GCKR gene transcripts and their detection reagents, methods and applications

Abstract

This invention discloses GCKR Gene transcripts and their detection reagents, methods, and applications. GCKR Gene transcripts are nucleic acid molecules capable of encoding proteins with amino acid sequences as shown in SEQ ID NO: 24 and / or SEQ ID NO: 25. This invention provides a novel... GCKR The gene transcripts sGCKR1 and sGCKR2 can effectively regulate GCK enzyme activity and have great application prospects in the preparation of products for regulating blood sugar.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving GCKR Gene transcripts and their detection reagents, methods and applications. Background Technology

[0002] GCKR (Glucokinase regulator) The gene, located on human chromosome 2 (2p23.3), encodes a protein that regulates glucokinase. Its encoded protein, GCKR, affects intracellular glucose and lipid metabolism by specifically binding to glucokinase (GCK). Genome-wide association studies (GWAS) have revealed… GCKR Single nucleotide polymorphism (SNP) sites are significantly associated with metabolic phenotypes, and this association exhibits pleiotropic effects, meaning it occurs in... GCKR Mutations at different sites can cause either elevated or decreased blood sugar levels, but the underlying mechanisms are currently unknown. Summary of the Invention

[0003] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a... GCKR New gene transcripts.

[0004] This invention also proposes biomaterials related to the aforementioned nucleic acid molecules.

[0005] The present invention also proposes the above. GCKR Applications of gene transcripts, nucleic acid molecules, and biomaterials.

[0006] The present invention also proposes a method for obtaining the above. GCKR Methods for gene transcription.

[0007] The present invention also proposes a method for detecting the above. GCKR Reagents for gene transcripts.

[0008] The present invention also proposes a method for detecting the above. GCKR Application of reagents for gene transcripts.

[0009] The present invention also proposes a method for detecting the above. GCKR Methods for gene transcription.

[0010] According to one aspect of the invention, a method is proposed. GCKR Gene transcripts, the GCKR Gene transcripts are nucleic acid molecules that encode one of the following proteins (A1)-A4): A1) Proteins with amino acid sequences as shown in SEQ ID NO: 24 and / or SEQ ID NO: 25; A2) A protein having the same function as the protein described in A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 24 and / or SEQ ID NO: 25. A3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence of A1) or A2); A4) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO:24 and / or SEQ ID NO:25 and has the same function as the protein shown in SEQ ID NO:24 and / or SEQ ID NO:25.

[0011] In some embodiments of the present invention, the at least 80% identity can be at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, or 81% identity.

[0012] In some embodiments of the present invention, the nucleic acid molecule sequence comprises: B1) A nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; or B2) A nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 that has undergone substitution and / or deletion and / or addition of one or more nucleotides, and has the same function as the nucleic acid molecule shown in SEQ ID NO:1 or SEQ ID NO:2; or B3) A nucleotide sequence that has at least 80% identity with SEQ ID NO:1 or SEQ ID NO:2 and has the same function as the nucleic acid molecule shown in SEQ ID NO:1 or SEQ ID NO:2.

[0013] In some embodiments of the present invention, the at least 80% identity can be at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, or 81% identity.

[0014] In some embodiments of the present invention, the GCKR The CDS fragment sequence of the gene transcript is shown in SEQ ID NO:22 or SEQ ID NO:23.

[0015] According to a second aspect of the present invention, a biomaterial related to the above-mentioned nucleic acid molecule is provided, which is any one of the following C1) to C7): C1) An expression cassette containing the above-mentioned nucleic acid molecules; C2) A recombinant vector containing the above-mentioned nucleic acid molecules; C3) A recombinant vector containing the expression cassette described in C1); C4) Recombinant microorganisms containing the above-mentioned nucleic acid molecules; C5) Recombinant microorganisms containing the expression cassette described in C1); C6) Recombinant microorganisms containing the recombinant vector described in C2); C7) Recombinant microorganisms containing the recombinant vector described in C3).

[0016] In some embodiments of the present invention, the recombinant vector originates from a vector including a GV366 vector, a GV140 vector, or a PCMV-3TAG-1A vector.

[0017] According to a third aspect of the invention, the above-mentioned provisions are made. GCKR Applications of gene transcripts, nucleic acid molecules, and biomaterials in any of the following: D1) Prepare products that regulate GCK enzyme activity; D2) Regulates blood sugar; D3) Prepare products to regulate blood sugar.

[0018] According to a fourth aspect of the invention, a method for obtaining the above-mentioned... GCKR A method for gene transcriptomics, the method comprising the following steps: reverse transcription of liver tissue RNA using a 5'RACE kit, followed by nested PCR to amplify the transcript as described in the first aspect. GCKR Gene transcripts; The nested PCR includes two rounds of amplification reactions. The primer sequences used in the first round of amplification reaction are shown in SEQ ID NO:3 or SEQ ID NO:4. The primer sequences used in the second round of amplification are shown in SEQ ID NO:5 or SEQ ID NO:6.

[0019] According to a fifth aspect of the present invention, a method for detecting the above-mentioned GCKR Reagents for detecting gene transcripts, said reagents comprising those for detecting the above GCKR Primer sets and / or probes for gene transcripts, and / or, used to detect the above GCKR Antibodies against proteins encoded by gene transcripts.

[0020] In some embodiments of the present invention, the sequences of the primer set are shown in any of the following sets: E1) Primer sequences as shown in SEQ ID NO:9 and SEQ ID NO:10; E2) Primer sequences as shown in SEQ ID NO:11 and SEQ ID NO:12; E3) Primer sequences as shown in SEQ ID NO:15 and SEQ ID NO:16; E4) Primer sequences as shown in SEQ ID NO:17 and SEQ ID NO:18.

[0021] In some embodiments of the present invention, the preparation of the method for detecting the above... GCKR The antigenic polypeptide sequence of the protein encoded by the gene transcript is shown in SEQ ID NO:19 or SEQ ID NO:20.

[0022] According to a sixth aspect of the present invention, a method for detecting the above-mentioned GCKR The application of reagents for gene transcripts in any of the following: F1) Preparation and Detection GCKR Products derived from the mRNA expression of different gene transcripts; F2) Preparation and Detection GCKR Products derived from the expression of proteins from different gene transcripts; F3) Prepare products that regulate GCK activity; F4) is used to prepare products for regulating blood sugar.

[0023] In some embodiments of the invention, the GCKR Gene transcripts include those that encode proteins with amino acid sequences as shown in SEQ ID NO:24. GCKR Gene transcripts, and / or proteins capable of encoding amino acid sequences as shown in SEQ ID NO:25 GCKR Gene transcripts.

[0024] According to a seventh aspect of the present invention, the present invention also provides a method for detecting the above-mentioned... GCKR A method for detecting gene transcripts, the method comprising the following steps: using the above-described method for detecting the above-described... GCKR Reagents for gene transcripts GCKR Gene transcripts were detected.

[0025] In some embodiments of the invention, the GCKR Gene transcripts include those that encode proteins with amino acid sequences as shown in SEQ ID NO:24. GCKR Gene transcripts, and / or proteins capable of encoding amino acid sequences as shown in SEQ ID NO:25 GCKR Gene transcripts.

[0026] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The present invention provides a novel... GCKR The gene transcripts sGCKR1 and sGCKR2 can effectively regulate GCK enzyme activity and have great application prospects in the preparation of products for regulating blood sugar.

[0027] This invention is determined for the first time GCKR The complete full-length cDNA sequences and amino acid sequences of the sGCKR1 and sGCKR2 transcripts were obtained. Specific primers were designed to detect their mRNA expression, and specific antibodies were prepared to detect their protein expression. The functions of these two novel transcripts were also preliminarily explored, thus contributing to a deeper understanding of their functions. GCKR The role of genes in regulating glucose and lipid metabolism pathways provides a new molecular basis. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 As described in the embodiments of the present invention GCKR Sequence alignment and gene structure diagram of the three transcript isoforms, where exons are represented by boxes, introns by lines, and new... GCKR Isomers are produced through intron retention: GCKR Representing the full-length transcript, sGCKR1 retains a portion of the sequence of intron 8, while sGCKR2 retains portions of the sequences of introns 9 and 16. Figure 2 As described in the embodiments of the present invention GCKR Comparison of three transcript sequences, where a is... GCKR The cDNA sequences of the three transcripts of the gene were compared and analyzed, with identical sequences represented by a uniform color scheme; b indicates a different sequence. GCKR The amino acid sequences of the three transcripts of the gene were compared and analyzed, with identical sequences represented by a uniform color scheme; Figure 3 As described in the embodiments of the present invention GCKR Agarose gel electrophoresis images of conventional PCR amplification using different transcript-specific primers, where a represents... GCKR agarose gel electrophoresis images of the full-length transcript and sGCKR1 transcript; b is the agarose gel electrophoresis image of the sGCKR2 transcript. Figure 4 RT-qPCR detection in this embodiment of the invention GCKR The graph shows the results of expression level detection of three transcripts, where a is... GCKR mRNA expression levels of three transcripts in human liver tissue; bd is GCKRFigure showing the expression results of the three transcripts in different human cell lines; Figure 5 The images show the results of Western blot (WB) detection of specific antibodies against sGCKR1 and sGCKR2 in this embodiment of the invention, where a represents the expression of the protein encoded by sGCKR1 in liver tissue; and b represents the results of the expression detection of the protein encoded by sGCKR2 in liver tissue. Figure 6 for GCKR Subcellular localization detection results of proteins encoded by three transcripts of the gene under low and high glucose conditions; Figure 7 Figure 1 shows the results of detecting the effects of three transcripts on GCK activity under different sugar concentrations. Figure 2a shows the results of co-transforming the GCKR transcript with GCK in 293T cells, detecting GCK activity under low and high sugar conditions, and comparing it with the control group of co-transforming an empty vector with GCK; Figure 3b shows the results of co-transforming the sGCKR1 transcript with GCK in 293T cells, detecting GCK activity under low and high sugar conditions, and comparing it with the control group of co-transforming an empty vector with GCK; Figure 4c shows the results of co-transforming the sGCKR2 transcript with GCK in 293T cells, detecting GCK activity under low and high sugar conditions, and comparing it with the control group of co-transforming an empty vector with GCK. "LG" indicates low sugar, "HG" indicates high sugar, "**" indicates p < 0.01, and "***" indicates p < 0.001. Detailed Implementation

[0029] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0030] Example 1 GCKR Obtaining sGCKR1 and sGCKR2 transcripts This embodiment prepared a GCKR The sequences of the sGCKR1 and sGCKR2 transcripts are as follows: sGCKR1 complete cDNA sequence (5'-3'): attgtgaccagaggggtttgtgtggctgaagaggcaggaggaacagtgtatccacagcgtgggaccatgccaggcacaaaacggtttcaacatgtcattgagaccccggagcctggcaagtgggagttgtctgggtacgaggcagctgtgccaatcacggagaagtcaaacccactgacccaggatctagacaaagcagatgctgagaacattgttcgactgctagggcaatgtgatgctgagatcttccaggaggaggggcaagccctgtccacataccagagactctacagcgaatccattctgaccaccatggtacaggtggctgggaaagttcaggaagtgctgaaggagccagatggggggctggttgtgctgagtggagggggcacctctggccggatggcattcctcatgtcggtgtcctttaatcagctgatgaaaggtctgggacagaaacctctttacacctacctcattgcaggtggtgacaggtctgtggtggcctctagggaggggacagaagatagtgccttgcacgggattgaggaactgaagaaggtggctgccgggaagaagagagtgattgtcattggcatttctgtgggactctctgctccctttgtggcaggccagatggactgctgcatgaacaacacagctgtcttcttgccagtcctggttggcttcaatccagtgagcatggccaggcacccctttcctcccccgaggatcctcagatctctcactgtcttcccttccctgagagcccctcattaccaaatcacctccctcttattctctatgtcagtggtcactcttatttcagaataaatcaacatattaaacca (SEQ ID NO:1).

[0031] The complete amino acid sequence of sGCKR1: MPGTKRFQHVIETPEPGKWELSGYEAAVPITEKSNPLTQDLDKADAENIVRLLGQCDAEIFQEEGQALSTYQRLYSESILTTMVQVAGKVQEVLKEPDGGLVVLSGGGTSGRMAFLMSVSFNQLMKGLGQKPLYTYLIAGGDRSVVASREGTEDSALHGIEELKKVAAGKKRVIVIGISVGLSAPFVAGQMDCCMNNTAVFLPVLVGFNPVSMARHPFPPPRILRSLTVFPSLRAPHYQITSLLFSMSVVTLISE (SEQ ID NO:24).

[0032] The complete cDNA sequence of sGCKR2 (5'-3'):

[0033] The complete amino acid sequence of sGCKR2: MKGGSATKILLETLLLAAHKTVDQGIAASQRCLLEILRTFERAHQVTYSQSPKIATLMKSVSTSLEKKGHVYLVGWQTLGIIAIMDGVECIHTFGADFRDVRGFLIGDHSDMFNQK AELTNQGPQFTFSQEDFLTSILPSLTEIDTVVFIFTLDDNLTEVQTIVEQVKEKTNHIQALAHSTVGQTLLIPLKKLFPSIISITWPLLFFEYEGNFIQRSGFSTLPRLFANS (SEQ ID NO:25).

[0034] The preparation process is as follows: 5' RACE kit was used to obtain... GCKR The complete cDNA sequences of the sGCKR1 and sGCKR2 gene transcripts are obtained through the following steps: 1. RNA extraction RNA was extracted from human liver tissue using a total RNA extraction kit and stored at -80℃ for later use. 2. First-strand cDNA synthesis (1) RNA denaturation and annealing Table 1

[0035] After thoroughly mixing the system shown in Table 1, briefly centrifuge, incubate at 72 °C for 3 min in a PCR instrument, and cool at 4 °C. After annealing, briefly centrifuge and immediately place on ice.

[0036] (2) cDNA synthesis Add reverse transcription reaction solution to the system obtained after annealing in (1). The reverse transcription system is shown in Table 2 below.

[0037] Table 2

[0038] Prepare the reaction solution shown in Table 2 on ice, mix thoroughly, and briefly centrifuge. Place the mixture in a PCR instrument and incubate under the following conditions: 42 °C for 90 min, 70 °C for 15 min, and 4 °C to obtain the cDNA synthesis product. Remove the first-strand cDNA synthesis product and place it on ice. Dilute 20 μL of cDNA product with 20 μL of Tricine-EDTA Buffer to obtain 5'RACE cDNA.

[0039] 3. Rapid amplification of cDNA ends (RACE amplification) (1) Nested PCR first amplification The 5' RACE cDNA obtained in the above steps was used as a PCR template, and nested first amplification was performed using the reagents in the RACE kit. The primer sequence used for sGCKR1 GSP amplification was: ATAAGAGGGAGGTGATTTGGTAATG (SEQ ID NO:3); the primer sequence used for sGCKR2 GSP amplification was: GGCTGTAGGTCACCTGATGAGCTCGC (SEQ ID NO:4). The annealing temperature of the sGCKR1 GSP primers was 63 ℃, and the annealing temperature of the sGCKR2 GSP primers was 65 ℃.

[0040] The components of the amplification system are shown in Table 3 below.

[0041] Table 3

[0042] Prepare the reaction system as shown in Table 3 on ice, mix thoroughly, and then briefly centrifuge; place in a PCR instrument and amplify according to the conditions in Table 4 below.

[0043] Table 4

[0044] (2) Nested PCR second amplification Take 5 μL of the PCR product obtained in (1) and add it to 245 μL of Tricine-EDTA Buffer. Dilute it 50 times, vortex and mix well. Use it as a template for the second round of nested amplification. The annealing temperature of sGCKR1 NGSP primer is 63 ℃, and the annealing temperature of sGCKR2 GSP primer is also 63 ℃. The sGCKR1 NGSP primer sequence is: GGAAGACAGTGAGAGATCTGAGGATC (SEQ ID NO:5); the sGCKR1 NGSP primer sequence is: AAGATTTCCAGGAGGCATCTTTGAG (SEQ ID NO:6). The components of the amplification system are shown in Table 5 below.

[0045] Table 5

[0046] Prepare on ice, mix thoroughly, and briefly centrifuge; place in a PCR instrument and amplify under the conditions shown in Table 6 below.

[0047] Table 6

[0048] 4. Sanger sequencing Two μL of the PCR product from the second nested PCR amplification was subjected to agarose gel electrophoresis. Amplification products matching the expected target fragment size were then subjected to Sanger sequencing. After confirming the amplified fragment as a fragment of the sGCKR1 / sGCKR2 transcript, the complete cDNA sequences of sGCKR1 / sGCKR2 and sGCKR1 were spliced ​​together.

[0049] Analysis of the cDNA sequences of sGCKR1, sGCKR2, and GCKR transcripts (NCBI sequence number: NM_001486.4) yielded the following exon compositions for the three GCKR transcripts: Figure 1 As shown.

[0050] Will GCKR The cDNA sequences of the three transcripts of the gene were compared, and the comparison results are shown below. Figure 2 Figure a shows the prediction of the complete open reading frames (ORFs) of sGCKR1 and sGCKR2 using NCBI's ORF Finder. The complete amino acid sequences of sGCKR1 and sGCKR2 were obtained, and the amino acid sequences of the three GCKR transcripts were compared. The results are shown in Figure a. Figure 2 As shown in Figure b.

[0051] Example 2: Identification and expression profiling of sGCKR1 / GCKR-S2 transcripts 1. RNA extraction and reverse transcription (1) RNA extraction Cellular RNA extraction: After discarding the culture medium in the culture dish, wash the cells twice with pre-chilled PBS. Add an appropriate amount of Trizol (calculated as 1 mL for a 6-well plate), lyse on ice for 5 min, mix thoroughly by pipetting, and transfer to a 1.5 mL centrifuge tube. Add 1 / 5 volume of chloroform, vortex for 15 s, and let stand for 5 min. Centrifuge at 12000 rpm, 4 ℃, for 15 min. Transfer the supernatant to a new centrifuge tube (avoiding the suspended precipitate in the middle), add the same volume of pre-chilled isopropanol as the supernatant, mix thoroughly, let stand for 10 min, and centrifuge at 4 ℃, 12000 rpm, for 15 min. Carefully discard the supernatant, leaving the precipitate. Prepare fresh 75% ethanol and pre-chill it. Add the same volume of pre-chilled 75% ethanol as the Trizol to the precipitate and mix thoroughly. Centrifuge at 7500 rpm, 4 ℃, for 5 min. Carefully discard the supernatant, open the centrifuge tube cap, invert the tube mouth downwards onto filter paper, and dry for 10 minutes. Add an appropriate amount of enzyme-free water, mix well by pipetting, measure the RNA concentration using a NANODROP 2000 UV spectrophotometer, and record the A260 / A80 value, ensuring that this value is around 1.8~2.0.

[0052] RNA was extracted from different human cell lines using the methods described above.

[0053] Tissue RNA extraction: Cut off a portion of fresh or frozen mouse tissue at -80 ℃, add an appropriate amount of Trizol according to the instructions, add grinding beads, grind in a grinder until no tissue is visible to the naked eye, transfer the tissue homogenate into a new centrifuge tube, and follow the same steps as for cells.

[0054] The method for extracting RNA from human liver tissue is the same as the method for extracting RNA from the tissues described above.

[0055] (2) Reverse transcription 1) Removal of gDNA The systems for removing gDNA are shown in Table 7.

[0056] Table 7

[0057] After mixing all components in the system shown in Table 7, briefly centrifuge at low speed. Treat at 42 ℃ for 2 min, then maintain at 4 ℃.

[0058] 2) Reverse transcription reaction Add the reagents shown in Table 8 to the 10 μL system as shown in Table 7 above.

[0059] Table 8

[0060] Mix the above system thoroughly and centrifuge briefly at low speed. Incubate at 37 ℃ for 15 min, 85 ℃ for 5 s, and maintain at 4 ℃ for reverse transcription to obtain cDNA.

[0061] 2. Identification of sGCKR1 / sGCKR2 transcripts by conventional PCR (1) PCR amplification The cDNA sequences of the three GCKR transcripts were compared using BLAST. Based on the specific regions of the sGCKR1 / sGCKR2 transcripts, different transcript-specific primers were designed. Using human liver tissue cDNA as a template, conventional PCR amplification was performed. The amplification systems and procedures for the sGCKR1 and GCKR transcripts are shown in Tables 9-10 and 11, respectively. The amplification system for sGCKR2 is shown in Table 12, and the amplification procedure differs from that in Table 10 only in that the annealing temperature is 55℃.

[0062] Table 9

[0063] Table 10

[0064] Table 11

[0065] Table 12

[0066] (2) The amplification products were subjected to agarose gel electrophoresis and Sanger sequencing. Take 5 μL of the PCR product obtained above and perform agarose gel electrophoresis. The electrophoresis results show that the amplification product matches the expected target fragment size. Figure 3 The target fragment was cut out and subjected to Sanger sequencing. The sequencing results showed that the sequence was error-free.

[0067] 3. RT-qPCR detection of sGCKR1 and sGCKR2 transcript expression We designed specific RT-qPCR primers for the three GCKR transcripts to quantitatively detect the mRNA expression of sGCKR1 and sGCKR2 transcripts in liver tissue and different cell lines.

[0068] The amplification conditions were as follows: cDNA was diluted 4-5 times with enzyme-free water, and 10 μL of the reaction mixture was prepared in a 384-well plate. After thorough mixing, the plate was centrifuged at 4 °C for 5 min. Amplification was performed using a real-time PCR instrument, with 3 auxiliary wells for each sample. The reaction system is shown in Table 13 below, and the primer sequences are shown in Table 14.

[0069] Table 13

[0070] Reaction program: Stage 1: 50 ℃ for 2 min, 95 ℃ for 10 min; Stage 2: 95 ℃ for 15 s, 60 ℃ for 1 min, 40 cycles; Melt curve: 95 ℃ for 15 s, 60 ℃ for 1 min.

[0071] Table 14

[0072] RT-qPCR detection of three types of cells and liver tissue GCKR Results of gene transcripts such as Figure 4 As shown in the figure, different transcripts are expressed at different levels in different human cells and liver tissues.

[0073] Example 3: Preparation of specific antibodies against sGCKR1 and sGCKR2 transcript-encoded proteins and Western blot detection. 1. Preparation of specific antibodies (1) Immunogen preparation First, peptide synthesis was performed. The protein sequences encoding the transcripts of sGCKR1 and sGCKR2 were analyzed. Candidate antigens were screened using the antigen design tool Optimum Antigen™ and prepared into peptides. The peptide sequence selected for the sGCKR1 antigen was PITEKSNPLTQDLDC (SEQ ID NO:19); the peptide sequence selected for the sGCKR1s2 antigen was ADFRDVRGFLIGDHC (SEQ ID NO:20). The synthesized peptides were conjugated with the carrier protein KLH to prepare the immunogen.

[0074] (2) Animal immunization Two rabbits were immunized with the target protein-KLH carrier protein conjugate. The first immunization was performed on day 0, and the second immunization was performed on day 14.

[0075] (3) Antibody purification After final immunization, antiserum from all rabbits was collected, yielding approximately 40 mL of antiserum per rabbit. An antigen affinity purification column was prepared using peptides, and the serum from two rabbits was mixed together for purification. The purified antibody was then stored in 0.02% Proclin 300.

[0076] 2. Western blotting to detect proteins encoded by sGCKR1 and sGCKR2 transcripts. (1) Protein extraction Human liver tissue samples frozen at -80℃ were cut into small pieces and added to RIPA lysis buffer. Grinding beads were added to the grinding tube and the mixture was ground until no tissue blocks were visible to the naked eye. The mixture was sonicated three times for 10 seconds each time, placed on ice and shaken for about 1 hour, centrifuged at 12,000 rpm for 10 minutes at 4℃, and the supernatant was collected as a protein solution.

[0077] (2) Protein WB Add 5× loading buffer to the prepared protein solution and boil in a metal bath at 95 °C for 10 min; perform SDS-PAGE polyacrylamide gel electrophoresis; perform wet transfer using the sandwich transfer method; block with skim milk at room temperature for 1 hour, and incubate the transferred PVDF membranes with the prepared specific antibodies for sGCKR1 and sGCKR2 (antibodies diluted with 1% BSA to a final concentration of 20 μg / mL), respectively, overnight on a shaker at 4 °C; the next day, incubate with secondary antibody at room temperature, and develop with ECL developing solution. The results are as follows. Figure 5 As shown.

[0078] As can be seen from the figure, the antibody prepared by this invention can be accurately used to detect sGCKR1 and sGCKR2 transcripts.

[0079] Example 4: Construction of exogenous overexpression plasmids of GCKR / sGCKR1 / sGCKR2 transcripts This embodiment constructs an exogenous overexpression plasmid of the GCKR gene transcript.

[0080] (1) GCKR transcript exogenous overexpression plasmid The GCKR plasmid was created using the PCMV-3TAG-1A vector, which is characterized by the fusion of a 3× flag tag protein; the selected restriction enzyme sites were BamHI / EcoRI; and a GCKR CDS fragment was inserted at two sites, with the inserted fragment sequence as follows:

[0081] (2) sGCKR1 transcript exogenous overexpression plasmid The sGCKR1 plasmid uses the GV140 vector, which is characterized by the fusion of two tag proteins, 6 × His and MYC; the selected restriction enzyme sites are XhoI / HindIII; and the sGCKR1 CDS fragment is inserted at two sites (SEQ ID NO:22).

[0082] (3) sGCKR2 transcript exogenous overexpression plasmid The sGCKR2 plasmid uses the GV366 vector, which is characterized by the fusion of the HA tag protein; select restriction enzyme sites: XhoI / HindIII; insert the sGCKR2 CDS fragment between the two sites, and the inserted fragment sequence is: atgaaaggtggaagtgccaccaagattctgctggaaaccctgttattagcagcccataagactgtggaccagggcattgcagcatctcaaagatgcctcctggaaatcttgcggacatttgagcgagctcatcaggtgacctacagccaaagccccaagattgccaccctgatgaagagtgtcagcaccagtctggagaagaaaggccacgtgtacctggttggctggcagaccctgggcatcattgccatcatggatggagtagagtgcatccacacctttggtgctgatttccgagatgtccgtggctttctcattggtgatcacagtgacatgtttaaccagaaggctgagctcaccaaccagggtccccagttcaccttctcccaggaggacttcctgacttccatccttccctctctcacggaaatcgatactgtggtcttcattttcaccctggatgacaacctcacggaggtgcagactatagtggagcaggtgaaagagaagaccaaccacatccaggccctggcacacagcaccgtgggtcagaccttgctgatccctctgaagaagctctttccctccatcatcagcatcacatggccactgcttttctttgaatatgaagggaacttcatccagagatcgggtttctccacattgcccaggctgtttgccaactcctag (SEQ ID NO:23).

[0083] Sterilized cell slides were placed in 24-well plates, and 293T cells were seeded onto the slides. Transfection began 24 hours after seeding, when the cell density reached 50%. The exogenous overexpression plasmids GCKR, sGCKR1, and sGCKR2 prepared above were transfected, with 1 μg of plasmid and 3 μL of transfection reagent added to each well. Eight hours after transfection, the medium was replaced with complete culture medium. Forty hours after transfection, the cells were subjected to low-glucose (final concentration 5 mM glucose) and high-glucose (final concentration 25 mM glucose) conditioned conditions for 8 hours. Immunofluorescence staining was used to observe the subcellular localization of the three transcripts under low-glucose and high-glucose conditions. The results are as follows: Figure 6 .

[0084] 293T cells were seeded in 12-well plates. Transfection began 24 h after seeding when the cell density reached 70%. Different GCKR transcripts (GCKR, sGCKR1, sGCKR2 transcripts) and the aforementioned exogenous overexpression plasmids were co-transfected with the GCK plasmid (1.2 μg of GCKR / sGCKR1 / sGCKR2 plasmid per well, 0.3 μg of GCK plasmid per well, and 4.5 μL of transfection reagent per well). Empty vector plasmids (corresponding to the aforementioned exogenous overexpression plasmids) were co-transfected with the GCK plasmid as a control group. Eight h after transfection, the medium was replaced with complete medium. Forty h later, cells were treated with low glucose (final concentration 5 mM glucose) and high glucose (final concentration 25 mM glucose) conditions for 8 hours. Forty-eight h later, cells were collected from the wells, GCK enzyme was extracted, and GCK activity was measured using a GCK activity assay kit.

[0085] The results are as follows Figure 7 As shown in the figure, GCKR transcripts reduce GCK enzyme activity under both low and high glucose conditions, indicating that GCKR has an inhibitory effect on GCK; sGCKR1 and sGCKR2 increase GCK activity under both low and high glucose conditions, indicating that sGCKR1 and sGCKR2 have a promoting effect on GCK. The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A kind GCKR Gene transcripts, characterized by, The GCKR Gene transcripts are nucleic acid molecules that encode one of the following proteins: A1)-A2): A1) Proteins with amino acid sequences as shown in SEQ ID NO: 24 or SEQ ID NO: 25; A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence of A1).

2. As described in claim 1 GCKR Gene transcripts, characterized by, The nucleic acid molecule comprises: B1) A nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; or B2) A nucleotide sequence in which SEQ ID NO:1 or SEQ ID NO:2 is substituted with one or more nucleotides, and has the same function as the nucleic acid molecule shown in SEQ ID NO:1 or SEQ ID NO:2; or B3) A nucleotide sequence that has at least 80% identity with SEQ ID NO:1 or SEQ ID NO:2 and has the same function as the nucleic acid molecule shown in SEQ ID NO:1 or SEQ ID NO:

2.

3. A biomaterial related to the nucleic acid molecule as described in claim 1, characterized in that, It is any one of C1) to C7) below: C1) An expression cassette containing the nucleic acid molecule as described in claim 1; C2) A recombinant vector containing the nucleic acid molecule described in claim 1; C3) A recombinant vector containing the expression cassette described in C1); C4) Recombinant microorganisms containing the nucleic acid molecules as described in claim 1; C5) Recombinant microorganisms containing the expression cassette described in C1); C6) Recombinant microorganisms containing the recombinant vector described in C2); C7) Recombinant microorganisms containing the recombinant vector described in C3).

4. The claim 1-2 GCKR The use of gene transcripts or the biomaterial of claim 3 in any of the following: D1) Prepare products that increase GCK enzyme activity; D2) Prepare products that lower blood sugar.

5. A method for obtaining the product as described in claim 1 GCKR The method for gene transcripts is characterized by, The method includes the following steps: after reverse transcription of liver tissue RNA using a 5'RACE kit, nested PCR is used to amplify the RNA obtained as described in claim 1. GCKR Gene transcripts; The nested PCR includes two rounds of amplification reactions. The primer sequences used in the first round of amplification reaction are shown in SEQ ID NO:3 or SEQ ID NO:

4. The primer sequences used in the second round of amplification are shown in SEQ ID NO:5 or SEQ ID NO:6.

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