CN103952367B - A kind of fermention medium and utilize the method for its production melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA - Google Patents
A kind of fermention medium and utilize the method for its production melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA Download PDFInfo
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Abstract
The present invention discloses a kind of fermention medium and utilizes the method for its production melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA. Every 1L high yield fermention medium contains: casein peptone 12g, yeast extract 16.61g, glycerine 3.05mL, NH4Cl1g, 0.17mol/L? KH2PO4And 0.72mol/L? K2HPO4Mixing solutions 100mL, MgSO40.185g, trace element 1mL, utilizing works bacterium E.coli? XL10-Gold/CAVA work seed and this fermention medium can obtain the high plasmid of volume product rate, it is achieved the fermentative production of melanoma therapeutic DNA vaccine pSVK-CAVA.
Description
The application is April 16 2013 applying date, and application number is 201310131566.3, and denomination of invention is point case application of " preparation method of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA and dedicated engineering bacteria and fermention medium " thereof patent application.
Technical field
The invention belongs to the preparation method of Plasmid DNA vaccines in biological technical field and dedicated engineering bacteria thereof and high yield fermention medium, particularly relate to the preparation method of a kind of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA and dedicated engineering bacteria thereof and high yield fermention medium.
Background technology
Plasmid DNA vaccines is the extremely potential novel vaccine of the one found after deliberation in recent years, receives the concern of scientist from the 90's of last century initial stage Plasmid DNA vaccines, develops nearly 30 years till now, has had significant progress. Plasmid DNA vaccines has high security, carrier free immunogenicity and is easy to the features such as production, therefore, compares with the vaccine of live virus with virus basis, and it has unique advantage. Hundreds of vaccines enter clinical experimental stage, existing 4 kinds of animal Plasmid DNA vaccines and product listing (MicheleAK at present, DavidBW.DNAvaccines:readyforprimetime? NatureReviewsGenetics, 2008,9:776-788.), people's Plasmid DNA vaccines and product are ready to appear. Therefore, along with the fast development of Plasmid DNA vaccines and product, the scale operation platform setting up the plasmid DNA meeting medicinal standard becomes a great research topic. The present invention is directed to the zymotechnique of Plasmid DNA vaccines to explore, be the foundation stone during Plasmid DNA vaccines scale operation develops, its importance is self-evident.
In Plasmid DNA vaccines scale operation, the seed bank setting up stable plasmid DNA (or being called for short plasmid) is very crucial, owing to the molecular weight ratio of plasmid DNA (pSVK-CAVA) is bigger, reach about 15kb, it is a kind of heavy burden for host bacterium, in the process of growth of host bacterium, very easily cause the unstable of plasmid DNA. the unstable of plasmid DNA, refers to that engineering bacteria plasmid DNA in process of growth changes, and result does not present original phenotypic characteristic. the instability of plasmid DNA can be divided into two classes: segregational instability and structure unstable (FilomenaAQ, FernandaCD.PlasmidDNAfermentationstratagies:influenceonp lasmidstabilityandcellphysiology.AppliedMicrobialBiotech nology, 2012,93:2571-2580.). separation is unstable to be referred in cell fission owing to defect distribution causes the loss of part or all of plasmid DNA, the unstable rule of structure is owing to the disappearance in plasmid DNA, insertion or rearrangement cause, plasmid DNA yield and quality that the distribution unstable of plasmid DNA and the instable result of structure all will make us can not get expection. if the separation that plasmid DNA occurs is unstable, due to the loss of part plasmid DNA, in fact the fermenting process of engineering bacteria is engineering bacteria and the mixed culture (ClarenceMO not containing the host bacterium of plasmid DNA, RaeleneP, DianeW, etal.CultivationofE.colicarryingaplasmid-basedMeaslesvac cineconstruct (4.2kbppcDNA3F) employingmediumoptimisationandpH-temperatureinductiontec hniques.MicrobialCellFactories, 2011, 10:16.doi:10.1186/1475-2859-10-16.), under non-selective condition, specific growth rate (the �� of the engineering bacteria containing plasmid DNA+) often it is less than the specific growth rate (�� of host cell-), after 25 generations, nutrient solution is nearly all the cell of plasmid-free DNA. It thus is seen that design and screen a kind of suitable engineering bacteria the productivity of plasmid DNA is extremely important to improve.
The stability of plasmid and plasmid quality are also played a part extremely important by substratum, Silva (Silva, Filomena, PassarinhaL, etal.InfluenceofgrowthconditionsonplasmidDNAproduction.J ournalofMicrobiologyandBiotechnology, 2009,19 (11): 1408-1414.) research shows that the loss frequency of plasmid when using glucose as carbon source is greater than take starch as the substratum of carbon source. FDA thinks that the result for the treatment of of open loop and linearization plasmid DNA is not as supercoiled plasmid DNA (FoodandDrugAdministration (FDA), Guidanceforindustry:considerationsforplasmiddeoxyribonuc leicacidvaccinesforinfectiousdiseaseindications, 2007, http://www.fda.gov/cber/gdlns/plasdnavac.htm.), but several forms of plasmid DNA are difficult to separately in the process of purifying, so should how to obtain being optimized (YingC in a high proportion of supercoiled plasmid DNA in fermenting process, RodriguezS, HebelH.DNAvaccinemanufacture:scaleandquality.ExpertRevie wsVaccines, 2009, 8 (9): 1277-1291.). it thus is seen that design and screen a kind of suitable engineering bacterium fermentation substratum the productivity of plasmid DNA is most important to improve.
Summary of the invention
An aspect, the engineering bacteria for the preparation of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA that the present invention provides a stability height, can repeatedly go down to posterity.
Engineering bacteria for the preparation of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA provided by the present invention, it is the intestinal bacteria XL10-Gold transforming and having melanoma therapeutic plasmid DNApSVK-CAVA (or claiming PSCK-2PFcGB), called after E.coliXL10-Gold/CAVA.
Melanoma therapeutic pSVK-CAVA plasmid DNA transformation intestinal bacteria XL10-Gold is obtained transformed bacteria XL10-Gold, and method for transformation includes but not limited to Hananhan method, Inoue method, Calcium Chloride Method and electric shock conversion method. Transformed bacteria XL10-Gold is coated LB solid plate substratum, 37 DEG C, cultivate 15 hours under 200rpm after choose and get mono-clonal bacterium colony; By mono-clonal colony inoculation in 5mLLB liquid nutrient medium, 37 DEG C, cultivate 12 hours under 200rpm after carry out continuous passage cultivation according to 1:100, switching in 12 hours is gone down to posterity 1 time, can continuous passage 30 generation; Mono-clonal bacterial strain and within 30 generations of going down to posterity all previous bacterial strain that goes down to posterity be the engineering bacteria of transformation of E. coli XL10-Gold.
With the engineering bacteria of Calcium Chloride Method transformation of E. coli XL10-Gold, method for transformation is: thawed in ice bath 10 minutes by the 100 �� l competent cell XL10-Gold that-70 DEG C preserve, then add pSVK-CAVA plasmid DNA 25-50ng, soft mixed even after, put in ice bath 30 minutes; After ice bath terminates, in 42 DEG C of water-baths, heat hits 90 seconds, is placed in rapidly ice bath afterwards and cools 3-5 minute, add the LB liquid nutrient medium of 800 �� l non-resistants, in 37 DEG C after mixed even, 200rpm shaking culture 1 hour, obtains the transformed bacteria XL10-Gold of pSVK-CAVA plasmid DNA.
On the other hand, the present invention provides a kind of high yield fermention medium for the preparation of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA.
This prepares the fermention medium ITB of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA, and every 1L substratum contains: casein peptone 10-14g, yeast extract 16-20g, glycerine 2.4-4mL, NH4Cl0.75-1g, 0.17mol/LKH2PO4And 0.72mol/LK2HPO4Mixing solutions 75-100mL, MgSO40.168-0.216g, trace element 0.75-1mL.
Preferably, every 1L substratum contains: casein peptone 12g, yeast extract 16.61g, glycerine 3.05mL, NH4Cl1g, 0.17mol/LKH2PO4And 0.72mol/LK2HPO4Mixing solutions 100mL, MgSO40.185g, trace element 1mL.
In described fermention medium ITB, trace element comprises: FeCl3��6H2O27g/L��ZnCl22g/L��CoCl2��6H2O2g/L��Na2MoO4��2H2O2g/L��CaCl2��2H2O1g/L, or anhydrous CaCl20.76g/L��CuCl2��2H2O1.27g/L��H3BO30.5g/L, is dissolved in 1.2mol/LHCl.
In described fermention medium ITB, K2HPO4And KH2PO4The compound method of mixing solutions is: with 90mL deionized water dissolving 2.31gKH2PO4And 12.54gK2HPO4, after dissolving completely, it is settled to 100mL with deionized water.
Another further aspect, the present invention provides a kind of effective method carrying out melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA production with above-mentioned engineering bacteria and fermention medium.
The production method of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA provided by the invention, it it is recovery described engineering bacteria E.coliXL10-Gold/CAVA work seed, 1:100 ratio is inoculated in 5mLLB liquid nutrient medium by volume, 37 DEG C, cultivate 12 hours under 200rpm, transfer again in the LB liquid nutrient medium of 100mL according to 1:100,37 DEG C, 200rpm cultivate 12 hours, obtain seed liquor;
The amount of seed liquor 1:100 being by volume inoculated in the 5L fermentor tank containing fermention medium ITB described in 3L, pH value controls 7.0 �� 0.1, and culture temperature is 37 DEG C, and dissolved oxygen controls about 30%, fermentation culture 6h; Add fermention medium ITB according to the flow velocity of 1mL/min afterwards, altogether add 500mL, ferment after 15 hours, terminate fermentation, receive bacterium, extract plasmid, obtain melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA.
Again on the one hand, the present invention provides construction process and the seed bank product of the engineering bacteria E.coliXL10-Gold/CAVA seed bank of production melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA.
The structure of this seed bank comprises:
1) original species word bank is prepared: be extended in 100mLLB liquid nutrient medium by described engineering bacteria E.coliXL10-Gold/CAVA bacterium liquid 0.5mL, 37 DEG C, cultivate 12 hours under 200rpm, adding 30% glycerine, packing 50 is managed and frozen in-70 DEG C of refrigerators, obtains original species word bank;
2) main seed bank is prepared: a primordial seed of recovering is inoculated in 12 pipe LB liquid nutrient mediums, little upgrading grain after going down to posterity for 2 times, choosing the highest bacterium liquid of plasmid content is extended in 100mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after add 30% glycerine, and packing 50 is in control main seed bank, frozen in-70 DEG C of refrigerators, obtain main seed bank;
3) preparation work seed bank: get 1 pipe bacterial classification from main seed bank, it is inoculated in 12 pipe LB liquid nutrient mediums, little upgrading grain after going down to posterity for 2 times, choosing the highest bacterium liquid of plasmid content is extended in 100mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after add 30% glycerine, packing 50 is managed and frozen in-70 DEG C of refrigerators, obtains work seed bank; Often prop up the limit of the bacterial classification in work seed bank and pass 5 substitutes in the production of claim 9 melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA.
More than build the seed bank and each seed that comprise primordial seed, main seed and work seed that obtain and it is content of the present invention.
The stability of plasmid DNA and engineering bacterium fermentation substratum are the important factors affecting yield plasmid, the present invention is in order to provide a stability height, the engineering bacteria for the preparation of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA that can repeatedly go down to posterity is to meet the demand of pilot process, first by experiment host bacterium is optimized screening, final discovery intestinal bacteria XL10-Gold is a host bacterium being more conducive to this plasmid DNA amplification, the engineering bacteria called after E.coliXL10-Gold/CAVA of plasmid pSVK-CAVA will be carried, when plasmid pSVK-CAVA is by host bacterium of intestinal bacteria XL10-Gold, show the stability similar with conventional plasmid DNA, can transfer goes down to posterity there is not restructuring or fragment loss 30 times yet, this may have relation with the characteristic of intestinal bacteria XL10-Gold, because this bacterium has an efficient transforming gene of macromolecule plasmid, except keeping stable in host bacterium of plasmid DNA to go down to posterity, an aspect very important in plasmid DNA production technique is the high yield problem of plasmid DNA, want to obtain higher product volume, the engineering bacteria carrying plasmid must be made to have higher biomass, generally to be realized through the composition of optimization culture base and special fermentation (fed-batch fermentation) strategy, and desirable fermention medium is the most important condition obtaining high yield, therefore, with one-factor-at-a-time methodology and response phase method its optimum medium screened respectively and optimized, one-factor-at-a-time methodology optimum result shows, TB substratum is its suitableeest basic medium, the volume product rate of plasmid is 9.9mg/L, obviously higher than LB and M9 substratum, the suitableeest Carbon and nitrogen sources is glycerine and casein peptone respectively, PB experimental result shows, 3 main affecting factors affecting yield plasmid are yeast extract, glycerine, MgSO4Eventually passing climbing the most suddenly and after CCD optimizes, it is determined that the composition of engineering bacteria E.coliXL10-Gold/CAVA fermention medium ITB and level, under the fermentation scale of 500mL shaking flask, the volume product rate of plasmid reaches 12.38mg/L, is more than 2 times of basic medium LB fermentation yield. In sum, the engineering bacteria of the present invention and substratum play a significant role in the fermentative production of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA, have a extensive future.
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 is engineering bacteria E.coliXL10-Gold/CAVA continuous passage 30 generation, every 5 generations extracting plasmid, carries out single endonuclease digestion with PVU I, and the enzyme carrying out double digestion with PVU I and Spe I cuts qualification result
Fig. 2 is engineering bacteria E.coliXL10-Gold/CAVA continuous passage 30 generation, every 5 generations extracting plasmid, and 1% agarose gel electrophoretogram of plasmid
Fig. 3 is the dull and stereotyped dibbling result of engineering bacteria E.coliXL10-Gold/CAVA
Fig. 4 is the gram stain microscopy result of engineering bacteria E.coliXL10-Gold/CAVA
Fig. 5 is the growing state of engineering bacteria E.coliXL10-Gold/CAVA in biochemistry detection substratum
Fig. 6 A is engineering bacteria E.coliXL10-Gold/CAVA changing conditions with incubation time strain density in 4 kinds of substratum
The structure 1% agarose gel electrophoresis detected result of plasmid in Fig. 6 B engineering bacteria that to be engineering bacteria E.coliXL10-Gold/CAVA grow at 4 kinds of substratum
Fig. 7 is the histogram that engineering bacteria E.coliXL10-Gold/CAVA yield plasmid (volume product rate) is affected by different carbon source
Fig. 8 is the histogram that engineering bacteria E.coliXL10-Gold/CAVA yield plasmid (volume product rate) is affected by different nitrogen sources
Fig. 9 is the curved surface figure that affected by yield plasmid (volume product rate) of three remarkable affecting genes interactions and isogram
Embodiment
The present inventor obtains early stage after deliberation has the outstanding original Novel black melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA based on Alphavirus vector (see document: Zhang Liang, Yan Jinqi, Wang Yue, Deng. the structure of replication competent type anti-tumor DNA vaccine PSCK-2PFcGB and Expression in Vivo and in Vitro [J]. Nanfang Medical Univ's journal, 2011, 3 (6): the 937-942. document are named the replication competent type anti-tumor DNA vaccine of " PSCK-2PFcGB " be melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA of the present invention, invent the popularization of artificial business to have carried out this vaccine in the present invention again naming), Pharmacodynamic research display, this vaccine can special cellular immunization and humoral immunoresponse(HI) in inducing mouse body effectively, can effectively suppress the growth of transplanted tumor. carrying out the pilot process of this vaccine and the foundation of quality control system at present, to promote further developing of this Novel black melanoma therapeutic vaccine. the general molecular weight of Plasmid DNA vaccines based on Alphavirus vector is all bigger, more than 14kb can be reached, and melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA molecular weight is close to 15kb, no matter the plasmid DNA (called after " pSVK-CAVA plasmid DNA " or " plasmid DNA pSVK-CAVA ") that molecular weight is so big is the design in upstream and structure, or extraction and the technique research to downstream, all has bigger challenge. the experiment in early stage finds: when taking bacillus coli DH 5 alpha as host bacterium, along with the increase of passage number, the situation that molecular weight diminishes can be there is in this Plasmid DNA vaccines, the more changeable little situation of algebraically is more serious, plasmid to last extraction has not been required object plasmid, and this is totally unfavorable for the development of the research of follow-up pilot process and product. contriver analyzes research further, in host bacterium, there occurs restructuring due to plasmid or lose, cause the reason of this kind of phenomenon may be due to the excessive problem of the load of host bacterium own, also may be that plasmid molecule amount is excessive or come from Alphavirus due to it, easily occur the self reasons such as restructuring or fragment loss to cause. plasmid in host bacterium is caused to occur restructuring or the fragment loss characteristic with plasmid itself and host bacterium etc. to have certain associating, first melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA is a kind of carrier based on virus, than the plasmid DNA of routine, restructuring or fragment loss more easily occurs, in addition, due to this plasmid pSVK-CAVA self relative molecular mass 15kb nearly, when copying in host bacterium, serious load can be caused to thalline, thus the unstable causing plasmid increases, and produces the above-mentioned situation truncating plasmid.
Wanting to solve the problem, contriver proposes: the most suitable host bacterium of screening Plasmid DNA vaccines pSVK-CAVA, and carries out the optimization of medium component.
Therefore, first aspect, the present invention provides a kind of engineering bacteria for the preparation of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA, and it is the intestinal bacteria XL10-Gold transforming and having melanoma therapeutic plasmid DNApSVK-CAVA, called after E.coliXL10-Gold/CAVA.
Available routine techniques well known to those skilled in the art, by melanoma therapeutic plasmid DNApSVK-CAVA transformation of E. coli XL10-Gold, method for transformation includes but not limited to Hananhan method (molecular cloning experiment guide (third edition) 87-92 page), Inoue method (molecular cloning experiment guide (third edition) 93-96 page), Calcium Chloride Method (molecular cloning experiment guide (third edition) 96-99 page), electric shock conversion method (molecular cloning experiment guide (third edition) 99-102 page).
On the other hand, the present invention also provides a kind of high yield fermention medium (ITB) for the preparation of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA. This substratum of every 1L contains: casein peptone 12g, yeast extract 16.61g, glycerine 3.05mL, NH4Cl1g, 0.17mol/LKH2PO4And 0.72mol/LK2HPO4Mixing solutions 100mL, Mg2SO40.185g, trace element 1mL, trace element comprises FeCl3��6H2O27g/L��ZnCl22g/L��CoCl2��6H2O2g/L��Na2MoO4��2H2O2g/L��CaCl2��2H2O1g/L or anhydrous CaCl20.76g/L��CuCl2��2H2O1.27g/L��H3BO30.5g/L, is dissolved in 1.2mol/LHCl; Described K2HPO4And KH2PO4The compound method of mixing solutions is: with 90mL deionized water dissolving 2.31gKH2PO4And 12.54gK2HPO4, after dissolving completely, it is settled to 100mL with deionized water.
Also on the one hand, the present invention also provides a kind of above-mentioned engineering bacteria and the method for high yield fermention medium production melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA. The method is:
1) preparation of seed liquor: work seeds from-70 DEG C of E.coliXL10-Gold/CAVA of recovering, be inoculated in the LB liquid nutrient medium of 5mL by 1:100,37 DEG C, 200rpm cultivate 12 hours. Transfer again in the LB liquid nutrient medium of 100mL according to 1:100,37 DEG C, 200rpm cultivate 12 hours, now obtain seed liquor;
2) fermentation culture: seed liquor is planted according to the amount of 1:100 and carries out fermentation culture in 5L fermentor tank (scale up test), the amount containing high yield fermention medium ITB in fermentor tank is 3L, and pH value controls (automatically to fill into H by fermentor tank 7.0 �� 0.12SO4And NH3.H20 regulates the pH value of substratum), culture temperature is 37 DEG C, and dissolved oxygen controls about 30% (when being reduced to this value when dissolved oxygen, regulating by supplementing pure oxygen and improve stirring velocity). After fermentation culture 6h, add high yield fermention medium ITB according to the flow velocity of 1mL/min, altogether add 500mL. Ferment after 15 hours, terminate fermentation, receive bacterium, extract plasmid, obtain melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA.
The present invention also builds multistage seed bank, and described E.coliXL10-Gold/CAVA works seed source in this seed bank. The building process of seed bank comprises:
1) original species word bank is prepared: be extended in 100mLLB liquid nutrient medium by E.coliXL10-Gold/CAVA engineering bacteria bacterium liquid 0.5mL, 37 DEG C, cultivate 12 hours under 200rpm, adding 30% glycerine, packing 50 is managed and frozen in-70 DEG C of refrigerators, obtains original species word bank;
2) main seed bank is prepared: a primordial seed of recovering is inoculated in 12 pipe LB liquid nutrient mediums, little upgrading grain after going down to posterity for 2 times, choosing the highest bacterium liquid of plasmid content is extended in 100mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after add 30% glycerine, and packing 50 is in control main seed bank, frozen in-70 DEG C of refrigerators, obtain main seed bank;
3) preparation work seed bank: get 1 pipe bacterial classification from main seed bank, it is inoculated in 12 pipe LB liquid nutrient mediums, little upgrading grain after going down to posterity for 2 times, choosing the highest bacterium liquid of plasmid content is extended in 100mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after add 30% glycerine, packing 50 is managed and frozen in-70 DEG C of refrigerators, obtains work seed bank, often prop up the limit of the bacterial classification in work seed bank and passed for 5 generations, for the production of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA.
In following embodiment, method therefor is ordinary method if no special instructions, concrete steps can be see: " MolecularCloning:ALaboratoryManual " (Sambrook, J., Russell, DavidW., MolecularCloning:ALaboratoryManual, 3rdedition, 2001, NY, ColdSpringHarbor).
Described percentage concentration is mass/volume (mg/100ml) percentage concentration or volume/volume (V/V) percentage concentration if no special instructions.
Embodiment is described to the acquirement approach of various biomaterials be only to provide a kind of approach testing acquisition to reach concrete disclosed object, it should not become the restriction to biological material source of the present invention. In fact, the source of used biomaterial is widely, and the biomaterial that any not contrary to law and moral ethics can obtain can be replaced according to the prompting in embodiment and use.
Embodiment is implemented under premised on technical solution of the present invention, gives detailed enforcement mode and concrete operating process, and embodiment will contribute to understanding the present invention, but protection scope of the present invention is not limited to following embodiment.
The screening of the high stability host bacterium of embodiment 1, melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA and Detection of Stability
Melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA plasmid DNA transforms competent escherichia coli cell DH5 ��, DH10 ��, Top10 and XL10-Gold of different genotype respectively, and method for transformation is Hananhan method (molecular cloning experiment guide (third edition) 87-92 page), Inoue method (molecular cloning experiment guide (third edition) 93-96 page), Calcium Chloride Method (molecular cloning experiment guide (third edition) 96-99 page), electric shock conversion method (molecular cloning experiment guide (third edition) 99-102 page).
Transform XL10-Gold for Calcium Chloride Method and carry out exemplary illustration:
The acquisition of pSVK-CAVA plasmid DNA is see document (Zhang Liang, Yan Jinqi, Wang Yue, etc. the structure of replication competent type anti-tumor DNA vaccine PSCK-2PFcGB and Expression in Vivo and in Vitro [J]. Nanfang Medical Univ's journal, 2011,3 (6): 937-942.). The document is named the replication competent type anti-tumor DNA vaccine of " PSCK-2PFcGB " be melanoma therapeutic Plasmid DNA vaccines " pSVK-CAVA " of the present invention, invent the popularization of artificial business and carried out again naming to it in the present invention. In the present invention pSVK-CAVA plasmid DNA be in document introduce PSCK-2PFcGB plasmid.
From-70 DEG C of refrigerator, get 100 �� l competent cell XL10-Gold, thaw in ice bath immediately 10 minutes, then add pSVK-CAVA plasmid DNA 25-50ng, soft mixed even after, put in ice bath 30 minutes. After ice bath terminates, in 42 DEG C of water-baths, heat hits 90 seconds, it is placed in rapidly ice bath afterwards and cools 3-5 minute, Xiang Guanzhong adds the LB liquid nutrient medium of 800 �� l non-resistants, mixed even after in 37 DEG C, 200rpm shaking culture 1 hour, makes bacterium restore normal growth state, and the antibiotics resistance gene of expression plasmid coding, now obtain the transformed bacteria XL10-Gold of pSVK-CAVA plasmid DNA.
Transformed bacteria XL10-Gold is coated LB (Luria-bertaniMedium) solid plate substratum (LB liquid culture based formulas: Tryptones 10g, yeast extract 5g, NaCl10g, add distilled water to dissolve, 5mol/LNaOH regulates pH to 7.0, being settled to 1000mL, after autoclaving, 4 DEG C save backup. LB solid plate substratum is then add agar (15g/L) in LB liquid nutrient medium, autoclaving, adding sulphuric acid kanamycin to final concentration when temperature is down to 50 DEG C is 20 �� g/mL, following substratum resistance level all for this reason), at 37 DEG C, choose after cultivating 15 hours under 200rpm and get mono-clonal bacterium colony, it is inoculated in 5mLLB liquid nutrient medium, at 37 DEG C, continuous passage cultivation (switching in 12 hours is gone down to posterity 1 time) is carried out according to 1:100 after cultivating 12 hours under 200rpm, 1mL bacterium liquid extracting plasmid is got before going down to posterity, by the form of 1% agarose gel electrophoresis detection plasmid and superhelix ratio, plasmid concentration measured by ultraviolet spectrophotometer, the stability of engineering bacteria is detected by the combined continuous method (every 12 hours switching 1 generation) that goes down to posterity.
Transform intestinal bacteria XL10-Gold engineering bacteria continuous passage 30 generation having pSVK-CAVA plasmid DNA, every 5 generations extracting plasmid, carrying out single endonuclease digestion with PVU I, carry out double digestion with PVU I and Spe I, enzyme cuts qualification result (M:DL15000Marker as shown in Figure 1; 1: plasmid pSVK-CAVA; 2:PVU I single endonuclease digestion fragment; 3:PVU I and Spe I double digestion fragment), singly cut with PVU I and obtain 14748bp linearization plasmid fragment, 12751bp and 1997bp two DNA fragmentations are obtained with PVU I and Spe I double digestion, consistent with expected results, show that the structural stability of plasmid is good. Transform and have intestinal bacteria XL10-Gold engineering bacteria continuous passage 30 generation of pSVK-CAVA plasmid DNA, every 5 generations extracting plasmid, 1% agarose gel electrophoretogram of plasmid as shown in Figure 2 (1: primary; 2:5 generation; 3:10 generation; 4:15 generation; 5:20 generation; 6:25 generation; 7:30 generation; M:DL15000Marker), engineering bacteria shows genetic stability well, plasmid copy number kept stable, maintains higher superhelix ratio simultaneously.
4 kinds of alternative host bacterium are screened by the method for the present embodiment example, and result can successfully grow mono-clonal; Choose and get mono-clonal and carry out Secondary Culture, wherein bacillus coli DH 5 alpha, DH10 ��, TOP10 all occur plasmid restructuring in various degree after 2 times and lose going down to posterity, and down continue to go down to posterity, this kind of restructuring and loss situation become more serious, go down to posterity and just almost do not have protoplasm grain after 5 times. But intestinal bacteria XL10-GOLD can be good at keeping plasmid to carry out stable copy, go down to posterity still very stable after 30 generations, the host bacterium being suitable as very much the melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA big up to 15kb, solves Large plasmid and restructuring easily occurs and loses this difficult problem.
This conversion is had the intestinal bacteria XL10-Gold of pSVK-CAVA plasmid DNA by the present invention, called after " melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA engineering bacteria E.coliXL10-Gold/CAVA ". Through qualification (see embodiment 2), this engineering bacteria shows genetic stability well, plasmid copy number kept stable, maintains higher superhelix ratio simultaneously.
This engineering bacteria is utilized to set up three grades of seed banks, E.coliXL10-Gold/CAVA bacterium liquid can be extended to 100mLLB liquid nutrient medium (formula: Tryptones 10g, yeast extract 5g, NaCl10g, add distilled water to dissolve, 5mol/LNaOH regulates pH to 7.0, is settled to 1000mL, and after autoclaving, 4 DEG C save backup. ) cultivate, 37 DEG C, cultivate 12 hours under 200rpm, add 30% glycerine, packing 50 is managed and frozen in-70 DEG C of refrigerators, obtains original species word bank. A primordial seed of recovering is inoculated in 12 pipe LB liquid nutrient mediums, little upgrading grain after going down to posterity for 2 times, choosing the highest bacterium liquid of plasmid content is extended in 100mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after add 30% glycerine, and packing 50 pipe is as main seed bank, frozen in-70 DEG C of refrigerators. Getting 1 pipe bacterial classification from main seed bank, operate as stated above, frozen 50 pipes, as work seed bank, are produced for routine. Often prop up the limit of the bacterial classification in work seed bank and passed for 5 generations.
The qualification of embodiment 2, melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA engineering bacteria E.coliXL10-Gold/CAVA
One, the seed bank genetic stability that works and structural stability detection
37 DEG C of heating in water bath are recovered 1 work seed, by being inoculated in 5mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after 1:100 go down to posterity, continuous passage 30 generation in this approach, select primary, 5 generations, 10 generations, 15 generations, 20 generations, 25 generations, 30 generation bacterium liquid extracting plasmid, cut the structural stability of detection work seed bank through 1% agarose gel electrophoresis and enzyme.
Result engineering bacteria E.coliXL10-Gold/CAVA continuous passage 30 generation, every 5 generations extracting plasmid, 1% agarose gel electrophoretogram (see Fig. 2) of plasmid shows that engineering bacteria shows genetic stability well, plasmid copy number kept stable, maintains higher superhelix ratio simultaneously; In addition, obtain 14748bp linearization plasmid fragment with mono-the cutting of PVU1, obtain 12751bp and 1997bp two DNA fragmentations with PVU I and Spe I double digestion, consistent with expected results, show that the structural stability of plasmid is good.
Two, the plasmid loss rate detection of work seed bank
To the 30th generation bacterium liquid detection plasmid loss rate, it is laid on not containing overnight incubation on the LB flat board of kantlex after getting the bacterium liquid dilution of the 30th generation, choose 100 mono-clonal dibblings in containing on the LB flat board of 20 �� g/mL kantlex, 37 DEG C, cultivate 15 hours under 200rpm, the mono-clonal colony number of counting growth, calculates plasmid loss rate: plasmid loss rate=(colony number that 100-grows on flat board)/100 �� 100%.
Result as shown in Figure 3, grown 100 points, and plasmid loss rate is 0, shows that this plasmid has good segregational stability in host bacterium, still keeps stable after 30 generations of going down to posterity.
Two, ne ar and gramstaining feature and biochemical character thereof
The gram stain microscopy figure of engineering bacteria E.coliXL10-Gold/CAVA is as shown in Fig. 4 (magnification 1000 times), according to mirror inspection display, engineering bacteria E.coliXL10-Gold/CAVA is bacillus, gramstaining is red (being shown as dark color in figure), and explanation is Gram-negative bacteria. Also investigate engineering bacteria E.coliXL10-Gold/CAVA containing the growing state in glucose, lactose, N.F,USP MANNITOL three kinds of different culture medium of carbohydrate, method is: with the method punctured, engineering bacteria E.coliXL10-Gold/CAVA is seeded in biochemistry detection substratum [formula: with Deng Heng Shi (Dunham) the peptone aqueous solution (peptone 1g, sodium-chlor 0.5g, water 100mL, pH7.6), 0.2% bromothymol blue that every 100mL adds 1.2mL makes indicator. Add agar in the medium and reach 0.5-0.7%, be sub-packed in test tube, autoclaving 10 minutes. 0.2% bromothymol blue solution joins method: bromothymol blue 0.2g, 0.1mol/LNaOH5mL, distilled water 95mL.
37 DEG C, cultivate 24 hours under 200rpm, observe its fermentation and produce gas situation, analysis of biochemical feature. Result is as shown in Figure 5, there is yellow (in figure light-colored part) in glucose and N.F,USP MANNITOL substratum in engineering bacteria E.coliXL10-Gold/CAVA, lactose medium does not occur yellow, test tube solution is shown as blueness (in figure dark parts), illustrate that engineering bacteria E.coliXL10-Gold/CAVA can glucose fermentation, N.F,USP MANNITOL, unfermentable lactose, the generation all not having bubble in two kinds of substratum turned yellow, illustrates in engineering bacteria E.coliXL10-Gold/CAVA fermenting process and does not produce gas.
Embodiment 3, different basic medium are on the impact of engineering bacteria E.coliXL10-Gold/CAVA growth and yield plasmid (volume product rate)
In order to find out the substratum that applicable engineering bacteria E.coliXL10-Gold/CAVA grows and plasmid is produced, engineering bacteria E.coliXL10-Gold/CAVA is cultivated in four kinds of different bacteria culture mediums:
LB fills a prescription: Tryptones 10g, yeast extract 5g, NaCl10g, adds distilled water and dissolves, and 5NNaOH regulates pH to 7.0, is settled to 1000mL, and after autoclaving, 4 DEG C save backup.
TB fills a prescription: casein peptone 12g, yeast extract 24g, glycerine 4mL, adds distilled water and dissolves, is settled to 900mL, and after autoclaving, 4 DEG C save backup. The 0.17mol/LKH that 100mL is aseptic is added when solution is cooled to 50 DEG C2PO4, 0.72mol/LK2HPO4Solution. The compound method of this solution is: with 90mL deionized water dissolving 2.31gKH2PO4And 12.54gK2HPO4, after dissolving completely, it is settled to 100mL with deionized water and autoclaving 20min.
M9 (glucose) fills a prescription: 5 �� M9 salts solution 200mL, 1mol/LMgSO42mL, 20% glucose solution 20mL, 1mol/LCaCl20.1mL, the distilled water of sterilizing is settled to 1000mL. The compound method of 5 �� M9 salts solution: dissolve following salt with distilled water, final volume is 1L:Na2HPO4.7H2O64g, KHPO415g, NaCl2.5g, NH4Cl5.0g, after autoclaving, 4 DEG C save backup.
M9 (glycerine) fills a prescription: 5 �� M9 salts solution 200mL, 1mol/LMgSO42mL, 20% glycerine solution 20mL, 1mol/LCaCl20.1mL, the distilled water of sterilizing is settled to 1000mL. The configuration method of 5 �� M9 salts solution: dissolve following salt with distilled water, final volume is 1L:Na2HPO4.7H2O64g, KHPO415g, NaCl2.5g, NH4Cl5.0g, after autoclaving, 4 DEG C save backup.
Cultural method is: an E.coliXL10-Gold/CAVA work seed of recovering, it is inoculated in 5mLLB substratum by 1:100,37 DEG C, 200rpm cultivate 12 hours, 1:100 goes down to posterity and is once inoculated in the above-mentioned four kinds of basic mediums of 100mL afterwards, 1mL bacterium night is got every 3h, a part for measuring the cell density A value OD600 of spectrophotometric determination 600nm wavelength (adopt) monitor thalli growth situation, a part is used for extracting plasmid in a small amount and measuring plasmid concentration, after getting 10mL bacterium liquid centrifuge washing after fermentation ends, dry and survey dry cell weight.
By analyzing, sky scale measures the dry weight of thalline, concrete operation: dry EP pipe, correct amount (G1), gets escherichia coli fermented broth 5mL, the centrifugal 10min of 8000rmp, abandoning supernatant, precipitation PBS washes twice, and together with centrifuge tube, precipitation is placed in baking oven, 80 DEG C are dried to constant weight (about 24h), being cooled to room temperature in moisture eliminator, correct amount (G2), dry cell weight is G2-G1. Finally get the mean value of each zymocyte liquid 3 parallel sample measuring result.
Fermention medium based on the substratum that selection thalli growth situation is best, plasmid volume product rate is the highest and plasmid specificity product rate is best. Cell density measurement result in different culture media is as shown in Figure 6A, Fungal biodiversity, plasmid volume product rate and specificity product rate are as shown in table 1, can see that engineering bacteria E.coliXL10-Gold/CAVA has embodied obvious growth vigor at TB substratum, particularly volume product rate, has had bigger lifting than LB substratum. In the engineering bacteria of different culture media growth, 1% agarose gel electrophoresis detected result of plasmid is such as Fig. 6 B (M:DL15000Marker; 1:LB substratum; 2:TB substratum; 3:M9 (glycerine) substratum; 4:M9 (glucose) substratum) shown in, the plasmid concentration of No. 2 swimming lanes (TB substratum) and the ratio of superhelix are all better than the plasmid of other swimming lane, in plasmid extraction process, carry out wash-out with the elutriant of same volume, namely describe and can obtain higher yield plasmid and higher superhelix ratio with the fermentation of TB substratum. Therefore, select substratum based on TB, carry out further research.
Table 1 Fungal biodiversity, plasmid volume product rate and specificity product rate
| Substratum | Biomass (g/L) | Volume production (mg/L) | Specificity output (mg/g) |
| LB | 1.1 | 6.2 | 5.7 |
| TB | 2.2 | 9.9 | 4.5 |
| M9 (glycerine) | 0.4 | 3.3 | 8.25 |
| M9 (glucose) | 0.2 | 1.4 | 6.95 |
Embodiment 4, different carbon source, nitrogenous source are on the impact of engineering bacteria E.coliXL10-Gold/CAVA fermentation and yield plasmid (volume product rate)
For engineering bacteria E.coliXL10-Gold/CAVA production melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA screens desirable carbon source, nitrogenous source, first conditions of flask fermentation is utilized, for engineering bacteria E.coliXL10-Gold/CAVA filters out best basic medium TB substratum from LB, TB, M9 (glycerine), M9 (glucose) four kinds of substratum. After basic medium filters out, compare different carbon sources (such as glucose, glycerine, N.F,USP MANNITOL) and different nitrogen sources respectively (such as Tryptones, soy peptone, casein peptone, peptone, ammonium chloride (NH4Cl)) engineering bacteria being produced the impact of pSVK-CAVA plasmid DNA, it is determined that optimum carbon source and nitrogenous source and their working concentration, to determine that it is on the impact of fermentation and yield plasmid, single time single-factor experimental technique is as follows:
One, different carbon source is on the impact of engineering bacteria E.coliXL10-Gold/CAVA growth and yield plasmid (volume product rate)
Substratum based on TB, wherein without carbon source composition, and the output of the growth of engineering bacteria and plasmid is had material impact by carbon source composition. This experimental selection glycerine, glucose and N.F,USP MANNITOL candidate target as carbon source, studies it and is grown by engineering bacteria E.coliXL10-Gold/CAVA and the impact of yield plasmid (volume product rate). Experimental technique is: an E.coliXL10-Gold/CAVA work seed of recovering, it is inoculated in 5mLTB substratum by 1:100,37 DEG C, 200rpm cultivate 12 hours, 1:100 goes down to posterity and is once inoculated in 100mL carbon source afterwards and is respectively in the 500mL Erlenmeyer flask of TB substratum that glycerine, glucose and N.F,USP MANNITOL, concentration are 4g/L, 37 DEG C, 200rpm shaking culture 15h, measure plasmid concentration after fermentation ends.
The growth that carbon source is engineering bacteria provides energy, is the factor that bacterium is rely and grows, and is also the element of the limiting nutrient in human antibody Fab of HBsAg usually. At present, glycerine is the most frequently used carbon source, and it is not only cheap but also effect is very good. On the basis that other medium component is constant, changing carbon source and cultivate, extracting plasmid after 15h, analyzes. As shown in Figure 7, glycerine is more conducive to the copy of plasmid to result, is significantly better than other two kinds of carbon sources, is suitable as the fermention medium carbon source of engineering bacteria E.coliXL10-Gold/CAVA. Binding experiment result, and consider from cost-effective angle, it is determined that using glycerine as final carbon source, working concentration is 4g/L.
Two, different nitrogen sources is on the impact of engineering bacteria E.coliXL10-Gold/CAVA growth and yield plasmid (volume product rate)
Some inorganic salt and organism all can be used as the nitrogenous source of microbial culture, have investigator to adopt CH in cultivating in a small amount4Cl or (NH4)SO4As nitrogenous source, but organism often can obtain better plasmid product rate as nitrogenous source. Substratum based on TB, wherein without nitrogenous source composition, and the output of the growth of engineering bacteria and plasmid is had material impact by nitrogenous source composition. This experimental selection Tryptones, soy peptone, peptone, casein peptone, ammonium chloride (NH4Cl) as the candidate target of nitrogenous source, it is studied on the impact of engineering bacteria E.coliXL10-Gold/CAVA growth and yield plasmid (volume product rate). Experimental technique is: an E.coliXL10-Gold/CAVA work seed of recovering, it is inoculated in 5mLTB substratum by 1:100,37 DEG C, 200rpm cultivate 12 hours, 1:100 goes down to posterity and is once inoculated in 100mL nitrogenous source afterwards and is respectively Tryptones, soy peptone, peptone, casein peptone, ammonium chloride (NH4Cl), concentration is in the 500mL Erlenmeyer flask of the TB substratum of 12g/L, and 37 DEG C, 200rpm shaking culture 15h, measure plasmid concentration after fermentation ends.
Experimental result is as shown in Figure 8, in 5 alternative nitrogenous sources of experiment, casein peptone demonstrates good plasmid product rate, and casein peptone is domestic analytical pure, relative cost is lower, therefore, selecting casein peptone as the fermention medium nitrogenous source of engineering bacteria E.coliXL10-Gold/CAVA, working concentration is 12g/L.
Embodiment 5, Plackett-Burman (PB) is utilized to design the remarkable affecting genes of screening culture medium
Application PB design screening affects the medium component (remarkable affecting genes) of engineering bacteria E.coliXL10-Gold/CAVA yield plasmid (volume product rate). Comprehensive single factor experiment result and literature survey, (formula: casein peptone 12g, yeast extract 24g, glycerine 4mL, adds distilled water and dissolves, be settled to 900mL, and after autoclaving, 4 DEG C save backup to have chosen TB substratum. The 0.17mol/LKH that 100mL is aseptic is added when solution is cooled to 50 DEG C2PO4��0.72mol/LK2HPO4Solution. The compound method of this solution is: with 90mL deionized water dissolving 2.31gKH2PO4And 12.54gK2HPO4, after dissolving completely, it is settled to 100mL with deionized water and autoclaving 20min. ) in casein peptone, yeast extract, glycerine, NH4Cl (ammonium chloride), K2HPO4And KH2PO4Mixture, trace element (microbial growth is had certain promoter action by trace element, is that microorganism growth is necessary), MgSO4(magnesium sulfate, adds inorganic salt and can improve yield plasmid, and magnesium sulfate adds as inorganic salt, to obtaining higher yield plasmid) 7 factors that may affect plasmid fermentation output are investigated. Adopting n=10 (wherein 3 dummy features), number of following an example is 1, and basis number of times is 13 times, and overall test number is 13,1 central points. In PB test, the normally high level of choosing of level of factor is low-level 1.25-1.5 times, adjusts according to test practical situation, the factor numbering that table 2 is this test and level selection.
PB experimental design is based on following linear function:
Y=�� 0+ �� �� i �� i
Wherein, Y is the response value of prediction, and �� 0 is the intercept of model, and �� i is linear coefficient, and �� i is independent variable(s). Often in group experiment, the volume product rate of plasmid carries out 3 detections, and the mean value getting 3 times is responsively worth.
Table 2Plackett-Burman test design level of factor scope
PB testing program and the results are shown in Table 3, plasmid volume product rate is the average of 3 times, utilize Minitab analytical data, result is as shown in table 4, can find out in the middle of 7 factors to be selected and 3 virtual factors, the P < 0.05 of these 5 factors of B, D, F, J, K from this table, namely the impact of yield plasmid had significance, wherein F, J item is virtual item, remaining 3 B (yeast extract), D (glycerine), K (MgSO4) for be continued the factor optimized.
Table 3PB testing program and test-results
Table 4PB result is analyzed
Regression analysis: Y and A, B, C, D, E, F, G, H, J, K
Regression equation is:
Y=155 3.21A 19.6B+0.05C 17.9D 0.39E+14.7F 1.43G 1.13H+6.56J 20.0K
| Independent variable(s) | Coefficient | Factor standard is by mistake | T | P |
| Constant | 155.246 | 1.128 | 137.59 | 0.000 |
| A | -3.213 | 1.174 | -2.74 | 0.112 |
| B | -19.585 | 1.174 | -16.68 | 0.004 |
| C | 0.050 | 1.174 | 0.04 | 0.970 |
| D | -17.912 | 1.174 | -15.25 | 0.004 |
| E | -0.388 | 1.174 | -0.33 | 0.772 |
| F | 14.708 | 1.174 | 12.52 | 0.006 |
| G | -1.428 | 1.174 | -1.22 | 0.348 |
| H | -1.132 | 1.174 | -0.96 | 0.437 |
| J | 6.562 | 1.174 | 5.59 | 0.031 |
| K | -20.025 | 1.174 | -17.05 | 0.003 |
S=4.06830R-Sq=99.8%R-Sq (adjustment)=98.8%
Embodiment 6, the most suddenly climbing method determine the central point of response surface level of factor
Method of the most suddenly climbing is utilized to approach the best response region of remarkable factor, climbing direction and step-length is determined according to the linear function that Plackett-Burman (PB) test obtains, if the remarkable factor filtered out presents positive-effect, then grope from toward high level, if negative effect is then grope toward minimum level, step-length is determined by coefficient, and the more big step-length of coefficient is more little. The value of PB test does not probably contain the best value of influence factor, thus masks the real case of influence factor, so before groping medium component content further, it is necessary to the remarkable factor selected is carried out the steepest hill climbing test. Regression equation shows, and 3 factors with remarkably influenced are negative effect, and therefore this research is to yeast extract, glycerine, MgSO4These factors carry out bearing to method of the most suddenly climbing, other substratum factor chooses central point.
The most suddenly method experimental result of climbing is as shown in table 5, along with successively decreasing of concentration, plasmid volume product rate embodies the trend increased progressively, and reaches extreme value at the 5th group, but the concentration of substratum factor continues plasmid volume product rate downward trend (the 6th group) just occur toward decline. Therefore, this experiment determines that the 5th group for best response region, and namely concentration is yeast extract 16g/L, glycerine 3.2mL/L, magnesium sulfate 0.192g/L.
Table 5 is climbed method experimental result the most suddenly
| Grouping | Yeast extract (g/L) | Glycerine (ml/L) | MgSO4(g/L) | Volume product rate (mg/L) |
| 1 | 20 | 4.0 | 0.240 | 4.5 |
| 2 | 19 | 3.8 | 0.228 | 6.6 |
| 3 | 18 | 3.6 | 0.216 | 7.6 |
| 4 | 17 | 3.4 | 0.204 | 8.9 |
| 5 | 16 | 3.2 | 0.192 | 10.1 |
| 6 | 15 | 3.0 | 0.180 | 9.2 |
Embodiment 7, response phase method determine the best value of remarkably influenced factor
Through PB test and method of the most suddenly climbing, pick out the remarkable factor of impact fermentation, and found out the best response region of remarkable factor, but can not determine that remarkable factor cuts value really. Remarkably influenced factor is carried out 3 factor 3 levels and gropes by central composite design (CCD) in this research employing response phase method, obtains quadratic equation, data are carried out Quadratic Regression Fitting, to obtain the best value of remarkably influenced factor. Regression analysis model is as follows:
Y=�� 0+ �� �� i �� i+ �� �� ii �� i2+ �� �� ij �� i �� j
Wherein, Y is the response value of prediction, and �� 0 is the intercept of model, and �� i is linear coefficient, and �� ii is 2 power coefficients, and �� ij is mutual term coefficient, and �� i and �� j represents independent variable(s). Often in group experiment, the volume product rate of plasmid carries out 3 detections, and the mean value getting 3 times is responsively worth. Experimental design and data analysis all utilize Minitab software. The present invention tests, according to PB experiment and method of the most suddenly climbing, the factor determined and level adopts central composite design (CCD) that plasmid fermention medium carries out the response surface analysis optimization of 3 factor 3 levels, each factor and level in table 6, CCD test design and the results are shown in Table 7.
The central composite design level of table 6 is selected
| Level | Yeast extract (g/L) | Glycerine (mL/L) | MgSO4(g/L) |
| 1.68 | 9.28 | 1.86 | 0.232 |
| -1 | 12 | 2.4 | 0.216 |
| 0 | 16 | 3.2 | 0.192 |
| 1 | 20 | 4 | 0.168 |
| 1.68 | 22.72 | 4.54 | 0.152 |
Table 7 is central composite design and result
Quadratic Regression Fitting and variance analysis
Data in his-and-hers watches 7 carry out two order polynomial regression fits, adopt t inspection to determine significance. By response surface regression process data analysis, setting up Quadratic response surface regression model, to seek optimal response level of factor, result is as shown in table 8. As can be seen from Table 8, the impact of response value is had significance by yeast extract, glycerine, yeast extract * yeast extract, glycerine * glycerine, magnesium sulfate * magnesium sulfate, yeast extract * glycerine, yeast extract * magnesium sulfate, wherein yeast extract and glycerine, yeast extract and magnesium sulfate have significant interaction, illustrate that these factors are all the key factors in plasmid fermentation process. After eliminating not remarkable item, it is possible to obtain one taking plasmid volume product rate as the regression equation of objective function:
Y=12.3205+0.2690A-0.3755B-1.1938A2-1.1779B2-1.3069C2-0.6250AB-0.7200AC
This model is carried out confidence level analysis, R2=0.981, illustrate that this model can explain 98.1% that response value changes, model-fitting degree is good. Model is carried out variance analysis, and the linear item of this model, square item and interaction item all have significance, and lose and intend item p=0.959, do not have significance, and this institute also just proved is very rational with the matching of model.
The regression analysis of table 8 regression model
Table 9 regression equation variance analysis
The optimization of remarkable level of factor
Utilize Minitab Software on Drawing response surface and equal-value map, result is as shown in Figure 9, the isogram (B1-B3 figure) of each response surface figure (A1-A3 figure) and correspondence thereof represents the interaction between two independent variables respectively, and the 3rd variable now remains on optimum level. As can be seen from the figure, response variable Y has maximum value, therefore can infer that A, B, C exist extreme point. Respectively the independent variable(s) of regression equation is sought first-order partial derivative, and make it equal 0, it is possible to obtain a ternary linear function group, thus obtain A (yeast extract), B (glycerine), C (MgSO4) optimum concn of three factors. The extreme point that can draw model by separating this system of equations is: A=16.61g/L, B=3.05mL/L, C=0.185g/L, the theoretical maximum of plasmid volume product rate is 12.38mg/L. Optimizing process before comprehensive, obtains medium component and the content of final optimal, and result is as shown in table 10, and every 1L substratum (ITB) contains: casein peptone 12g, yeast extract 16.61g, glycerine 3.05mL, NH4Cl1g, 0.17mol/LKH2PO4And 0.72mol/LK2HPO4Mixing solutions 100mL, Mg2SO40.185g, trace element 1mL, trace element comprises FeCl3��6H2O27g/L��ZnCl22g/L��CoCl2��6H2O2g/L��Na2MoO4��2H2O2g/L��CaCl2��2H2O1g/L or anhydrous CaCl20.76g/L��CuCl2��2H2O1.27g/L��H3BO30.5g/L is dissolved in 1.2mol/LHCl. Described K2HPO4And KH2PO4The compound method of mixing solutions is: with 90mL deionized water dissolving 2.31gKH2PO4And 12.54gK2HPO4, after dissolving completely, it is settled to 100mL with deionized water and autoclaving 20min. Whether consistent with desired value in order to verify the substratum after optimization, substratum has been prepared according to table 10, carry out 3 groups of repeated authentication tests, obtained plasmid volume product rate and be respectively: 12.45mg/L, 12.51mg/L, 11.88mg/L, substantially identical with the maximum value of expection.
Table 10 optimize after medium component and content
Embodiment 8, prepare melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA with engineering bacteria E.coliXL10-Gold/CAVA and high yield fermention medium and yield plasmid (volume product rate) compares
Melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA is prepared with engineering bacteria E.coliXL10-Gold/CAVA and high yield fermention medium (ITB), in contrast, comprise the following steps with TB, LB, M9 (glycerine) and M9 (glucose) substratum;
1) original species word bank is prepared: be extended in 100mLLB liquid nutrient medium by E.coliXL10-Gold/CAVA engineering bacteria bacterium liquid 0.5mL, 37 DEG C, cultivate 12 hours under 200rpm, adding 30% glycerine, packing 50 is managed and frozen in-70 DEG C of refrigerators, obtains original species word bank;
2) main seed bank is prepared: a primordial seed of recovering is inoculated in 12 pipe LB liquid nutrient mediums, little upgrading grain after going down to posterity for 2 times, choosing the highest bacterium liquid of plasmid content is extended in 100mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after add 30% glycerine, and packing 50 is in control main seed bank, frozen in-70 DEG C of refrigerators;
3) preparation work seed bank: get 1 pipe bacterial classification from main seed bank, it is inoculated in 12 pipe LB liquid nutrient mediums, little upgrading grain after going down to posterity for 2 times, choosing the highest bacterium liquid of plasmid content is extended in 100mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after add 30% glycerine, packing 50 is managed and frozen in-70 DEG C of refrigerators, obtains work seed bank, produce for routine, often prop up the limit of the bacterial classification in work seed bank and passed for 5 generations;
4) recovery E.coliXL10-Gold/CAVA work seed, it is inoculated in 5mL high yield fermention medium ITB or TB, LB, M9 (glycerine) and M9 (glucose) substratum by 1:100,37 DEG C, 200rpm cultivate 12 hours, 1:100 goes down to posterity and is once inoculated in the 500mL Erlenmeyer flask containing 100mL high yield fermention medium afterwards, at 37 DEG C, shaking culture 15h under 200rpm, after cultivation terminates, upgrading grain, measures plasmid concentration.
Plasmid concentration detected result is as shown in table 11, result shows, the yield plasmid (volume product rate) of TB substratum can reach 9.9mg/L, improves 60% than LB substratum, is also better than M9 (glycerine) substratum and M9 (glucose) substratum significantly; 12.38mg/L can be reached by the ITB substratum yield plasmid (volume product rate) of above statistical method optimization, 25% is improved than TB substratum, 2 times to LB substratum, illustrate that finally optimizing the ITB substratum obtained can improve yield plasmid (volume product rate) significantly, shows that ITB is high yield fermention medium.
Table 11 engineering bacteria E.coliXL10-Gold/CAVA and different fermentations medium preparing melanoma therapeutic
Yield plasmid (the volume product rate) comparative result of Plasmid DNA vaccines pSVK-CAVA
| Substratum title | Yield plasmid (volume product rate) (mg/L) |
| ITB | 12.38 |
| TB | 9.9 |
| LB | 6.2 |
| M9 (glycerine) | 3.3 |
| M9 (glucose) | 1.1 |
Embodiment 9: the scale up test carrying out melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA with engineering bacteria E.coliXL10-Gold/CAVA and high yield fermention medium
The preparation of seed liquor: work seeds from-70 DEG C of E.coliXL10-Gold/CAVA of recovering, be inoculated in the LB liquid nutrient medium of 5mL by 1:100,37 DEG C, 200rpm cultivate 12 hours. Transfer again in the LB liquid nutrient medium of 100mL according to 1:100,37 DEG C, 200rpm cultivate 12 hours, now obtain seed liquor.
Pilot scale fermentation: seed liquor being planted according to the amount of volume ratio 1:100 and carry out fermentation culture (amount containing high yield fermention medium ITB in fermentor tank is 3L) in 5L fermentor tank, pH value controls 7.0 �� 0.1, automatically fills into H by fermentor tank2SO4And NH3.H20 regulates the pH value of substratum. Culture temperature is 37 DEG C, and dissolved oxygen controls about 30%, when dissolved oxygen is reduced to this value, regulates by supplementing pure oxygen and improve stirring velocity. After fermentation culture 6h, add high yield fermention medium ITB according to the flow velocity of 1mL/min, altogether add 500mL. Ferment after 15 hours, terminate fermentation, receive bacterium, extract plasmid, measure plasmid concentration.
Being identified by pilot product: carry out pilot scale fermentation with engineering bacteria E.coliXL10-Gold/CAVA and high yield fermention medium ITB and prepare melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA, yield plasmid can reach 150mg/L-200mg/L (volume product rate).
And use the yield plasmid (volume product rate) that identical seed liquor is fermented in LB liquid nutrient medium to be only about 80mg/L-110mg/L, show that the ITB that the present invention determines is high yield fermention medium.
Claims (7)
1. the production method of a melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA, it it is recovery engineering bacteria E.coliXL10-Gold/CAVA work seed, 1:100 ratio is inoculated in 5mLLB liquid nutrient medium by volume, 37 DEG C, cultivate 12 hours under 200rpm, transfer again in the LB liquid nutrient medium of 100mL according to 1:100,37 DEG C, 200rpm cultivate 12 hours, obtain seed liquor;
The amount of seed liquor 1:100 being by volume inoculated in the 5L fermentor tank containing 3L fermention medium ITB, pH value controls 7.0 �� 0.1, and culture temperature is 37 DEG C, and dissolved oxygen controls 30%, fermentation culture 6h; Add fermention medium ITB according to the flow velocity of 1mL/min afterwards, altogether add 500mL, ferment after 15 hours, terminate fermentation, receive bacterium, extract plasmid, obtain melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA;
Fermention medium ITB described in every 1L contains: casein peptone 10-14g, yeast extract 16-20g, glycerine 2.4-4mL, NH4Cl0.75-1g, 0.17mol/LKH2PO4And 0.72mol/LK2HPO4Mixing solutions 75-100mL, MgSO40.168-0.216g, trace element 0.75-1mL;
Described engineering bacteria E.coliXL10-Gold/CAVA is that melanoma therapeutic pSVK-CAVA plasmid DNA transformation intestinal bacteria XL10-Gold is obtained transformed bacteria XL10-Gold; Transformed bacteria XL10-Gold is coated LB solid plate substratum again, 37 DEG C, cultivate 15 hours under 200rpm after choose and get mono-clonal bacterium colony; By mono-clonal colony inoculation in 5mLLB liquid nutrient medium, 37 DEG C, cultivate 12 hours under 200rpm after carry out continuous passage cultivation according to 1:100, switching in 12 hours is gone down to posterity 1 time, continuous passage 30 generation; Mono-clonal bacterial strain and within 30 generations of going down to posterity all previous bacterial strain that goes down to posterity be the engineering bacteria of transformation of E. coli XL10-Gold.
2. production method according to claim 1, it is characterised in that: fermention medium ITB described in every 1L contains: casein peptone 12g, yeast extract 16.61g, glycerine 3.05mL, NH4Cl1g, 0.17mol/LKH2PO4And 0.72mol/LK2HPO4Mixing solutions 100mL, MgSO40.185g, trace element 1mL.
3. production method according to claim 2, it is characterised in that: trace element comprises: FeCl3��6H2O27g/L��ZnCl22g/L��CoCl2��6H2O2g/L��Na2MoO4��2H2O2g/L��CaCl2��2H2O1g/L, or anhydrous CaCl20.76g/L��CuCl2��2H2O1.27g/L��H3BO30.5g/L, is dissolved in 1.2mol/LHCl.
4. production method according to claim 3, it is characterised in that: K in fermention medium ITB2HPO4And KH2PO4The compound method of mixing solutions is: with 90mL deionized water dissolving 2.31gKH2PO4And 12.54gK2HPO4, after dissolving completely, it is settled to 100mL with deionized water.
5. production method according to claim 1 or 2 or 3 or 4, it is characterized in that: described engineering bacteria E.coliXL10-Gold/CAVA, for the engineering bacteria with Calcium Chloride Method transformation of E. coli XL10-Gold, method for transformation comprises: thawed in ice bath 10 minutes by the 100 �� l competent cell XL10-Gold that-70 DEG C preserve, then pSVK-CAVA plasmid DNA 25-50ng is added, soft mixed even after, put in ice bath 30 minutes; After ice bath terminates, in 42 DEG C of water-baths, heat hits 90 seconds, is placed in rapidly ice bath afterwards and cools 3-5 minute, add the LB liquid nutrient medium of 800 �� l non-resistants, in 37 DEG C after mixed even, 200rpm shaking culture 1 hour, obtains the transformed bacteria XL10-Gold of pSVK-CAVA plasmid DNA.
6. production method according to claim 1 or 2 or 3 or 4, it is characterised in that: described E.coliXL10-Gold/CAVA engineering bacteria work seed obtains by the following method:
1) original species word bank is prepared: be extended in 100mLLB liquid nutrient medium by described engineering bacteria E.coliXL10-Gold/CAVA bacterium liquid 0.5mL, 37 DEG C, cultivate 12 hours under 200rpm, adding 30% glycerine, packing 50 is managed and frozen in-70 DEG C of refrigerators, obtains original species word bank;
2) main seed bank is prepared: a primordial seed of recovering is inoculated in 12 pipe LB liquid nutrient mediums, little upgrading grain after going down to posterity for 2 times, choosing the highest bacterium liquid of plasmid content is extended in 100mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after add 30% glycerine, and packing 50 is in control main seed bank, frozen in-70 DEG C of refrigerators, obtain main seed bank;
3) preparation work seed bank: get 1 pipe bacterial classification from main seed bank, it is inoculated in 12 pipe LB liquid nutrient mediums, little upgrading grain after going down to posterity for 2 times, choosing the highest bacterium liquid of plasmid content is extended in 100mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after add 30% glycerine, packing 50 is managed and frozen in-70 DEG C of refrigerators, obtains work seed bank; Often prop up the limit of the bacterial classification in work seed bank and pass 5 substitutes in the production of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA.
7. production method according to claim 5, it is characterised in that: described E.coliXL10-Gold/CAVA engineering bacteria work seed obtains by the following method:
1) original species word bank is prepared: be extended in 100mLLB liquid nutrient medium by described engineering bacteria E.coliXL10-Gold/CAVA bacterium liquid 0.5mL, 37 DEG C, cultivate 12 hours under 200rpm, adding 30% glycerine, packing 50 is managed and frozen in-70 DEG C of refrigerators, obtains original species word bank;
2) main seed bank is prepared: a primordial seed of recovering is inoculated in 12 pipe LB liquid nutrient mediums, little upgrading grain after going down to posterity for 2 times, choosing the highest bacterium liquid of plasmid content is extended in 100mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after add 30% glycerine, and packing 50 is in control main seed bank, frozen in-70 DEG C of refrigerators, obtain main seed bank;
3) preparation work seed bank: get 1 pipe bacterial classification from main seed bank, it is inoculated in 12 pipe LB liquid nutrient mediums, little upgrading grain after going down to posterity for 2 times, choosing the highest bacterium liquid of plasmid content is extended in 100mLLB substratum, 37 DEG C, cultivate 12 hours under 200rpm after add 30% glycerine, packing 50 is managed and frozen in-70 DEG C of refrigerators, obtains work seed bank; Often prop up the limit of the bacterial classification in work seed bank and pass 5 substitutes in the production of melanoma therapeutic Plasmid DNA vaccines pSVK-CAVA.
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| CN1449442A (en) * | 2000-08-31 | 2003-10-15 | 第一三得利制药株式会社 | Process for producing biopterins |
| CN102369838A (en) * | 2010-08-11 | 2012-03-14 | 宝生物工程株式会社 | Manufacturing Method Of Liquid Seed Fungus Of Mushroom |
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| CN1449442A (en) * | 2000-08-31 | 2003-10-15 | 第一三得利制药株式会社 | Process for producing biopterins |
| CN102369838A (en) * | 2010-08-11 | 2012-03-14 | 宝生物工程株式会社 | Manufacturing Method Of Liquid Seed Fungus Of Mushroom |
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