CN107119072B - Over-expression ZEB2 gene plasmid and construction method and application thereof - Google Patents

Abstract

The invention discloses an overexpression ZEB2 gene plasmid, which is constructed by recombining a ZEB2 gene CDS sequence and a pEGFP-C2 eukaryotic expression vector; the nucleotide is shown as SEQ ID NO. 1 of the sequence table, and is positioned at the 134-position 3778 of the ZEB2 gene; the amino acid sequence is shown in a sequence table SEQ ID NO. 2. The invention also discloses a construction method and application of the over-expression ZEB2 gene plasmid. The invention has the advantages that: (1) the method is helpful for researching the function of the ZEB2 gene and provides a useful molecular biological tool for researching the effect of the ZEB2 gene on the AKI course; (2) lays a foundation for further researching the function of ZEB2 and the gene therapy of AKI.

Description

Over-expression ZEB2 gene plasmid and construction method and application thereof

Technical Field

The invention relates to the technical field of biological engineering, in particular to an over-expression ZEB2 gene plasmid and a construction method and application thereof.

Background

Acute Kidney Injury (AKI) is one of the most common critical conditions in various clinical departments and can be caused by various reasons, and the occurrence mechanism of the AKI is always an important scientific problem concerned in the field of kidney diseases. AKI is also currently thought to be an inflammatory response mediated disease, but the mechanism by which ischemia reperfusion initiates the inflammatory response in renal tissue is not fully understood. From mild elevation of blood creatinine to severe anuresis AKI, the extent and clinical manifestations of AKI are variable, and AKI is closely associated with patient mortality, the occurrence of Chronic Kidney Disease (CKD), and maintenance dialysis rates. Epidemiological survey data show that the incidence of AKI increases year by year, from 610 cases (per million) in 1988 to 2888 cases (per million) in 2009, with AKI incidence rates of 3% -5% in common hospitalized patients and 30% -50% higher in ICU; meanwhile, the prognosis and the outcome of AKI are more deeply known. Therefore, it is important to prevent and treat the occurrence and development of AKI and to find effective means and drugs for treating AKI.

Genetic engineering, also known as gene recombination technology, is a technology developed in the seventies of the twentieth century to manipulate DNA in vitro, and is widely used for basic research of diseases, gene therapy, gene immunization, gene vaccines, and the like. The three elements are respectively as follows: enzyme, target gene and vector. The eukaryotic expression vector is a vector used for constructing an eukaryotic gene expression system in genetic engineering, and in recent decades, various scholars conduct related researches on various diseases, genes and the like around the eukaryotic expression vector, do a great deal of work in the aspect of genetic engineering, and open up a new way for medical science research.

ZEB2 is also called ZFHX1B, SIP1, etc., belongs to the E-box binding zinc finger protein (zinc finger E-box binding homeobox) family member of zinc finger structure transcription factor, is a necessary transcription factor in embryonic development, and the first report about ZEB2 is that the mRNA of ZEB2 is found in Xenopus laevis embryo. The ZEB2 gene is located on chromosome 2q22.3 and is encoded by ZFHX1B gene, and comprises 9 exons and 8 introns, and the CDS region is 3645 bp. ZEB2 consists of 1215 amino acid residues, has a molecular weight of approximately 136kD, and comprises 2 zinc finger clusters (zinc finger cluster) and a variable sequence linking the two zinc finger clusters. Each zinc finger in the N-and C-termini is capable of independently binding to the 5' -CACCT sequence in the regulated region of the gene of interest. ZEB2 belongs to a transcription factor, and the initial study considered to be located in the nucleus, and the recent study showed that it is expressed in both cytoplasm and nucleus. It is involved in the regulation of various vital activities such as cell growth, differentiation, apoptosis, embryonic development and inflammatory reaction.

The human ZEB2 gene is registered in GenBank under the registration number BC-127102.1, and is positioned at 2q22.3 and has the full length of 3912bp, wherein a CDS sequence 3645bp encodes 1215 amino acids. Tissue expression profiling analysis of the human ZEB2 gene showed: the gene is expressed in a plurality of tumor (gastric cancer, breast cancer, nasopharyngeal cancer and the like) cells and mast cells of human, and the research finds that the ZEB2 gene is also expressed in rat abdominal cavity macrophages and human monocyte THP-1.

Therefore, in order to further study the function of the ZEB2 gene, a useful molecular biological tool which is helpful for the functional study of the ZEB2 gene and provides a research basis for the role of the ZEB2 gene in the AKI course is urgently needed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and provides an over-expressed ZEB2 gene plasmid which is beneficial to the functional research of ZEB2 gene and provides a research basis for the function of the ZEB2 gene in the AKI disease process, and a construction method and application thereof.

The invention is realized by the following technical scheme: an overexpression ZEB2 gene plasmid is constructed by recombining a ZEB2 gene CDS sequence and a pEGFP-C2 eukaryotic expression vector; the nucleotide is shown as SEQ ID NO. 1 of the sequence table, and is positioned at the 134-position 3778 of the ZEB2 gene; the amino acid sequence is shown in a sequence table SEQ ID NO. 2.

A method for constructing the ZEB2 gene overexpression plasmid comprises the following steps:

(1) cracking HK-2 cells, extracting RNA, and performing reverse transcription to obtain ZEB2 cDNA;

(2) a CDS sequence fragment of the artificial ZEB2 gene is amplified by PCR;

(3) purifying a CDS sequence fragment of the ZEB2 gene;

(4) carrying out double enzyme digestion on the obtained gene fragment and a pEGFP-C2 eukaryotic expression vector by using BamH I and Xba I, and connecting;

(5) the ligation product was transformed into E.coli TG1, and positive clones were picked out for culture, followed by extraction of the plasmid.

In a preferred embodiment of the present invention, after the RNA is extracted in the step (1), the RNA is denatured, specifically including the steps of: the RNA was allowed to stand at 65 ℃ for 5-10min, and then transferred to ice water.

As one of the preferable modes of the invention, the reverse transcription reaction in the step (1) is carried out according to the product instruction of the RT Master Mix reverse transcription kit, and the reaction system of the reverse transcription comprises: 5 × RT Master Mix, RNA, nucleic-free Water; the method comprises the following specific steps:

(1) preparing a reaction system: 5 × RT Master Mix 2 μ L + RNA 2 μ L + Nuclear-free Water6 μ L;

(2) standing the reaction system at 37 deg.C for 15 min;

(3) transferring the reaction system to 50 ℃ and acting for 5 min;

(4) transferring the reaction system to 98 ℃, and acting for 5 min;

(5) transferring the reaction system to 4 ℃, cooling and freezing to obtain the cDNA.

In a preferred embodiment of the present invention, the sequences of the upstream and downstream primers required for PCR amplification of the CDS sequence fragment in step (2) are as follows:

an upstream primer: ZEB2-F: 5'-GGGGTACCCCATGAAGCAGCCGATCAT-3'

A downstream primer: ZEB2-R: 5'-GCTCTAGAGCTCACATGCCATCTTCC-3'.

In a preferred embodiment of the present invention, the PCR amplification reaction in step (2) is performed according to the instruction manual of PrimeSTAR Max amplification enzyme product, and the reaction system of the PCR comprises: 2 x PrimeSTAR Max, cDNA sample, upstream and downstream primers and high-purity water; the method comprises the following specific steps:

(1) preparing a reaction system: 2 XPrimeSTAR Max 25. mu.L + cDNA 1. mu.L + primers each 1. mu.L + high purity water 22. mu.L;

(2) the reaction system is gently mixed and then put into a PCR instrument, and the following procedures are set for operation;

(3) carrying out 1.2% agarose gel electrophoresis on the PCR product;

(4) observing the result and recovering the target DNA fragment.

As one of the preferable modes of the invention, the double enzyme cutting reaction of the PCR product of ZEB2 and pEGFP-C2 vector in the step (4) is carried out according to the instruction of restriction enzyme products, wherein the enzyme cutting system is as follows: BamH I restriction enzyme, Xba I restriction enzyme, PCR product of 10 XBuffer Tango, ZEB2 or pEGFP-C2 vector; the method comprises the following specific steps:

(1) preparing a reaction system: 10 x Buffer Tango 2. mu.L + PCR product of BamH I and Xba I0.5. mu.L + ZEB2 each or 7. mu.L each with pEGFP-C2 vector;

(2) placing the reaction system in a water bath kettle at 37 ℃ and acting for 4-6 h;

(3) carrying out 1-1.2% agarose gel electrophoresis on the enzyme digestion product;

(4) observing the result and recovering the target DNA fragment.

As one preferable embodiment of the present invention, the connection method in the step (4) is specifically as follows:

(1) preparing a dephosphorylation system with the volume of 10 mu L: the vector is dephosphorylated by CIAP1 mu l, 10 XCIAP buffer solution 1 mu l and pEGFP-C2 vector 8 mu l;

(2) prepare 10 μ L of ligation: after dephosphorylation of CIAP, 0.5. mu.l of pEGFP-C2 vector, 1. mu.l of 10 XT 4DNA ligase buffer solution, 1. mu.l of T4DNA ligase and 7.5. mu.l of PCR enzyme digestion purified product;

(3) the reaction system is placed in a 0.5ml centrifuge tube and mixed evenly, and water bath is carried out at 16 ℃ overnight.

As one of the preferred modes of the invention, the specific steps of transforming the ligation product into Escherichia coli TG1 in the step (5) are as follows:

(1) taking out TG1 competent cell stored at-80 deg.C, thawing in ice bath, adding 10 μ l ligation product, mixing, and standing on ice for 30 min;

(2) performing heat shock at 42 deg.C for 90s, rapidly taking out, and standing on ice for 3 min;

(3) adding 500 μ l LB culture solution, and performing shake culture at 37 deg.C and 250rpm for 45 min;

(4) about 100. mu.l of the transformation solution was spread on LB solid medium with the corresponding resistance and cultured upside down at 37 ℃ for 8-12 hours.

An application of the plasmid for overexpressing ZEB2, wherein the plasmid for overexpressing ZEB2 gene is transfected into a human HK-2 cell line to inhibit the secretion of inflammatory factors in human HK-2 cells.

Compared with the prior art, the invention has the advantages that:

(1) the method is helpful for researching the function of the ZEB2 gene and provides a useful molecular biological tool for researching the effect of the ZEB2 gene on the AKI course;

(2) by applying a gene recombination technology, aiming at the ZEB2 gene, a recombinant eukaryotic expression vector pEGFP-C2-ZEB2 is constructed and transfected into a human HK-2 cell strain, the influence of the overexpression of ZEB2 on the expression of inflammatory factors in HK-2 cells is researched, and a foundation is laid for further researching the function of ZEB2 and the gene therapy of AKI.

Drawings

FIG. 1 is a schematic structural diagram of a eukaryotic expression vector pEGFP-C2 overexpressing ZEB2 gene plasmid in example 1;

FIG. 2 is a schematic structural diagram of a eukaryotic recombinant vector pEGFP-C2-ZEB2 of the construction method of the plasmid for overexpressing ZEB2 gene in example 2;

FIG. 3 is a restriction enzyme identification map of a eukaryotic recombinant vector pEGFP-C2-ZEB2 of the construction method of the plasmid for over-expressing ZEB2 gene in example 2;

FIG. 4 is a graph showing the expression of the eukaryotic recombinant vector pEGFP-C2-ZEB2 in HEK293T cells during the expression of a plasmid overexpressing ZEB2 gene in example 3;

FIG. 5 is a graph showing the expression levels of mRNA for inflammatory factors of HK-2 cells (IL-6, TNF-. alpha.) in the transfection group employing the plasmid overexpressing ZEB2 gene in example 4;

FIG. 6 is a graph showing the expression levels of the HK-2 cytokines (IL-6, TNF-. alpha.) protein in the transfection group for the application of one of the plasmids overexpressing ZEB2 gene in example 4.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

The overexpression ZEB2 gene plasmid is constructed by recombining a CDS sequence of a ZEB2 gene and a pEGFP-C2 eukaryotic expression vector; the nucleotide is shown as SEQ ID NO. 1 of the sequence table, and is positioned at the 134-position 3778 of the ZEB2 gene; the amino acid sequence is shown as a sequence table SEQ ID NO. 2; in addition, the structure of the pEGFP-C2 eukaryotic expression vector is shown in FIG. 1.

Example 2

The construction method of the over-expression ZEB2 gene plasmid comprises the following steps:

1. extraction of ZEB2cDNA

(1) Removing the cell culture dish, discarding the culture medium, and washing the cell surface of the cultured HK-2 cells with 1ml of PBS buffer (2-3 times);

(2) adding 1ml of Trizol to lyse the cells for 10min, gently mixing the cells by a pipette, transferring the cell lysate into a centrifuge tube, reversing the mixing, standing at room temperature for 5 min;

(3) adding 1/5 amount of trichloromethane of Trizol (precooling at 4 ℃), mixing vigorously, standing at room temperature for 5min, and centrifuging at high speed and low temperature for 15min (4 ℃, 12000 r/min);

(4) transferring 0.4ml of supernatant liquid into a new centrifuge tube, adding isopropanol with the same volume, reversing, uniformly mixing, standing for 60min (-20 ℃), centrifuging at high speed and low temperature for 15min (4 ℃, 12000r/min), and then removing supernatant;

(5) adding 1ml of 75% ethanol solution, shaking, mixing, centrifuging at high speed and low temperature for 5min (4 deg.C, 12000r/min), removing supernatant, and drying for 5 min;

(6) adding 20 μ L of non-enzyme water to dissolve the precipitate, and performing reverse transcription, or freezing at-80 deg.C;

(7) denaturation of RNA

The extracted RNA is denatured, and the denaturation steps are as follows: after RNA was allowed to stand at 65 ℃ for 5min, it was quickly transferred to ice water and allowed to stand for 2 min.

The reverse transcription reaction system (10. mu.L) was as follows:

the process comprises the following steps:

placing the reaction system at 37 ℃ and acting for 15 min;

② transferring the reaction system to 50 ℃ and acting for 5 min;

thirdly, transferring the reaction system to 98 ℃ and acting for 5 min;

fourthly, transferring the reaction system to 4 ℃, and freezing and storing the reaction system after cooling to obtain the cDNA.

2. Cloning of ZEB2 Gene

(1) Based on the CDS sequence of ZEB2, an upstream primer and a downstream primer are designed, and the primer sequences are respectively as follows:

ZEB2-F: 5'-GGGGTACCCCATGAAGCAGCCGATCAT-3' (containing BamH I cleavage site)

ZEB2-R: 5'-GCTCTAGAGCTCACATGCCATCTTCC-3' (containing Xba I cleavage site)

(2) The enzyme used in the PCR process is preferably PrimeSTAR Max Hi enzyme from Takara, and the reaction system and reaction program are as follows:

reaction system (50 μ L):

and (2) uniformly mixing the reaction system, putting the reaction system into a PCR instrument, and setting the following program to operate:

(3) carrying out 1.2% agarose gel electrophoresis on the PCR product;

(4) observing the result and recovering the target DNA fragment;

the PCR product is subjected to agarose gel electrophoresis, and a comparison with a DNA Marker shows that a band is obtained at 3645bp, and the size of the band is basically consistent with that of a ZEB2 target fragment (including an enzyme cutting site), so that the CDS sequence of the human ZEB2 gene is successfully amplified.

3. Purification of ZEB2 mesh fragment

The kit used for purification is preferably a glue recovery kit from Esika, and the purification steps are as follows:

(1) the agarose gel containing the desired DNA was cut under an ultraviolet lamp, and the gel surface liquid was aspirated off with filter paper and minced. Calculating the weight of the gel, which is equal to 100. mu.l volume per 100 mg;

(2) adding 3 gel volumes of Buffer DE-A, uniformly mixing, heating at 75 ℃ (heating low-melting-point agarose gel at 40 ℃), and intermittently mixing (every 2-3 min) until the gel block is completely melted;

(3) 0.5 volume of Buffer DE-A added to the mixture was mixed well. When the separated DNA fragment is less than 400bp, adding 1 gel volume of isopropanol;

(4) the mixture in step 3 was aspirated, transferred to a DNA preparation tube (placed in a 2ml centrifuge tube), and centrifuged at 12,000rpm for 1 min. Discarding the filtrate;

(5) placing the preparation tube back into a 2ml centrifuge tube, adding 500 μ l Buffer W1, centrifuging at 12,000rpm for 30s, and discarding the filtrate;

(6) placing the preparation tube back to a 2ml centrifuge tube, adding 700 μ l Buffer W2, centrifuging at 12,000rpm for 30s, and discarding the filtrate; the same procedure was followed with a further 700. mu.l Buffer W2 wash for 1min at 12,000 rpm;

(7) placing the prepared tube back into a 2ml centrifuge tube, and centrifuging at 12,000rpm for 1 min;

(8) the preparation tube was placed in a clean 1.5ml centrifuge tube, and 30. mu.l of Eluent or deionized water was added to the center of the preparation membrane, and allowed to stand at room temperature for 1 min. The DNA was eluted by centrifugation at 12000rpm for 1 min.

4. Double digestion of PCR products and eukaryotic expression vectors

The PCR product of ZEB2 and pEGFP-C2 vector are subjected to double enzyme digestion of BamH I and Xba I, and the enzyme digestion system and method are as follows:

enzyme digestion system (10 μ L):

the enzyme digestion method comprises the following steps: the reaction system is put into a 0.5ml centrifuge tube to be mixed evenly, water bath is carried out for 6h at 37 ℃ (the temperature and the time are selected according to the specification of the endonuclease), the result is observed rapidly after 1.2% agarose gel electrophoresis, the target DNA fragment is recovered, and ultraviolet long-term irradiation is avoided.

5. Ligation of the cleavage products

Connecting the products of the double enzyme digestion of ZEB2 and pEGFP-C2, wherein the enzyme used for connecting is preferably T4 ligase; before the connection, in order to avoid the self-cyclization of the carrier, the carrier is subjected to dephosphorylation treatment;

dephosphorylation system (10 μ L): 1. mu.l of CIAP, 1. mu.l of 10 XCIAP buffer solution and 8. mu.l of pEGFP-C2 vector;

ligation system (10 μ L): 0.5 mul of pEGFP-C2 vector after dephosphorylation of CIAP, 1 mul of 10 XT 4DNA ligase buffer solution, 1 mul of T4DNA ligase and 7.5 mul of PCR enzyme digestion purified product;

the connection method comprises the following steps: the reaction system is placed in a 0.5ml centrifuge tube and mixed evenly, and water bath is carried out at 16 ℃ overnight.

6. Conversion of ligation products

Coli competence used for transformation was: TG1, transformation procedure was as follows:

(1) taking out TG1 competent cells stored at-80 deg.C, thawing in ice bath, adding 10 μ l ligation product, mixing, and standing on ice for 30 min;

(2) heat shock at 42 deg.C for 90s (without shaking), rapidly taking out and placing on ice for 3 min;

(3) adding 500 μ l LB culture solution, and performing shake culture at 37 deg.C and 250rpm for 45 min;

(4) about 100. mu.l of the transformation solution was spread on LB solid medium with the corresponding resistance and cultured in an inverted state at 37 ℃ for 12 hours.

7. Small extraction of plasmids

Selecting 8 positive clones, adding the positive clones into a centrifuge tube containing 2ml of LB culture medium (ampicillin resistance), carrying out shaking culture at 37 ℃ and 250rpm for 12h, and then carrying out plasmid extraction on bacterial liquid, wherein the used kit is a small plasmid extraction kit of Aisika company, and the extraction steps are as follows:

(1) picking a monoclonal of bacteria by using a sterilized toothpick, putting the monoclonal into a 15ml centrifuge tube containing 2ml of LB culture solution containing corresponding antibiotics, and carrying out shaking culture at 37 ℃ and 250rpm for 12 h;

(2) transferring about 1ml of the bacterial liquid into a 1.5ml centrifuge tube, centrifuging at 4 ℃ and 12,000rpm for 1min, and collecting supernatant;

(3) adding 250 mul Buffer S1 to suspend the bacteria for even suspension without small bacteria;

(4) adding 250 μ l Buffer S2, gently and fully turning over for 6 times to fully crack the thallus until a transparent solution is formed;

(5) adding 350 μ l Buffer S3, mixing gently and turning up and down sufficiently for 8 times, and centrifuging at 12,000rpm for 10 min;

(6) sucking the centrifugal supernatant obtained in the step 5, transferring the centrifugal supernatant into a preparation tube (placed in a 2ml centrifugal tube), centrifuging at 12000rpm for 1min, and removing the filtrate;

(7) placing the prepared tube back into the centrifuge tube, adding 500 μ l Buffer W1, centrifuging at 12,000rpm for 1min, and discarding the filtrate;

(8) placing the prepared tube back into the centrifuge tube, adding 700 μ l Buffer W2, centrifuging at 12,000rpm for 1min, and discarding the filtrate; in the same manner, 700. mu.l of Buffer W2 was washed once more. Discarding the filtrate;

(9) placing the prepared tube back into a 2ml centrifuge tube, and centrifuging at 12,000rpm for 1 min;

(10) transferring the preparation tube into a new 1.5ml centrifuge tube, adding 80 μ l sterile water in the center of the preparation tube membrane, standing at room temperature for 1min, centrifuging at 12,000rpm for 1min, and discarding the preparation tube;

(11) the sample is placed at-20 ℃ for standby, and FIG. 2 is a structural schematic diagram of a eukaryotic recombinant vector pEGFP-C2-ZEB 2.

8. Carrying out enzyme digestion identification and sequencing on plasmids

Carrying out double enzyme digestion identification of BamH I and Xba I on the extracted plasmid, wherein an enzyme digestion system and a method are as follows:

enzyme digestion system (10 μ L):

the enzyme digestion method comprises the following steps: placing the reaction system in a 0.5ml centrifuge tube, mixing uniformly, performing enzyme digestion for 6h at 37 ℃, performing agarose gel electrophoresis, observing by an ultraviolet spectrometer, photographing by a gel imager, storing, and sending plasmids with correct enzyme digestion identification to a company of biologies for sequencing; wherein, the enzyme digestion identification map of the eukaryotic recombinant vector pEGFP-C2-ZEB2 is shown in figure 3, wherein the mark 1 is pEGFP-C2 double enzyme digestion, the mark 2 is PCR product of ZEB2, and the mark 3 is pEGFP-C2-ZEB2 double enzyme digestion;

the sequencing result is compared by BLAST, and is found to be consistent with the sequence published in GencBank, and the length of the target gene fragment is 3645 bp.

Example 3

Expression of a plasmid overexpressing ZEB2 gene, wherein the plasmid overexpressing ZEB2 gene is obtained according to example 2 and then is expressed, and the specific steps are as follows:

1. cell culture

The cells required to be cultured by the invention are as follows: HEK, 293T, THP-1, rat peritoneal macrophages, HK-2 cells, were first revived and cultured in a cell incubator containing DMEM high-glucose medium containing 10% fetal bovine serum by volume at 37 ℃ and 5% CO2, and timely changed and passaged.

(1) Cell recovery: taking out the cells frozen in the liquid nitrogen, quickly immersing the cells into warm water at 37 ℃, slightly shaking the cells until the cells are completely melted (the rewarming time is controlled within two minutes), quickly transferring cell suspension into a centrifuge tube, adding 5ml of culture medium, removing supernatant after low-speed centrifugation (1000r/min, 2min), adding 5ml of culture medium, slightly suspending the cells, and inoculating the cells into a culture dish to start culture;

(2) cell liquid change: the old cell culture medium was gently discarded, and after gentle washing with PBS, fresh complete medium (high-glucose DMEM containing 10% fetal bovine serum) was added to continue the culture (note: PBS and fresh medium need to be pre-warmed at 37 ℃);

(3) cell passage: adding 1ml of pancreatin into cells, then placing the cells in an incubator for digestion, observing the morphological change of the cells under a microscope, adding a complete culture medium in time to terminate the digestion, transferring cell suspension to a centrifuge tube, centrifuging at a low speed (1000r/min, 2min), then discarding supernatant, washing the cells once by PBS, then adding 0.2ml of culture medium to suspend the cells, and re-inoculating the cells into a culture dish for continuous culture;

(4) freezing and storing cells: and (3) centrifuging the cell suspension in the passage process at a low speed (1000r/min, 2min), discarding the supernatant, adding 1ml of cell cryopreservation liquid (formula), slightly suspending the cells, subpackaging the mixture into cell cryopreservation tubes, and performing fractional cryopreservation, namely 0.5-1h at 4 ℃, 1-2h at-20 ℃ and about 24h at-80 ℃ and performing liquid nitrogen cryopreservation.

2. Plasmid transient transfection (Liposome method)

(1) Diluting 5. mu.l of liposomes and a total of about 2. mu.g of DNA with 250. mu.l of Opti-MEM, gently mixing, and standing at room temperature for 5 min;

(2) the two solutions in the steps are mixed gently and evenly, and are kept stand for 20min at room temperature to form a DNA-liposome compound;

(3) discarding the culture solution in the culture dish, adding 1ml of Opti-MEM transfection solution, slowly dripping the DNA-liposome compound in the step (2) into the culture dish, and slightly shaking the culture dish to mix uniformly;

(4)37℃、5％CO₂after 6h incubation, the cells were washed once with pre-warmed PBS and incubated for about 24h after changing fresh complete medium.

3. Immunoblotting

(1) And (3) total protein extraction: centrifuging to collect cells, washing with PBS for 2-3 times, adding 0.5ml cell lysate, lysing on ice for 10min, freezing at high speed and centrifuging for 10min, collecting 0.4ml supernatant, adding sample buffer, boiling in water bath for 5min, and standing on ice for electrophoresis;

(2) mounting a rubber plate and detecting leakage, firstly preparing 10% separation rubber, pressing a line with 1ml of isopropanol, preparing concentrated rubber after full solidification (about 1 hour), inserting a required comb, and using after full solidification;

(3) installing an electrophoresis system, adding 500ml of electrophoresis buffer solution into an electrophoresis tank, adding 10 mu L of sample into a loading hole, and starting electrophoresis (80V electrophoresis for 0.5h +100V electrophoresis for 1.5 h);

(4) after electrophoresis is finished, cutting off SDS-PAGE gel, and performing wet rotation by using a sandwich mode (sequentially from bottom to top, filter paper, gel, PVDF membrane and filter paper);

(5) installing an electric rotating system, adding 500ml of precooled electric rotating buffer solution into an electric rotating tank, putting the whole electric rotating tank into ice water, and starting electric rotating (100V electric rotating for 2 h);

(6) after the electrotransformation is finished, gently taking down the PVDF membrane, dyeing with ponceau red to identify whether the membrane is successfully transfected, washing off ponceau red by TBST after the membrane is successfully transfected, and sealing at room temperature for at least 1 h;

(7) after blocking, washing the membrane with TBST (3 times, 5min), adding primary antibody solution, and incubating overnight at 4 ℃;

(8) the next day, after washing the membrane with TBST (3 times, 10min), the secondary antibody solution was added and incubated for 2h at room temperature. The membrane was then washed with TBST (3 times, 20 min);

(9) bands on PVDF were detected with ECL luminophores and developed with X-ray film darkroom.

The experimental result shows that the eukaryotic recombinant expression vector pEGFP-C2-ZEB2 can be normally expressed in eukaryotic cells, and lays a foundation for subsequent research; FIG. 4 is a graph showing the expression of the eukaryotic recombinant vector pEGFP-C2-ZEB2 in HEK293T cells, wherein 1 is an untransfected 293T cell lysate and 2 is a 293T cell lysate transfected with pEGFP-C2-ZEB 2.

Example 4

The application of the plasmid for over-expressing ZEB2 gene is characterized in that the plasmid for over-expressing ZEB2 gene is transfected into a human HK-2 cell line, and the influence of over-expressing ZEB2 gene on the secretion of inflammatory factors (IL-6, TNF-alpha) of the human HK-2 cell line is researched; the method comprises the following specific steps:

1. passaging cells in a logarithmic growth phase to obtain a cell suspension, inoculating the cells into a 6-well plate at a proper density (about 1 x 108. L < -1 >), culturing for about 24 hours until the cell density is about 80%, performing transient transfection on pEGFP-C2-ZEB2, and collecting the cell suspension after a period of transfection;

2. total RNA was isolated from cultured HK-2 cells using Trizol reagent according to the manufacturer's instructions, eluted with RNase-free water and stored at-80 ℃;

3. cDNA was synthesized using the transcript first strand cDNA synthesis kit and real-time PCR was performed using a detection system with SYBR-Green MasterMix, q-PCR primers were obtained from Invitrogen in duplicate, with at least three replicates per measurement, and the q-PCR results are shown in figure 5;

analysis of qRT-PCR of figure 5A shows: transfection of pEGFP-C2-ZEB2 up-regulated the expression of ZEB 2; analysis of the qRT-PCR of figure 5B shows: transfection of pEGFP-C2-ZEB2 down-regulated the expression of TNF-. alpha.and IL-6 (. P <0.05 and. P <0.01 vs. normal group; # P <0.05 and # P <0.01 vs. empty plasmid group);

the combination of the above results shows that: compared with the level of the mRNA of the inflammatory factors (IL-6 and TNF-alpha) of the cells of the untransfected group, the expression of the mRNA of the inflammatory factors IL-6 and TNF-alpha in the HK-2 cells of the pEGFP-C2-ZEB2 transfected group is obviously reduced, and the difference has statistical significance (P is less than 0.05); the over-expression of the ZEB2 gene is proved to be capable of obviously reducing the mRNA level of inflammatory factors (IL-6, TNF-alpha) so as to inhibit inflammatory reaction.

In addition, total protein was extracted and concentration was checked, gel electrophoresis was performed, after wet-converting nitrocellulose membrane, blocking with 5% skim milk for 60min, and 1: 200 dilution of ZEB2 rabbit antibody, 1: after diluting IL-6 and TNF-alpha by 1000 and Beta-Actin internal reference primary antibody by 1: 1000, incubating overnight at 4 ℃, rinsing 3 times by TBST, adding secondary antibody with corresponding concentration, standing for 60min at room temperature, rinsing 3 times by TBST, adding ECL Plus, developing under scotopic condition, analyzing protein bands by Quantity One software, and the Western blot result is shown in figure 6;

analysis of the Western blot of FIG. 6A shows: transfection of pEGFP-C2-ZEB2 up-regulated the expression of ZEB 2; western blot analysis of FIG. 6B showed that transfection of pEGFP-C2-ZEB2 down-regulated the expression of TNF-. alpha.and IL-6 (. SP <0.05 and. SP <0.01 vs. normal; and. SP <0.05 and. SP # 0.01 vs. vector);

the combination of the above results shows that: compared with the levels of the inflammatory factor (IL-6 and TNF-alpha) protein of the cells of the untransfected group, the inflammatory factor (IL-6 and TNF-alpha) protein of the cells of the pEGFP-C2-ZEB2 group is obviously reduced, and the difference is statistically significant (P is less than 0.05); the over-expression of the ZEB2 gene is proved to be capable of obviously reducing the protein level of inflammatory factors (IL-6, TNF-alpha) so as to inhibit inflammatory reaction.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

SEQUENCE LISTING

<110> university of medical in Anhui

<120> overexpression ZEB2 gene plasmid and construction method and application thereof

<130>2017

<160>1

<170>PatentIn version 3.3

<210>1

<211>3645

<212>DNA

<213>human

<400>1

atgaagcagc cgatcatggc ggatggcccc cggtgcaaga ggcgcaaaca agccaatccc 60

aggaggaaaa acgtggtgaa ctatgacaat gtagtggaca caggttctga aacagatgag 120

gaagacaagc ttcatattgc tgaggatgac ggtattgcca accctctgga ccaggagacg 180

agtccagcta gtgtgcccaa ccatgagtcc tccccacacg tgagccaagc tctgttgcca 240

agagaggaag aggaagatga aataagggag ggtggagtgg aacacccctg gcacaacaac 300

gagattctac aagcctctgt agatggtcca gaagaaatga aggaagacta tgacactatg 360

gggccagaag ccacgatcca gaccgcaatt aacaatggta cagtgaagaa tgcaaattgc 420

acatcagatt ttgaggaata ctttgccaaa agaaaactgg aggaacgcga tggtcatgca 480

gtcagcatcg aggagtacct tcagcgcagt gacacagcca ttatttaccc agaagcccct 540

gaggagctgt ctcgccttgg cacgccagag gccaatgggc aagaagaaaa tgacctgcca 600

cctggaactc cagatgcttt tgcccaactg ctgacctgcc cctactgcga ccggggctac 660

aagcgcttga catcactgaa ggagcacatc aagtaccgcc acgagaagaa tgaagagaac 720

ttttcctgcc ctctctgtag ctacacgttt gcctaccgca cccagctcga gcggcatatg 780

gtgacacaca agccagggac agatcagcac caaatgctaa cccaaggagc aggtaatcgc 840

aagttcaaat gcacagagtg tggcaaggcc ttcaaatata aacaccatct gaaagaacac 900

ctgcgaattc acagtggtga aaaaccttac gagtgcccaa actgcaagaa acgtttctcc 960

cattctggtt cctacagttc gcacatcagc agcaagaaat gtattggttt aatctctgta 1020

aatggccgaa tgagaaacaa tatcaagacg ggttcttccc ctaattctgt ttcttcttct 1080

cctactaatt cagccattac ccagttaaga aacaagttgg agaatggaaa accacttagt 1140

atgtctgaac agacaggctt acttaaaatt aaaacagaac cactagactt caatgactat 1200

aaagttctta tggctacaca cgggtttagt ggcactagtc cctttatgaa tggtgggctt 1260

ggagccacca gccctttagg agttcatcca tctgctcaga gtccaatgca gcacttaggt 1320

gtagggatgg aagccccttt acttgggttt cccaccatga atagtaattt aagtgaggta 1380

caaaaggttc tacagattgt ggacaatact gtttccaggc aaaaaatgga ctgcaaggct 1440

gaagaaattt caaagttgaa aggttatcac atgaaggatc catgctctca acctgaggaa 1500

caaggagtta cttctcctaa tattccgcct gtcggtcttc cggtagtgag tcataatggt 1560

gccactaaaa gtattattga ctatacgttg gaaaaagtca atgaagccaa agcttgcctc 1620

cagagcttga ctactgactc aaggagacag atcagtaata taaagaaaga gaagctacgt 1680

actttaatag atttggtcac tgatgacaaa atgattgaga accacaacat atccactcca 1740

ttttcatgcc agttctgtaa agaaagtttt cctggcccca tccctttgca tcagcatgaa 1800

cgttaccttt gtaagatgaa tgaagagatc aaggcggtcc tgcagcctca tgaaaacata 1860

gtccccaaca aagccggagt ttttgttgat aataaagccc tcctcttgtc atctgtactt 1920

tctgagaaag gaatgacaag ccccatcaac ccatacaagg accacatgtc tgtactcaaa 1980

gcatactatg ctatgaacat ggagcccaac tccgatgaac tgctgaaaat ttccattgct 2040

gtgggccttc ctcaggaatt tgtgaaggaa tggtttgaac aacgaaaagt ctaccagtac 2100

tcaaattcca ggtccccatc cctggaaaga agctccaagc cgttagctcc caacagtaac 2160

cctcccacaa aagactcttt attacccagg tctcctgtaa aacctatgga ctccataaca 2220

tcaccatcta tagcagaact ccacaacagt gttacgaatt gtgatcctcc tctcaggcta 2280

acaaaacctt cccattttac caatattaaa ccagttgaaa aattggacca ctccaggagt 2340

aatactcctt ctcccttaaa tctttcctcc acatcttcta aaaactccca cagtagttca 2400

tacactccaa acagcttctc ttctgaggag ctccaggctg agcctttaga cttgtcatta 2460

ccaaaacaaa tgaaagaacc caaaagtatt atagccacaa agaacaaaac aaaagctagt 2520

agcatcagtt tagatcataa cagtgtttct tcctcatctg aaaactcaga tgagcctctg 2580

aacttgactt ttatcaagaa ggaattttca aattcaaata atctggacaa caaaagcact 2640

aacccagtgt tcagcatgaa cccatttagt gccaaacctt tatacacagc tcttccacct 2700

caaagcgcat ttccccctgc tactttcatg ccaccagtcc agaccagtat tcctgggcta 2760

cgaccatacc caggactgga tcagatgagc ttcctaccac atatggccta cacctaccca 2820

actggagcag ctacttttgc tgatatgcag caaaggagaa agtaccagcg gaaacaagga 2880

tttcagggag aattgcttga tggagcacaa gactacatgt caggcctaga tgatatgaca 2940

gactccgact cctgtctgtc tcgcaaaaag atcaagaaga cagagagtgg catgtatgca 3000

tgtgacttat gtgacaagac attccagaaa agcagttccc ttctgcgaca taaatacgaa 3060

cacacaggaa aaagaccaca tcagtgtcag atttgtaaga aagcgtttaa acacaagcac 3120

caccttatcg agcactcaag gcttcactcg ggcgagaagc cctatcagtg tgataaatgt 3180

ggcaagcgct tctcacactc gggctcgtac tcgcagcaca tgaatcacag gtattcctac 3240

tgcaagcggg aggcggagga gcgggaagcg gcggagcgcg aggcgcgcga gaaagggcac 3300

ttggaaccca ccgagctgct gatgaaccgg gcttacttgc agagcattac ccctcagggg 3360

tactctgact cggaggagag ggagagtatg ccgagggatg gcgagagcga gaaggagcac 3420

gagaaagaag gcgaggatgg ctacgggaag ctgggcagac aggatggcga cgaggagttc 3480

gaggaggaag aggaagaaag tgaaaataaa agtatggata cggatcccga aacgatacga 3540

gatgaagaag agactggaga tcactccatg gacgatagtt cggaggatgg gaaaatggaa 3600

accaaatcag accacgagga agacaatatg gaagatggca tgtaa 3645

<210>2

<211>1214

<212>PRT

<213>human

<400>2

Met Lys Gln Pro Ile Met Ala Asp Gly Pro Arg Cys Lys Arg Arg Lys

1 5 10 15

Gln Ala Asn Pro Arg Arg Lys Asn Val Val Asn Tyr Asp Asn Val Val

20 25 30

Asp Thr Gly Ser Glu Thr Asp Glu Glu Asp Lys Leu His Ile Ala Glu

35 40 45

Asp Asp Gly Ile Ala Asn Pro Leu Asp Gln Glu Thr Ser Pro Ala Ser

50 55 60

Val Pro Asn His Glu Ser Ser Pro His Val Ser Gln Ala Leu Leu Pro

65 70 75 80

Arg Glu Glu Glu Glu Asp Glu Ile Arg Glu Gly Gly Val Glu His Pro

85 90 95

Trp His Asn Asn Glu Ile Leu Gln Ala Ser Val Asp Gly Pro Glu Glu

100 105 110

Met Lys Glu Asp Tyr Asp Thr Met Gly Pro Glu Ala Thr Ile Gln Thr

115 120 125

Ala Ile Asn Asn Gly Thr Val Lys Asn Ala Asn Cys Thr Ser Asp Phe

130 135 140

Glu Glu Tyr Phe Ala Lys Arg Lys Leu Glu Glu Arg Asp Gly His Ala

145 150 155 160

Val Ser Ile Glu Glu Tyr Leu Gln Arg Ser Asp Thr Ala Ile Ile Tyr

165 170 175

Pro Glu Ala Pro Glu Glu Leu Ser Arg Leu Gly Thr Pro Glu Ala Asn

180 185 190

Gly Gln Glu Glu Asn Asp Leu Pro Pro Gly Thr Pro Asp Ala Phe Ala

195 200 205

Gln Leu Leu Thr Cys Pro Tyr Cys Asp Arg Gly Tyr Lys Arg Leu Thr

210 215 220

Ser Leu Lys Glu His Ile Lys Tyr Arg His Glu Lys Asn Glu Glu Asn

225 230 235 240

Phe Ser CysPro Leu Cys Ser Tyr Thr Phe Ala Tyr Arg Thr Gln Leu

245 250 255

Glu Arg His Met Val Thr His Lys Pro Gly Thr Asp Gln His Gln Met

260 265 270

Leu Thr Gln Gly Ala Gly Asn Arg Lys Phe Lys Cys Thr Glu Cys Gly

275 280 285

Lys Ala Phe Lys Tyr Lys His His Leu Lys Glu His Leu Arg Ile His

290 295 300

Ser Gly Glu Lys Pro Tyr Glu Cys Pro Asn Cys Lys Lys Arg Phe Ser

305 310 315 320

His Ser Gly Ser Tyr Ser Ser His Ile Ser Ser Lys Lys Cys Ile Gly

325 330 335

Leu Ile Ser Val Asn Gly Arg Met Arg Asn Asn Ile Lys Thr Gly Ser

340 345 350

Ser Pro Asn Ser Val Ser Ser Ser Pro Thr Asn Ser Ala Ile Thr Gln

355 360 365

Leu Arg Asn Lys Leu Glu Asn Gly Lys Pro Leu Ser Met Ser Glu Gln

370 375 380

Thr Gly Leu Leu Lys Ile Lys Thr Glu Pro Leu Asp Phe Asn Asp Tyr

385 390 395 400

Lys Val Leu Met AlaThr His Gly Phe Ser Gly Thr Ser Pro Phe Met

405 410 415

Asn Gly Gly Leu Gly Ala Thr Ser Pro Leu Gly Val His Pro Ser Ala

420 425 430

Gln Ser Pro Met Gln His Leu Gly Val Gly Met Glu Ala Pro Leu Leu

435 440 445

Gly Phe Pro Thr Met Asn Ser Asn Leu Ser Glu Val Gln Lys Val Leu

450 455 460

Gln Ile Val Asp Asn Thr Val Ser Arg Gln Lys Met Asp Cys Lys Ala

465 470 475 480

Glu Glu Ile Ser Lys Leu Lys Gly Tyr His Met Lys Asp Pro Cys Ser

485 490 495

Gln Pro Glu Glu Gln Gly Val Thr Ser Pro Asn Ile Pro Pro Val Gly

500 505 510

Leu Pro Val Val Ser His Asn Gly Ala Thr Lys Ser Ile Ile Asp Tyr

515 520 525

Thr Leu Glu Lys Val Asn Glu Ala Lys Ala Cys Leu Gln Ser Leu Thr

530 535 540

Thr Asp Ser Arg Arg Gln Ile Ser Asn Ile Lys Lys Glu Lys Leu Arg

545 550 555 560

Thr Leu Ile Asp Leu Val ThrAsp Asp Lys Met Ile Glu Asn His Asn

565 570 575

Ile Ser Thr Pro Phe Ser Cys Gln Phe Cys Lys Glu Ser Phe Pro Gly

580 585 590

Pro Ile Pro Leu His Gln His Glu Arg Tyr Leu Cys Lys Met Asn Glu

595 600 605

Glu Ile Lys Ala Val Leu Gln Pro His Glu Asn Ile Val Pro Asn Lys

610 615 620

Ala Gly Val Phe Val Asp Asn Lys Ala Leu Leu Leu Ser Ser Val Leu

625 630 635 640

Ser Glu Lys Gly Met Thr Ser Pro Ile Asn Pro Tyr Lys Asp His Met

645 650 655

Ser Val Leu Lys Ala Tyr Tyr Ala Met Asn Met Glu Pro Asn Ser Asp

660 665 670

Glu Leu Leu Lys Ile Ser Ile Ala Val Gly Leu Pro Gln Glu Phe Val

675 680 685

Lys Glu Trp Phe Glu Gln Arg Lys Val Tyr Gln Tyr Ser Asn Ser Arg

690 695 700

Ser Pro Ser Leu Glu Arg Ser Ser Lys Pro Leu Ala Pro Asn Ser Asn

705 710 715 720

Pro Pro Thr Lys Asp Ser Leu Leu ProArg Ser Pro Val Lys Pro Met

725 730 735

Asp Ser Ile Thr Ser Pro Ser Ile Ala Glu Leu His Asn Ser Val Thr

740 745 750

Asn Cys Asp Pro Pro Leu Arg Leu Thr Lys Pro Ser His Phe Thr Asn

755 760 765

Ile Lys Pro Val Glu Lys Leu Asp His Ser Arg Ser Asn Thr Pro Ser

770 775 780

Pro Leu Asn Leu Ser Ser Thr Ser Ser Lys Asn Ser His Ser Ser Ser

785 790 795 800

Tyr Thr Pro Asn Ser Phe Ser Ser Glu Glu Leu Gln Ala Glu Pro Leu

805 810 815

Asp Leu Ser Leu Pro Lys Gln Met Lys Glu Pro Lys Ser Ile Ile Ala

820 825 830

Thr Lys Asn Lys Thr Lys Ala Ser Ser Ile Ser Leu Asp His Asn Ser

835 840 845

Val Ser Ser Ser Ser Glu Asn Ser Asp Glu Pro Leu Asn Leu Thr Phe

850 855 860

Ile Lys Lys Glu Phe Ser Asn Ser Asn Asn Leu Asp Asn Lys Ser Thr

865 870 875 880

Asn Pro Val Phe Ser Met Asn Pro Phe Ser AlaLys Pro Leu Tyr Thr

885 890 895

Ala Leu Pro Pro Gln Ser Ala Phe Pro Pro Ala Thr Phe Met Pro Pro

900 905 910

Val Gln Thr Ser Ile Pro Gly Leu Arg Pro Tyr Pro Gly Leu Asp Gln

915 920 925

Met Ser Phe Leu Pro His Met Ala Tyr Thr Tyr Pro Thr Gly Ala Ala

930 935 940

Thr Phe Ala Asp Met Gln Gln Arg Arg Lys Tyr Gln Arg Lys Gln Gly

945 950 955 960

Phe Gln Gly Glu Leu Leu Asp Gly Ala Gln Asp Tyr Met Ser Gly Leu

965 970 975

Asp Asp Met Thr Asp Ser Asp Ser Cys Leu Ser Arg Lys Lys Ile Lys

980 985 990

Lys Thr Glu Ser Gly Met Tyr Ala Cys Asp Leu Cys Asp Lys Thr Phe

995 1000 1005

Gln Lys Ser Ser Ser Leu Leu Arg His Lys Tyr Glu His Thr Gly

1010 1015 1020

Lys Arg Pro His Gln Cys Gln Ile Cys Lys Lys Ala Phe Lys His

1025 1030 1035

Lys His His Leu Ile Glu His Ser Arg Leu His Ser Gly Glu Lys

1040 1045 1050

Pro Tyr Gln Cys Asp Lys Cys Gly Lys Arg Phe Ser His Ser Gly

1055 1060 1065

Ser Tyr Ser Gln His Met Asn His Arg Tyr Ser Tyr Cys Lys Arg

1070 1075 1080

Glu Ala Glu Glu Arg Glu Ala Ala Glu Arg Glu Ala Arg Glu Lys

1085 1090 1095

Gly His Leu Glu Pro Thr Glu Leu Leu Met Asn Arg Ala Tyr Leu

1100 1105 1110

Gln Ser Ile Thr Pro Gln Gly Tyr Ser Asp Ser Glu Glu Arg Glu

1115 1120 1125

Ser Met Pro Arg Asp Gly Glu Ser Glu Lys Glu His Glu Lys Glu

1130 1135 1140

Gly Glu Asp Gly Tyr Gly Lys Leu Gly Arg Gln Asp Gly Asp Glu

1145 1150 1155

Glu Phe Glu Glu Glu Glu Glu Glu Ser Glu Asn Lys Ser Met Asp

1160 1165 1170

Thr Asp Pro Glu Thr Ile Arg Asp Glu Glu Glu Thr Gly Asp His

1175 1180 1185

Ser Met Asp Asp Ser Ser Glu Asp Gly Lys Met Glu Thr Lys Ser

1190 1195 1200

Asp His Glu Glu Asp Asn Met Glu Asp Gly Met

1205 1210

Claims (9)

1. An overexpression ZEB2 gene plasmid is characterized by being constructed by recombining a ZEB2 gene CDS sequence and a pEGFP-C2 eukaryotic expression vector; the nucleotide is shown as SEQ ID NO. 1 of the sequence table, and is positioned at the 134-position 3778 of the ZEB2 gene; the amino acid sequence is shown as a sequence table SEQ ID NO. 2;

the method for over-expressing ZEB2 gene plasmid comprises the following steps:

(1) cracking HK-2 cells, extracting RNA, and performing reverse transcription to obtain ZEB2 cDNA;

(2) a CDS sequence fragment of the artificial ZEB2 gene is amplified by PCR;

(3) purifying a CDS sequence fragment of the ZEB2 gene;

(4) carrying out double enzyme digestion on the obtained gene fragment and a pEGFP-C2 eukaryotic expression vector by using BamH I and Xba I, and connecting;

(5) the ligation product was transformed into E.coli TG1, and positive clones were picked out for culture, followed by extraction of the plasmid.

2. The plasmid for overexpressing ZEB2 gene according to claim 1, wherein the RNA extracted in step (1) is denatured by the following steps: the RNA was allowed to stand at 65 ℃ for 5-10min, and then transferred to ice water.

3. The overexpression ZEB2 gene plasmid according to claim 1, wherein the reverse transcription reaction in step (1) is performed according to the product instruction of RT Master Mix reverse transcription kit, and the reaction system of reverse transcription comprises: 5 × RTMaster Mix, RNA, nucleic-free Water; the method comprises the following specific steps:

(1) preparing a reaction system: 5 × RT Master Mix 2. mu.L + RNA 2. mu.L + nucleic-free Water 6. mu.L;

(2) standing the reaction system at 37 deg.C for 15 min;

(3) transferring the reaction system to 50 ℃ and acting for 5 min;

(4) transferring the reaction system to 98 ℃, and acting for 5 min;

(5) transferring the reaction system to 4 ℃, cooling and freezing to obtain the cDNA.

4. The plasmid for overexpressing ZEB2 gene in accordance with claim 1, wherein the sequences of the upstream and downstream primers required for PCR amplification of CDS sequence fragment in the step (2) are as follows:

an upstream primer: ZEB2-F: 5'-GGGGTACCCCATGAAGCAGCCGATCAT-3'

A downstream primer: ZEB2-R: 5'-GCTCTAGAGCTCACATGCCATCTTCC-3'.

5. The plasmid for overexpressing ZEB2 gene according to claim 1, wherein the PCR amplification reaction in the step (2) is performed according to PrimeSTAR Max amplification enzyme product instruction, and the reaction system of PCR comprises: 2 x PrimeSTAR Max, cDNA sample, upstream and downstream primers and high-purity water; the method comprises the following specific steps:

(1) preparing a reaction system: 2 XPrimeSTAR Max 25. mu.L + cDNA 1. mu.L + primers each 1. mu.L + high purity water 22. mu.L;

(2) the reaction system is gently mixed and then put into a PCR instrument, and the following procedures are set for operation;

(3) carrying out 1.2% agarose gel electrophoresis on the PCR product;

(4) observing the result and recovering the target DNA fragment.

6. The plasmid for overexpressing ZEB2 gene in accordance with claim 1, wherein the PCR product of ZEB2 and pEGFP-C2 vector in step (4) are subjected to double digestion reaction of BamH I and Xba I according to the restriction enzyme product instruction as follows: BamH I restriction enzyme, Xba I restriction enzyme, PCR product of 10 XBuffer Tango, ZEB2 or pEGFP-C2 vector; the method comprises the following specific steps:

(1) preparing a reaction system: 10 x Buffer Tango 2. mu.L + PCR product of BamH I and Xba I0.5. mu.L + ZEB2 each or 7. mu.L each with pEGFP-C2 vector;

(2) placing the reaction system in a water bath kettle at 37 ℃ and acting for 4-6 h;

(3) carrying out 1-1.2% agarose gel electrophoresis on the enzyme digestion product;

(4) observing the result and recovering the target DNA fragment.

7. The plasmid overexpressing ZEB2 gene according to claim 1, wherein the ligation method in the step (4) is specifically as follows:

(1) preparing a dephosphorylation system with the volume of 10 mu L: the vector is dephosphorylated by CIAP1 mu l, 10 XCIAP buffer solution 1 mu l and pEGFP-C2 vector 8 mu l;

(2) prepare 10 μ L of ligation: after dephosphorylation of CIAP, 0.5. mu.l of pEGFP-C2 vector, 1. mu.l of 10 XT 4DNA ligase buffer solution, 1. mu.l of T4DNA ligase and 7.5. mu.l of PCR enzyme digestion purified product;

(3) the reaction system is placed in a 0.5ml centrifuge tube and mixed evenly, and water bath is carried out at 16 ℃ overnight.

8. The plasmid for overexpressing ZEB2 gene in accordance with claim 1, wherein the specific steps of the transformation of the ligation product into E.coli TG1 in step (5) are as follows:

(1) taking out TG1 competent cell stored at-80 deg.C, thawing in ice bath, adding 10 μ l ligation product, mixing, and standing on ice for 30 min;

(2) performing heat shock at 42 deg.C for 90s, rapidly taking out, and standing on ice for 3 min;

(3) adding 500 μ l LB culture solution, and performing shake culture at 37 deg.C and 250rpm for 45 min;

(4) about 100. mu.l of the transformation solution was spread on LB solid medium with the corresponding resistance and cultured upside down at 37 ℃ for 8-12 hours.

9. Use of the plasmid overexpressing ZEB2 gene according to claim 1, wherein the transfection of the plasmid overexpressing ZEB2 gene into human HK-2 cell line inhibits the secretion of inflammatory factors in human HK-2 cells.

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