IRES-hINS recombinant gene, lentivirus, and their construction methods and applications

Abstract

The present invention discloses a recombinant insulin gene containing a furin protease cleavage site. The sense strand of the oligonucleotide sequence of the recombinant insulin gene containing the furin protease cleavage site is shown as SEQ ID NO:1, and its antisense strand sequence is shown as SEQ ID NO:2. The present invention also discloses the construction methods and applications of the IRES-hINS recombinant gene, expression cassette, recombinant vector, recombinant cell, lentivirus and its recombinant vector. The present invention also discloses the application of the recombinant gene, etc. in recombinant insulin-related therapeutic drugs. The IRES-hINS sequence designed by the present invention can respond to changes in blood glucose concentration in the culture medium after transfection of cells, thereby increasing or decreasing the synthesis level of insulin, which is of great significance for the treatment of diabetes.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the IRES-hINS recombinant gene, lentivirus, its construction method, and its application. Background Art

[0002] Diabetes is gradually becoming the leading cause of disease threatening human health and a major public health concern for countries worldwide. According to global statistics from the International Diabetes Federation in 2017, the prevalence of diabetes among adults worldwide was 9.1%. In the past 30 years, the prevalence of diabetes in China has increased from less than 1% in 1980 to 10.9% in 2013. Diabetes is caused by the pancreas's inability to produce enough insulin (type 1 diabetes) or by the body's own cells' inability to respond appropriately to insulin (type 2 diabetes). Both type 1 and type 2 diabetes eventually lead to pancreatic failure, resulting in insufficient insulin. The chronic hyperglycemia in diabetic patients can cause chronic damage and functional impairment to various tissues, particularly the eyes, kidneys, heart, blood vessels, and nerves.

[0003] If blood glucose levels are strictly maintained within the normal range over a long period, complications such as diabetic foot, diabetic kidney failure, and diabetic heart failure can be avoided. Despite extensive research into treatments for diabetic hyperglycemia, supplementation with exogenous insulin or its analogues remains the primary means of controlling blood sugar levels. For type 1 diabetes with absolute insulin deficiency, the core treatment is to administer exogenous insulin via manual injection or automated delivery systems such as islet pumps, following a prescribed procedure. Type 2 diabetes, due to its diverse etiologies and the progressive deterioration of pancreatic beta cells, offers various treatment options, such as using glucagon-like peptide-1 (GLP-1) to enhance the glucose sensitivity of damaged beta cells and increase insulin secretion. Exogenous insulin can typically maintain the health of diabetic patients for more than 10 years, significantly extending their lifespan. However, these treatments severely impact patients' quality of life and carry the risk of life-threatening hypoglycemia due to incorrect administration. Islet or whole pancreas transplantation can restore endogenous insulin secretion, precisely control blood sugar levels, prevent hypoglycemia, and reduce dependence on exogenous insulin, thus improving patients' quality of life. The protocol has been gradually developed and improved since the 1980s and is now quite mature. However, islet or whole pancreas transplantation has problems such as surgical risks, immune rejection, and donor scarcity, so it cannot cope with the increasingly severe epidemic trend of the disease.

[0004] If we could use the patient's own cells to prepare insulin-secreting cells that can dynamically respond to blood glucose levels, we could perfectly overcome the current limitations of islet transplantation (including donor shortage and allogeneic graft immune response), making it an ideal solution for treating diabetes.

[0005] Current methods for preparing autologous insulin-secreting cells generally involve overexpressing PDX1 to induce adult cells or stem cells into pancreatic cell lineages, followed by chemically induced differentiation to activate downstream transcription factors (such as NGN3, NEUROD1, and MAFA) or transgenic transcription to highly express one or more of these transcription factors, promoting further differentiation and maturation of insulin-secreting cells. For example, Berneman-Zeitouni et al. used a combination of three pancreatic transcription factors, PDX1, PAX4, and MAFA, to enhance the differentiation of mature β-cells. However, this method typically requires complex cell processing and culture differentiation, resulting in a low proportion of terminally differentiated functional cells with poor activity, making it difficult to achieve the desired insulin secretion efficiency. Therefore, finding a gene sequence that can directly guide functional insulin synthesis and respond to changes in blood glucose levels, and transfecting cells with this sequence, can avoid the drawbacks of traditional methods and efficiently produce insulin-secreting cells, which is of great significance for the treatment of diabetes. Summary of the Invention

[0006] Purpose of the invention: In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a recombinant insulin gene containing furin cleavage sites.

[0007] Another technical problem that this invention aims to solve is to provide the IRES-hINS recombinant gene.

[0008] Another technical problem that this invention aims to solve is to provide expression cassettes, recombinant vectors, recombinant cells, and lentiviruses.

[0009] Another technical problem that this invention aims to solve is to provide a recombinant vector, its construction method, and its application.

[0010] The final technical problem to be solved by this invention is to provide the IRES-bINS recombinant gene, the expression cassette, the recombinant vector, the recombinant cells, and the application of the lentivirus in the preparation of recombinant insulin-related therapeutic drugs.

[0011] Technical solution: In order to solve the above technical problems, the present invention provides a recombinant insulin gene containing a furin protease cleavage site. The sense strand of the oligonucleotide sequence of the recombinant insulin gene containing the furin protease cleavage site is shown in SEQ ID NO: 1, and its antisense strand sequence is shown in SEQ ID NO: 2.

[0012] The sense strand of the oligonucleotide sequence is as follows:

[0013] The antispeech chain is:

[0014]

[0015] The present invention also includes the IRES-bINS recombinant gene, which includes the recombinant insulin gene containing furin cleavage site and the sugar-responsive IRES gene. The sense strand of the oligonucleotide sequence of the sugar-responsive IRES gene is shown in SEQ ID NO: 3, and its antisense strand sequence is shown in SEQ ID NO: 4.

[0016] The sense strand of the oligonucleotide sequence regulating the IRES sequence is:

[0017]

[0018] The antispeech chain is:

[0019]

[0020]

[0021] The nucleotide sequence of the IRES-hINS recombinant gene is shown in SEQ ID NO: 5.

[0022] The present invention also includes an expression cassette, a recombinant vector, and recombinant cells containing the IRES-hINS recombinant gene described above.

[0023] The recombinant vector includes, but is not limited to, pLVX-IRES-hINS.

[0024] The present invention also includes a method for constructing the recombinant vector, wherein the recombinant vector is obtained by sequentially linking the sugar-responsive IRES gene, the P2A protein cleavage sequence, and the recombinant insulin gene sequence containing furin cleavage sites to the recombinant vector.

[0025] Furthermore, the lentiviral expression vector can be obtained by cloning a synthesized IRES-bINS sequence (shown in SEQ ID NO: 5) into the lentiviral expression vector pLVX using homologous recombination.

[0026] The present invention also includes a lentivirus containing the IRES-hINS recombinant gene or the recombinant vector described above.

[0027] The present invention also includes the IRES-hINS recombinant gene, the expression cassette, the recombinant vector, the recombinant cells, and the application of the lentivirus in the preparation of recombinant insulin-related therapeutic drugs.

[0028] The present invention also includes the IRES-hINS recombinant gene, the expression cassette, the recombinant vector, the recombinant cells, and the application of the lentivirus in the preparation of drugs for treating diabetes, hyperglycemia, diabetic foot, or diabetic complications.

[0029] The present invention also includes a drug for treating diabetes, comprising the recombinant insulin gene containing a furin cleavage site or the IRES-hINS recombinant gene.

[0030] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: Based on the mRNA sequence of the insulin gene and its maturation process after the precursor cleavage of the C-peptide, this invention designs and synthesizes a recombinant insulin gene sequence containing a furin cleavage site. The introduction of the furin cleavage site allows for the cleavage of mRNA to form the α and β monomers of insulin in the presence of furin in the cell, ensuring the production of active insulin. Simultaneously, to ensure that insulin production can dynamically respond to changes in blood glucose levels, we ultimately screened for the IRES sequence and constructed a glucose-responsive IRES-hINS oligonucleotide sequence. Unexpectedly, we found that this sequence, after being packaged into lentivirus using a lentiviral vector and infecting human fibroblasts, could respond to changes in glucose concentration in the culture medium. The IRES-hINS sequence designed in this invention can respond to changes in blood glucose concentration in the culture medium after transfection into cells, thereby increasing or decreasing insulin synthesis levels, which is of great significance for the treatment of diabetes. This invention provides a theoretical basis and solution for new gene therapy strategies for diabetes, and is expected to be applied to the preparation of highly efficient, sensitive, and low-side-effect gene drugs for diabetes treatment. The development of gene drugs is of great significance and has enormous social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It represents the concentration of insulin released by IRES-hINS-293T cells, hINS-293T cells, and hINSnf-293T cells in response to different concentrations of glucose. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can determine the basic features of the invention and can make various modifications and changes to the invention without departing from its spirit and scope to make it suitable for various uses and conditions.

[0033] In the following examples, some of the materials and reagents used were sourced from the following sources:

[0034] Lentiviral packaging vectors pMdlg, RSV-REV, VSV-G, and lentiviral knockout plasmid vector pLVX were all purchased from Takara Bio Inc. Human 293FT cells used in this invention were purchased from the American Type Culture Collection (ATCC). Lipofectamine 2000 reagent was purchased from Thermo Fisher Scientific, USA. OPTI-MEM medium was purchased from Gibico, USA.

[0035] Unless otherwise specified in the examples, all conditions were performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products. Unless otherwise specified, all techniques employed in this invention are prior art in the field.

[0036] Example 1: Construction of Lentiviral Vector

[0037] The mRNA sequences of the A and B strands, P2A sequence, Puro sequence, and ires sequence of the human insulin gene were obtained from the GenBank database (http: / / www.ncbi.nlm.nih.gov / genbank).

[0038] We further modified the human insulin gene to create a gene sequence containing the A and B chains and furin cleavage sites for the insulin α and β chains. The sense strand of this hINS oligonucleotide sequence is as follows:

[0039]

[0040]

[0041] The antispeech chain is:

[0042]

[0043] P2A:

[0044] The chain of justice is as follows:

[0045]

[0046] The antispeech chain is:

[0047]

[0048] Ires(IRES FGF ):

[0049] The chain of justice is as follows:

[0050]

[0051] The antispeech chain is:

[0052]

[0053] The sequence described in this invention was synthesized by Sangon Biotech (Shanghai) Co., Ltd. in the order Ires-P2A-Insulin. The above-mentioned oligonucleotide fragment was ligated into the lentiviral vector pLVX (Addgene, USA) via homologous recombination to obtain the IRES-hINS recombinant gene. The IRES-hINS recombinant gene sequence (including IRES...) FGF The base sequence of Puro, P2A, bINS is:

[0054]

[0055]

[0056] The ligation product was transformed into DH5α *E. coli* competent cells using a heat shock method. The cells were then incubated on LB solid medium containing ampicillin at 37°C for 16 h. Single colonies were picked and cultured in LB liquid medium containing ampicillin at 37°C and 220 rpm for 16 h. The bacterial cells were collected, and plasmids were extracted. The plasmids were verified by enzyme digestion. Positive plasmids were sent to BGI Genomics for sequencing verification. Sequencing showed that the constructed lentiviral plasmid was successfully used for subsequent experiments. Using the above method, the lentiviral vector pLVX-IRES-hINS (SEQ ID NO: 5) containing the target plasmid was constructed. To illustrate the effect of introducing furin cleavage sites and IRES into the recombinant insulin in this invention, pLVX-hINS and pLVX-hINSnf vectors without the IRES sequence were also prepared using the above method. The insulin sequence in the pLVX-hINS vector contains furin cleavage sites, while the insulin in the pLVX-hINSnf vector does not contain furin cleavage sites.

[0057] Example 2: Preparation of IRES-hINS, hINS, and hINSnf lentiviruses

[0058] Lentiviral viruses were packaged according to the Lipofectamine 2000 reagent instructions. Cells were transfected with the lentiviral vector prepared in Example 1 as experimental groups, while cells transfected with the pLVX lentiviral empty vector served as control groups.

[0059] Following the instructions of the lipofectmin2000 reagent, the target plasmid and helper plasmids pMdlg, RSV-REV, and VSV-G were transfected into 293FT cells. Viruses were prepared and collected, and then 293FT cells were infected with lentiviruses containing IRES-hINS, bINS, and hINSnf sequences. Cells with resistance were screened with puromycin for use in experiments.

[0060] The specific steps are as follows: Add 0.75 μg pMdlg, 0.35 μg RSV-REV, 0.49 μg VSV-G, and 0.61 μg lentiviral vectors Plvx-RES-hINS, pLVX-hINS, and pLVX-hINSnf to 0.5 mL of low-serum OPTI-MEM medium, mix gently, and incubate at room temperature for 5 min to obtain solution A, which is ready for use. Add 9 μL of liposome Lipofectmin 2000 to 0.5 mL of OPTI-MEM medium, mix gently, and incubate at room temperature for 5 min to obtain solution B, which is ready for use. Mix solution A and solution B at a 1:1 volume ratio to obtain a mixed solution, and incubate at room temperature for 20 min; incubate with TrypLE (Gibco, USA) at room temperature for 5 min to digest 293FT cells, count the cells under a microscope, and adjust the cell density to 10T. 5 Cells / ml. Gently and thoroughly mix the 293FT cell suspension with the mixing solution at a 1:1 volume ratio, transfer to a culture dish, and gently shake to even out the bottom. Incubate the dish in an incubator for 12 hours, then change the medium and continue culturing. After two days of culture, aspirate the cell supernatant using a 5mL sterile syringe, filter through a 0.45μm microporous membrane to remove impurities, and collect the virus to obtain IRES-hINS, hINS, and hINSnf lentiviruses.

[0061] Example 3: Transfection of 293T cells with IRES-hINS, hINS, and hINSnf lentiviruses

[0062] The IRES-hINS, hINS, and hINSnf lentiviruses collected in Example 2 were used to transfect 293FT cells: On the first day, cells were seeded, counted, and the cell density was adjusted to one-third of the bottom area of ​​a 6cm culture dish; on the second day, the virus was infected, the 6cm culture dish was removed, the supernatant was discarded, the virus and 293FT cell culture medium were mixed at a volume ratio of 1:1 and added, and 8 mg / mL polybrene was added to make the effective concentration 8 μg / mL. The dish was placed in an incubator and incubated for 8 hours; after 8 hours of incubation, the supernatant was discarded, the cell medium was changed, and the dish was incubated for another two days; after two days of culture, the supernatant was discarded, and the cells were diluted with 1000 mg / mL puromycin at a volume ratio of 1:1000 and screened for 2-3 days; the cells were reseeded: the cells after two days of screening were digested and reseeded into the culture dish to obtain resistant IRES-hINS-293T, hINS-293T, and hINSnf-293T cells for subsequent experiments.

[0063] Example 4: Glucose shock test

[0064] To evaluate the role of the furin cleavage site and the function of IRES in response to glucose concentration, we performed a glucose shock assay. The method was as follows: IRES-hINS-293T, hINS-293T, and hINSnf-293T cells were seeded into 6-well plates. After 1 week of culture, the cells were washed and incubated for 3 h in 10% FBS / DMEM supplemented with low glucose (3.3 mM D-glucose), followed by incubation for 1 h in DMEM medium containing 0.1% BSA. Finally, the medium was changed to DMEM medium containing 0 mM / L, 5 mM / L, 15 mM / L, or 25 mM glucose (high glucose) and 0.1% BSA, and incubated for 1.5 h. The supernatant was then collected, and the amount of insulin released was determined by ELISA.

[0065] Figure 1 This refers to the concentration of insulin released by IRES-hINS-293T cells, hINS-293T cells, and hINSnf-293T cells in response to different concentrations of glucose. For example... Figure 1 As shown, under glucose stimulation, insulin secretion was detectable in both IRES-hINS-293T and hINS-293T cells. However, no insulin secretion was detected in the culture supernatant of hINSnf-293T cells, which lack a furin protease cleavage site, at various blood glucose concentrations. This suggests that hINSnf-293T cells cannot secrete insulin, indicating that the furin protease cleavage site is essential for the synthesis of mature insulin in this invention. Figure 1As shown, IRES-hINS-293T cells can respond differently to different glucose concentrations, and the amount of insulin released varies with blood glucose concentration. However, hINS-293T cells, lacking the glucose-responsive IRES sequence, maintain a relatively constant insulin secretion in culture media with varying glucose concentrations. Therefore, the IRES-hINS sequence designed in this invention can respond to changes in blood glucose concentration in the culture medium after cell transfection, increasing or decreasing insulin synthesis levels. sequence list <110> Southeast University <120> IRES-hINS recombinant genes, lentiviruses, their construction methods and applications <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 333 <212> DNA <213> Artificial Sequence of Recombinant Insulin Gene <400> 1 atggccctgt ggatgcgcct cctgcccctg ctggcgctgc tggccctctg gggacctgac 60 ccagccgcag cctttgtgaa ccaacacctg tgcggctcac acctggtgga agctctctac 120 ctagtgtgcg gggaacgagg cttcttctac acacccagga ccaagcggga ggcagaggac 180 ctgcaggtgg ggcaggtgga gctgggcggg ggccctggtg caggcagcct gcagcccttg 240 gccctggagg gatcccggca gaagcgtggc attgtggaac aatgctgtac cagcatctgc 300 tccctctacc agctggagaa ctactgcaac tag 333 <210> 2 <211> 333 <212> DNA <213> Antisense strand of recombinant insulin gene (Artificial Sequence) <400> 2 ctagttgcag tagttctcca gctggtagag ggagcagatg ctggtacagc attgttccac 60 aatgccacgc ttctgccggg atccctccag ggccaagggc tgcaggctgc ctgcaccagg 120 gcccccgccc agctccacct gccccacctg caggtcctct gcctcccgct tggtcctggg 180 tgtgtagaag aagcctcgtt ccccgcacac taggtagaga gcttccacca ggtgtgagcc 240 gcacaggtgt tggttcacaa aggctgcggc tgggtcaggt ccccagaggg ccagcagcgc 300 cagcaggggc aggaggcgca tccacagggc cat 333 <210> 3 <211> 570 <212> DNA <213> Sense strand of IRES gene (Artificial Sequence) <400> 3 tccctccccc ccccntaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt 60 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc 120 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca 180 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac 240 gtctgtagcg accctttgca ggcagcgga ccccccacct ggcgacaggt gcctctgcgg 300 360. ccaaagcca cgtgtataag atacacctgc aaaggcggca caccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtacacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt 540 ggttttcctt tgaaaacac throws <210> 4 <211> 570 <212> DNA <213> IRES Enhanced Sequence (Artificial Sequence) <400> 4 tattatcatc gtgtttttca aaggaaacc acgtccccgt ggttcggggg gcctagcgt ttttttaacc tcgactaaac acatgtaaag catgtgtacc gaggccccag atcagatccc attackggg gtaccttctg ggcatccttc agccccttgt tgaatacgct tgaggagagc 240. catttgactc tttccacaac tatccaactc acaacgtggc actggggttg tgccgccttt gcaggtgtat cttatacacg tggcttttgg ccgcagaggc acctgtcgcc aggtgggggg 300 ttccgctgcc tgcaaagggt cgctacagac gttgtttgtc ttcaagaagc ttccagagga 360 actgcttcct tcacgacatt caacagacct tgcattcctt tggcgagagg ggaaagaccc 420 ctaggaatgc tcgtcaagaa gacagggcca ggtttccggg ccctcacatt gccaaaagac 480 ggcaatatgg tggaaaataa catatagaca aacgcacacc ggccttattc caagcggctt 540 cggccagtaa cgttangggg gggggaggga 570 <210> 5 <211> 1577 <212> DNA <213> IRES-hINS Recombinant Gene (Artificial Sequence) <400> 5 tccctccccc ccccntaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt 60 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc 120 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca 180 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac 240 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg 300 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt 360 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct 420 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtacacatg 480 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt 540 ggttttcctt tgaaaaacac gatgataata tggaattcat gaccgagtac aagcccacgg 600 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg 660 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg 720 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg 780 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt 840 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac 900 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg 960 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag 1020 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc 1080 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac 1140 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgagg atccggcgca acaaacttct 1200 ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accgatggcc ctgtggatgc 1260 gcctcctgcc cctgctggcg ctgctggccc tctggggacc tgacccagcc gcagcctttg 1320 tgaaccaaca cctgtgcggc tcacacctgg tggaagctct ctacctagtg tgcggggaac 1380 gaggcttctt ctacacaccc aggaccaagc gggaggcaga ggacctgcag gtggggcagg 1440 tggagctggg cgggggccct ggtgcaggca gcctgcagcc cttggccctg gagggatccc 1500 ggcagaagcg tggcattgtg gaacaatgct gtaccagcat ctgctccctc taccagctgg 1560 agaactactg caactag 1577 <210> 6 <211> 57 <212> DNA <213> Forward strand of P2A gene (Artificial Sequence) <400> 6 gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccg 57 <210> 7 <211> 57 <212> DNA <213> P2A gene antisense strand (Artificial Sequence) <400> 7 cggtccagga ttctcttcga catctccggc ttgtttcagc agagagaagt ttgttgc 57

Claims

1. The IRES-hINS recombinant gene, characterized in that, The IRES-hINS recombinant gene includes a recombinant insulin gene containing a furin protease cleavage site and a sugar-responsive IRES gene. The sense strand of the oligonucleotide sequence of the recombinant insulin gene containing the furin protease cleavage site is shown as SEQ ID NO:1, and its antisense strand sequence is shown as SEQ ID NO:

2. The sense strand of the oligonucleotide sequence of the sugar-responsive IRES gene is shown as SEQ ID NO:3, and its antisense strand sequence is shown as SEQ ID NO:

4. The nucleotide sequence of the IRES-hINS recombinant gene is shown as SEQ ID NO:

5.

2. An expression cassette, which contains the IRES-hINS recombinant gene described in claim 1.

3. A recombinant vector, which contains the IRES-hINS recombinant gene described in claim 1.

4. A recombinant cell, which contains the IRES-hINS recombinant gene described in claim 1.

5. The recombinant vector according to claim 3, wherein The recombinant vector is pLVX-IRES-hINS.

6. The method for constructing the recombinant vector according to claim 5, characterized in that, The recombinant vector is obtained by successively connecting a sugar-responsive IRES gene, a P2A protein cleavage sequence, a recombinant insulin gene sequence containing a furin protease cleavage site to the recombinant vector.

7. A lentivirus, which contains the IRES-hINS recombinant gene described in claim 1 or contains the recombinant vector described in claim 3 or 5.

8. Use of the IRES-hINS recombinant gene described in claim 1, the expression cassette described in claim 2, the recombinant vector described in claim 3 or 5, the recombinant cell described in claim 4, or the lentivirus described in claim 7 in the preparation of a gene recombinant insulin-related therapeutic drug.

9. Use of the IRES-hINS recombinant gene described in claim 1, the expression cassette described in claim 2, the recombinant vector described in claim 3 or 5, the recombinant cell described in claim 4, or the lentivirus described in claim 7 in the preparation of a drug related to the treatment of diabetes or diabetic complications.

10. Use of the IRES-hINS recombinant gene described in claim 1, the expression cassette described in claim 2, the recombinant vector described in claim 3 or 5, the recombinant cell described in claim 4, or the lentivirus described in claim 7 in the preparation of a drug for the treatment of hyperglycemia.

11. Use of the IRES-hINS recombinant gene described in claim 1, the expression cassette described in claim 2, the recombinant vector described in claim 3 or 5, the recombinant cell described in claim 4, or the lentivirus described in claim 7 in the preparation of a drug for the treatment of diabetic foot.

12. A drug for treating diabetes, characterized in that, It contains the IRES-hINS recombinant gene described in claim 1.

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