Fusion gene for modifying mesenchymal stem cells, plasmid, stem cells obtained through modification and preparation method

A technology of mesenchymal stem cells and gene fusion, applied in the field of genetic engineering, can solve the problems that the role has not been fully explored

Pending Publication Date: 2021-04-23
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is clear that mesenchymal stem cells have a regenerative role in injured tissue,...

Method used

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  • Fusion gene for modifying mesenchymal stem cells, plasmid, stem cells obtained through modification and preparation method
  • Fusion gene for modifying mesenchymal stem cells, plasmid, stem cells obtained through modification and preparation method
  • Fusion gene for modifying mesenchymal stem cells, plasmid, stem cells obtained through modification and preparation method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Modified fusion gene (Klotho-FGF23) of mesenchymal stem cells, including Klotho nucleic acid artificial sequence, self-cleaving polypeptide T2A nucleic acid artificial sequence and FGF23 nucleic acid artificial sequence, and such as figure 1 As shown, the artificial nucleic acid sequence of Klotho, the artificial nucleic acid sequence of the self-cleaving polypeptide T2A and the artificial nucleic acid sequence of FGF23 are concatenated in sequence, and its nucleic acid sequence is shown in SEQ ID NO.1. Wherein, the artificial nucleic acid sequence of Klotho is listed as SEQ ID NO.2; the artificial nucleic acid sequence of the self-cleaving polypeptide T2A is SEQ ID NO.3; the artificial nucleic acid sequence of FGF23 is SEQ ID NO.3.

Embodiment 2

[0045] A plasmid containing a fusion gene for modifying mesenchymal stem cells. The plasmid is prepared by the following method: the fusion gene of the modified mesenchymal stem cell is synthesized, inserted into the pAV-EF1α-GFP vector, transformed into E.coli (TOP10), and after the sequencing is correct, the plasmid extraction kit is used to extract and purify Plasmids, to obtain recombinant expression vector plasmids.

[0046] In more detail, the preparation method provided in this embodiment includes the following steps:

[0047] The nucleic acid artificial sequence of the fusion gene Klotho-FGF23 was entrusted to Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize its entire expression frame and insert it into the standard vector pUC, so it was named pUC-Klotho-FGF23, and pUC-Klotho-FGF23 and The pAV-EF1α-GFP vector was digested with Fast Digest BamHI (purchased from ThermoFisher) and Fast Digest HindIII (purchased from ThermoFisher) at 37° C. for 1 hour. 100μl enz...

Embodiment 3

[0052] Preparation of umbilical cord mesenchymal stem cells

[0053] Take the neonatal umbilical cord donated by the hospital, disinfect it twice with 75wt% alcohol in the ultra-clean workbench, put it in a petri dish, use tweezers to peel off the Fahrenheit jelly tissue, and cut it to 0.5mm with scissors 2 Small pieces of size. Transfer the shredded Wahrenheit jelly tissue to a culture bottle, add UltraCULTURE medium containing plasma substitute for culture, and observe with a microscope every day. When the stem cells that climbed out covered 80% of the bottom of the bottle, they were subcultured. After subculture, the cell growth rate was accelerated, and they were passed to the P4 generation for experiments every 2-3 days (see figure 2 ).

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Abstract

The invention discloses a fusion gene for modifying mesenchymal stem cells, a plasmid, stem cells obtained through modification and a preparation method. The fusion gene comprises a Klotho nucleic acid artificial sequence, a self-shearing polypeptide T2A nucleic acid artificial sequence and an FGF23 nucleic acid artificial sequence, and the Klotho nucleic acid artificial sequence, the self-shearing polypeptide T2A nucleic acid artificial sequence and the FGF23 nucleic acid artificial sequence are sequentially connected in series. The mesenchymal stem cells modified by the fusion gene prepared by the invention are obviously higher than normal stem cells Klotho and FGF23 in expression quantity, and the continuous expression effect on the Klotho and the FGF23 can be realized.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a fusion gene and a plasmid for modifying mesenchymal stem cells, stem cells obtained through modification and a preparation method. Background technique [0002] Chronic kidney disease (CKD) is a growing international problem, affecting 753 million people worldwide in 2016, including 417 million women and 336 million men. Kidney Klotho RNA is reduced in CKD patients, a clinical observation confirmed in many preclinical models. Moreover, unilateral nephrectomy and contralateral ischemia-reperfusion injury can down-regulate the expression of renal Klotho protein and mRNA. Klotho expression was also reduced in the chronic glomerulonephritis model. Klotho overexpression improves renal function and improves renal histology. Coronary artery calcification is very prevalent in CKD patients, increasing cardiovascular morbidity and mortality. Klotho-FGF23 plays an importan...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/861C12N5/10C12N5/0775A61K35/28A61K48/00A61K38/47A61P3/10A61P9/10A61P11/00A61P13/12A61P17/00A61P25/28A61P29/00A61P35/00A61P37/02A61P39/06
CPCC07K14/50C12N9/2402C12N15/86C12N5/0668C12Y302/01031A61K35/28A61K48/0008A61K48/005A61K38/47A61P29/00A61P35/00A61P13/12A61P25/28A61P9/10A61P3/10A61P37/02A61P11/00A61P39/06A61P17/00C07K2319/00C12N2710/10343C12N2800/107C12N2510/00C12N2509/10A61K2300/00
Inventor 刘明录王立新韩庆梅冯建海金海锋卢永灿许淼万磊张传鹏
Owner SHANDONG XINRUI BIOTECH CO LTD
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