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Plasmid vector capable of simultaneously expressing PLAUR and GPLD1 genes and construction method of plasmid vector

A plasmid vector and construction method technology, applied in the field of biomedicine, can solve problems such as instability and achieve high repeatability

Active Publication Date: 2021-12-07
GUANGDONG BEATING ORIGIN REGENERATIVE MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

uPAR and uPA have been shown to participate in a wide variety of pathophysiological processes, including: proteolysis, inflammation, atherosclerosis, angiogenesis, plaque instability, and fibrinolysis; The occurrence and development of the disease are closely related, but the specific mechanism remains to be further studied

Method used

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  • Plasmid vector capable of simultaneously expressing PLAUR and GPLD1 genes and construction method of plasmid vector
  • Plasmid vector capable of simultaneously expressing PLAUR and GPLD1 genes and construction method of plasmid vector
  • Plasmid vector capable of simultaneously expressing PLAUR and GPLD1 genes and construction method of plasmid vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of PLAUR-T2A-GPLD1-T2A-Puro-T2A-EGFP plasmid

[0033] The commercial pCW-Cas9-Blast plasmid (Addgen, 83481); the original plasmid of green fluorescent protein (EGFP) was purchased from Addgene; the original plasmid of Puro resistance was purchased from Addgene.

[0034] All restriction enzymes and T4 ligase were purchased from Thermo Fisher Scientific; plasmid mini-extraction and purification kits, DNA gel recovery and purification kits were purchased from Axygen; chemically competent cells DH5α strain: purchased from Shanghai Sangong Bioengineering Ltd.

[0035] 1.1 Transformation of pCW-Cas9-Blast vector

[0036] The reason for choosing pCW-Cas9-Blast as the vector is that NheI, AgeI, SbfI, EcorI, NsiI, MluI, AvrII, and BamhI are single enzyme cutting sites in this vector, and it has a tetracycline-induced expression system.

[0037] Artificially synthesize a piece of DNA, the fragment contains the restriction endonuclease NheI-AgeI-SbfI-Ecor...

Embodiment 2

[0064] Example 2: Packaging of PLAUR-T2A-GPLD1-T2A-Puro-EGFP lentivirus

[0065] In the following examples, the culture medium used has:

[0066] The culture medium of HEK293T cells is composed of: 10% fetal bovine serum (purchased from HyClone, SH30396.03); 1% PS (purchased from Invitrogen, 10378016); 1% non-essential amino acid NEAA (purchased from Invitrogen, 11140050) ; The balance is DMEM (purchased from Invitrogen, 11965).

[0067] HEK293T cells were inoculated into six-well plates, cultured in DMEM medium containing 10% fetal bovine serum, and transfected when the confluence of the cells reached 70%-80%.

[0068] PLAUR-T2A-GPLD1-T2A-Puro-EGFP (20 μg), pVSVG (10 ug), and psPAX2 (15 ug) were co-transfected into HEK293T cells according to the instructions of Lipofectamine 2000.

[0069] After 6 h, the culture medium was replaced with DMEM containing 10% fetal bovine serum.

[0070] After continuing to cultivate for 60 hours, the culture solution was taken and centrifuge...

Embodiment 3

[0073] Example 3: Infection of HEK293T cells

[0074] Inoculate HEK293T cells into a six-well plate, culture with DMEM medium containing 10% fetal bovine serum, and add virus to infect when the cell confluence reaches 70%-80%. After 24 hours of infection, the culture medium was replaced with fresh DMEM culture medium containing 10% fetal bovine serum (containing tetracycline hydrochloride Dox+puromycin Puro at a final concentration of 2ug / ml) for screening. After 2-3 days of selection, a transformation efficiency of about 30% can be obtained. Then, the cell line expressing EGFP was obtained by sorting by flow cytometry, that is, the PLAUR-GPLD1 cell line was obtained.

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Abstract

The invention discloses a plasmid vector capable of simultaneously expressing PLAUR and GPLD1 genes and a preparation method of the plasmid vector. In the construction process of the plasmid vector, a segment of DNA containing NheI-AgeI-SbfI-EcorI-NsiI-MluI-AvrII-BamhI restriction enzyme cutting sites is firstly designed, the NheI and BamhI are subjected to double restriction enzyme cutting, then fragments are recovered, the recovered fragments are cloned to a pCW-Cas9-Blast vector subjected to double restriction enzyme cutting through the same restriction enzyme cutting sites, so that a new cloning vector pCW-DNA-Blast is obtained. Amplifying the new cloning vector pCW-DNA-Blast to obtain PLAUR and GPLD1 gene segments with the restriction enzyme cutting sites, and connecting the PLAUR and GPLD1 gene segments with pCW-DNA-Blast after the same restriction enzyme cutting treatment. According to the method, the PLAUR gene and the GPLD1 gene are simultaneously expressed through the plasmid vector for the first time, so that a large amount of PLUAR protein is obtained from cell culture supernatant.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a plasmid vector for simultaneously expressing PLAUR and GPLD1 genes and a construction method thereof. Background technique [0002] Myocardial infarction (AMI) refers to myocardial ischemic necrosis caused by acute and persistent ischemia and hypoxia. Coronary atherosclerotic lesions and myocardial lesions can be seen in most patients. The occurrence of myocardial infarction is the result of the joint action of genes and environmental factors. Genetic and environmental factors affect the occurrence and development of atherosclerosis, the morphology and metabolism of coronary plaque, the instability of plaque, the balance of coagulation and fibrinolytic system, and the formation of thrombus. [0003] Urokinase-type plasminogen activator receptor (uPAR, also known as PLAUR) is a glycoprotein with a molecular weight of 55-60kD, consisting of 313 amino acid residue...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/12C12N15/55C12N15/65C12N15/66
CPCC12N15/86C07K14/70596C12N9/16C12N15/65C12N15/66C12Y301/0405C12N2800/107C12N2740/15043
Inventor 林彬林泽斌许恒王萍周丽诗
Owner GUANGDONG BEATING ORIGIN REGENERATIVE MEDICINE CO LTD
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