Muscle-derived cell blood glucose response type expression SIA promoter, recombinant vector and construction method and application thereof

A recombinant vector and construction method technology, applied in the field of biomedicine, can solve the problems of short cell life cycle and cannot achieve long-term therapeutic effect, and achieve the effects of avoiding hypoglycemia side effects, simple construction method and convenient operation.

Active Publication Date: 2022-07-29
SICHUAN UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

And the microcapsule encapsulation technology adopted for cell implantation in the body, the short life cycle of cells cannot achieve long-term therapeutic effect

Method used

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  • Muscle-derived cell blood glucose response type expression SIA promoter, recombinant vector and construction method and application thereof
  • Muscle-derived cell blood glucose response type expression SIA promoter, recombinant vector and construction method and application thereof
  • Muscle-derived cell blood glucose response type expression SIA promoter, recombinant vector and construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0040] The present invention will be further described below in conjunction with the accompanying drawings and examples. The protection scope of the present invention is not limited to the following: Example 1: pGREMS promoter cell specificity experiment

[0041] 1. Preparation of Reagents

[0042] C2C12 mouse myoblasts, 293T human embryonickidney cells, HEK-293 human embryonic kidney cells (Human Embryonic Kidney 293), NIH3T3 mouse embryo fibroblast cell All were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The reagent formulations used are shown in Table 1.

[0043] Table 1 Reagent formula

[0044]

[0045] 2. Construction of the plasmid

[0046] A blood glucose-responsive enhanced muscle-specific promoter pGREMS was designed, and the pGREMS sequence was cloned by overlapping PCR and cloned into the pSC plasmid, and the sequence was checked to ensure that the sequence was completely accurate. Plasmi...

Embodiment 2

[0067] Example 2: SIA plasmid in vivo experiment

[0068] C57BL / 6J male mice, purchased at 6 weeks old, were adaptively fed for 1 week. Establishment of type 1 diabetes mouse model: The model mice were injected with STZ solution (60 mg / kg) on ​​an empty stomach for 5 consecutive days. For injection, STZ was dissolved in citrate buffer (pH 4.5) and prepared on ice protected from light. After the injection, fast for 6 hours. From the third day after the injection, the blood of the mice was collected by the tail clipping method, the tail tip was cut off by 1-2 mm, the first drop was discarded, and the blood glucose was detected with a Roche blood glucose meter. It was considered that the diabetes model was constructed successfully.

[0069] The successfully modeled mice were randomly divided into groups: T1D group (modeling, no treatment), Exp1 group (modeling, injected with pGREMS-SIA3), Exp2 group (modeling, injected with pGREMS-SIA4) and Exp3 group ( Modeling, injection of...

Embodiment 3

[0090] Example 3: Detection of oxidative stress markers

[0091] 1 Detection of reduced glutathione (GSH) content in kidney and serum of mice

[0092] GSH detection According to the instructions of the Solebao reduced glutathione (GSH) content detection kit (Cat: BC1175) operation 2 Malondialdehyde (MDA) content detection

[0093] MDA detection was performed according to the instructions of Solebao Malondialdehyde (MDA) Content Detection Kit (Cat: BC0025) 3 Superoxide dismutase (SOD) activity detection

[0094] SOD detection was performed according to the instructions of Solebao Superoxide Dismutase (SOD) Activity Detection Kit (Cat: BC0175) 4 Total Antioxidant Capacity (T-AOC) Detection

[0095] The T-AOC test was performed in accordance with the instructions of the Solebao Total Antioxidant Capacity (T-AOC) Test Kit (Cat: BC1315)

[0096] The contents of four oxidative stress markers GSH, T-AOC, MDA and SOD in serum and kidney of mice in each group were detected 60 days af...

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Abstract

The invention discloses a muscle-derived cell blood glucose response type expression SIA promoter, a recombinant vector and a construction method and application thereof, and belongs to the technical field of biological medicine.The blood glucose response type muscle specific promoter pGREMS is used for regulating and controlling expression of SIA, high-activity single-chain insulin analogues SIA3 and SIA4 are obtained, and the expression of the SIA is regulated and controlled. SIA7 is specifically and efficiently expressed at the skeletal muscle part and secreted into blood, so that the effect of effectively reducing blood sugar can be achieved. Meanwhile, the designed and screened promoter has a blood glucose responsive function, so that insulin expression can be closed when the blood glucose is relatively low, and the side effect of hypoglycemia caused by overexpression of insulin is avoided. The recombinant expression vector provided by the invention can be applied to preparation of medicines for treating diabetes mellitus and is particularly suitable for type I diabetes mellitus, and the provided recombinant expression vector is simple in construction method, convenient to operate and suitable for industrial large-scale production.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a promoter, a recombinant vector and a construction method and application of myogenic cell blood sugar-responsive expression of SIA. Background technique [0002] Insufficient insulin secretion is the core cause of diabetes, and the treatment of type 1 diabetes (T1D) requires lifelong use of exogenous insulin. In the past 100 years, the development of insulin therapy has made great strides, with the development of new insulin formulations and delivery methods including new insulins and insulin analogs, continuous subcutaneous insulin infusion without and with sensors, prediction of hypoglycemia Suspension function and closed loop system that continuously adjusts automatically based on patient continuous glucose monitoring readings. The development of these technologies has expanded the range of products, giving clinicians the possibility to achieve tight glycemi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/66C12N15/85A61K38/28A61K48/00A61P3/10
CPCC12N15/113C12N15/66C12N15/85A61K48/0058A61K38/28A61P3/10C12N2800/107Y02A50/30
Inventor 王刚谢丽芳卢宛翎邓璐张嫣菡
Owner SICHUAN UNIV
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