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A kind of overexpression zeb2 gene plasmid and its construction method and application
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An overexpression and gene technology, applied in the field of bioengineering, can solve problems such as incomplete understanding of the mechanism
Active Publication Date: 2020-10-27
ANHUI MEDICAL UNIV
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At present, it is believed that AKI is also a disease mediated by inflammatory response, but the mechanism by which ischemia-reperfusion initiates renal tissue inflammatory response is not fully understood
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Embodiment 1
[0057] An overexpression ZEB2 gene plasmid of this embodiment is constructed by recombination of the ZEB2 gene CDS sequence and the pEGFP-C2 eukaryotic expression vector; its nucleotide is as shown in the sequence table SEQ ID NO: 1, located at the 134th position of the ZEB2 gene -position 3778; the amino acid sequence is shown in the sequence table SEQ ID NO: 2; in addition, the structure of the pEGFP-C2 eukaryotic expression vector is as follows figure 1 shown.
Embodiment 2
[0059] A method for constructing an overexpression ZEB2 gene plasmid of the present embodiment comprises the following steps:
[0060] 1. Extraction of ZEB2 cDNA
[0061] (1) Take out the cell culture dish and discard the clean medium, wash the cell surface of the cultured HK-2 cells with 1ml of PBS buffer solution (2-3 times);
[0062] (2) Add 1ml of Trizol to lyse the cells for 10 minutes, mix the cells gently with a pipette, transfer the cell lysate to a centrifuge tube, invert and mix well, and let stand at room temperature for 5 minutes;
[0063] (3) Add 1 / 5 of Trizol in chloroform (pre-cooled at 4°C), mix vigorously, let stand at room temperature for 5 minutes, and centrifuge at high speed and low temperature for 15 minutes (4°C, 12000r / min);
[0064] (4) Transfer 0.4ml of the upper liquid to a new centrifuge tube, add an equal volume of isopropanol, invert and mix well, let stand for 60min (-20°C), centrifuge at high speed and low temperature for 15min (4°C, 12000r / min...
Embodiment 3
[0136] An expression of an overexpression ZEB2 gene plasmid, after obtaining the overexpression ZEB2 gene plasmid according to Example 2, it is expressed, and the specific steps are as follows:
[0138] The cells required to be cultured in the present invention are: HEK, 293T, THP-1, rat peritoneal macrophages, HK-2 cells, first resuscitated cells, and cultured with DMEM high glucose containing 10% fetal bovine serum based on 37 ℃, 5% CO2 in a cell cultureincubator, and timely replacement of medium and passage.
[0139] (1) Cellrecovery: Take out the cells frozen in liquid nitrogen, quickly immerse them in warm water at 37°C and shake gently until they are completely melted (rewarming time should be controlled within two minutes), and quickly transfer the cell suspension to a centrifuge tube Add 5ml medium, centrifuge at low speed (1 000r / min, 2min), discard the supernatant, add 5ml medium to gently suspend the cells, inoculate them into a petri dis...
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Abstract
The invention discloses an overexpression ZEB2 geneplasmid which is obtained by reconstructing a ZEB2 gene CDS and a pEGFP-C2 eukaryotic expression vector. A nucleotide sequence of the plasmid is shown in SEQ ID NO (sequence identifier number): 1 in a sequence table and located in the 134-3778 site of a ZEB2 gene, and an amino acid sequence of the plasmid is shown in SEQ ID NO: 2 in the sequence table. The invention further discloses a construction method and an application of the overexpression ZEB2 gene plasmid. The plasmid, the method and the application have the advantages that (1) research of a function of the ZEB2 gene is facilitated; a useful molecular biology tool is provided for researching an effect of the ZEB2 gene on inflammation in a course of an AKI (acute kidney injury), and (2) a foundation is laid for further research of the function of ZEB2 and gene therapy of the AKI.
Description
technical field [0001] The invention relates to the technical field of bioengineering, in particular to a plasmid for overexpressing ZEB2 gene and its construction method and application. Background technique [0002] Acute kidney injury (acute kidney injury, AKI) is one of the most common critical illnesses in clinical departments, which can be caused by a variety of reasons. The mechanism of AKI has always been an important scientific issue in the field of nephrology. At present, it is believed that AKI is also a disease mediated by inflammatory response, but the mechanism by which ischemia-reperfusion initiates inflammatory response in renal tissue is not fully understood. From mildly elevated serumcreatinine to severe anuric AKI, the degree and clinical manifestations of AKI vary. AKI is closely related to the mortality of patients, the occurrence of chronic kidneydisease (CKD) and the rate of maintenance dialysis. Epidemiological survey data show that the incidence o...
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