present invention can be used for antibody detection in clinical serum samples.

[0051] The human GAD65 recombinant protein fragment of the present invention is recombinantly constructed on the basis of human GAD65, and the hydrophobic helices and disordered regions of different lengths at the N-terminus are removed to retain the highly immunogenic extracellular region fragment.

[0052] The present invention also provides an expression vector comprising the polynucleotide of the present invention. That is, the present invention provides an expression vector encoding the recombinant protein of the present invention. In some embodiments, the backbone vector of the expression vector is selected from the pFastBac1 vector.

[0053] The virus vector of the present invention is a baculovirus, and the cell strain containing the polynucleotide or the expression vector adopts an insect cell that can be transfected with a baculovirus and express a protein.

[0054] In the present invention, the host cell is a eukaryotic insect cell, and the expression vector containing the polynucleotide is expressed in an insect cell. In some embodiments, the host insect cell is a sf9 or High Five cell line.

[0055] The present invention also provides a method for preparing the recombinant protein, comprising: culturing the host cells, transfecting the host cells with baculovirus and expressing the protein.

[0056] In some embodiments, the method for preparing the recombinant protein further comprises the steps of crushing, extracting and purifying the cells after expression. The crushing is performed by high pressure homogenization. The extraction and purification are performed by Ni metal chelate chromatography and DEAE ion exchange chromatography.

[0057] The recombinant protein, the polynucleotide, the expression vector, the cell line, the host cell or the product obtained by the preparation method of the present invention are used in the detection of serum-specific GAD65 antibodies.

[0058] The nucleotide sequence encoding the human GAD65 recombinant protein is shown in SEQ ID NO: 1:

[0059]

[0060] The amino acid sequence of human glutamate decarboxylase 65 recombinant protein is shown in SEQ ID NO: 2:

[0061] MASPGSGFWSFGSEDGSGDSENPGTARAWCQVAQKFTGGIGNKLCALLYGDAEKPAESGGSQPPRAAARKAACACDQKPCSCSKVDVNYAFLHATDLLPACDGERPTLAFLQDVMNILLQYVVKSFDRSTKVIDFHYPNELLQEYN WELADQPQNLEEILMHCQTTLKYAIKTGHPRYFNQLSTGLDMVGLAADWLTSTANTNMFTYEIAPVFVLLEYVTLKKMREIIGWPGGSGDGIFSPGGAISNMYAMMIARFKMFPEVKEKGMAALPRLIAFTSEHSHFSLKKGAAAL GIGTDSVILIKCDERGKMIPSDLERRILEAKQKGFVPFLVSATAGTTVYGAFDPLLAVADICKKYKIWMHVDAAWGGGLLMSRKHKWKLSGVERANSVTWNPKMMGVPLQCSALLVREEGLMQNCNQMHASYLFQQDKHYDLSYD TGDKALQCGRHVDVFKLWLMWRAKGTTGFEAHVDKCLELAEYLYNIIKNREGYEMVFDGKPQHTNVCFWYIPPSLRTLEDNEERMSRLSKVAPVIKARMMEYGTTMVSYQPLGDKVNFFRMVISNPAATHQDIDFLIEEIERLGQDL

[0062] The sequence of nucleotides 53-585 encoding the human GAD65 recombinant protein is shown in SEQ ID NO: 3:

[0063]

[0064] The sequence of nucleotides 72-585 encoding the human GAD65 recombinant protein is shown in SEQ ID NO:4:

[0065]

[0066] The sequence of nucleotides 93-585 encoding the human GAD65 recombinant protein is shown in SEQ ID NO:5:

[0067]

[0068] The sequence of nucleotides 136-585 encoding the human GAD65 recombinant protein is shown in SEQ ID NO:6:

[0069]

[0070] The sequence of nucleotides 155-585 encoding the human GAD65 recombinant protein is shown in SEQ ID NO:7:

[0071]

[0072] The sequence of nucleotides 180-585 encoding the human GAD65 recombinant protein is shown in SEQ ID NO: 8:

[0073]

[0074] The full-length GAD65 protein is only expressed in trace amounts in insect cells. This patent screens and retains the main immunogenic fragments of GAD65, which can be efficiently expressed in the host and can be used for glutamic acid decarboxylase antibody detection.

[0075] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0076] Unless otherwise specified, the truncated form of recombinant human glutamate decarboxylase 65 provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0077] The present invention will be further described below in conjunction with embodiments:

[0078] Example 1 Gene Synthesis

[0079] The recombinant GAD65 protein gene (NCBI accession number: NM_000818) was synthesized by GENEWISE after adding a 10*his tag to the C-terminus and codon optimization (SEQ ID NO: 1).

[0080] Example 2 Preparation of pFastbac-GAD65 plasmid

[0081] Take 1 μg of GAD65 polynucleotide aqueous solution and 2 μg of pFastbac1 plasmid synthesized in Example 1 and add 100 units of BamHI and 100 units of HindⅢ restriction enzymes respectively to react at 37°C for 30 minutes. After the reaction is completed, remove the excess enzymes with a purification kit (purchased from omega, item number D2005-02). Mix the GAD65 and pFastBac1 plasmids after enzyme digestion and add 100 units of T4 ligase. After incubation at 25°C±5°C for 2 hours, remove the excess T4 enzyme with a purification kit. The purified pFastBac-GAD65 construction plasmid was transferred into electrocompetent Escherichia coli DH10BacTM through the electroporator Bio-Rad Gene PulserXcell, added 1 mL of SOC rehabilitation culture medium and cultured at 37°C for 1 hour, and then coated on LB agarose culture plates containing 100 μg / mL ampicillin and 70 μg / mL gentamicin. After overnight culture, positive clones were selected and inoculated into SB culture medium containing 100 μg / mL ampicillin and 70 μg / mL gentamicin. The bacteria were cultured to the logarithmic growth phase, and the pFastBac-GAD65 plasmid was purified using the QIAGEN Maxi Prep plasmid extraction kit.

[0082] Example 3 Expression of human glutamate decarboxylase 65 recombinant protein

[0083] (1) Preparation of sf9 cells:

[0084] Sf9 insect cells were cultured in Grace insect culture medium supplemented with 10% calf serum until the logarithmic growth phase (1.5-2.5×10 6 2 mL of Grace insect culture medium was added to each well of a 6-well culture plate. Without adding antibiotics, 8×10 5 sf9 cells.

[0085] (2) Preparation of Cellfectin II and pFastBac-GAD65 plasmid mixture:

[0086] Add 8 μL Cellfectin II (purchased from Thermo Fisher Scientific) to 100 μL Grace insect culture medium without additives, and dilute 1 μg pFastBac-GAD65 to 100 μL Grace insect culture medium without additives. Mix the diluted Cellfectin II and pFastBac-GAD65 plasmids and mix at 25℃±5℃ for 15~30 minutes.

[0087] (3) Transfect sf9 cells with pFastBac-GAD65 plasmid to prepare human glutamate decarboxylase 65 recombinant protein:

[0088] The mixture of Cellfectin II and pFastBac-GAD65 plasmid obtained in (2) was added dropwise to the sf9 cells cultured in (1) in a 6-well cell culture plate and incubated at 27 degrees for 3 to 5 hours. After removing the transfection mixture, 2 mL of Grace insect culture medium containing 10% fetal bovine serum was added to each culture well, and 100 μg / mL ampicillin and 70 μg / mL gentamicin were added to the culture medium.

[0089] After culturing sf9 cells in culture medium for 72 hours (late infection period) until a large number of cells can be observed floating, the cells were resuspended and centrifuged at 500 g for 5 min to remove cells and debris, the supernatant was aspirated, and the remaining residues were centrifuged again at 5000 x g for 5 minutes to obtain the P1 generation virus.

[0090] The P1 virus was passaged, a 6-well cell culture plate was prepared, 2 x 10^6 sf9 insect cells were added to each well, and incubated at room temperature for 1 hour to allow the cells to adhere to the culture plate.

[0091] Select an appropriate amount of P1 virus and add it to each culture well. The amount of virus added should be controlled between 0.05 and 0.1 as needed. The cells and virus were incubated at 27 degrees Celsius under humid conditions for 48 hours.

[0092] After 48 hours, 2 mL of culture medium was aspirated from each culture well, all the culture medium was mixed and centrifuged at 5000xg for 5 minutes, and the supernatant was collected. The collected virus is the P2 generation, and its virus content must reach 1x10^8 pfu / mL before it can be used to infect cells again and express proteins.

[0093] Select an appropriate amount of P2 virus and add it to each culture bottle. The amount of virus added should be controlled between 0.05 and 0.1 as needed. The cells and virus were incubated at 27 degrees Celsius under humid conditions for 48 hours.

[0094] After 48 hours, all cell culture medium was transferred into a 1000 ml centrifuge cup and centrifuged at 8000 rpm and 4°C for 20 min to collect the culture supernatant containing human glutamic acid decarboxylase 65 recombinant protein.

[0095] Example 4 Screening of Glutamate Decarboxylase 65 Protein Expression Fragments

[0096] The human glutamate decarboxylase 65 recombinant protein fragment is recombinantly constructed on the basis of human glutamate decarboxylase 65, and GAD65 polypeptide sequences of different lengths (sequence 1: 1-585, sequence 2: 53-585, sequence 3: 72-585, sequence 4: 93585, sequence 5: 136-585, sequence 6: 155-585, sequence 7: 180-585) are expressed by the experimental methods in Examples 2 and 3, and fragments with high expression levels are screened for process research, and sequence 3 is preferably expressed.

[0097] Table 1 Expression levels of GAD65 polypeptide sequences of different lengths in sf9 insect cells

[0098]

[0099] Example 5 Purification and preparation of human glutamate decarboxylase 65 recombinant protein

[0100] (1) Bacterial disruption: Take the bacteria expressing human glutamate decarboxylase 65 recombinant protein, centrifuge the cells at 7000 rpm for 20 min, discard the supernatant, collect the cell pellet, resuspend it in buffer (20 mM PBS + 1 mM PMSF + 5 mM EDTA + 0.1% N-acetyl-Cys, pH 7.4; add 250 ml buffer to 2 L of cell supernatant), and disrupt it using a high-pressure homogenizer at 800 bar, and homogenize 3 times.

[0101] (2) Centrifugation: After high-pressure homogenization, the sample is placed in a 500 ml centrifuge cup, balanced and centrifuged at 4°C, 10,000 rpm, 30 min. After centrifugation, the supernatant is immediately poured into a clean beaker.

[0102] (3) Filtration: Use an ultrafiltration cup with a pore size of 0.45 μm and collect the filtrate into another clean beaker.

[0103] (4) Use AKTA pure for Ni metal chelate chromatography. Washing: After loading and rebalancing, use 20 mM PBS + 30 mM imidazole pH 7.4 to wash away impurities. Observe the trend of the UV curve. When the UV absorbance value rises, place the collection tube in a clean beaker to collect the eluate. When the UV value is level, stop collecting and put the waste liquid tube back into the waste liquid bucket.

[0104] (5) Dissociation of target protein: Use 20 mM PBS + 500 mM imidazole pH 7.4 to elute the target protein, observe the changing trend of the UV curve, start collecting samples when the UV value rises, end collection when the UV value is level, and then put the waste liquid tube back into the waste liquid bucket.

[0105] (6) Fluid exchange: Use a dialysis bag with a molecular cutoff of 14 KD to exchange the fluid. The storage buffer is 20 mM PB + 2% glycerol pH 7.4. Dialyze twice, each time for no less than 3 hours.

[0106] (7) Perform DEAE ion exchange chromatography using AKTA pure. Washing: After loading and rebalancing, use 20 mM PB + 50 mM Nacl pH 7.4 to wash away impurities. Observe the trend of the UV curve. When the UV absorbance value rises, place the waste liquid tube in a clean beaker to collect the eluate. When the UV value is level, stop collecting and put the waste liquid tube back into the waste liquid bucket.

[0107] (8) Dissociation of target protein: Use 20 mM PB + 200 mM Nacl pH 7.4 to elute the target protein, observe the change trend of the UV curve, start collecting samples when the UV value rises, end collection when the UV value is level, and then put the waste liquid tube back into the waste liquid bucket.

[0108] (9) Treatment of target protein: Use a dialysis bag with a molecular cutoff of 14 KD to change the liquid, and the storage buffer is 20 mM Hepes + 250 mM NaCl + 0.2% TritonX100 + 0.05 mM 5-pyridoxal phosphate + 20% glycerol, pH 7.5.

[0109] Example 6 Application of human glutamate decarboxylase recombinant protein in serum sample detection

[0110] The antigen expressed by sequence 3 in Example 4 was purified using the method of Example 5, and the positive samples were detected using the double antigen sandwich method. A gradient was detected for the positive samples, and clinical positive and negative samples could be distinguished. The results are shown in Table 2.

[0111] Double antigen sandwich detection steps:

[0112] 1. Add 20μL of the diluted sample to be tested, 50μL of magnetic particles coated with GAD65 antigen (5μg / mL) and 50μL of biotin-labeled antigen to the test tube, mix well and react at 37℃ for 15 minutes. 2. Transfer the test tube to the magnetic test tube rack, let it stand for 3 minutes, tilt it to pour out the supernatant, and gently tap it on absorbent paper. 3. Remove the test tube rack from the magnetic plate, add 500μL of cleaning solution, vortex mix to resuspend the magnetic particles, then place it on the magnetic plate and let it stand for 3 minutes, tilt it to pour out the supernatant, and gently tap it on absorbent paper. Repeat this step twice. 4. Add 135μL of enzyme conjugate, mix well and react at 37℃ for 15 minutes. 5. Repeat steps 2 and 3. 6. Add 200μL of substrate solution to each tube, react at 37℃ for 5 minutes, and then put it into the chemiluminescence analyzer to measure the luminescence intensity.

[0113] Table 2 Human glutamic acid decarboxylase recombinant protein as antigen detection serum samples

[0114]

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A truncated form of recombinant human glutamate decarboxylase 65, characterized in that: Include any of the following: (I) a truncated sequence having the amino acid sequence as shown in SEQ ID NO: 2 with the 1st to (52-154) amino acids replaced or deleted; or (II) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or (III) A sequence having a homology of 90% or more to the amino acid sequence shown in (I) or (II).

2. The truncated body according to claim 1, characterized in that: The truncated body is linked to a tag for detection or purification.

3. Biomaterial, characterized in that Include any of the following: (i) a nucleic acid molecule encoding the truncated form of claim 1 or 2; or (ii) an expression vector comprising the nucleic acid molecule described in (i); or (iii) a virus comprising the expression vector described in (ii); or (iv), transducing (iii) the virus, transforming or transfecting (ii) the expression vector, or a host cell having the nucleic acid molecule (i) integrated into its genome.

4. The biomaterial according to claim 3, characterized in that The nucleic acid molecule has a nucleotide sequence as shown in any of SEQ ID NOs: 3 to 7.

5. The biomaterial according to claim 3 or 4, characterized in that The expression vector includes a pFastBac1 backbone vector.

6. The biomaterial according to any one of claims 3 to 5, characterized in that The viruses include baculoviruses.

7. The biomaterial according to any one of claims 3 to 6, characterized in that The host includes insect cells.

8. A culture product obtained by culturing the host cells in the biomaterial according to any one of claims 3 to 7.

9. Use of any of the following in the preparation of a GAD65 antibody detection kit: (A) A truncated body according to claim 1 or 2; and / or (B) a biomaterial according to any one of claims 3 to 7; and / or (C) The culture product according to claim 8.

10. A GAD65 antibody detection kit, characterized in that: Include any of the following: (A) A truncated body according to claim 1 or 2; and / or (B) a biomaterial according to any one of claims 3 to 7; and / or (C) The culture product according to claim 8.

Images