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IRES-hINS recombinant gene, lentivirus, and construction method and application of IRES-hINS recombinant gene

A technology of recombining genes and genes, applied in the direction of reverse transcription RNA virus, application, virus, etc., can solve the problems of low proportion of functional cells, poor activity, difficult to achieve insulin secretion efficiency, etc., achieving huge social and economic benefits, and small side effects. Effect

Pending Publication Date: 2021-12-14
SOUTHEAST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method generally requires complex cell processing and cell culture differentiation processes, and the proportion of terminally differentiated functional cells obtained in the end is low, with poor activity, and it is difficult to achieve the ideal insulin secretion efficiency.

Method used

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  • IRES-hINS recombinant gene, lentivirus, and construction method and application of IRES-hINS recombinant gene
  • IRES-hINS recombinant gene, lentivirus, and construction method and application of IRES-hINS recombinant gene
  • IRES-hINS recombinant gene, lentivirus, and construction method and application of IRES-hINS recombinant gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The construction of embodiment 1 lentiviral vector

[0038] According to the GenBank database (http: / / www.ncbi.nlm.nih.gov / genbank), the mRNA sequence, P2A sequence, Puro sequence and ires sequence of A chain and B chain of human insulin gene were obtained.

[0039] We further transformed the human insulin gene into gene sequences of insulin α and β chains containing A, B chains and furin cleavage sites. The sense chain of the oligonucleotide sequence of the hINS sequence is:

[0040]

[0041]

[0042] The antisense strand is:

[0043]

[0044] P2A:

[0045] The justice chain is:

[0046]

[0047] The antisense strand is:

[0048]

[0049] Ires (IRES FGF ):

[0050] The justice chain is:

[0051]

[0052]

[0053] The antisense strand is:

[0054]

[0055] The sequence described in the present invention was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. according to the sequence of Ires-P2A-Insulin. The above chain oligonucleotid...

Embodiment 2I

[0059] The preparation of embodiment 2IRES-hINS, hINS and hINSnf lentivirus

[0060] The lentivirus was packaged according to the instructions of Lipofectamine 2000 reagent. The cells transfected with the lentiviral vector prepared in Example 1 were used as the experimental group, while the cells transfected with the pLVX lentiviral empty vector were used as the control group.

[0061] Referring to the lipofectmin2000 reagent instructions, transfer the target plasmid and helper plasmids pMdlg, RSV-REV, and VSV-G into 293FT cells, prepare and collect the virus, and then infect 293FT with lentivirus containing IRES-hINS, bINS, and hINSnf sequences, and use Puromycin Puromycin was used to select resistant cells for experiments.

[0062] The specific steps are as follows: 0.75 μg pMdlg, 0.35 μg RSV-REV, 0.49 μg VSV-G, 0.61 μg lentiviral vectors Plvx-RES-hINS, pLVX-hINS and pLVX-hINSnf were added to 0.5 mL of low serum OPTI-MEM In the culture medium, mix gently and incubate at ro...

Embodiment 3I

[0063] Example 3 IRES-hINS, hINS and hINSnf lentiviral transfection of 293T cells

[0064] Use the IRES-hINS, hINS and hINSnf lentiviruses collected in Example 2 to transfect 293FT cells respectively: on the first day, plant the plate, count the cells, and adjust the cell density to one-third of the bottom area of ​​the 6cm culture dish; on the second day , virus infection, take out the 6cm petri dish, discard the supernatant, mix the virus and 293FT cell culture medium at a volume ratio of 1:1, and add 8mg / mL polybrene (polybrene) to make the effect concentration 8ug / mL, put in Incubate for 8 hours in an incubator; after 8 hours of incubation, discard the supernatant, change the medium for the cells, and continue to culture in the incubator for two days; after culturing for two days, discard the supernatant and dilute with 1000 mg / mL puromycin at a volume ratio of 1:1000 , screening for 2-3 days; re-seeding: Digest the cells after two days of screening and plant them back int...

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Abstract

The invention discloses a recombinant insulin gene containing a furin enzyme cutting site, the positive-sense strand of the oligonucleotide sequence of the recombinant insulin gene containing the furin enzyme cutting site is shown as SEQ ID NO: 1, and the antisense strand sequence of the recombinant insulin gene containing the furin enzyme cutting site is shown as SEQ ID NO: 2. The invention also discloses an IRES-hINS recombinant gene, an expression cassette, a recombinant vector, a recombinant cell, a lentivirus and a construction method and application of the IRES-hINS recombinant gene. The invention also discloses application of the recombinant gene and the like in recombinant insulin-related therapeutic drugs. The IRES-hINS sequence designed by the invention can respond to the change of the blood sugar concentration in a culture medium after cells are transfected, so that the synthesis level of insulin is increased or reduced, and the IRES-hINS sequence has very important significance on treatment of diabetes mellitus.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to an IRES-hINS recombinant gene, a lentivirus and a construction method and application thereof. Background technique [0002] Diabetes has gradually become the number one disease threatening human health, and it is a public health issue that is of great concern to all countries in the world. According to the 2017 global statistics of the "International Diabetes Federation", the proportion of adults with diabetes worldwide is 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 results from the inability of the pancreas to produce enough insulin (type 1 diabetes) or the failure of its own tissue cells to respond properly to insulin (type 2 diabetes). Both type 1 and type 2 diabetes eventually cause the patient's pancreas to fail, resulting in insufficient insulin. Long-term hyperglycemia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/867C12N5/10A61K38/28A61P3/10A61P17/02
CPCC07K14/62C12N15/86A61K38/28A61P3/10A61P17/02C07K2319/50C12N2740/15043
Inventor 孙博肖忠党贾法尔.阿里何聪李裕民
Owner SOUTHEAST UNIV
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