CN103352051A - PcDNA3.1(+)-Fbw7 (F-box and WD repeat domain-containing 7) recombinant plasmid as well as construction method and application of PcDNA3.1(+)-Fbw7 recombinant plasmid - Google Patents

Abstract

The invention provides a PcDNA3.1(+)-Fbw7 recombinant plasmid as well as a construction method and an application of the PcDNA3.1(+)-Fbw7 recombinant plasmid and relates to a recombinant plasmid. The construction method comprises the following steps: (1) comparing the spectrogram information of a PSG5-Myc-Fbw7 plasmid with the spectrogram information of a PcDNA3.1(+) to determine the double-enzyme cleavage sites of EcoR I and Not I; (2) cleaving the PSG5-Myc-Fbw7 plasmid with two enzymes; (3) cleaving the carrier of the PcDNA3.1(+) plasmid with two enzymes; (4) identifying the efficiency of the double-enzyme cleavage through agarose gel electrophoresis, and connecting the carrier of the PcDNA3.1(+) plasmid with the whole length of the target gene of Fbw7; (5) transforming the recombinant plasmid into competent bacteria, screening out positive clones, performing identification through sequencing, and extracting the PcDNA3.1(+)-Fbw7 recombinant plasmid. The PcDNA3.1(+)-Fbw7 recombinant plasmid can be used for preparing medicine for curing the tumor of bile duct cancer of people.

Description

PcDNA3.1 (+)-Fbw7 recombinant plasmid and construction process and application

Technical field

The present invention relates to a kind of recombinant plasmid, especially relate to PcDNA3.1 (+)-Fbw7 recombinant plasmid and construction process thereof and the application in preparation treatment human bile duct cancer medicine.

Background technology

FBW7(F-box and WD repeat domain-containing7) be found in the earliest in the intestinal bacteria, initial called after Cdc4, claiming again FBXW7, SEL-10, hAgo, hCDC4, is SCF (SKP1/CUL1/F-box) mixture type ubiquitin ligase, often participates in the ubiquitin of substrate.

So-called ubiquitin refers to substrate protein is transformed into the chemical reaction of ubiquitin, causes substrate afunction and degraded.Ubiquitin has important life meaning, in recent years, become the integral part (1.Philip Cohen and Marianna Tcherpakov.Will the Ubiquitin System Furnish as Many Drug Targets as Protein Kinases Cell.2010.143:686-693) of tumor research from the genesis of the angle research tumour of ubiquitin.Understanding ubiquitin by ubiquitin ligase is the integral part of this research.FBW7 plays a role in escherichia coli splitting.

Other studies confirm that, but also cell cycle regulation, Growth of Cells, differentiation etc. of FBW7, and FBW7 sudden change or afunction can cause that chromosome instability reaches the pathology of cell surely, even canceration.Bibliographical information, the FBW7 mutation rate is the highest in cholangiocarcinoma, reach 35% (2.Shahab Akhoondi, Dahui Sun, Natalie von der Lehr, et al.FBXW7/hCDC4is a General Tumor Suppressor in Human Cancer.Cancer Res.2007.67 (19): 9006-12), the mutation rate in T-shaped acute lymphoblastic leukemia, mammary cancer, colorectal carcinoma, uterine endometrium, carcinoma of the pancreas and cancer of the stomach is all greater than 6%.FBW7+ in mouse/-heterozygote than FBW7+ /+homozygote has higher tumorigenesis rate (3.Welcker M, Clurman BE.Ubiquitin ligase:a tumour suppressor at the crossroads of cell division, growth and differentiation.Nat Rev Cancer.2008.8:83-93).

This shows, FBW7 sudden change and relation between tumor are close.FBW7 effect substrate is very extensive, has plenty of proto-oncogene, has plenty of cyclin, and a lot of all have substantial connection with generation and the development of tumour.

Now existing document confirms, undesired even the apoptosis that will directly cause growth of cancer cells at the high expression level of cell levels FBW7, proved that FBW7 is exactly cancer suppressor gene (4.Crusio KM, King B, Reavie LB, Aifantis I.The ubiquitous natureof cancer:the role of the SCF (Fbw7) complex in development and transformation.Oncogene.2010.29:4865 – 73).

The disappearance of FBW7 can promote resistance (the 5.Zhiwei Wang of tumour in addition, Hidefumi Fukushima, Daming Gao, Hiroyuki Inuzuka, Lixin Wan, Alan W.Lau, Pengda Liu and Wenyi Wei.The two faces of FBW7in cancer drug resistance.Bioessays.2011.33:851 – 859).In mammary cancer, the relation of FBW7 and mTOR has been reported, knock out the rising (6.Jian-Hua Mao, et al.FBXW7Targets mTOR for Degradation and Cooperates with PTEN in Tumor Suppression.Science.2008.321:1499-1502) that FBW7 will cause the mTOR expression amount.In addition, C/EBP δ can suppress the expression of FBW7 in mammary cancer, thereby strengthens the activity of mTOR/AKT/HIF-1 signal path, is beneficial to generation and the development of mammary cancer; FBW7 and NOTCH1 signal path also have important relation in mammary cancer, the disappearance of FBW7 further causes the dependent MYC of NOTCH1 to regulate unsuccessfully so that the NOTCH1 signal is strengthened, and the AKT high expression level.MYC also is subject to the adjusting of the direct ubiquitin of FBW7 in mammary cancer, so the NOTCH1/AKT/mTOR signal path all has close relationship (7.Jinhua Xu with FBW7 in the mammary cancer, Yinghua Chen, and Olufunmilayo I.Olopade.MYC and Breast Cancer.Genes﹠amp; Cancer.2010.1 (6): 629-640).

In addition, the antagonist of FBW7 removes ubiquitin enzyme Usp28 high expression level usually in mammary cancer, and is favourable to the genesis of mammary cancer, and the proto-oncogene of the mammary cancer such as KLF5 all is subject to the ubiquitin degraded of FBW7.In cholangiocarcinoma, have been found that the undesired expression of mTOR and NOTCH1, with cholangiocarcinoma (8.Wang Z in close relations, Zheng T, Wu Q, Wang J, Wu C, Wang J.Immunohistochemical analysis of the mTOR pathway in intrahepatic cholangiocarcinoma.Neoplasma.2012.59 (2): 137-41; 9.Hyun Ah Yoon, Myung Hwan Noh, Byung Geun Kim, Ji Sun Han, Jin Seok Jang, Seok Ryeol Choi, Jin Sook Jeong, Jin Ho Chun.Clinicopathological signiicance of altered Notch signaling in extrahepatic cholangiocarcinoma and gallbladder carcinoma.World J Gastroenterol.2011.17 (35): 4023-4030).

Summary of the invention

The object of the present invention is to provide a kind of PcDNA3.1 (+)-Fbw7 recombinant plasmid and construction process and application.

Described PcDNA3.1 (+)-Fbw7 recombinant plasmid is by extracting people Fbw7 full length gene, screen PcDNA3.1 (+) plasmid of label as carrier to contain Liu Suanyan NEOMYCIN SULPHATE, insert the Fbw7 full length gene PcDNA3.1's (+) and between restriction enzyme site EcoR I and the Not I, structure a kind of contains the PcDNA3.1 (+) of Fbw7 full length gene and Liu Suanyan NEOMYCIN SULPHATE screening label-Fbw7 recombinant plasmid.

The construction process of described PcDNA3.1 (+)-Fbw7 recombinant plasmid may further comprise the steps:

1) PSG5-Myc-Fbw7 plasmid spectrogram information and PcDNA3.1 (+) plasmid spectrogram information are compared, determine double enzyme site EcoR I and Not I;

2) double digestion PSG5-Myc-Fbw7 plasmid, concrete reaction system (20 μ L) is as follows:

PSG5-Myc-Fbw71 μ g, EcoR I 1.0 μ L, Not I 1.0 μ L, 1 * H2.0 μ L, BSA2.0 μ L, the sterilization ultrapure water is mended to 20 μ L, behind the each component mixing, is placed on 2h in 37 ℃ of water-baths;

3) double digestion PcDNA3.1 (+) plasmid vector, concrete reaction system (20 μ L) is as follows:

PcDNA3.1 (+) 1 μ g, EcoR I 1.0 μ L, Not I 1.0 μ L, 1 * H2.0 μ L, BSA2.0 μ L, the sterilization ultrapure water is mended to 20 μ L, behind the each component mixing, is placed on 2h in 37 ℃ of water-baths;

4) identify double digestion efficient by agarose gel electrophoresis, plasmid vector PcDNA3.1 (+) is connected with goal gene Fbw7 total length, concrete reaction system (10 μ L) is as follows:

10 * reaction buffer, 1.0 μ L, PCDNA3.1 (+) 2.0 μ L, Fbw76.0 μ L, T4DNA ligase enzyme 1.0 μ L behind the each component mixing, connect in 16 ℃ of metal baths and spend the night;

5) with the recombinant plasmid transformed competence colibacillus bacterium, screening positive clone, and the evaluation of checking order, such as the positive clone of qualification result, extracting PcDNA3.1 (+)-Fbw7 recombinant plasmid then;

6) with step 5) PcDNA3.1 (+) of described extracting-Fbw7 recombinant plasmid carries out the dna sequencing analysis, choose two-way order-checking pattern, use PcDNA3.1 (+) plasmid universal primer across the one by one base detection of multiple clone site district of plasmid, detected result and Fbw7 sequence are compared.

In step 5), the concrete grammar that described order-checking is identified can be:

Carrying out colony PCR amplification with the 1st pair of primer, the rear products therefrom that increases is carried out agarose gel electrophoresis, is 244bp such as gained dna fragmentation length, then is accredited as positive colony, and described the 1st pair of primer is:

Forward primer: 5 '-ctacccaaaagtaatcatcttaagtg-3 ';

Reverse primer: 5 '-cccaaccatgacaagattttcc-3 ';

Carrying out colony PCR amplification with the 2nd pair of primer, the rear products therefrom that increases is carried out agarose gel electrophoresis, is 278bp such as gained dna fragmentation length, then is accredited as positive colony, and described the 2nd pair of primer is:

Forward primer: 5 '-tttctgtttctccctctg-3 ';

Reverse primer: 5 '-gagcatttaagggagagataagag-3 '.

In step 6) in, described carry out the dna sequencing analysis can be with step 5) PcDNA3.1 (+) of described extracting-Fbw7 recombinant plasmid delivers to American I nvitrogen company and carries out the dna sequencing analysis; The result that described Selected Inspection survey result and Fbw7 sequence are compared is as follows:

The result is presented at 914 sites the sudden change of one synonym occurs: CUC sports CUU, because there is the degeneracy phenomenon in biological genetic codon, coded amino acid is still leucine, can not affect the structure and function of the protein of its translation, qualification result is positive, finishes the structure of PcDNA3.1 (+)-Fbw7 recombinant plasmid.

The present invention uses with the cholangiocarcinoma cell strain (being the cholangiocarcinoma cell strain of stably express Fbw7) of the plasmid of Fbw7 and studies ubiquitin ligase Fbw7 to the impact of cholangiocarcinoma cell, and described PcDNA3.1 (+)-Fbw7 recombinant plasmid can be used for preparing the medicine for the treatment of human bile duct cancer.

Below provide the establishment method of the cell strain of stable transfection PcDNA3.1 (+)-Fbw7 plasmid, concrete steps are:

1) will be with the PcDNA3.1 (+) of neomycin resistance screening-Fbw7 plasmid with liposome 2000 reagent transfected with human cholangiocarcinoma cell strains, the transfection concrete steps are as follows:

(1) treats that Growth of Cells carries out transfection when density is 70%～90%, use the opti-MEM of 150 μ L to dilute 3 μ g plasmids;

(2) opti-MEM with 150 μ L dilutes 10 μ L's Then room temperature effect 5min;

(3) behind the 5min mixed solution in the step (1) added to mixed solution in the step (2), blow and beat gently mixing, room temperature effect c;

(4) during the 20min, the cell of transfection is used the phosphoric acid buffer rinsing once, be replaced by unparalleled anti-substratum;

(5) shake up about the transfection liquid adding cell with above-mentioned mixing behind the 20min;

(6) cell cultures detects two days later transfection efficiency and prepares the beginning screening operation;

2) after the transfection, will be transferred in the Tissue Culture Dish after the digestion of human bile duct cancer cell strain, nutrient solution can be 1640 substratum;

3) in nutrient solution, add the G418(neomycin analog) screen; Be 3～4 days the pitch time of changing nutrient solution during the screening;

4) with cell dilution to 1000 cell/ml, in the G418 concentration range of 100 μ g/ml～1mg/ml, screen, select the minimum G418 concentration that in 10～14 days, make the whole death of cell and carry out next step shaker test;

5) because gene transfection wants for some time just can give expression to protein after cell is interior, after transfection 24h, add the G418 screening, all to change the screening liquid that once contains G418 in per 3～5 days, at this moment drug level can be down to 1000 μ g/mL;

6) select monoclonal optimization, the disadvantageous effect that causes to positive colony for the death that as far as possible reduces negative clone and the yield that increases positive colony can be used cover around-France or scrape division and be combined with the limit dilution method and come screening positive clone; After the dosing, under high power lens, strike off recessive clone, continue screening and culturing behind the digestion positive colony; Or entangle positive colony with the collar, in the collar, add trypsinase or EDTA digestion, Digestive system is drawn onto in the new hole of another one cultivates, with limiting dilution assay positive colony is screened in 96 orifice plates more at last;

7) through the screening about 4 weeks, the positive colony that obtains is all more stable, foreign gene is not incorporated into goal gene in the genome and can causes the volume production of the target protein of expressing to give birth to very big-difference, continuity along with incubation time, those cells of having lost the cell of foreign gene and seldom having expressed goal gene can occupy advantage, the cell of strongly expressed target protein can be fewer and feweri, through being secreted by force target protein, the cell clone of inheritance stability after the screening more than 2 times.

The experiment discovery, cholangiocarcinoma patients Fbw7 expression of nucleic acid level significantly is lower than the normal people, Fbw7 is transfected in the cholangiocarcinoma cell strain, result's demonstration, Fbw7 can suppress propagation and the transfer ability of cholangiocarcinoma cell, and the mediation cholangiocarcinoma cell is to the tolerance of medicine.

The invention provides construction process and the application of PcDNA3.1 (+)-Fbw7 recombinant plasmid in preparation treatment human bile duct cancer tumour medicine of a kind of PcDNA3.1 (+)-Fbw7 recombinant plasmid.The damping fluid wiring solution-formings such as this recombinant plasmid water soluble or TE, or make dry powder with freeze-drying.This recombinant plasmid can be at propagation and the transfer ability that can suppress at cell levels the human bile duct cancer cell, and the cell anti-apoptotic genes expression of reduction and Tumor-assaciated is expressed, blocking-up tumour cell cycle related developments, and the resistance of the antitumor drug of reduction human bile duct cancer cell.For the treatment of human bile duct cancer provides new target spot and treatment thinking, promote the human bile duct cancer progress of research.

Description of drawings

Fig. 1 is the ability that recombinant plasmid PcDNA3.1 (+)-Fbw7 suppresses cholangiocarcinoma cell strain propagation.In Fig. 1, X-coordinate is fate, and ordinate zou is light absorption value (560nm); Mark ■ is vector, ● be Fbw7.

Fig. 2 is the ability that recombinant plasmid PcDNA3.1 (+)-Fbw7 suppresses cholangiocarcinoma cell strain migration.In Fig. 2, ordinate zou is cell quantity (10 ⁴).

Fig. 3 is that Fbw7 is on the impact of cis-platinum mediation QBC939 propagation.In Fig. 3, ordinate zou is light absorption value (490nm); Mark a is 8uM, and b is 16uM.

Fig. 4 is pcDNA3.1 (+) plasmid map.

Fig. 5 is the pSG5-Myc plasmid map.

Embodiment

Embodiment 1

Recombinant plasmid PcDNA3.1 (+)-Fbw7 is on the impact of cholangiocarcinoma cell strain multiplication capacity, and concrete operation step is as follows:

The cholangiocarcinoma of taking the logarithm vegetative period carries out transfection, to counting behind the cell dissociation behind the transfection 24h, be inoculated in 96 orifice plates with 5000 cells in every hole, each group is set up 8 multiple holes, final volume 200 μ L/ holes, and establish row and acellularly 200 μ L nutrient solutions are only arranged as the blank hole, marginal pore is filled with PBS, puts into 5%CO ₂37 ℃ are spent the night adherent in the incubator.Add MTT by experimental design requirement lucifuge after cultivating different time, every sky removes substratum before adding, every hole adds 60 μ L substratum and 10 μ L concentration are the MTT solution of 5mg/ml, hatch careful sucking-off MTT behind the 6h for 37 ℃, every hole adds 100 μ L dimethyl sulfoxide (DMSO), and lucifuge low-frequency oscillation 10min fully dissolves crystallisate, measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument 560nm, judge cell proliferation or survival condition by comparing light absorption value.

Recombinant plasmid PcDNA3.1 (+)-Fbw7 suppresses the ability of cholangiocarcinoma cell strain propagation, the results are shown in Figure 1, can find out in cholangiocarcinoma cell QBC939, compare with the cell of the unloaded plasmid of transfection, transfection recombinant plasmid PcDNA3.1 (+)-Fbw7 can suppress cell proliferation.

Embodiment 2

Recombinant plasmid PcDNA3.1 (+)-Fbw7 is on the impact of cholangiocarcinoma cell strain transfer ability, and concrete operation step is as follows:

The cell serum starvation is processed 24h, DMEM serum-free medium aquation Migration/Invasion cell: the cell upper strata adds 500 μ l nutrient solutions, and lower floor adds 700 μ l nutrient solutions, places 37 ℃ of cell culture incubators to hatch 2h, cell dissociation, counting are diluted to 1 * 10 with serum-free DMEM is resuspended ⁵/ mL removes the DMEM nutrient solution of cell, adds the DMEM nutrient solution of 700 μ l10%FBS in cell lower floor, the upper strata adds 500 μ l cell suspensions, incubator is hatched 48h, removes nutrient solution, wipes the cell of upper chamber with cotton swab, methyl alcohol is 20min fixedly, PBS washes 2 times, Viola crystallina dye liquor dyeing 30min, and PBS washes 3 times, microscopically is taken pictures, upper 8 visuals field, cut-off footpath counting.

Recombinant plasmid PcDNA3.1 (+)-Fbw7 suppresses the ability of cholangiocarcinoma cell strain migration, the results are shown in Figure 2, can find out in cholangiocarcinoma cell Sk-chA-1, the cell migration ability of transfection recombinant plasmid PcDNA3.1 (+)-Fbw7 is lower than cellular control unit, difference has statistical significance, illustrates that recombinant plasmid PcDNA3.1 (+)-Fbw7 can suppress the migration of cell.

Embodiment 3

Recombinant plasmid PcDNA3.1 (+)-Fbw7 is on the impact of cholangiocarcinoma cell Susceptibility, and concrete operation step is as follows:

After using recombinant plasmid PcDNA3.1 (+)-Fbw7 effect cholangiocarcinoma cell strain QBC939, be worth up and down two concentration cultivation 48h with chemotherapeutics cis-platinum Lethal Dose 50 IC50, mtt assay detects the survival rate (step is referring to embodiment 1) of cell, the result as shown in Figure 3, show that recombinant plasmid PcDNA3.1 (+)-Fbw7 can increase cholangiocarcinoma cell to the susceptibility of chemotherapeutics cis-platinum, the collaborative chemotherapeutics that promotes suppresses the propagation of cholangiocarcinoma cell QBC939.

Embodiment 4

Studies show that in a large number, Fbw7 expresses at kinds cancers such as liver cancer, mammary cancer, uterus carcinoma, prostate cancer, cancer of the stomach, colorectal carcinoma, carcinoma of the pancreas, esophageal squamous cell carcinoma and liver cancer, and to the genesis important role of mammary cancer, uterus carcinoma and prostate cancer.Recent research discloses FBW7 promotes cell by many A signal pathways such as NOTCH, C-Myc, NF-κ B apoptosis, and the expression that suppresses cell interior inhibitor of apoptosis protein Bcl-2, Bcl-xL and cFLIPL reduces apoptosis.In addition, research finds that FBW7 also has important regulating and controlling effect aspect the active oxygen of cell, thereby affects the apoptosis of cell, so FBW7 important role aspect the regulating cell proliferation and apoptosis.FBW7 and substrate, inhibitor and and the research of the mutual relationship of other signal paths also have a lot of blank, believe along with going deep into that FBW7 is understood, the establishment of FBW7 function in normal and cancerous tumor cell is for FBW7 provides possibility as drug development.

The full length sequence information of recombinant plasmid collection of illustrative plates details and FBW7 is shown in Figure 4 and 5.

Materials and methods

1, material

1) main agents

The plasmid extraction test kit is available from German QIAGEN company; Glue reclaims test kit available from TIANGEN company; Restriction enzyme EcoR I, Not I, T4DNA ligase enzyme are all available from Dalian Takara bio-engineering corporation; Transfection reagent Lipofectamine 2000 is available from American I nvitrogen company; The medicine cis-platinum is available from the gloomy medicine company of Jiangsu Province, China person of outstanding talent limited-liability company; Calf serum, substratum are available from U.S. Hyclone company; Tissue Culture Plate is available from U.S. Corning company; Primer is synthetic, sequencing analysis is finished by American I nvitrogen company.

2) carrier, bacterial strain, cell strain

Expression vector PcDNA3.1 (+), PSG5-Myc-Fbw7 plasmid, bacillus coli DH 5 alpha, human bile duct cancer cell QBC939 are this laboratory and preserve.

3) key instrument

TE2000 fluorescence inverted microscope (Nikon), 1284 Biohazard Safety Equipments (Thermo), 3110 CO2 incubators (Thermo), Multiskan Mk3 microplate reader (Thermo), horizontal gel-electrophoretic apparatus (Beijing 6 1), Tannon-2500R gel imaging system (Tannon).

2, experimental technique

1) design of primers

Use biosoftware Primer5, follow two pairs of goal gene primers of fundamental principle design of design of primers, the primer is as shown in table 1.

Table 1

Detect the expression contents of gene with the SYBR Ex Taq test kit of Takara company, concrete experimental procedure is as follows:

Reaction conditions is 95 ℃ of denaturation 20s; 95 ℃ of sex change 10s, 60 ℃ of annealing 20s, 72 ℃ are extended 20s, circulate altogether 45 times, detect fluorescence at annealing stage; The melt curve analysis condition is 50 ℃ of 15s, and 51 ℃ are warming up to 99 ℃, and every rising was once stopping 5s; Data analysis is undertaken by relative quantification-Δ Δ Ct method.

2) extraction of plasmid

Picking list bacterium colony contains the antibiotic LB nutrient solution in 100mL from flat board, 37 ℃ of shaking table overnight incubation, with overnight culture with 3, the centrifugal 10min of 500rpm, abandon supernatant, add subsequently 5ml P1, vortex concussion suspension bacterium, guarantee that bacterium all hangs, room temperature adds 5ml P2 after placing 2～3min, softly put upside down back and forth rapidly mixing, room temperature adds 5ml P3 after placing 3～5min, put upside down rapidly mixing, place 5min on ice, then with the centrifugal 5min of the speed of 12,000rpm, recentrifuge 10min all removes to guarantee precipitation behind the collection supernatant, then supernatant liquor is drawn in the tip-100 pillar (pillar being carried out balance with 4ml QBT buffer), flow to end rear adding 10ml QC buffer washing until liquid, again with 10ml QC buffer washing once, add subsequently 5ml QF buffer eluted dna and collect elutriant after flowing to end, elutriant adds 3.5ml isopropanol precipitating DNA plasmid, with 4 ℃ of centrifugal 30min of speed of 13,000rpm, supernatant discarded, precipitation 1ml70% washing with alcohol, place in the Bechtop and dry, then add 200 μ l Tris-HCl(pH8.0) dissolution precipitation, gained DNA plasmid can be used for the cell transfecting experiment.

3) acquisition of purpose fragment

Cut the vector plasmid that comprises purpose fragment full length fragment with restriction enzyme EcoR I and Not I while enzyme, endonuclease reaction system and parameter are as follows:

Behind the each component mixing, be placed on 2h in 37 ℃ of water-baths;

4) purifying of product

The purifying of product reclaims the operation of test kit specification sheets by company's glue, and concrete operations are as follows:

A, dna fragmentation separate with 2% agarose gel electrophoresis;

B, corresponding molecular weight Marker downcut the gel strips that reclaims in advance fragment, put into centrifuge tube, add the long-pending PN of triploid, and water-bath is placed, until blob of viscose dissolves fully;

C, previous step gained solution is added among the adsorption column CA1, the centrifugal 30sed of 13,000rpm outwells the waste liquid in the collection tube, and adsorption column is reentered in the collection tube;

D, add 700uL rinsing liquid PW in the adsorption column, the centrifugal 30sed of 13,000rpm outwells waste liquid, and adsorption column is reentered in the collection tube;

E, add 500uL rinsing liquid PW in the adsorption column, the centrifugal 30sed of 13,000rpm outwells waste liquid, and adsorption column CA1 is reentered in the collection tube, and the centrifugal rinsing liquid of as far as possible going out places room temperature number minute with adsorption column, thoroughly dries;

F, adsorption column is put in the clean centrifuge tube, to the elution buffer of an amount of 65～70 ℃ of water-bath preheatings of the unsettled dropping in adsorption column mid-way, room temperature is placed 2min, and the centrifugal 1min of 13,000rpm collects dna solution;

G, in order to improve the DNA yield, in the solution of centrifugal gained again add-back centrifuge tube adsorption column, repeating step F.

5) purpose fragment and vector plasmid are cut, connected to enzyme

Cut each purpose fragment product and carrier with restriction enzyme EcoR I and Not I while enzyme, endonuclease reaction system and parameter are as follows:

Behind the each component mixing, be placed on 2h in 37 ℃ of water-baths;

Behind the each component mixing, be placed on 2h in 37 ℃ of water-baths;

37 ℃ of effect 2h after agarose electrophoresis identifies that enzyme is cut efficient, cut plasmid and purpose fragment after glue recovery enzyme is cut, quantitatively.Purpose fragment after enzyme is cut has identical sticking end with linear PcDNA3.1 (+), connects through the T4DNA ligase enzyme.The ligation system:

Behind the each component mixing, connect in 16 ℃ of metal baths and spend the night;

6) preparation of competence bacterium and conversion

The picking bacillus coli DH 5 alpha is preserved bacterial strain, do not containing streak inoculation on the antibiotic LB flat board, 37 ℃ of overnight incubation, picking list bacterium colony is in 5ml LB substratum subsequently, (37 ℃ of incubated overnight on shaking table, 200rpm), getting the 0.5ml overnight culture receives in the 50ml LB substratum, 37 ℃ of shaking tables are cultured to bacterium liquid OD600 and bacterium liquid were transferred in the 50ml centrifuge tube in 0.2～0.4 o'clock, place ice bath 20min on ice, then with 3,000rpm speed is collected bacterium in 4 ℃ of centrifugal 10min, the bacterial sediment MgSO4(0.1M of 10ml precooling) soft resuspension places 10min on ice, again with 3,000rpm speed is in 4 ℃ of centrifugal 10min, abandon supernatant, add the CaCl2(0.1M of 8ml precooling) resuspended, place 4 ℃ of refrigerator overnight, add the 2ml50% sterile glycerol, be distributed into the 50ul/ pipe stand-by in-80 ℃ of preservations.

From-80 ℃, take out competent cell, place and treat that on ice bacterium liquid dissolves, in Bechtop, add DNA, finger springing mixing is placed 42 ℃ of water-bath heat shock 90s behind the 30min on ice, places fast 1～2min on ice after the heat shock, then add 200 μ L LB substratum recovery 45min on 37 ℃ of shaking tables, at last with conversion fluid coating with contain on the antibiotic LB flat board, in 37 ℃ of overnight incubation, choose single bacterium colony and in containing antibiotic LB substratum, cultivate also upgrading grain.

7) cultivation of cell and going down to posterity

Cell cultures places 37 ℃, the incubator of the saturated humidity environment of 5%CO2+95% air to cultivate in DMEM+10%FBS or RPMI1640+10%FBS substratum.

Suck first the nutrient solution of attached cell when going down to posterity, the rinsing of PBS damping fluid once, adding an amount of trysinization liquid digests, add subsequently a small amount of nutrient solution and stop the pancreatin effect, transfer pipet piping and druming forms the individual cells suspension for several times, then by blood counting chamber cell is counted, according to the cell of experiment needs inoculation different concns, or suspension inoculation continued to cultivate in other are preinstalled with the Tissue Culture Dish of fresh culture.

8) cell transfecting

Use Lipofectamine2000 transfected with human cholangiocarcinoma QBC939 cell, concrete steps are: 24h accesses cell in the culture dish of 24 orifice plates or 35mm before the transfection, keep cell density to be about 60～70%, then 1h is changed to unparalleled anti-nutrient solution with cell before the transfection, suck old nutrient solution, carefully add the nutrient solution of 37 ℃ of preheatings; Then prepare transfection liquid; take the culture dish of 35mm as example; the opti-MEM that adds 250 μ l in the centrifuge tube of a 1.5ml; then add 4 μ g plasmid mixings; add the opti-MEM of 250 μ l in the centrifuge tube of another 1.5ml, subsequently 10 μ l Lipofectamine2000 transfection reagents are added, room temperature is placed 5min behind the mixing; then the transfection reagent after will diluting and plasmid mixing, room temperature dropwise adds the transfection liquid for preparing in the cell after placing 20min.Different size culture dish compound method is as shown in table 2.

Table 2

9) cytotoxicity experiment

Cell is inoculated in 96 orifice plates, every hole 100 μ l, and establish that 2 holes are acellular only to have 100 μ l nutrient solutions as the blank hole, cultivating behind the different time every hole, to add 10 μ l concentration be 5mg/ml MTT solution, every hole adds 100 μ l10%SDS-0.01M HCl dissolving and spends the night behind 37 ℃ of reaction 4h, and microplate reader is measured OD ₅₆₀, by comparing OD ₅₆₀Value is judged cell proliferation or survival condition.

10) cell migration experiment

The cell serum starvation is processed 24h; The cell upper strata adds 500 μ l serum-free mediums before the experiment, and lower floor adds 700 μ l serum-free mediums, places 37 ℃ of cell culture incubators to hatch 2h the Migration/Invasion cell is carried out aquation; Cell dissociation, counting after hungry the processing are with the resuspended 1 * 105/ml that is diluted to of serum-free medium; Remove the aquation nutrient solution of cell, contain the 10%FBS nutrient solution what cell lower floor added 700 μ l10%FBS, the upper strata adds 500 μ l cell suspensions, and incubator is hatched 48h; Remove nutrient solution, wipe the cell of upper chamber with cotton swab; Methyl alcohol is 20min fixedly, and PBS washes 2 times, Viola crystallina dye liquor dyeing 30min, and PBS washes 3 times, and microscopically is taken pictures, upper 8 visuals field, cut-off footpath counting.

11) data processing

Data are the mean value of many experiments in the experimental result, and represent with mean value+standard deviation; With SPSS11.0 data are carried out the significance difference opposite sex and analyze, take P＜0.05 as significant difference.

Claims (6)

1. (1.PcDNA3.1+)-Fbw7 recombinant plasmid, it is characterized in that by extracting people Fbw7 full length gene, screen PcDNA3.1 (+) plasmid of label as carrier to contain Liu Suanyan NEOMYCIN SULPHATE, insert the Fbw7 full length gene PcDNA3.1's (+) and between restriction enzyme site EcoR I and the Not I, structure a kind of contains the PcDNA3.1 (+) of Fbw7 full length gene and Liu Suanyan NEOMYCIN SULPHATE screening label-Fbw7 recombinant plasmid.
2. As claimed in claim 1 PcDNA3.1 (+) ?the construction process of Fbw7 recombinant plasmid, it is characterized in that may further comprise the steps:

   1) PSG5 ?Myc ?Fbw7 plasmid spectrogram information and PcDNA3.1 (+) plasmid spectrogram information are compared, determine double enzyme site EcoR I and Not I;

   2) double digestion PSG5 ?Myc ?the Fbw7 plasmid, concrete reaction system is as follows:

   PSG5 ?Myc ?Fbw71 μ g, EcoR I 1.0 μ L, Not I 1.0 μ L, 1 * H2.0 μ L, BSA2.0 μ L, the sterilization ultrapure water is mended to 20 μ L, behind the each component mixing, is placed on 2h in 37 ℃ of water-baths;

   3) double digestion PcDNA3.1 (+) plasmid vector, concrete reaction system is as follows:

   PcDNA3.1 (+) 1 μ g, EcoR I 1.0 μ L, Not I 1.0 μ L, 1 * H2.0 μ L, BSA2.0 μ L, the sterilization ultrapure water is mended to 20 μ L, behind the each component mixing, is placed on 2h in 37 ℃ of water-baths;

   4) identify double digestion efficient by agarose gel electrophoresis, plasmid vector PcDNA3.1 (+) is connected with goal gene Fbw7 total length, concrete reaction system is as follows:

   10 * reaction buffer, 1.0 μ L, PCDNA3.1 (+) 2.0 μ L, Fbw76.0 μ L, T4DNA ligase enzyme 1.0 μ L behind the each component mixing, connect in 16 ℃ of metal baths and spend the night;

   5) with the recombinant plasmid transformed competence colibacillus bacterium, screening positive clone, and the evaluation of checking order, such as the positive clone of qualification result, extracting PcDNA3.1 (+)-Fbw7 recombinant plasmid then;

   6) with step 5) PcDNA3.1 of described extracting (+) ?the Fbw7 recombinant plasmid carry out the dna sequencing analysis, choose two-way order-checking pattern, use PcDNA3.1 (+) plasmid universal primer across the one by one base detection of multiple clone site district of plasmid, detected result and Fbw7 sequence are compared.
3. As claimed in claim 2 PcDNA3.1 (+) ?the construction process of Fbw7 recombinant plasmid, it is characterized in that in step 5), the concrete grammar that described order-checking is identified is:

   Carrying out colony PCR amplification with the 1st pair of primer, the rear products therefrom that increases is carried out agarose gel electrophoresis, is 244bp such as gained dna fragmentation length, then is accredited as positive colony, and described the 1st pair of primer is:

   Forward primer: 5 '-ctacccaaaagtaatcatcttaagtg-3 ';

   Reverse primer: 5 '-cccaaccatgacaagattttcc-3 ';

   Carrying out colony PCR amplification with the 2nd pair of primer, the rear products therefrom that increases is carried out agarose gel electrophoresis, is 278bp such as gained dna fragmentation length, then is accredited as positive colony, and described the 2nd pair of primer is:

   Forward primer: 5 '-tttctgtttctccctctg-3 ';

   Reverse primer: 5 '-gagcatttaagggagagataagag-3 '.
4. 4. the construction process of PcDNA3.1 (+)-Fbw7 recombinant plasmid as claimed in claim 2, it is characterized in that in step 6) in, it is described that to carry out the dna sequencing analysis be with step 5) PcDNA3.1 (+) of described extracting-Fbw7 recombinant plasmid delivers to American I nvitrogen company and carries out the dna sequencing analysis.
5. 5. the as claimed in claim 1 application of PcDNA3.1 (+)-Fbw7 recombinant plasmid in preparation treatment human bile duct cancer medicine.
6. Stable transfection PcDNA3.1 (+) ?the establishment method of cell strain of Fbw7 plasmid, it is characterized in that its concrete steps are:

   1) will with the PcDNA3.1 of neomycin resistance screening (+) ?the Fbw7 plasmid with liposome 2000 reagent transfected with human cholangiocarcinoma cell strains, the transfection concrete steps are as follows:

   (1) treat that Growth of Cells carries out transfection when density is 70%～90%, use 150 μ L opti ?MEM dilute 3 μ g plasmids;

   (2) with the opti of 150 μ L ?MEM dilute 10 μ L's

   

   2000, room temperature effect 5min then;

   (3) behind the 5min mixed solution in the step (1) added to mixed solution in the step (2), blow and beat gently mixing, room temperature effect c;

   (4) during the 20min, the cell of transfection is used the phosphoric acid buffer rinsing once, be replaced by unparalleled anti-substratum;

   (5) shake up about the transfection liquid adding cell with above-mentioned mixing behind the 20min;

   (6) cell cultures detects two days later transfection efficiency and prepares the beginning screening operation;

   2) after the transfection, will be transferred in the Tissue Culture Dish after the digestion of human bile duct cancer cell strain, nutrient solution can be 1640 substratum;

   3) in nutrient solution, add the G418(neomycin analog) screen; Be 3～4 days the pitch time of changing nutrient solution during the screening;

   4) with cell dilution to 1000 cell/ml, in the G418 concentration range of 100 μ g/ml～1mg/ml, screen, select the minimum G418 concentration that in 10～14 days, make the whole death of cell and carry out next step shaker test;

   5) because gene transfection wants for some time just can give expression to protein after cell is interior, after transfection 24h, add the G418 screening, all to change the screening liquid that once contains G418 in per 3～5 days, at this moment drug level can be down to 1000 μ g/mL;

   6) select monoclonal optimization, the disadvantageous effect that causes to positive colony for the death that as far as possible reduces negative clone and the yield that increases positive colony can be used cover around-France or scrape division and be combined with the limit dilution method and come screening positive clone; After the dosing, under high power lens, strike off recessive clone, continue screening and culturing behind the digestion positive colony; Or entangle positive colony with the collar, in the collar, add trypsinase or EDTA digestion, Digestive system is drawn onto in the new hole of another one cultivates, with limiting dilution assay positive colony is screened in 96 orifice plates more at last;

   7) through the screening about 4 weeks, the positive colony that obtains is all more stable, foreign gene is not incorporated into goal gene in the genome and can causes the volume production of the target protein of expressing to give birth to very big-difference, continuity along with incubation time, those cells of having lost the cell of foreign gene and seldom having expressed goal gene can occupy advantage, the cell of strongly expressed target protein can be fewer and feweri, through being secreted by force target protein, the cell clone of inheritance stability after the screening more than 2 times.

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