Truncated recombinant human glutamate decarboxylase 65 and uses thereof

Abstract

The present application relates to the technical field of molecular biology, in particular to a truncated form of recombinant human glutamate decarboxylase 65 and application thereof. The present application screens and retains a main immunogenic fragment of GAD65, and constructs the truncated form of recombinant human glutamate decarboxylase 65. The truncated form provided by the present application can be efficiently expressed in insect cells, and a stable antigen can be obtained after purification and preparation of the expression product, and the stable antigen can be used for glutamate decarboxylase antibody detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to a truncated form of recombinant human glutamate decarboxylase 65 and its application. BACKGROUND

[0002] Glutamate decarboxylase (GAD) is a 5'-phosphopyridoxal (PLP) dependent enzyme, which is widely present in organisms, and in an acidic environment, it irreversibly catalyzes the alpha-decarboxylation of L-glutamic acid or glutamate to generate γ-aminobutyric acid (GABA).

[0003] In 1991, human pancreatic islet GAD65 was cloned and sequenced, and it was found that GAD has two isoforms, GAD65 and GAD67. GAD65 gene is located on chromosome 10p11, and GAD67 is located on chromosome 2q31, confirming that GAD65 and GAD67 are derived from a single isolated gene. GAD67 is mainly distributed in the cytoplasm of neurons, and the GABA catalyzed to be synthesized does not play a role as a neurotransmitter; while GAD65 is mainly concentrated in the pre-nodal nerve endings, catalyzing the generation of inhibitory neurotransmitter GABA. In human pancreatic islets, only GAD65 is expressed, mainly in B cells. Therefore, GAD65 is closely related to human diabetes. In recent years, GAD antibodies have received more and more extensive attention and have become a hot issue in the study of diabetes.

[0004] Both GAD subtypes form functional dimers. According to the primary structure sequence of GAD gene, GAD monomer is composed of three parts: C-terminal domain, 5'-phosphopyridoxal binding region and N-terminal domain. The N-terminal domain has two parallel α-helices corresponding to the 5'-phosphopyridoxal binding domain of itself and another monomer; the 5'-phosphopyridoxal binding region adopts a 1-type 5'-phosphopyridoxal domain containing 3 α-helices and a 2-sheet anti-parallel β-fold. The two catalytic active centers of GAD dimer are located in the center of the 5'-phosphopyridoxal binding domain, the inner surface of the dimer. The active site of GAD67 is covered by an extended catalytic loop, while in GAD65, its catalytic loop has high mobility, allowing the auxiliary factor 5'-phosphopyridoxal to enter and exit, controlling the enzyme activity. The active sites of the two isozymes both have a lysine residue contacting PLP (Lys405 in GAD67; Lys396 in GAD65), and are covered by a catalytic loop.

[0005] The high-sensitivity GAD antibody can be used to screen as many as possible high-risk population of type I diabetes from normal population, and then the ICA determination can be used to increase the specificity of the screening.

[0006] GAD65 is composed of 585 amino acids, and has a molecular weight of 65kDa, the sequence of the N terminal is hydrophobic, and the protein is unstable and difficult to express and purify. SUMMARY

[0007] Therefore, the application provides a truncated form of recombinant human glutamate decarboxylase 65 and application thereof. The application selects and retains a main immunogenic fragment of GAD65 to construct the truncated form of recombinant human glutamate decarboxylase 65.

[0008] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0009] The application provides a truncated form of recombinant human glutamate decarboxylase 65, which includes any of the following:

[0010] (I) a truncated sequence with the amino acid sequence shown in SEQ ID NO: 2, wherein the amino acids at positions 1 to 52 to 154 are replaced or deleted; or

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

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

[0013] The application provides a truncated form of recombinant human glutamate decarboxylase 65, which includes any of the following:

[0014] (I) a truncated sequence with the amino acid sequence shown in SEQ ID NO: 2, wherein the amino acids at positions 1 to 52 to 154 are deleted; or

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

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

[0017] In some embodiments of the present application, the truncation is linked to a tag for detection or purification.

[0018] In some embodiments of the present application, the truncation comprises a truncated sequence having the amino acid sequence set forth in SEQ ID NO: 2 with substitution or deletion of amino acids 1-71.

[0019] In some embodiments of the present application, the truncation comprises a truncated sequence having the amino acid sequence set forth in SEQ ID NO: 2 with substitution or deletion of amino acids 1-52, 1-71, 1-92, 1-135, or 1-154.

[0020] Preferably, the truncation comprises a truncated sequence having the amino acid sequence set forth in SEQ ID NO: 2 with substitution or deletion of amino acids 1-71.

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

[0022] The present application also provides a biological material comprising any of:

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

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

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

[0026] (iv) a host cell transduced with the virus of (iii), transformed or transfected with the expression vector of (ii), or having integrated into its genome the nucleic acid molecule of (i).

[0027] In some embodiments of the present application, the nucleic acid molecule has a nucleotide sequence set forth in any of SEQ ID NOs: 3-7.

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

[0029] In some embodiments of the present application, the expression vector comprises a pFastBacl backbone vector.

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

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

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

[0033] The present application also provides a culture product of the host cells in the biological material after cultivation.

[0034] In some specific embodiments of the present application, the method for preparing the truncation includes: cultivating the host cells, and extracting and purifying the truncation after crushing.

[0035] In some specific embodiments of the present application, the crushing includes crushing the host cells by high-pressure homogenization; and the extracting and purifying includes extracting and purifying by Ni metal chelate chromatography and DEAE ion exchange chromatography.

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

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

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

[0039] (C), the culture product.

[0040] The present application also provides a GAD65 antibody detection kit, which includes any of the following:

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

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

[0043] (C), the culture product.

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

[0045] Full-length GAD65 protein is only expressed in a small amount in insect cells. The present application screens and retains the main immunogenic fragment of GAD65, and constructs the truncation of the recombinant human glutamate decarboxylase 65. The truncation provided by the present application can be efficiently expressed in insect cells, and the expression product is stable antigen after purification and preparation, and can be used for glutamate decarboxylase antibody detection. BRIEF DESCRIPTION OF DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced.

[0047] Figure 1 SDS-PAGE detection results of the recombinant human glutamate decarboxylase 65 protein according to the present application. DETAILED DESCRIPTION

[0048] The present application discloses a truncated form of recombinant human glutamate decarboxylase 65 and its application. Those skilled in the art can improve the process parameters according to the content of the present application. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application to realize and apply the present application technology.

[0049] Therefore, the present application aims to provide a GAD65 recombinant protein expressed in insect cells and its application.

[0050] The present application can be used for clinical serum sample antibody detection by improving the stability of GAD65 sequence and weakening the unstable factors for protein stable and efficient expression.

[0051] The human GAD65 recombinant protein fragment of the present application is constructed by recombination based on human GAD65. By removing the N-terminal hydrophobic helix and disordered region of different lengths, the extracellular fragment with high immunogenicity is retained.

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

[0053] The virus vector of the present application is baculovirus, and the cell strain containing the polynucleotide or the expression vector is an insect cell capable of transfecting baculovirus and expressing protein.

[0054] In the present application, 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 sf9 or High Five cell strain.

[0055] The present application also provides a preparation method of the recombinant protein, comprising: culturing the host cell, transfecting the host cell with baculovirus and expressing protein.

[0056] In some embodiments, the preparation method of the recombinant protein further comprises the steps of breaking and extracting, purifying the cells after expression. The breaking uses high-pressure homogenization method. The extraction and purification use Ni metal chelate chromatography and DEAE ion exchange chromatography.

[0057] The recombinant protein, the polynucleotide, the expression vector, the cell strain, the host cell or the product prepared by the preparation method described in the application are applied in serum specific GAD65 antibody detection.

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

[0059]

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

[0061] MASPGSGFWSFGSEDGSGDSENPGTARAWCQVAQKFTGGIGNKLCALLYGDAEKPAESGGSQPPRAAARKAACACDQKPCSCSKVDVNYAFLHATDLLPACDGERPTLAFLQDVMNILLQYVVKSFDRSTKVIDFHYPNELLQEYNWELADQPQNLEEILMHCQTTLKYAIKTGHPRYFNQLSTGLDMVGLAADWLTSTANTNMFTYEIAPVFVLLEYVTLKKMREIIGWPGGSGDGIFSPGGAISNMYAMMIARFKMFPEVKEKGMAALPRLIAFTSEHSHFSLKKGAAALGIGTDSVILIKCDERGKMIPSDLERRILEAKQKGFVPFLVSATAGTTVYGAFDPLLAVADICKKYKIWMHVDAAWGGGLLMSRKHKWKLSGVERANSVTWNPHKMMGVPLQCSALLVREEGLMQNCNQMHASYLFQQDKHYDLSYDTGDKALQCGRHVDVFKLWLMWRAKGTTGFEAHVDKCLELAEYLYNIIKNREGYEMVFDGKPQHTNVCFWYIPPSLRTLEDNEERMSRLSKVAPVIKARMMEYGTTMVSYQPLGDKVNFFRMVISNPAATHQDIDFLIEEIERLGQDL

[0062] The nucleotide sequence encoding the human GAD65 recombinant protein is shown in SEQ ID NO: 3:

[0063]

[0064] The nucleotide sequence encoding the human GAD65 recombinant protein from position 72 to 585 is shown in SEQ ID NO: 4:

[0065]

[0066] The nucleotide sequence encoding the human GAD65 recombinant protein from position 93 to 585 is represented by SEQ ID NO: 5:

[0067]

[0068] The nucleotide sequence encoding the human GAD65 recombinant protein from position 136 to 585 is represented by SEQ ID NO: 6:

[0069]

[0070] The nucleotide sequence encoding the human GAD65 recombinant protein from position 155 to 585 is represented by SEQ ID NO: 7:

[0071]

[0072] The nucleotide sequence encoding the human GAD65 recombinant protein from position 180 to 585 is represented by SEQ ID NO: 8:

[0073]

[0074] Full length GAD65 protein is only expressed in trace amounts in insect cells. The present patent screens and retains the main immunogenic fragment of GAD65 which can be efficiently expressed in hosts and can be used for glutamate decarboxylase antibody detection.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present patent, the phrase "a species of genus" means one or more members of a genus. For definitions of common terms in the art and terms of describing chemical instruments and methods, the reader is directed to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are the standard three letter and / or one letter codes used in the art to designate one of the 20 commonly occurring L-amino acids.

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

[0077] The present application is further illustrated by the following examples:

[0078] Example 1 Gene synthesis

[0079] The GAD65 recombinant protein gene (NCBI accession number: NM_000818) containing a 10*his tag added at the C-terminal and codon optimized was synthesized by Jinweizhi (SEQ ID NO: 1).

[0080] Example 2 Preparation of pFastbac-GAD65 plasmid

[0081] GAD65 and pFastBac1 plasmid were mixed after the restriction enzyme digestion and ligation was performed with 100 units of T4 ligase. After incubation at 25°C±5°C for 2 hours, the excess T4 ligase was removed by using a purification kit. The purified pFastBac-GAD65 plasmid was transformed into electrocompetent E. coli DH10Bac™ by using a Bio-Rad Gene Pulser Xcell electroporator. After incubation at 37°C for 1 hour in 1 mL of SOC recovery medium, the cells were plated on LB agar plates containing 100 μg / mL ampicillin and 70 μg / mL gentamicin. After overnight incubation, positive clones were selected and inoculated into SB medium containing 100 μg / mL ampicillin and 70 μg / mL gentamicin. The cells were cultured to the logarithmic growth phase, and the purified pFastBac-GAD65 plasmid was obtained by using a QIAGEN Maxi Prep plasmid extraction kit.

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

[0083] (1) Preparation of sf9 cells:

[0084] The sf9 insect cells were cultured in Grace insect medium supplemented with 10% calf serum to the logarithmic growth phase (1.5-2.5 x 10 6 cells per milliliter of culture medium and a survival rate of more than 95%). In each well of a 6-well culture plate, 2 mL of Grace insect medium was added, and 8 x 10 5 sf9 cells were added to each well without additional antibiotics.

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

[0086] 8 μL of Cellfectin II (purchased from Thermo Fisher Scientific) was added to 100 μL of Grace insect medium without supplements, and 1 μg of pFastBac-GAD65 was diluted in 100 μL of Grace insect medium without supplements. The diluted Cellfectin II and pFastBac-GAD65 plasmid were mixed and incubated at 25°C±5°C 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 the 6-well cell culture plate in (1), and incubated at 27°C for 3-5 hours. Then the transfection mixture was removed, and 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 the sf9 cells were cultured in the culture medium for 72 hours (post-infection), a large number of cells were observed to float, the cells were resuspended, centrifuged at 500 g for 5 min to remove the cells and debris, the supernatant was aspirated, and the remaining residues were centrifuged again at 5000xg for 5 min to obtain P1 generation virus.

[0090] The P1 generation virus was passaged, and a 6-well cell culture plate was prepared, 2x10^6 sf9 insect cells were added to each well, and the cells were incubated at room temperature for 1 hour to adhere to the culture plate.

[0091] An appropriate amount of P1 generation virus was added to each culture well, and the amount of virus added was controlled to be between 0.05 and 0.1 according to the required MOI. The cells and virus were incubated at 27°C under humid conditions for 48 hours.

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

[0093] An appropriate amount of P2 generation virus was added to each culture flask, and the amount of virus added was controlled to be between 0.05 and 0.1 according to the required MOI. The cells and virus were incubated at 27°C under humid conditions for 48 hours.

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

[0095] Example 4 Screening of glutamate decarboxylase 65 protein expression fragments

[0096] The human glutamate decarboxylase 65 recombinant protein fragment is constructed by recombination based on human glutamate decarboxylase 65, and different lengths of GAD65 polypeptide sequences (sequence 1: 1-585, sequence 2: 53-585, sequence 3: 72-585, sequence 4: 93-585, sequence 5: 136-585, sequence 6: 155-585, sequence 7: 180-585) are expressed by the experimental methods in Examples 2 and 3, and the fragment with high expression amount is screened for process research, and sequence 3 is preferably expressed.

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

[0098]

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

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

[0101] (2) Centrifugation: After high-pressure homogenization, the sample is loaded into a 500 ml centrifuge cup, and after balancing, centrifugation is performed at 4°C, 10000 rpm, 30 min. Immediately after centrifugation, the supernatant is poured into a clean beaker.

[0102] (3) Filtration: Use a 0.45 μm pore size filter, and collect the filtrate into another clean beaker.

[0103] (4) Use AKTA pure for Ni metal chelation chromatography. Wash the miscellaneous: After loading and rebalancing, use 20 mM PBS+30 mM imidazole pH 7.4 to wash away the miscellaneous proteins, observe the trend of ultraviolet curve change, when the ultraviolet value rises, put the collection tube into a clean beaker to collect the eluent, and when the ultraviolet value is level, end the collection and then put the waste liquid tube back into the waste liquid barrel.

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

[0105] (6) Liquid exchange: use a dialysis bag with a molecular weight cut-off of 14 KD to exchange the liquid, and the storage buffer is 20 mM PB + 2% glycerol pH 7.4, dialysis for 2 times, each time not less than 3 hours.

[0106] (7) DEAE ion exchange chromatography was performed using AKTA pure. Washing: after loading and rebalancing, the impurities were washed away using 20 mM PB + 50 mM Nacl pH 7.4, and the trend of ultraviolet curve change was observed. When the ultraviolet value rose, the waste liquid pipe was put into a clean beaker to collect the eluate, and when the ultraviolet value was level, the collection was ended and then the waste liquid pipe was put back into the waste liquid barrel.

[0107] (8) Dissociation of target protein: the target protein was eluted using 20 mM PB + 200 mM Nacl pH 7.4, and the trend of ultraviolet curve change was observed. When the ultraviolet value rose, the sample collection was started, and when the ultraviolet value was level, the collection was ended and then the waste liquid pipe was put back into the waste liquid barrel.

[0108] (9) Treatment of target protein: use a dialysis bag with a molecular weight cut-off of 14 KD to exchange the liquid, and the storage buffer is 20 mM Hepes + 250 mM NaCl + 0.2% Triton X100 + 0.05 mM 5-phosphorylated pyridoxal + 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 by the method of Example 5, and the positive samples were detected by using double antigen sandwich method. The positive samples were detected with gradient, and the clinical positive and negative samples could be distinguished. The results are shown in Table 2.

[0111] Double antigen sandwich detection steps:

[0112] 1. Add the diluted sample to be detected 20 μL, the magnetic microparticles coated with GAD65 antigen (5 μg / mL) 50 μL and the biotin-labeled antigen 50 μL in turn in the test tube, mix well and react at 37°C for 15 minutes. 2. Transfer the test tube to the magnetic test tube rack, stand for 3 minutes, tilt and pour off the supernatant, and gently pat dry on the absorbent paper. 3. Take the test tube rack off the magnetic plate, add 500 μL of washing solution, vortex to mix and resuspend the magnetic microparticles, then place them on the magnetic plate and stand for 3 minutes, tilt and pour off the supernatant, and gently pat dry on the absorbent paper. Repeat this step 2 times. 4. Add 135 μL of enzyme conjugate, mix well and react at 37°C for 15 minutes. 5. Repeat steps 2 and 3. 6. Add 200 μL of substrate solution to each tube, react at 37°C for 5 minutes, and then place it in a chemiluminescence analyzer to measure the luminescence intensity.

[0113] Table 2. Human glutamate decarboxylase recombinant protein as antigen for detection of serum samples

[0114]

[0115] The above merely preferred embodiments of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered within the scope of the present application.

Claims

1. A truncated form of recombinant human glutamate decarboxylase 65, characterized in that, The amino acid sequence of the truncation is the amino acid sequence of positions 72-585 of the amino acid sequence set forth in SEQ ID NO:

2.

2. The truncation of claim 1, wherein The truncation is linked to a tag for detection or purification.

3. Biomaterials characterized in that, Any of the following: (i) a nucleic acid molecule encoding the truncation of claim 1 or 2; or (ii) an expression vector comprising the nucleic acid molecule of (i); or (iii) a virus comprising the expression vector of (ii); or (iv) a host cell transduced with the virus of (iii), transformed or transfected with the expression vector of (ii), or having integrated into its genome the nucleic acid molecule of (i).

4. The biomaterial of claim 3, wherein, The nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:

4.

5. The biomaterial of claim 3, wherein The expression vector comprises a pFastBacl backbone vector.

6. The biomaterial of claim 3, wherein The virus comprises a baculovirus.

7. The biomaterial of claim 3, wherein The host comprises an insect cell.

8. A culture product obtained from a host cell of any of claims 3 to 7 after culturing the host cell.

9. Use of any of the following in the preparation of a GAD65 antibody detection kit: (A) the truncation of claim 1 or 2; and / or (B) the biological material of any of claims 3 to 7; and / or (C) the culture product of claim 8.

10. A GAD65 antibody test kit characterized in that, Any of the following: (A) the truncation of claim 1 or 2; and / or (B) the biological material of any of claims 3 to 7; and / or (C) the culture product of claim 8.

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