CN107119072A

CN107119072A - One kind is overexpressed ZEB2 gene plasmids and its construction method and application

Info

Publication number: CN107119072A
Application number: CN201710315809.7A
Authority: CN
Inventors: 张磊; 徐涛; 王啸; 潘林鑫; 丁琪; 汪洋
Original assignee: Anhui Medical University
Current assignee: Anhui Medical University
Priority date: 2017-05-08
Filing date: 2017-05-08
Publication date: 2017-09-01
Anticipated expiration: 2037-05-08
Also published as: CN107119072B

Abstract

ZEB2 gene plasmids are overexpressed the invention discloses one kind, are formed by ZEB2 gene C DS sequences and the recombination to construct of pEGFP C2 carrier for expression of eukaryon；Its nucleotides such as sequence table SEQ ID NO:1, positioned at the 134th 3778 of ZEB2 genes；Amino acid sequence such as sequence table SEQ ID NO:Shown in 2.The invention also discloses a kind of construction method of overexpression ZEB2 gene plasmids and application.The advantage of the invention is that：(1) contribute to study ZEB2 gene functions, and useful biology tool is provided to effect of the inflammation in the AKI courses of disease to study it；(2) laid a good foundation for further research ZEB2 function, and AKI gene therapy.

Description

One kind is overexpressed ZEB2 gene plasmids and its construction method and application

Technical field

The present invention relates to technical field of bioengineering, more particularly to a kind of overexpression ZEB2 gene plasmids and its construction method And application.

Background technology

Acute injury of kidney (acute kidney injury, AKI) is one of most common Severe acute disease in clinical departments room, Can be caused by many reasons, about AKI mechanism be always kidney trouble field concern important scientific issues.Recognize at present For AKI is also a kind of disease of inflammatory reaction mediation, but ischemia-reperfusion starts the mechanism of nephridial tissue inflammatory reaction not completely It is clear.Serious anuria AKI is increased to from slight serum creatinine, AKI degree and clinical manifestation are numerous, and AKI dies of illness with patient Rate, the generation of CKD (CKD) and maintenance dialysis rate are closely related.Epidemiological survey data display, the AKI incidences of disease Increase year by year, increase to 2 888 (per million people) of 2009 by 610 (per million people) of 1988, commonly living The AKI incidences of disease are 3%-5% in institute patient, and 30%-50% is more up in ICU；Prognosis simultaneously for AKI and lapse to compared with There is more deep understanding in the past.Therefore, AKI generation, development and the means for finding effective treatment AKI how is prevented and treated It is particularly important with medicine.

Genetic engineering, also known as gene recombination technology, are entering in vitro to DNA of growing up nineteen seventies A special kind of skill of row operation, is widely used in disease basic research, gene therapy, genetic immunization, gene vaccine etc..Its three elements point It is not：Enzyme, target gene, carrier.And carrier for expression of eukaryon is to be used in genetic engineering build the one of eukaryotic gene expression system Carrier is planted, in recent decades, scholars have carried out the correlative studys such as various diseases, gene around carrier for expression of eukaryon, in base It is that medical scientific opens brand-new route because being done a lot of work in terms of engineering.

ZEB2 is called ZFHX1B, SIP1 etc., the E box joint zinc finger proteins (zinc belonged in zinc fingers transcription factor Finger E-box binding homeobox) family member, be required transcription factor in embryonic development, first on ZEB2 report is the mRNA that ZEB2 is found that in the embryo of Africa xenopus.The ZEB2 assignments of genes gene mapping on chromosome 2q22.3, By ZFHX1B coded by said gene, comprising 9 extrons and 8 intrones, CDS areas are 3645bp.ZEB2 is by 1215 amino acid Residue constitute, its molecular weight is about 136KD, comprising 2 zinc finger clusters (zinc finger cluster) and connection the two zinc Refer to the variable sequence of cluster.Each zinc fingers in N-terminal and C-terminal can be in the conditioned zone of independent binding purpose gene 5 '-CACCT sequences.ZEB2 belongs to transcription factor, and initial research is thought to be positioned in core, it has recently been demonstrated that in born of the same parents There is expression in slurry and karyon.It participates in a variety of vital movements such as cell growth, differentiation, apoptosis, embryonic development and inflammatory reaction Regulation and control.

People source ZEB2 genes are registered in GenBank, and number of registration BC-127102.1 is positioned at 2q22.3, total length 3912bp, wherein CDS sequences 3645bp, encode 1215 amino acid.The tissue expression analysis of spectrum of people source ZEB2 genes is shown：Should Gene has expression in many tumours (stomach cancer, the breast cancer, nasopharyngeal carcinoma etc.) cell and mast cell of people, and research is found, ZEB2 genes also have expression in rat peritoneal macrophages, person monocytic cell THP-1.

Therefore, in order to further study ZEB2 gene functions, being badly in need of one kind at present contributes to ZEB2 gene functional research, and And the useful biology tool of Research foundation is provided effect of the inflammation in the AKI courses of disease for it.

The content of the invention

Contribute to ZEB2 gene functional research it is an object of the invention to overcome the deficiencies of the prior art and provide one kind, And for its effect of the inflammation in the AKI courses of disease is provided Research foundation overexpression ZEB2 gene plasmids and its construction method and Using.

The present invention is achieved by the following technical solutions：One kind is overexpressed ZEB2 gene plasmids, by ZEB2 gene C DS sequences The recombination to construct arranged with pEGFP-C2 carrier for expression of eukaryon is formed；Its nucleotides such as sequence table SEQ ID NO:1, positioned at ZEB2 bases 134-3778 of cause；Amino acid sequence such as sequence table SEQ ID NO:Shown in 2.

A kind of method for building above-mentioned overexpression ZEB2 gene plasmids, comprises the following steps：

(1) HK-2 cells are cracked, RNA progress reverse transcriptions is extracted and obtains ZEB2cDNA；

(2) PCR amplifies the CDS sequence fragments of artificial ZEB2 genes；

(3) purifying of the CDS sequence fragments of ZEB2 genes；

(4) BamH I and Xba I double digestions are carried out to the genetic fragment and pEGFP-C2 carrier for expression of eukaryon that are obtained, and It is attached；

(5) connection product is converted into e. coli tg1, and chooses positive colony and cultivated, then extract plasmid.

Extracted in one of preferred embodiment as the present invention, the step (1) after RNA, denaturation treatment is carried out to it, specifically Step is：RNA is placed after 5-10min in 65 DEG C, is transferred in frozen water and places.

One of preferred embodiment as the present invention, reverse transcription reaction is reversed according to RT Master Mix in the step (1) The product description for recording kit is carried out, and the reaction system of reverse transcription includes：5×RT Master Mix、RNA、Nuclease- free Water；Comprise the following steps that：

(1) reaction system is prepared：5×RT Master Mix 2μL+RNA 2μL+Nuclease-free Water 6μL；

(2) above-mentioned reaction system is placed 37 DEG C, and acts on 15min；

(3) above-mentioned reaction system is transferred to 50 DEG C, and acts on 5min；

(4) above-mentioned reaction system is transferred to 98 DEG C, and acts on 5min；

(5) above-mentioned reaction system is transferred to 4 DEG C, frozen after cooling, that is, obtain cDNA.

As one of preferred embodiment of the present invention, PCR expands required upper and lower during CDS sequence fragments in the step (2) Swim primer sequence as follows：

Sense primer：ZEB2-F:5’-GGGGTACCCCATGAAGCAGCCGATCAT-3’

Anti-sense primer：ZEB2-R:5’-GCTCTAGAGCTCACATGCCATCTTCC-3’.

One of preferred embodiment as the present invention, pcr amplification reaction expands according to PrimeSTAR Max in the step (2) Increase enzyme product specification to carry out, PCR reaction system includes：2 × PrimeSTAR Max, cDNA sample, upstream and downstream primer, height Pure water；Comprise the following steps that：

(1) reaction system is prepared：Each μ L of 1 μ L+ high purity waters 22 of the μ L+ primers of 2 × PrimeSTAR Max, 25 μ L+cDNA 1；

(2) PCR instrument device is put into after above-mentioned reaction system is gently mixed, following program operation is set；

(3) above-mentioned PCR primer is subjected to 1.2% agarose gel electrophoresis；

(4) observe result and reclaim target DNA fragment.

One of preferred embodiment as the present invention, ZEB2 PCR primer and pEGFP-C2 carriers are carried out in the step (4) BamH I and Xba I double digestion reaction are carried out according to restriction enzyme product description, and digestion system is as follows：BamH I are limited Property restriction endonuclease processed, Xba I restriction enzymes, 10 × Buffer Tango, ZEB2 PCR primer or pEGFP-C2 carriers；Tool Body step is as follows：

(1) reaction system is prepared：Each 0.5 μ L+ZEB2 of μ L+BamH I and Xba I of 10 × Buffer Tango 2 PCR productions Thing or with each 7 μ L of pEGFP-C2 carriers；

(2) above-mentioned reaction system is placed in 37 DEG C of water-baths, and acts on 4-6h；

(3) above-mentioned digestion products are subjected to 1-1.2% agarose gel electrophoresis；

(4) observe result and reclaim target DNA fragment.

One of preferred embodiment as the present invention, connection method is specific as follows in the step (4)：

(1) the μ L of dephosphorylation system 10 are prepared：CIAP1 μ l, 10 × CIAP buffer solutions, 1 μ l, pEGFP-C2 carrier 8 μ l are right Carrier carries out dephosphorylation process；

(2) the μ L of linked system 10 are prepared：The μ l of pEGFP-C2 carriers 0.5,10 × T4 DNA ligases after CIAP dephosphorylations The μ l of 1 μ l, T4DNA ligase of buffer solution, 1 μ l, PCR digestions purified product 7.5；

(3) above-mentioned reaction system is placed in 0.5ml centrifuge tube and mixed, 16 DEG C of water-baths are stayed overnight.

One of preferred embodiment as the present invention, connection product converts the tool into e. coli tg1 in the step (5) Body step is as follows：

(1) the TG1 competent cells for being stored in 80 DEG C of ﹣ are taken out, ice bath adds 10 μ l connection products after thawing, and mixes, ice Upper standing 30min；

(2) 42 DEG C of heat shock 90s, rapid take out puts 3min on ice；

(3) 500 μ l LB nutrient solutions, 37 DEG C, 250rpm shaken cultivations 45min are added；

(4) take out about 100 μ l conversion fluids to be applied on the LB solid mediums with corresponding resistant, 37 DEG C are inverted culture 8- 12h。

A kind of application using above-mentioned overexpression ZEB2 gene plasmids, people is transfected into by the overexpression ZEB2 gene plasmids In HK-2 cell lines, suppress the secretion of inflammatory factor in people's HK-2 cells.

The advantage of the present invention compared with prior art is：

(1) contribute to study ZEB2 gene functions, and effect of the inflammation in the AKI courses of disease has been provided to study it Use biology tool；

(2) gene recombination technology is used, for ZEB2 genes, recombinant eukaryon expression vector pEGFP-C2-ZEB2 is built, and Transfected into people's HK-2 cell lines, research ZEB2 is overexpressed the influence to inflammatory Cytokines Expression in HK-2 cells, to enter one Step research ZEB2 function, and AKI gene therapy are laid a good foundation.

Brief description of the drawings

The structure that Fig. 1 is a kind of carrier for expression of eukaryon pEGFP-C2 of overexpression ZEB2 gene plasmids in embodiment 1 is shown It is intended to；

Fig. 2 is a kind of eucaryon recombinant vector pEGFP- of the construction method of overexpression ZEB2 gene plasmids in embodiment 2 C2-ZEB2 structural representation；

Fig. 3 is a kind of eucaryon recombinant vector pEGFP- of the construction method of overexpression ZEB2 gene plasmids in embodiment 2 C2-ZEB2 digestion identification collection of illustrative plates；

Fig. 4 be a kind of overexpression ZEB2 gene plasmids in embodiment 3 expression during eucaryon recombinant vector pEGFP- Expression figures of the C2-ZEB2 in HEK 293T cells；

Fig. 5 be a kind of transfection group of the application of overexpression ZEB2 gene plasmids in embodiment 4 HK-2 cellular inflammations because The situation map of sub (IL-6, TNF-α) mRNA expressions；

Fig. 6 be a kind of transfection group of the application of overexpression ZEB2 gene plasmids in embodiment 4 HK-2 cellular inflammations because The situation map of sub (IL-6, TNF-α) protein expression level.

Embodiment

Embodiments of the invention are elaborated below, the present embodiment is carried out lower premised on technical solution of the present invention Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following implementations Example.

Embodiment 1

The a kind of of the present embodiment is overexpressed ZEB2 gene plasmids, by ZEB2 gene C DS sequences and pEGFP-C2 eukaryotic expressions The recombination to construct of carrier is formed；Its nucleotides such as sequence table SEQ ID NO:1, positioned at 134-3778 of ZEB2 genes；Ammonia Base acid sequence such as sequence table SEQ ID NO:Shown in 2；In addition, the structure of pEGFP-C2 carrier for expression of eukaryon is as shown in Figure 1.

Embodiment 2

A kind of construction method of overexpression ZEB2 gene plasmids of the present embodiment, comprises the following steps：

1st, ZEB2cDNA extraction

(1) take out Tissue Culture Dish and abandon net culture medium, the cell of the HK-2 cells of culture is cleaned with 1ml PBS Face (2-3 times)；

(2) 1ml Trizol cell lysis 10min are added, are gently mixed cell pyrolysis liquid turn with pipettor after cell Move in centrifuge tube, 5min is stored at room temperature after reverse mixing；

(3) chloroform (4 DEG C of precoolings) of Trizol 1/5 amount is added, 5min is stored at room temperature after acutely mixing, high speed is low Temperature centrifugation 15min (4 DEG C, 12000r/min)；

(4) 0.4ml supernatant liquids are transferred in new centrifuge tube, add isometric isopropanol, overturn quiet after mixing Supernatant is abandoned after putting 60min (- 20 DEG C), high-speed low temperature centrifugation 15min (4 DEG C, 12000r/min)；

(5) vibrate and mix after the ethanol solution for adding 1ml75%, after high-speed low temperature centrifugation 5min (4 DEG C, 12000r/min) Supernatant is abandoned, and dries 5min；

(6) after adding 20 μ L without enzyme water dissolving precipitation, for reverse transcription, it can also freeze in -80 degree；

(7) RNA denaturation

The RNA extracted is denatured, denaturing step is as follows：RNA is placed after 5min in 65 DEG C, is transferred quickly to 2min is placed in frozen water.

Reverse transcription reaction system (10 μ L) is as follows：

Processing step includes：

1. above-mentioned reaction system is placed 37 DEG C, and acts on 15min；

2. above-mentioned reaction system is transferred to 50 DEG C, and acts on 5min；

3. above-mentioned reaction system is transferred to 98 DEG C, and acts on 5min；

4. above-mentioned reaction system is transferred to 4 DEG C, frozen after cooling, that is, obtain cDNA.

2nd, the clone of ZEB2 genes

(1) according to ZEB2 CDS sequences, upstream and downstream primer is designed, primer sequence difference is as follows：

ZEB2-F:5 '-GGGGTACCCCATGAAGCAGCCGATCAT-3 ' (restriction enzyme sites of I containing BamH)

ZEB2-R:5 '-GCTCTAGAGCTCACATGCCATCTTCC-3 ' (restriction enzyme sites of I containing Xba)

(2) enzyme used in the process of PCR is preferably the PrimeSTAR Max high-fidelity enzymes of Takara companies, its reactant System and response procedures are as follows：

Reaction system (50 μ L)：

PCR instrument device is put into after above-mentioned reaction system is mixed, following program operation is set：

(4) observe result and reclaim target DNA fragment；

PCR primer is entered into row agarose gel electrophoresis, by the contrast with DNA Marker, as a result finds to go out in 3645bp A band is obtained, size and the size of ZEB2 purpose fragments (including restriction enzyme site) are basically identical, show that Successful amplification goes out people The CDS sequences of ZEB2 genes.

3rd, the purifying of ZEB2 purpose fragments

Purifying kit used is preferably that love is pursued progress the glue reclaim kit of company, and purification step is as follows：

(1) Ago-Gel containing target DNA is cut under uviol lamp, gel surface liquid is exhausted with filter paper and cuts It is broken.Calculated for gel weight, 100 μ l volumes are equal to per 100mg；

(2) the Buffer DE-A of 3 gel volumes are added, is well mixed and heats (low melting-point agarose gel after 75 DEG C Heated in 40 DEG C), interruption mixing (every 2~3min), until gel piece is completely melt；

(3) add the Buffer DE-B of 0.5 Buffer DE-A volume, be well mixed.When the DNA fragmentation of separation is less than During 400bp, the isopropanol of 1 gel volume need to be added；

(4) mixed liquor in aspiration step 3, is transferred to DNA and prepares in pipe (being placed in 2ml centrifuge tubes), 12,000rpm centrifugations 1min.Abandon filtrate；

(5) pipe will be prepared and put back into 2ml centrifuge tubes, plus 500 μ l Buffer W1,12,000rpm centrifugation 30s, filtrate is abandoned；

(6) pipe will be prepared and put back into 2ml centrifuge tubes, plus 700 μ l Buffer W2,12,000rpm centrifugation 30s, filtrate is abandoned；With Same method washed once 12,000rpm centrifugations 1min with 700 μ l Buffer W2 again；

(7) pipe will be prepared to put back into 2ml centrifuge tubes, 12,000rpm centrifugation 1min；

(8) pipe will be prepared to be placed in the 1.5ml centrifuge tubes of cleaning, is preparing film center plus 30 μ l Eluent or deionization Water, is stored at room temperature 1min.12000rpm centrifuges 1min eluted dnas.

4th, the double digestion of PCR primer and carrier for expression of eukaryon

PCR primer and pEGFP-C2 carriers to ZEB2 carry out BamH I and Xba I double digestion, digestion system and method It is as follows：

Digestion system (10 μ L)：

Enzymatic cleavage methods：Above reaction system is placed in 0.5ml centrifuge tube and mixed, 37 DEG C of water-bath 6h (temperature and times Selection gist restriction endonuclease specification depending on), rapid observation result and reclaim target DNA after 1.2% agarose gel electrophoresis Fragment, it is to avoid ultraviolet long-term irradiation.

5th, the connection of digestion products

ZEB2 and pEGFP-C2 double digestion product is attached, connection enzyme used is preferably T4 ligases；Even Before connecing, to avoid the recirculation of carrier, therefore dephosphorylation process is carried out to carrier；

Dephosphorylation system (10 μ L)：The μ l of CIAP 1, the μ l of 10 × CIAP buffer solutions, 1 μ l, pEGFP-C2 carrier 8；

Linked system (10 μ L)：The μ l of pEGFP-C2 carriers 0.5,10 × T4DNA connection enzyme buffers after CIAP dephosphorylations The μ l of 1 μ l, T4DNA ligase of liquid, 1 μ l, PCR digestions purified product 7.5；

Connection method：Above-mentioned reaction system is placed in 0.5ml centrifuge tube and mixed, 16 DEG C of water-baths are stayed overnight.

6th, the conversion of connection product

Converting E. coli competent used is：TG1, step of converting is as follows：

(1) the TG1 competent cells for being stored in 80 DEG C of ﹣ are taken out, ice bath adds 10 μ l connection products after thawing, gently mixed It is even, 30min is stood on ice；

(2) 42 DEG C of heat shock 90s (should not rock), rapid take out puts 3min on ice；

(4) take out about 100 μ l conversion fluids to be applied on the LB solid mediums with corresponding resistant, 37 DEG C are inverted culture 12h.

7th, a small amount of extractings of plasmid

Choose 8 positive colonies to add in the centrifuge tubes containing 2ml LB culture mediums (ammonia benzyl resistance), 37 DEG C, 250rpm shakes Culture 12h is swung, plasmid extraction is then carried out to bacterium solution, kit used is pursued progress the small amount plasmid extraction agent of company for love Box, extraction steps are as follows：

(1) with the monoclonal of sterilizing toothpick picking bacterium, it is put into the 15ml for containing the LB nutrient solutions that 2ml contains corresponding antibiotic In centrifuge tube, 37 DEG C, 250rpm shaken cultivations 12h；

(2) about 1ml bacterium solutions are transferred in 1.5ml centrifuge tubes, 4 DEG C, 12,000rpm centrifugation 1min, supernatant；

(3) 250 μ l Buffer S1 suspended bacterials precipitation is added, suspending, it is uniform to need, and should not leave small fungus block；

(4) it is plus 250 μ l Buffer S2, gentle and fully spinning upside down 6 times makes thalline fully crack, until being formed thoroughly Bright solution；

(5) add 350 μ l Buffer S3, spin upside down gently and fully mixing 8 times, 12,000rpm centrifugation 10min；

(6) centrifugation supernatant in aspiration step 5 is simultaneously transferred to preparation pipe (being placed in 2ml centrifuge tubes), 12000rpm centrifugations 1min, abandons filtrate；

(7) pipe will be prepared and put back into centrifuge tube, plus 500 μ l Buffer W1,12,000rpm centrifugation 1min, filtrate is abandoned；

(8) pipe will be prepared and put back into centrifuge tube, plus 700 μ l Buffer W2,12,000rpm centrifugation 1min, filtrate is abandoned；With same The method of sample washed once with 700 μ l Buffer W2 again.Abandon filtrate；

(9) pipe will be prepared to put back into 2ml centrifuge tubes, 12,000rpm centrifugation 1min；

(10) pipe will be prepared to move into new 1.5ml centrifuge tubes, is preparing periosteum center plus 80 μ l sterilized waters, be stored at room temperature 1min, 12,000rpm centrifugation 1min, abandons preparation pipe；

(11) sample put -20 DEG C it is standby, Fig. 2 be eucaryon recombinant vector pEGFP-C2-ZEB2 structural representation.

8th, digestion identification is carried out to plasmid and send sequencing

BamH I and Xba I double digestion identification are carried out to the plasmid extracted, digestion system and method are as follows：

Digestion system (10 μ L)：

Enzymatic cleavage methods：Above-mentioned reaction system is placed in 0.5ml centrifuge tube and mixed, after 37 DEG C of digestion 6h, agarose coagulates Preservation that gel electrophoresis, ultraviolet spectrometer are observed, gel imager is taken pictures, and correct plasmid Song Sheng works biotech firm is identified into digestion Sequencing；Wherein, eucaryon recombinant vector pEGFP-C2-ZEB2 digestion identification collection of illustrative plates is shown in Fig. 3, wherein sign 1 is pEGFP-C2 double Digestion, sign 2 is ZEB2 PCR primer, and sign 3 is pEGFP-C2-ZEB2 double digestions；

Sequencing result is compared by BLAST, target gene piece segment length consistent with the sequence announced in GencBank is found Spend actually 3645bp.

Embodiment 3

A kind of expression of overexpression ZEB2 gene plasmids, obtains being overexpressed after ZEB2 gene plasmids, to it according to embodiment 2 Expressed, comprised the following steps that：

1st, cell culture

The cell of culture is needed for of the invention：HEK, 293T, THP-1, rat peritoneal macrophages, HK-2 cells, first Recovery cell, and with containing volume ratio for 10% hyclone DMEM high glucose mediums in 37 DEG C, 5%CO2 cell culture Cultivated in case, and change liquid and passage in time.

(1) cell recovery：The cell frozen in liquid nitrogen is taken out, immerses rapidly in 37 DEG C of warm water and jiggles to complete Complete to melt (rewarming time was controlled within two minutes), rapid transfer cell suspension is into centrifuge tube and adds 5ml culture mediums, Abandon most supernatant after low-speed centrifugal (1 000r/min, 2min), add 5ml culture mediums and be gently seeded in culture dish after suspension cell Start culture；

(2) cell changes liquid：Old cell culture medium is gently discarded, PBS adds fresh complete medium after gently cleaning (DMEM in high glucose containing 10% hyclone) continues to cultivate (note：PBS and fresh culture medium need 37 DEG C of preheatings)；

(3) passage：It is placed in incubator and is digested after the pancreatin that 1ml is added in cell, is seen under the microscope Cellular morphology change is examined, complete medium is added in time and terminates digestion, transfer cell suspension low-speed centrifugal (1 into centrifuge tube 000r/min, 2min) after abandon supernatant, 0.2ml culture mediums suspension cell is added after washed once with PBS and culture is seeded to again Continue to cultivate in ware；

(4) cell cryopreservation：Supernatant will be abandoned after cell suspension low-speed centrifugal (1 000r/min, 2min) in succeeding generations, Add 1ml cells frozen storing liquids (formula is shown in) and gently dispense into cell cryopreservation tube after suspension cell and carry out classification and freeze, i.e., 4 DEG C 0.5-1h, -20 DEG C of 1-2h, -80 DEG C of about 24h, liquid nitrogen cryopreservations.

2nd, plasmid transiently transfects (liposome method)

(1) DNA that 5 μ l liposome and total amount are about 2 μ g is diluted with 250 μ L Opti-MEM, gentle to mix, room temperature is quiet Put 5min；

(2) two kinds of solution in above-mentioned steps are gently mixed, is stored at room temperature 20min, to form DNA- lipid bluk recombinations Thing；

(3) nutrient solution in culture dish is discarded, 1ml Opti-MEM transfection liquids are added, the DNA- liposomes in (2) are answered Compound is slowly dropped in culture dish, jiggles culture dish mixing；

(4) 37 DEG C, 5%CO₂Cultivate after 6h, renew and continue to train after fresh complete culture solution with the PBS of pre-temperature once Support about 24h.

3rd, immunoblotting analysis

(1) total protein extraction：Cell is collected by centrifugation, after PBS 2-3 times, 0.5ml cell pyrolysis liquids are added, on ice 10min is cracked, then high speed refrigerated centrifuge 10min, collect 0.4ml supernatants, add sample-loading buffer, boiling water bath 5min is put Put and treat electrophoresis on ice；

(2) glue plate is installed and hunted leak, 10% separation gel is prepared first, with 1ml isopropanol line balls, after after abundant solidification (about 1h), prepares concentration glue, and comb needed for insertion is used after after abundant solidification；

(3) electrophoresis system is installed, 500ml electrophoretic buffers are added in electrophoresis tank, 10 μ L samples are added behind loading hole Start electrophoresis (80V electrophoresis 0.5h+100V electrophoresis 1.5h)；

(4) after electrophoresis terminates, cut SDS-PAGE glue, using sandwich mode (be successively from the bottom up filter paper+glue+ Pvdf membrane+filter paper) carry out wet turn；

(5) electricity is installed and transfers from one department to another system, the electricity that 500ml precoolings are added in electric turn trough turns buffer solution, and whole electric turn trough is put Enter in frozen water, start electricity and turn (100V electricity turns 2h)；

(6) whether electricity turns after terminating, and gently removes pvdf membrane, succeeded with Ponceaux dyeing identification transferring film, after transferring film success, Ponceaux, room temperature closing at least 1h are washed away with TBST；

(7) after having closed, washed with TBST after film (3 times, 5min), add 4 DEG C of overnight incubations of primary antibody solution；

(8) next day, washed with TBST after film (3 times, 10min), add two corresponding anti-solution incubation at room temperature 2h.Then film is washed with TBST (3 times, 20min)；

(9) with the band on ECL luminous agents detection PVDF, and X-ray film scotography is used.

Experimental result shows, recombinant eukaryotic expression plasmid pEGFP-C2-ZEB2 in eukaryotic can normal expression, be Follow-up research is laid a good foundation；Fig. 4 is expressions of the eucaryon recombinant vector pEGFP-C2-ZEB2 in HEK293T cells In figure, figure, sign 1 is the 293T cell pyrolysis liquids of untransfected, the 293T cells cracking that sign 2 is transfection pEGFP-C2-ZEB2 Liquid.

Embodiment 4

A kind of application of overexpression ZEB2 gene plasmids, people's HK-2 cells are transfected into by the overexpression ZEB2 gene plasmids In system, research is overexpressed the influence of ZEB2 gene pairs people's HK-2 cell lines inflammatory factors (IL-6, TNF-α) secretion；Specific steps It is as follows：

1st, the cell of exponential phase is passed on, obtains cell suspension, and with suitable density (about 1 × 108L-1) Seed cells into 6 orifice plates, cultivate about 24h, until cell density is 80% or so, carry out the instantaneous of pEGFP-C2-ZEB2 Transfection, cell suspension is collected after transfection a period of time；

2nd, according to the specification of manufacturer, total serum IgE is separated from culture HK-2 cells using Trizol reagents, always Water elutions of the RNA without RNase, and it is stored in -80 DEG C；

3rd, cDNA is synthesized using transcripton the first chain cDNA synthetic agent box, and using with SYBR-Green Master Mix detecting system carries out real-time PCR, and q-PCR primers are obtained from Invitrogen, and two parts of sample, every time measurement is repeated At least three times, q-PCR results are as shown in Figure 5；

Fig. 5 A qRT-PCR analysis shows：ZEB2 expression has been raised in pEGFP-C2-ZEB2 transfection；Fig. 5 B's QRT-PCR analysis shows：TNF-α and IL-6 expression (* P have been lowered in pEGFP-C2-ZEB2 transfection<0.05 and * *, P< 0.01vs normal groups；#P<0.05 and ##P<0.01vs empty plasmids group)；

Find based on the above results：Compared with inflammatory factor (IL-6, TNF-α) mRNA level in-site of untransfected group cell, turn Having contaminated the expression of inflammatory factor IL-6 and TNF-α mRNA in the HK-2 cells of pEGFP-C2-ZEB2 groups has obvious reduction, and poor It is different to there is statistical significance (P ＜ 0.05)；Illustrate that inflammatory factor (IL-6, TNF-α) can substantially be reduced by being overexpressed ZEB2 genes MRNA level in-site, so as to suppress inflammatory reaction.

In addition, extracting total protein and detectable concentration, gel electrophoresis is carried out, it is wet to turn after nitrocellulose membrane, use 5% skim milk 60min is closed, 1 is added：The ZEB2 rabbit-antis of 200 dilutions, 1：IL-6, TNF-α and the Beta- of 1: 1 000 dilution of 1000 dilutions After Actin internal reference primary antibodies, 4 DEG C of overnight incubations are rinsed 3 times with TBST, add the secondary antibody of respective concentration, 60min is placed at room temperature, TBST is rinsed 3 times, adds development under the conditions of ECL Plus, scotopia, protein band is analyzed with Quantity One softwares, Western blot results are as shown in Figure 6；

Fig. 6 A Western blot analysis shows：ZEB2 expression has been raised in pEGFP-C2-ZEB2 transfection；Fig. 6 B Western blot analysis shows, pEGFP-C2-ZEB2 transfection lowered TNF-α and IL-6 expression (* P<0.05 With * * P<0.01 compared with normal group；#P<0.05 and ##P<0.01 compared with vector groups)；

Find based on the above results：Compared with inflammatory factor (IL-6, TNF-α) protein level of untransfected group cell, turn Having contaminated inflammatory factor (IL-6, TNF-α) albumen of the cell of pEGFP-C2-ZEB2 groups has obvious reduction, and difference has system Meter learns meaning (P ＜ 0.05)；Illustrate that inflammatory factor (IL-6, TNF-α) protein level can substantially be reduced by being overexpressed ZEB2 genes, So as to suppress inflammatory reaction.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.

SEQUENCE LISTING

<110>Medical University Of Anhui

<120>One kind is overexpressed ZEB2 gene plasmids and its construction method and application

<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 Cys Pro 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 Ala Thr 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 Thr Asp 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 Pro Arg 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 Ala Lys 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

1. one kind is overexpressed ZEB2 gene plasmids, it is characterised in that carried by ZEB2 gene C DS sequences and pEGFP-C2 eukaryotic expressions The recombination to construct of body is formed；Its nucleotides such as sequence table SEQ ID NO:1, positioned at 134-3778 of ZEB2 genes；Amino Acid sequence such as sequence table SEQ ID NO:Shown in 2.

2. a kind of build the method as claimed in claim 1 for being overexpressed ZEB2 gene plasmids, it is characterised in that including following step Suddenly：

(2) PCR amplifies the CDS sequence fragments of artificial ZEB2 genes；

(3) purifying of the CDS sequence fragments of ZEB2 genes；

(4) BamH I and Xba I double digestions are carried out to the genetic fragment and pEGFP-C2 carrier for expression of eukaryon that are obtained, and carried out Connection；

3. the construction method of overexpression ZEB2 gene plasmids according to claim 2, it is characterised in that the step (1) After middle extraction RNA, denaturation treatment is carried out to it, concretely comprised the following steps：RNA is placed after 5-10min in 65 DEG C, frozen water is transferred to It is middle to place.

4. the construction method of overexpression ZEB2 gene plasmids according to claim 2, it is characterised in that the step (1) Middle reverse transcription reaction is carried out according to the product description of RT Master Mix Reverse Transcriptase kits, the reaction system bag of reverse transcription Include：5×RT Master Mix、RNA、Nuclease-free Water；Comprise the following steps that：

(2) above-mentioned reaction system is placed 37 DEG C, and acts on 15min；

5. the construction method of overexpression ZEB2 gene plasmids according to claim 2, it is characterised in that the step (2) Required upstream and downstream primer sequence is as follows during middle PCR amplifications CDS sequence fragments：

Sense primer：ZEB2-F:5’-GGGGTACCCCATGAAGCAGCCGATCAT-3’

Anti-sense primer：ZEB2-R:5’-GCTCTAGAGCTCACATGCCATCTTCC-3’.

6. the construction method of overexpression ZEB2 gene plasmids according to claim 2, it is characterised in that the step (2) Middle pcr amplification reaction is carried out according to PrimeSTAR Max amplification enzymes product description, and PCR reaction system includes：2× PrimeSTAR Max, cDNA sample, upstream and downstream primer, high purity water；Comprise the following steps that：

(4) observe result and reclaim target DNA fragment.

7. the construction method of overexpression ZEB2 gene plasmids according to claim 2, it is characterised in that the step (4) Middle ZEB2 PCR primer and pEGFP-C2 carriers carries out BamH I and Xba I double digestion reaction according to restriction enzyme production Product specification is carried out, and digestion system is as follows：BamH I restriction enzymes, Xba I restriction enzymes, 10 × Buffer Tango, ZEB2 PCR primer or pEGFP-C2 carriers；Comprise the following steps that：

(1) reaction system is prepared：Each 0.5 μ L+ZEB2 of μ L+BamH I and Xba I of 10 × Buffer Tango 2 PCR primer or With each 7 μ L of pEGFP-C2 carriers；

(4) observe result and reclaim target DNA fragment.

8. the construction method of overexpression ZEB2 gene plasmids according to claim 2, it is characterised in that the step (4) Middle connection method is specific as follows：

(1) the μ L of dephosphorylation system 10 are prepared：CIAP1 μ l, the μ l of 10 × CIAP buffer solutions, 1 μ l, pEGFP-C2 carrier 8, to carrier Carry out dephosphorylation process；

(2) the μ L of linked system 10 are prepared：The μ l of pEGFP-C2 carriers 0.5,10 × T4DNA ligase buffer solutions after CIAP dephosphorylations The μ l of 1 μ l, T4DNA ligase, 1 μ l, PCR digestions purified product 7.5；

9. the construction method of overexpression ZEB2 gene plasmids according to claim 2, it is characterised in that the step (5) Middle connection product converts comprising the following steps that into e. coli tg1：

(1) the TG1 competent cells for being stored in 80 DEG C of ﹣ are taken out, ice bath adds 10 μ l connection products after thawing, and mixes, quiet on ice Put 30min；

(2) 42 DEG C of heat shock 90s, rapid take out puts 3min on ice；

(4) take out about 100 μ l conversion fluids to be applied on the LB solid mediums with corresponding resistant, 37 DEG C are inverted culture 8-12h.

10. a kind of use the application as claimed in claim 1 for being overexpressed ZEB2 gene plasmids, it is characterised in that by the mistake Expression ZEB2 gene plasmids are transfected into people's HK-2 cell lines, suppress the secretion of inflammatory factor in people's HK-2 cells.