CN102657879A - Application of yeast expressing product of gentiana straminea beta-amyrin synthetase gene GsAS in pharmacy - Google Patents

Abstract

The invention discloses application of a yeast expressing product of gentiana straminea beta-amyrin synthetase gene GsAS in preparing a hepatoma cell resistance medicament. The concentration of the yeast expressing product of gentiana straminea beta-amyrin synthetase gene GsAS, which can effectively inhibit growth of hepatoma cell HepG2 and SMMC7721, is between 1 and 25mu g/mL. Compared with a triterpenoid which is directly extracted from Chinese herbal medicaments, the yeast expressing product of gentiana straminea beta-amyrin synthetase gene GsAS is purer, and a specific triterpenoid can be acquired more conveniently, so that a novel method and resources are provided to preparation of liver cancer resistance medicaments, and foundation is established for research and development of liver cancer resistance medicaments.

Description

The application of yeast expression product in pharmacy of Flos Cannabis Macrophylla β-amyrin synthase gene GsAS

Technical field

The present invention relates to the preparation of cancer therapy drug, relate in particular to the application of yeast expression product in the preparation medicines resistant to liver cancer of a kind of Flos Cannabis Macrophylla β-amyrin synthase gene GsAS.

Background technology

High and cold Tibetan medicine Flos Cannabis Macrophylla is Gentianaceae (Gentianaceae), Gentiana (Gentiana); Mainly be distributed in the extremely frigid zones of Tibet, Qinghai, Gansu height above sea level 2500-4700 rice, perennial herb, herb and root are used as medicine; Owing to its minute spreading; Trophophase is long, and medical material is collected very difficulty, thereby has become Endangered species.Cure mainly jaundice type hepatitis, rheumatic arthritis, tuberculosis hectic fever, asthma, tic, shock, anaphylaxis.Be rich in the pentacyclic triterpene secondary metabolites, β-amyrin (beta-amyrin) is one type of important triterpenoid compound wherein.

β-amyrin is one of modal triterpenoid compound in the plant.Its five carbocyclic ring skeletons are folded into chair conformation, are by 2, and 3-oxidized keratin zamene forms under β-amyrin synzyme catalysis through series reaction.And its synzyme is a very important rate-limiting enzyme in this approach, is controlling the synthetic first step reaction of these medicinal ingredient precursors, and therefore the clone to this gene is extremely important.Oleanolic acid is the end product in this reaction, and β in gentianaceae plant Flos Cannabis Macrophylla-amyrin synthase gene has very high facilitation for the accumulation of anti-hepatitis effective ingredient oleanolic acid.

Till now, from many dicotyledons, be separated to β-amyrin synthase gene, like the PNY1 in the dicotyledonous Radix Ginseng, PNY2; GgbAS1 in the Radix Glycyrrhizae; PSY in the Semen Pisi sativi; Also have the shape Herba Medicaginis of cutting and Betula platyphylla Suk. etc.: the AsbAS1 in the monocotyledon Herba bromi japonici.And there is article to report checking to its function.Although the existing report of the clone of β in the Flos Cannabis Macrophylla-amyrin synthase gene GsAS and functional verification,, utilize Flos Cannabis Macrophylla β-yeast expression product of amyrin synthase gene GsAS to prepare medicines resistant to liver cancer and also do not appear in the newspapers.

Summary of the invention

To the deficiency of prior art, the problem that the present invention will solve provides the application of yeast expression product in the preparation medicines resistant to liver cancer of a kind of Flos Cannabis Macrophylla β-amyrin synthase gene GsAS.

The application of the yeast expression product of Flos Cannabis Macrophylla β according to the invention-amyrin synthase gene GsAS in the preparation medicines resistant to liver cancer, wherein: said HCC is HepG2 HCC or SMMC7721 HCC; The yeast expression production concentration that effectively suppresses the Flos Cannabis Macrophylla β-amyrin synthase gene GsAS of HepG2 HCC or SMMC7721 liver cancer cell growth is 1～25 μ g/mL, and preferred concentration is 10 μ g/mL.

The yeast expression product of Flos Cannabis Macrophylla β provided by the invention-amyrin synthase gene GsAS is laid a good foundation for developing medicines resistant to liver cancer.

In order to understand essence of the present invention better, yeast expression product pharmacological evaluation and the result with Flos Cannabis Macrophylla β-amyrin synthase gene GsAS explains its application in the medicines resistant to liver cancer preparation below.

The preparation of the yeast expression product of Flos Cannabis Macrophylla β according to the invention-amyrin synthase gene GsAS:

Utilize Flos Cannabis Macrophylla cDNA to be template, the clone obtains β-amyrin synthase gene GsAS, makes up Yeast expression carrier pPICZA-GsAS, and transformed yeast can form methanol evoked (Mut after the yeast conversion ⁺) and methanol responsive type (Mut ^s) bacterial strain, growth on the MM culture medium and the bacterial strain of on the MD culture medium, not growing is Mut ^sThe type yeast strain, and on two kinds of culture medium, can both grow for Mut ⁺The type yeast strain.Contrast pPICZA-GsAS yeast transformant filters out Mut at the growing state that contains on MM (containing the methanol culture medium) and MD (the no methanol culture medium) culture medium ⁺Yeast transformant.Select Mut ⁺Yeast in 25mL MGY culture medium 28 ℃ of shaken cultivation to OD ₆₀₀=2-6 is transferred in the 100-200mL MM culture medium, makes its OD ₆₀₀=1.0.Continue 28 ℃ of shaken cultivation, added a methanol (final concentration 0.5%) in per 24 hours, 4d is cultivated in concussion, and it is subsequent use to collect thalline.

Get in the centrifuge tube of 4 days yeast soln of methanol induction adding 50mL, centrifugal thalline is used the liquid nitrogen grinding thalline; Adding 3-4mL volume ratio is methanol-chloroform (chromatographically pure) of 2: 1, supersonic wave wall breaking 30min; 10000rpm, centrifugal, supernatant is used chloroform extraction, and with washing, repetitive operation.Collect chloroform layer solution, water-bath distillation concentrates, and reclaims solvent and gets extractum.

The pharmacological evaluation of the yeast expression product of β-amyrin synthase gene GsAS:

Normal group: on the DMEM culture medium culturing of 10% calf serum exponential phase normal adherent growth HepG2 HCC or SMMC7721 HCC; Matched group: compare with solvent methanol; Experimental group: extractum is used dissolve with methanol, and the concentration that is used to handle HepG2 HCC or SMMC7721 HCC is 0.5,1,5,10,25,50,75,100 μ g/mL.

The result shows: compare with solvent control group with normal group; After handling 72h, 1,5; 10; The processed group of 25 μ g/mL is compared all with the cellular control unit form and is changed, and the cellular morphology of 10 μ g/mL processed group changes obviously (seeing accompanying drawing 1), and its cell appearance by typical adherent growth before becomes and justifies, comes off, necrosis.And a little less than the variation relatively of the cellular morphology of other processed group in the experimental group.The survival rate that the yeast expression product of GsAS is handled HepG2 HCC and SMMC7721 HCC has a significant effect.HepG2 HCC and SMMC7721 HCC are with the yeast expression product (0.5,1,5,10,25 of the Flos Cannabis Macrophylla β-amyrin synthase gene GsAS of variable concentrations; 50,75,100 μ g/mL) behind the processing 72h, 1,5; The survival rate of HCC HepG2 HCC and SMMC7721 HCC was respectively under 10,25 μ g/mL yeast expression products were handled: 72.8%, 67.4%, 52.5%; 49.8% (seeing accompanying drawing 2) and 74%, 68.8%, 49.5%, 47.8% (seeing accompanying drawing 3).

Sum up: the yeast expression product of Flos Cannabis Macrophylla β-amyrin synthase gene GsAS has inhibitory action to the growth of liver cancer cell growth.The yeast expression production concentration that effectively suppresses the Flos Cannabis Macrophylla β-amyrin synthase gene GsAS of HepG2 HCC or SMMC7721 liver cancer cell growth is 1～25 μ g/mL, and preferred concentration is 10 μ g/mL.

The present invention uses the yeast transformant of the gene that the clone obtains from Chinese herbal medicine to cultivate, induce, extract; Obtain the pentacyclic triterpene chemical compound of anti-hepatocarcinoma; Compare with the triterpenoid that from Chinese herbal medicine, directly extracts; Product is purer, and the acquisition of specific triterpenoid is more convenient, for the preparation of medicines resistant to liver cancer provides a kind of new method and resource.

Description of drawings

Fig. 1: the observed HepG2 HCC of microscopically normal group, solvent control group and 10 μ g/mL processed group are cultivated the cellular morphology behind the 72h.Normal group and the normal adherent growth of solvent control group cell, 10 μ g/mL processed group cellular morphology generation significant changes, the cell appearance becomes and justifies, comes off, necrosis (result of SMMC7721 HCC is similar).

Fig. 2: HCC HepG2 is 1,5, the survival change under the yeast expression product treatment conditions of 10,25 μ g/mL Flos Cannabis Macrophylla β-amyrin synthase gene GsAS.Data are the meansigma methods of three repeated experiments, utilize t-test check carry out the significant difference analysis (* P＜0.05, n=3).

Fig. 3: HCC SMMC7721 is 1,5, the survival change under the yeast expression product treatment conditions of 10,25 μ g/mL Flos Cannabis Macrophylla β-amyrin synthase gene GsAS.Data are the meansigma methods of three repeated experiments, utilize t-test check carry out the significant difference analysis (* P＜0.05, n=3).

The specific embodiment

The clone of embodiment 1, GsAS

1.1 the extraction of the total RNA of Flos Cannabis Macrophylla

(1) Flos Cannabis Macrophylla material is put into the mortar of pre-cooling, adds liquid nitrogen and grinds to form uniform powder rapidly.Note to guarantee that material is immersed in the liquid nitrogen in the process of lapping.

(2) treat after the liquid nitrogen volatilization material to be changed over to rapidly in the centrifuge tube of pre-cooling, every 50-100mg organization material adds 1mL TRIZOL solution, behind the mixing, places 5min in room temperature, 12000r/min, and 2-8 ℃ of centrifugal 10min goes deposition.

(3) add the 0.2mL chloroform in every 1mL TRIZOL solution, the abundant mixing of vibration 15sec is placed 5min, 12000r/min, 2-8 ℃ of centrifugal 15min in room temperature.

(4) get supernatant, every 1mL TRIZOL adds the isopropyl alcohol of 0.5mL, mixing, and room temperature is placed 10～20min.

(5) the 2-8 ℃ of centrifugal 10min of 12000r/min abandons supernatant.

(6) add 1mL 75% washing with alcohol deposition 2 times, 4 ℃ are no more than the centrifugal 5min of 7500r/min, abandon supernatant.

(7) after room temperature is placed and dried, add the distilled water dissolving RNA that 15 μ L DEPC handle.

1.2 reverse transcription synthesizes the first chain cDNA

(1) in the 0.2mL Eppendorf pipe, add following ingredients:

Above-mentioned total RNA (0.1 μ g/ μ L) 2.0 μ L with the DNaseI purification

Olige(dT)anchored?primer(2μM) 2.0μL

(2) 0 ℃ of water-bath degeneration 10 minutes place ice bath immediately.

(3) in 10 μ L reaction systems, add following ingredients: 2.0 μ L, 5 * MMLV RT buffer; 1.0 μ L 250 μ mol/L dNTP mix; 0.25 μ L Rnasin; 0.5 μ L (200u/ μ L) MMLV reverse transcriptase; The above-mentioned RNA degeneration of 2 μ L liquid.42 ℃ are carried out reverse transcription reaction 1hr.The reverse transcription first chain cDNA is used for the reaction of back.

1.3PCR

The synthetic first chain cDNA is a template with above-mentioned reverse transcription, PCR method amplification gene fragment.

(1) the PCR primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd

Primer P0:5 '-ATGTGGAGGCTAAAAATCGG-3 '

Primer P1:5 '-CGTCTCCTACGGCACTTGCTTGCG-3 '

(2) PCR reaction system

(3) PCR response procedures

94 ℃ of 5min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 2min, 35 circulations; Last 72 ℃ are extended 7min.PCR product electrophoresis is as shown in Figure 1.

1.4?5′-RACE

According to procedure shown in TaKaRa 5 '-Full RACE Core Set description.Electrophoresis 5 '-the RACE product.

1.5 the segmental recovery of purpose

(1) downcuts the adhesive tape that contains the purpose band in the above-mentioned electrophoresis result with blade and place the 1.5mLEppendorf pipe.

(2) weigh, add the DR-I Buffer of 3-4 times of volume, 65 ℃ of heating 10min melt blob of viscose, and every separated 2min mixing guarantees that once blob of viscose can fully melt.

(3) the DR-II Buffer of 1/2 volume of adding DR-I Buffer amount, uniform mixing.

(4) Spin Cloumn is placed on the Collection Tube, above-mentioned solution is transferred among the Spin Column, the centrifugal 1min of 5000rpm abandons filtrating.

(5) 500 μ L Rinse A are added among the Spin Column, the centrifugal 1min of 5000rpm abandons filtrating.

(6) 700 μ L Rinse B are added among the Spin Column, the centrifugal 1min of 5000rpm abandons filtrating.

(7) repeating step 6, then the centrifugal again 1min of 12000rpm.

(8) Spin Cloumn is placed on the centrifuge tube of new 1.5mL, adds water or the elution buffer of the 25 μ L that are preheating to 60 ℃ in the centre of Spin Cloumn film, room temperature leaves standstill 1min.

(9) the centrifugal 1min eluted dna of 12000rpm.

(10) repeating step is 9,2 times, with all eluent centralized collection in a pipe, mixing, the electrophoretic examinations organic efficiency that takes a morsel, all the other add the 3mol/L NaAc (pH5.3) of 1/10 volume and the dehydrated alcohol of 2 times of volumes ,-20 ℃ precipitate 1hr.

(11) then 4 ℃, the centrifugal 15min of 12000rpm with 70% cold washing with alcohol deposition, abandons most liquid, with 40 μ L sterilized water dissolution precipitations.The fragment that reclaims is used for the coupled reaction of back.

1.6 connect

Connect with above-mentioned recovery purpose fragment and about 1: the 1 in molar ratio ratio of pMD-18T carrier (available from precious biological engineering company limited, down together), linked system is following:

Mixing is also of short duration centrifugal, and 16 ℃ of connections are spent the night.Obtain recombiant plasmid pMD18T-AS1, be used for the reaction of back.

1.7CaCl2 legal system is equipped with competent cell

(1) the single colony inoculation of picking DH10B is in 5mL LB fluid medium, and 37 ℃ of shaken cultivation are spent the night.

(2) in 1: 100-1: 50 ratio, get 1mL bacterium liquid and be inoculated in the 100mL LB fluid medium, 37 ℃ of shaken cultivation to bacterium liquid OD600 are 0.3-0.6.

(3) with bacterium liquid ice bath 10min, 4 ℃ of centrifugal 10min of 4000rpm collect thalline.

(4) after deposition adds the 0.1M CaCl2 suspension of 10mL pre-cooling, ice bath 30min.

(5) 4 ℃ of centrifugal 10min of 4000rpm.Pellet resuspended is preserved in mixing and the ice-water bath in the 0.1M of 1mL pre-cooling CaCl2, and is subsequent use or add 15% glycerol and place-70 ℃ of preservations.

1.8 thermal shock method Transformed E coli DH10B

(1) under aseptic condition, draw 100 μ L competent cells, add in the aseptic Eppendorf pipe of 1.5mL pre-cooling, add 10 μ L and connect product (recombiant plasmid in 1.6), mixing places 30min on ice immediately gently.

(2) heat shock 90sec (accurately clocking) in 42 ℃ of waters bath with thermostatic control.

(3) ice bath 3-5min.

(4) add 800 μ L and do not contain antibiotic LB fluid medium, mixing, 37 ℃ of shaken cultivation 45-60min.

(5) of short duration centrifugal collection thalline with the resuspended thalline of 150 μ L LB fluid mediums, goes on the LB solid plate that contains antibiotic Amp, X-gal, IPTG, with aseptic spreading rod coating.

(6) flat board is placed 15-30min in 37 ℃ of forwards and be absorbed to liquid, inversion is dull and stereotyped, in 37 ℃ of cultivation 12-16hr, observes flat board and has blue white macula.The plasmid that white macula is used for the back extracts.

1.9 alkaline lysis method of extracting escherichia coli plasmid

(1) the single bacterium colony of picking white, access contains in the 3mL LB fluid medium of antibiotic Amp (50mg/L), and the white single bacterium colony that also will preserve institute's picking simultaneously is in the solid LB flat board that contains antibiotic Amp (50mg/L), and 12-16h are cultivated in 37 ℃ of concussions;

(2) the centrifugal 30sec of 12000rpm collects the thalline in the culture, abandons most supernatant as far as possible;

(3) add the solution I that 100 μ L ice pre-cooling, on vortice, make thalline fully resuspended;

(4) add 200 μ L solution II, immediately centrifuge tube is slowly put upside down for several times ice bath 5-10min;

(5) add the solution III that 150 μ L ice pre-cooling, slowly put upside down centrifuge tube and fully form ice bath 5-10min until white precipitate for several times;

(6) the centrifugal 3min of 12000rpm gets supernatant and changes in another microcentrifugal tube, adds 2 times of volume 95% ethanol, and behind the mixing, room temperature leaves standstill 3min; The centrifugal 3min of 12000rpm makes the DNA deposition;

(7) abandon most supernatant, get 200 μ L TE solution dissolution precipitations;

(8) add the 5mol/L LiCl solution that equal-volume is iced pre-cooling, ice bath 5min precipitates a large amount of RNA;

(9) 12000rpm, centrifugal 3min;

(10) shift supernatant in another centrifuge tube, add 95% ethanol of 2 times of volumes, leave standstill 3min in room temperature behind the mixing, the centrifugal 3min of 12000rpm makes the plasmid deposition;

(11) abandon behind the supernatant with 1mL 70% washing with alcohol deposition, abandon most liquid;

(12) after the drying at room temperature, get the TE solution dissolution precipitation that 20 μ L contain RNaseA (20 μ g/mL), 37 ℃ of water-bath 30～60min digestion RNA.

(13) for reducing the loss, can with TE cumulative volume be supplemented to 200 μ L earlier, add isopyknic phenol-chloroform-isoamyl alcohol, the 5-10min that fully vibrates, the centrifugal 5min of 12000rpm;

(14) get supernatant, add isopyknic chloroform-isoamyl alcohol, repeat above operation;

(15) get supernatant, add the 3mol/L sodium acetate (pH5.3) of 1/10 volume and the dehydrated alcohol of 2 times of volumes, place 15min for-20 ℃ behind the mixing, the centrifugal 3min of 12000rpm makes the plasmid deposition;

(16) abandon supernatant with 1mL 70% washing with alcohol deposition, abandon liquid, with 40 μ L TE or sterilized water dissolution precipitation.Plasmid is used for the reaction of back.

1.10 plasmid enzyme restriction checking

With the above-mentioned plasmid of EcoRI enzyme action, the endonuclease reaction system is as shown in the table:

Recombiant plasmid pMD18T-AS1 16 μ L

EcoRI restricted enzyme 2 μ L

10×Buffer 2μL

With above-mentioned reaction system mixing and the centrifugal 1min of 5000rpm, 37 ℃ of water-baths spend the night, and carry out 1% TAE agarose gel electrophoresis detection then.

1.11DNA order-checking

The positive single bacterium colony that picking contains recombiant plasmid shakes with the liquid LB that contains Amp (50mg/L) and spends the night; Serve the order-checking of Hai Boya Bioisystech Co., Ltd then; Obtain sequencing result, analyze the PNY1 homology of above-mentioned cDNA sequence and Radix Ginseng through NCBI blast; What the proof experiment obtained is the β-amyrin synthase gene of Flos Cannabis Macrophylla, called after GsAS.

The Yeast expression carrier of embodiment 2, Flos Cannabis Macrophylla β-amyrin synthase gene GsAS makes up

2.1 the structure of Yeast expression carrier

2.1.1 the acquisition of genes of interest

(1) design of primers: design one couple of PCR amplified band EcoRI and XhoI restriction enzyme site be the GsAS gene all.

P01：5’-GAATTCATGTGGAGGCTAAAAATCGG-3’

EcoRI

P12：5’-CTCGAGCGTCTCCTACGGCACTTGCTTGCG-3’

XhoI

(2) PCR reaction system:

(3) the PCR reaction condition does

94 ℃ of 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 2min, 35 circulations; Last 72 ℃ are extended 7min.After reclaiming, PCR product electrophoresis is used for the reaction of back.

2.1.2 the construction step of expression vector

Introduce Yeast expression carrier pPICZA (available from precious biological engineering company limited), contain EcoR and two restriction enzyme sites of XhoI in containing on this carrier.

To have the cDNA of EcoR and XhoI restriction enzyme site and the double digestion that plasmid carries out EcoR and XhoI respectively, connect more than 16 hours under the catalysis of T4 ligase according to 5: 1 ratio again.Reaction system and condition are following:

(1) cDNA endonuclease reaction system and condition:

37℃16h；

(2) plasmid enzyme restriction reaction system and condition:

37℃16h；

Coupled reaction is carried out with T4DNA Ligase, and system and condition are with the linked system and the condition of T carrier.With connecting the product transformed into escherichia coli, from the bacterium colony of kalamycin resistance, filter out recon, obtain Yeast expression carrier pPICZA-GsAS.The plasmid that extracts positive colony pPICZA-GsAS carries out PCR evaluation and double digestion evaluation, and reaction system and condition are with identifying recon.

2.2DNA importing yeast

(1) monoclonal of picking yeast strain (Pichia sp.) is seeded to 10mL liquid YPD culture medium [20g/L peptone, 10g/l yeast extract, glucose 2% (singly join singly and add)], and 30 ℃, 200rpm cultivated 2-3 days;

(2) according to 1: 50 ratio bacterium liquid is transferred in the 50mL YPD fluid medium 30 ℃, 200rpm cultivates 3～5h, the bacterium liquid OD of this moment ₆₀₀=0.4～0.6.

(3) bacterium liquid branch is filled in the aseptic centrifuge tube of 10mL every pipe 5mL;

(4) the centrifugal 5min of 3000rpm, collecting cell.With the resuspended thalline of the aseptic ddH2O of 2.5mL;

(5) the centrifugal 5min of 3000rpm, collecting cell.With the resuspended thalline of 0.1mL 100mM (1 *) LiAc/TE buffer;

(6) the centrifugal 1～2min sedimentation cell of 3000rpm (only need with thalline is centrifugal at the bottom of centrifuge tube, get final product) carefully siphons away supernatant with micropipettor;

(7), bacteria suspension is moved in the 1.5mL Eppendorf pipe of sterilization with the resuspended thalline of 50 μ L 100mM (1 *) LiAc/TE buffer;

(8) 1mL strand carrier DNA is boiled 5min, in frozen water, cool off fast; (annotate: all boil carrier DNA when need not to use, it is frozen to be distributed into aliquot at every turn; Should boil again after the freeze thawing 3～4 times, should remain on ice after the taking-up)

(9) sample of step 7 being prepared, the centrifugal 1～2min sedimentation cell of 3000rpm carefully siphons away supernatant with microscale sampler;

(10) add by following order: 240 μ L PEG (50%w/v) (add the back and blow and beat up and down, thalline is suspended as far as possible) with the rifle head; 36 μ L 1M (10 *) LiAc; 25 μ L strand carrier DNA (2.0mg/mL), 50 μ L water and DNA (0.1～10 μ g) (generally speaking, plasmid adding 10～40 μ L get final product);

(11) vortex vibration Eppendorf pipe 1min, up to the complete mixing of cell, 30 ℃ of water-bath 30min;

Heat shock 20～25min in (12) 42 ℃ of water-baths;

(13) the centrifugal 1～2min of 5000rpm removes the conversion mixed liquor with micropipettor;

(14) draw 0.2～1.0mL sterilized water and be added in each reaction tube, with pipettor gently extracting with the deposition that suspends;

(15) get 50 μ L conversional solution, be uniformly coated on the culture medium that contains kalamycin resistance, cultivate 2～4d for 30 ℃.Do contrast with unconverted yeast.

2.3 the evaluation of yeast recon

2.3.1 bacterium colony PCR identifies primary dcreening operation

(1) bacterium colony on the flat board is long to naked eyes when visible.

(2) other PCR reactant liquor components except template are ready to, and packing.

(3) with a sterilization toothpick picking colony, in the PCR pipe, survey, put into the 1.5ml centrifuge tube of a sterilization, PCR pipe and 1.5ml centrifuge tube numbering.

(4) pcr amplification, 0.8% agarose gel electrophoresis.To the clone that pcr amplification manifests specific band, place the half toothpick of 1.5ml centrifuge tube to throw away the 5mlYPD culture medium, 30 ℃ of cultivations were extracted DNA, and were carried out PCR with DNA again as template and further confirm in 24 ± 1 hours.

PCR reaction system and condition are following:

The PCR reaction condition is:

94 ℃ of 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 2min, 35 circulations; Last 72 ℃ are extended 7min.

2.3.2 being template, the total DNA of yeast carries out the secondary evaluation

The extraction of yeast genes group

(1) inoculation reorganization and empty plasmid transformant 30 ℃, were cultivated 16-18 hour in 5ml YPD culture medium;

(2) under the room temperature, the centrifugal 5-10min of 1500g collects thalline;

(3) the resuspended back of 100uL TE (pH7.0) the centrifugal 5-10min of 1500g collects thalline;

(4) resuspended with the SCED (PH7.5) of 2mL;

(5) add 1～3mg Snailase, 37 ℃, 50min;

(6) add 2ml 1%SDS, the jog mixing places 5min on ice then;

(7) add 1.5mL 5M potassium acetate, mixing gently;

(8) the centrifugal 5-10min of 10000g abandons supernatant;

(9) add 2 times of volume dehydrated alcohol, room temperature is placed 15min;

(10) the centrifugal 20min of 10000g abandons supernatant;

(11) add the 0.7mLTE buffer;

(12) add isopyknic phenol: chloroform (1: 1V/V);

(13) the 7.5M Spirit of Mindererus. of adding 1/2 volume;

(14) dehydrated alcohol of 2 times of volumes of adding is placed 1hr for-20 ℃;

(15) the centrifugal 20min of 10000g;

(16) add 1mL 75% ethanol rinsing deposition once;

(17) after the drying, add TE or the H20 dissolving of 50ul ,-20 ℃ subsequent use.

The composition of PCR reaction system and reaction condition be with bacterium colony PCR, the 0.8% agarose gel electrophoresis analysis of PCR product.

The cultivation of embodiment 3, yeast transformant, the extraction of abduction delivering and expression product.

3.1 the phenotypic screen of yeast transformant

3.1.1 the reagent of screening yeast phenotype: MM culture medium, MD culture medium

(1) MM culture medium: 1.34%YNB 4 * 10 ^-5% biotin 0.5% methanol

With 890mL water high temperature sterilize, add 100mL10 * YNB when being cooled to 60 ℃ earlier, 2mL500 * biotin and 10mL100 * methanol add the 15g agar powder in the solid medium.

(2) MD culture medium: 1.34%YNB 4 * 10 ^-5% biotin 2% glucose

With 800mL water high temperature sterilize 20min, add 100mL 10 * YNB when being cooled to 60 ℃ earlier, 2mL 500 * biotin, 100mL10 * glucose adds the 15g agar powder in the solid medium.

(3) 10 * YNB (the sulfur acid ammonium does not contain amino acid whose yeast unit): 134g YNB dissolves with 1000mL, fully dissolves the after-filtration sterilization, 4 ℃ of preservations;

(4) 500 * biotin: the 20mg biotin is dissolved in the 100mL water, filtration sterilization, 4 ℃ of preservations;

(5) 100 * methanol: 50mL methanol and 100mL water mixing, filtration sterilization, subsequent use;

(6) 10 * glucoses; Dissolving 200g glucose in the 1000mL water, high temperature sterilize 15min or filtration sterilization;

3.1.2 the phenotypic screen of yeast transformant

(1) can form methanol evoked (Mut after the yeast conversion ⁺) and methanol responsive type (Mut ^s) bacterial strain, going up the bacterial strain of growing and on MD culture medium (no methanol culture medium), not growing in MM culture medium (containing the methanol culture medium) is Mut ^sThe type yeast strain, and on two kinds of culture medium, can both grow for Mut ⁺The type yeast strain;

The positive bacterium colony that (2) will filter out is being put on MM culture medium and MD culture medium by corresponding successively order, leaves standstill under 30 ℃ of conditions to cultivate 2-3d;

(3) contrast pPICZA-GsAS yeast transformant filters out the Mut that on two kinds of culture medium, can both grow at the growing state that contains on MM and the MD culture medium ⁺The type yeast strain.

3.2 the cultivation of yeast transformant and abduction delivering

3.2.1 the cultivation of yeast transformant and the culture medium of abduction delivering and reagent:

(1) MGY culture medium

10 * glycerol: 100mL glycerol is with 900mL water mixing, and high temperature sterilize or filtration sterilization are subsequent use;

MGY culture medium: 1.34%YNB 1% glycerol 4 * 10-5% biotin

With 100mL 10 * YNB, 2mL 500 * biotin and 100mL 10 * glycerol mix, 4 ℃ of preservations with the 800mL aquesterilisa.

(2) MM culture medium

(3) methanol

3.2.1 the cultivation of yeast transformant and abduction delivering

(1) selects Mut ⁺Yeast is inoculated in the 100mL triangular flask that contains 25mL MGY culture medium, and 28 ℃ of shaken cultivation are to OD ₆₀₀=2-6;

(2) all be transferred in the 100-200mL MM culture medium what cultivate in the MGY culture medium, make its OD ₆₀₀=1.0;

(3) yeast in the MM culture medium continues 30 ℃ of shaken cultivation, adds a methanol (final concentration 0.5%) in per 24 hours, and 4d is cultivated in concussion;

(4) 12,000rpm, centrifugal 10min, it is subsequent use to collect thalline.

3.3 expression product extracts

(1) Mut ⁺Behind the type yeast transformant abduction delivering 4d, 12, the centrifugal collection thalline of 000rpm is used the liquid nitrogen grinding thalline;

(2) adding 3-4mL volume ratio is methanol-chloroform (chromatographically pure) of 2: 1, supersonic wave wall breaking 30min (excusing from death 15s, 10s at interval);

(3) 10,000rpm are centrifugal;

(4) supernatant is used chloroform extraction, and with washing, repetitive operation 3 times;

(5) collect chloroform layer solution, water-bath distillation concentrates, and reclaims solvent and gets extractum.

Embodiment 4, yeast transformant expression product active anticancer detect

4.1 the yeast transformant expression product detects the pharmacotoxicological effect of HepG2 HCC

4.1.1HepG2 the cultivation of HCC

The HepG2 HCC is with containing the DMEM culture medium culturing of 10% calf serum, in 37 ℃, saturated humidity, 5%CO ₂Incubator in cultivate, 2-3d goes down to posterity 1 time.

4.1.2 processing to the HepG2 HCC

(1) the take the logarithm attached cell of trophophase, also digestion of PBS flushing is mixed with 2.5 * 10 with the culture medium that contains 10% calf serum ⁴ML ^-1Cell suspension;

(2) extractum that the yeast transformant that obtains among the embodiment 3 extracts is used dissolve with methanol;

(3) with cell inoculation in 96 well culture plates, every hole 200 μ L; Add the yeast conversion seed extract with the methanol preparation respectively, the final concentration that makes extract is 0.5,1,5,10,25,50,75,100 μ g/mL; The groups of cells that adds methanol is a matched group; The groups of cells of normal growth is made blank control group; 3 groups of each concentration, experiment repetition 3 times.

4.1.3HepG2 HCC metamorphosis and survival rate are observed

Respectively at 12h, 24h, 48h, 72h examine under a microscope the metamorphosis of HCC, add up its survival rate.

4.1.4 statistical analysis as a result

The result finds: compare with solvent control group with normal group; After handling 72h, 1,5; 10; The processed group of 25 μ g/mL is compared all with the cellular morphology of matched group HepG2 HCC and is changed, and the cellular morphology of 10 μ g/mL processed group changes obviously (seeing accompanying drawing 1), and its cell appearance by typical adherent growth before becomes and justifies, comes off, necrosis.And a little less than the variation relatively of the cellular morphology of other processed group in the experimental group.The survival rate that the yeast expression product of GsAS is handled the HepG2 HCC has a significant effect.After the yeast expression product of 1,5,10,25 μ g/mL Flos Cannabis Macrophylla β-amyrin synthase gene GsAS was handled 72h, HCC HepG2 HCC survival rate was respectively: 72.8%, 67.4%, 52.5%, 49.8% (seeing accompanying drawing 2).The yeast expression product of GsAS is handled the influence to the survival rate of HepG2 HCC, and along with concentration rising and the growth of time, extract becomes big to the survival rate influence of HepG2 HCC under 1～25 μ g/mL extract-treated; Behind the extract-treated 24h of 5 μ g/mL, significant change just takes place in the cytoactive of HepG2, and cell survival rate obviously reduces; Behind the extract-treated 48h of 5 μ g/mL, the survival rate of the cell of HepG2 is reduced to about 60%; And after handling 72h, under the extract-treated condition of 1 μ g/mL, obvious reduction (like Fig. 2) also takes place in the viability of HepG2 cell.

4.2 the yeast transformant expression product detects the pharmacotoxicological effect of SMMC7721 HCC

4.1.1SMMC7721 the cultivation of HCC

The SMMC7721 HCC is with containing the DMEM culture medium culturing of 10% calf serum, in 37 ℃, saturated humidity, 5%CO ₂Incubator in cultivate, 2-3d goes down to posterity 1 time.

4.1.2 processing to the SMMC7721 HCC

(1) the take the logarithm attached cell of trophophase, also digestion of PBS flushing is mixed with 2.5 * 10 with the culture medium that contains 10% calf serum ⁴ML ^-1Cell suspension;

(2) extractum that the yeast transformant that obtains among the embodiment 3 extracts is used dissolve with methanol,

(3) with cell inoculation in 96 well culture plates, every hole 200 μ L; Add the yeast conversion seed extract with the methanol preparation respectively, the final concentration that makes extract is 0.5,1,5,10,25,50,75,100 μ g/mL; The groups of cells that adds methanol is a matched group; The groups of cells of normal growth is made blank control group; 3 groups of each concentration, experiment repetition 3 times.

4.1.3SMMC7721 HCC metamorphosis and survival rate are observed

Respectively at 12h, 24h, 48h, 72h examine under a microscope the metamorphosis of HCC, add up its survival rate.

4.1.4 statistical analysis as a result

The result finds: compare with solvent control group with normal group; The processing of the yeast expression product of GsAS is similar with the form influence to the HepG2 HCC to the influence of SMMC7721 HCC form; 1,5,10; The processed group of 25 μ g/mL is compared with the cellular morphology of matched group SMMC7721 HCC and is changed, and the cellular morphology of 10 μ g/mL processed group changes obviously; The survival rate that the yeast expression product of GsAS is handled the SMMC7721 HCC also has a significant effect.After the yeast expression product of 1,5,10,25 μ g/mL Flos Cannabis Macrophylla β-amyrin synthase gene GsAS was handled 72h, SMMC7721 HCC survival rate was respectively: 74%, 68.8%, 49.5%, 47.8% (seeing accompanying drawing 3).

Claims (4)

1. the application of the yeast expression product of Flos Cannabis Macrophylla β-amyrin synthase gene GsAS in the preparation medicines resistant to liver cancer.

2. application as claimed in claim 1 is characterized in that: said HCC is HepG2 HCC or SMMC7721 HCC.

3. according to claim 1 or claim 2 application, it is characterized in that: the yeast expression production concentration that effectively suppresses the Flos Cannabis Macrophylla β-amyrin synthase gene GsAS of HepG2 HCC or SMMC7721 liver cancer cell growth is 1～25 μ g/mL.

4. application as claimed in claim 3 is characterized in that: the concentration of yeast expression product that effectively suppresses the Flos Cannabis Macrophylla β-amyrin synthase gene GsAS of HepG2 HCC or SMMC7721 liver cancer cell growth is 10 μ g/mL.

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