CN103387946A - Preparation method for competent host cell, and method for rapid and highly efficient transfer of exogenous substances into host cell and application thereof - Google Patents
Preparation method for competent host cell, and method for rapid and highly efficient transfer of exogenous substances into host cell and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method for a competent host cell and a method for rapid and highly efficient transfer of exogenous substances into a host cell and application thereof. Core points of the invention are as follows: a conventional vacuum freeze drying technology is employed to treat a host cell so as to obtain the competent host cell, an aqueous solution containing the exogenous substances is used for rehydration of the competent host cell, and the competent host cell having undergone rehydration is placed in a constant temperature incubator for culture and then transferred to a conditioned medium for screening so as to obtain an exogenous substance transformed bacterium. According to the invention, rapid transformation of the host cell by using the exogenous substance is realized, and the advantages of simple operation, small damage, low cost and the like are obtained; through implementation of the method, batch competent cells can be obtained in a simple and convenient mode, and the method is applicable to the application fields like research on activity and functions of the exogenous substances.
Description
Technical field
The invention belongs to biological product and biological technology application, be specifically related to allogenic material is changed over to the method for host cell.
Background technology
at biological technical field, the gene horizontal transfer, refer to biological the genetic material level is passed to other cells but not vertical transmission to the process of its filial generation, it is the important means of present Study of Exogenous gene function, particularly in the microorganism level (as virus, bacterium, fungi, vegetable cell, zooblast etc.) very common, thereby a kind of bacterium often obtains new proterties by from other bacteriums, obtaining the part genetic material, for example a large amount of microbiotic that use cause coding can decompose the transverse dispersion between different bacterium of antibiotic gene afterwards at present, thereby formation resistant organism.And in laboratory, often utilize this genetic material can propagate and spread between Different Individual property research foreign DNA and the function of other biological active substance.Yet, the cell that comprises bacterium, fungi (as yeast), vegetable cell, zooblast etc. has cytolemma usually, entering of strict restriction allogenic material, therefore, how these cells being changed into it the state that can accept allogenic material by certain technical finesse is competence, is an important research content of biotechnology and biological technical field.
present commonly used promoting mainly contains the method for allogenic material transformed host cell: utilize divalent-metal ion (as calcium ion) under certain temperature condition (as 42 ℃), can make host cell become large because thermal stimulus causes permeability of cell membrane, thereby be easy to take in allogenic material (as DNA) (Mei Yunjun, Chen XiangDong, the .Ca such as Xie Zhixiong ~ (2+) on inducing the impact of intestinal bacteria picked-up foreign DNA. Wuhan University Journal: Edition, 2012, 58 (4): 354-358.), perhaps adopt high-voltage electric field to impel the method (Zhang Yang such as host cell membrane moment perforation, Wang Zhiqiang, the .DH10B bacterial strain efficient electric conversion conditions such as Liu Bin are probed into. the biotechnology journal, 2007, 23 (2): 347-351.).But the shortcomings such as all there is complicated operation in these methods, large to cell injury, running cost is high, transformation efficiency is low.How setting up a kind of method that allogenic material is transformed (changing over to) host cell rapidly and efficiently is one of Important Problems of present biotechnology and biological technical field research.
Summary of the invention
Based on above problem, the object of the invention is to provide a kind of preparation method of competence host cell rapidly and efficiently, and a kind of method that allogenic material is changed over to host cell.
The preparation method of competence host cell provided by the present invention comprises the following steps:
1) purifying, recovery and pre-treatment host cell;
2) Vacuum Freezing ﹠ Drying Technology is processed host cell, obtains the competence host cell.
The method that allogenic material is changed over to host cell provided by the present invention, further comprising the steps:
3) prepare the aqueous solution of allogenic material to be changed over to;
4) to step 2) the competence host cell that obtains carries out fast rehydrating and changes allogenic material over to host cell, and transformed the host cell of allogenic material with the conditioned medium screening, obtain the allogenic material transformed bacteria.
In aforesaid method, the host cell in described step 1) can be any one protokaryon or eukaryotic host cell, as bacterial cell, fungal cell, vegetable cell and zooblast etc.Due to bacterium (the most frequently used is intestinal bacteria) and yeast has that genetic background is clear, the target gene expression level is high, expression system ripe perfect, be easy to cultivate, low cost and other advantages, used intestinal bacteria and yeast in the embodiment of the present invention.
Described purifying host cell refers to former host cell streak culture at the solid culture primary surface, obtains mono-clonal; The recovery host cell refers to the host cell of purifying is cultivated certain hour again, makes it from stationary state, reenter growth conditions; The pre-treatment host cell refers to the host cell enlarged culturing of recovery to respective concentration.Particularly, the process of described step 1) purifying, recovery and pre-treatment host cell is as follows:
(1) purifying host cell: with the original host cell of transfering loop picking, sectional streak is inoculated in solid medium (LB solid medium (the being fit to intestinal bacteria) formula: Tryptones 10g/L that is applicable to cultivate host cell, yeast extract paste 5g/L, NaCl10g/L, agar powder 15g/L; YPD solid medium (be fit to yeast) formula: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, agar powder 20g/L), (37 ℃ of intestinal bacteria, 12～16h under optimum conditions; 30 ℃ of yeast culture, 48h) cultivate, and obtains the host cell mono-clonal;
(2) recovery host cell: picking host cell mono-clonal is inoculated into liquid nutrient medium (LB substratum (the being fit to intestinal bacteria) formula: Tryptones 10g/L, yeast extract paste 5g/L, NaCl10g/L that 5mL is applicable to host cell; YPD substratum (being fit to yeast) formula: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L) in, (37 ℃ of intestinal bacteria under optimal temperature; 30 ℃ of yeast culture) 220rpm recovery spend the night (12～16 hours);
(3) pre-treatment host cell: the host cell through recovery is inoculated into the fresh liquid nutrient medium of 100mL (LB substratum (being fit to intestinal bacteria) formula: Tryptones 10g/L, yeast extract paste 5g/L, NaCl10g/L according to the ratio of volume ratio 1:100; YPD substratum (being fit to yeast) formula: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L) in, (37 ℃ of intestinal bacteria under optimal temperature; 30 ℃ of yeast culture) 220rpm shakes bacterium to OD
600=0.6～1.0; Draw 500 μ l bacterium liquid and divide and be filled in 1.5mL EP pipe ,-80 ℃ of refrigerator precoolings are more than 12～24 hours.
In aforesaid method, described step 2) in, the method for vacuum lyophilization host cell is: adopt on market any vacuum freeze drier or the vacuum freeze drier of processing voluntarily, host cell after step 1) is processed is used vacuum freeze drier precooling 30～120 minutes, until temperature reach-54 ℃ and be stabilized in this temperature after, vacuum lyophilization host cell 12～24 hours or more than.
In aforesaid method, in described step 3), allogenic material used comprises the biologically active substances such as nucleic acid (thymus nucleic acid (DNA), Yeast Nucleic Acid (RNA)), protein, oligopeptides, amino acid.
The described allogenic material aqueous solution uses ddH
2O。The consumption of allogenic material can change according to concrete research purpose that (for example the consumption of plasmid DNA is not less than 0.01ng; For other allogenic materials to be transformed, those skilled in the art can determine according to reference open source literature the consumption of allogenic material), the allogenic material aqueous solution for preparing was 37 ℃ of insulations 30～60 minutes.
In aforesaid method, the method of in described step 4), the competence host cell being carried out fast rehydrating is: with step 2) the competence host cell add rapidly step 3) to prepare and be incubated the aqueous solution that contains allogenic material of processing,, in 37 ℃ of insulations 30 minutes, be placed in (37 ℃ of intestinal bacteria under optimal temperature after mixing gently; 30 ℃ of yeast culture) 220rpm shaking culture 45～60 minutes in thermostat container; Contain the consumption of the aqueous solution of allogenic material and the host cell equal-volume before lyophilize.
In step 4), the conditioned medium of screening use is: LB solid medium (being fit to intestinal bacteria), and filling a prescription is: Tryptones 10g/L, yeast extract paste 5g/L, NaCl10g/L, agar powder 15g/L, the microbiotic that is suitable for allogenic material of final concentration 100-200 μ g/mL; YPD solid medium (being fit to yeast), filling a prescription is: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, agar powder 20g/L, the microbiotic that is suitable for allogenic material of final concentration 100-200 μ g/mL.Antibiotic selection is relevant with the allogenic material kind, and those skilled in the art can be according to allogenic material with reference to open source literature is definite.
In step 4), the process of screening competence host cell is: the competence host cell bacterium liquid of rehydration is inoculated or is applied to described conditioned medium, (37 ℃ of intestinal bacteria under optimal temperature; 30 ℃ of yeast culture) constant temperature culture grew mono-clonal after 12～16 hours, and the host cell that namely obtains transforming allogenic material is the allogenic material transformed bacteria.
The competence host cell and the allogenic material transformed bacteria that prepare in aforesaid method also belong to content of the present invention.And described competence host cell can still keep biological activity at 0-4 ℃ of temperature, be convenient to storage and transport, and the competent cell of this area can only transport under dry ice (70 ℃) condition at present.
The present invention utilizes Vacuum Freezing ﹠ Drying Technology to prepare the competence host cell, and can rapidly and efficiently allogenic material be transformed (changing over to) to this competence host cell, and its core is to adopt Vacuum Freezing ﹠ Drying Technology and rehydration technology.At first the present invention carries out lyophilize to host cell and processes acquisition competence host cell under vacuum state, then the aqueous solution that will intend the allogenic material that transforms joins the competence host cell and carries out rehydration, after host cell after rehydration is placed in the constant incubator cultivation, transfer in the conditioned medium that contains resistance pressure and screen, and whether can detect the conclusive evidence conversion by biochemical indicator successful.Utilize the present invention not only can realize exogenous nucleic acid rapid conversion host cell, and can realize biologically active substance and other material rapid conversion host cells such as exogenous protein, amino acid, carbohydrate, and the method has simple to operate, damaging little, low cost and other advantages.By enforcement of the present invention, not only can easy quick acquisition competence host cell in batch (namely through cryodesiccated host cell, be in the state that acceptant foreign DNA and other allogenic materials enter), can be used for the research of follow-up biological function, the transmembrane transport that also can be allogenic material provides important technical study means, and then, for the functional study of allogenic material provides gordian technique, can significantly save research cost, so the present invention's prospect that is widely used.
Below in conjunction with specific embodiment, the present invention is described in further details.
Description of drawings
Fig. 1 is that the present invention utilizes Vacuum Freezing ﹠ Drying Technology quickly and efficiently with the schema of the method for allogenic material transformed host cell
Fig. 2 A is prokaryotic expression carrier pET28b-Tat rehydration conversion results under 37 ℃ of conditions
Fig. 2 B is prokaryotic expression carrier pET28b-Tat rehydration conversion results under 42 ℃ of conditions
Fig. 3 is that pET28b-Tat-EGFP rehydration under 37 ℃ of conditions transforms rear growth curve of bacteria detected result
Fig. 4 A is 2 hours SDS-PAGE of pET28b-Tat-EGFP abduction delivering and immunoblot assay detected result
Fig. 4 B is 6 hours SDS-PAGE of pET28b-Tat-EGFP abduction delivering and immunoblot assay detected result
Fig. 5 is that plasmid pUC19 rehydration under 37 ℃ of conditions transforms bacillus coli DH 5 alpha competence result
Fig. 6 is that plasmid pUC19 rehydration under 37 ℃ of conditions transforms Pichia pastoris GS115 competence result
Embodiment
The shortcoming such as all there is complicated operation in the method for in view of existing calcium ion and high-voltage etc., carrying out the gene horizontal transfer, large to cell injury, running cost is high, transformation efficiency is low, the present invention make great efforts to probe into to obtain a kind of rapidly and efficiently with the method for allogenic material transformed host cell.
The present invention utilizes Vacuum Freezing ﹠ Drying Technology to realize the conversion of allogenic material to host cell.
Vacuum Freezing ﹠ Drying Technology is the process that the principle of utilization distillation realizes biological tissue under the vacuum and low temperature condition in, solid water is directly sublimed into gas evaporation, this process not only can keep the integrity of biological sample, and can keep activity (the Kang MS of biological sample, Jang H, Kim MC, Kim MJ, Joh SJ, Kwon JH, Kwon YK:Development of a stabilizer for lyophilization of an attenuated duck viral hepatitis vaccine.Poult Sci2010,89 (6): 1167-1170; Chen C, Han D, Cai C, Tang X:An overview of liposome lyophilization and its future potential.J Control Release2010,142 (3): 299-311.).At present, Freeze Drying Technique has been widely used in foodstuffs industry, drying and prolonged preservation (the Liu JH of food, biomass cells, histoorgan and medicine etc. in a plurality of fields such as biology and pharmacy, Zhou J, Wang DM, Ouyang XL, Xing YC, Lu FQ:[Experimental study on lyophilization of platelets] .Zhongguo Shi Yan Xue Ye Xue Za Zhi2007,15 (5): 1098-1101; Matejtschuk P:Lyophilization of proteins.Methods Mol Biol 2007,368:59-72; Taniwaki L, Mendonca R, Cunha-Junior AS, Faraco AA, Ribeiro JA, Scott IU, Jorge R:Effect of lyophilization on the in vitro biological activity of bevacizumab.Eye (Lond) 2010).Utilize Freeze Drying Technique not only can realize the lyophilize of prokaryote and rehydration bring back to life (Renoux G:[Lyophilization of Brucella strains.] .Ann Inst Pasteur (Paris) 1962,103:290-292), and can realize that the lyophilize of eukaryotic cells and rehydration bring back to life, as (Guibert L, Brechot P:[Lyophilization of yeasts such as yeast cell and mouse sperm cells; First part.] .Ann Inst Pasteur (Paris) 1955,88 (6): 750-768; Atkin L, Moses W, Gray PP:The preservation of yeast cultures by lyophilization.J Bacteriol1949,57 (6): 575-578; Leirner AA, Tattini V, Jr., Pitombo RN:Prospects in lyophilization of bovine pericardium.Artif Organs2009,33 (3): 221-229).the researchist finds to adopt Freeze Drying Technique can realize the long-term preservation of room temperature of mice organs tissue by research, its amplifying nucleic acid and protein are not degraded with the prolongation in storage time, become bulk and can keep structural integrity (Yonghong Wu after rehydration after organizing lyophilize, Min Wu, Yanchun Zhang, Weiguang Li, Yan Gao, Zhihui Li, Zhaoyan Wang, Gert Lubec, Chenggang Zhang.Lyophilization is suitable for storage and shipment of fresh tissue samples without altering RNA and protein levels stored at room temperature.Amino Acids.2012, 43 (3): 1383-1388).
Vacuum Freezing ﹠ Drying Technology can not only keep integrity and the activity of biological sample, and the contriver also finds under study for action: after bacterial cell (for example intestinal bacteria) being carried out the lyophilize processing, the hole of some amount will appear in cytolemma.If after intending the aqueous solution of the allogenic material (for example thymus nucleic acid (DNA), Yeast Nucleic Acid (RNA), protein, biologically active substance and other material) that transforms this moment and joining bacterial cell after the vacuum lyophilization processing, allogenic material just can be along with moisture enters bacterial cell together fast, thereby realized allogenic material is changed over to the purpose of cell.On this basis, the contriver proposes following mechanism: adopt Freeze Drying Technique can realize lyophilize and the rehydration resurrection of Bacillus coli cells; Intestinal bacteria after lyophilize become bulk, and fenestra is loose, and after rehydration, fenestra is closed rapidly; Intestinal bacteria after lyophilize in reconstitution process along with closing of fenestra can, with allogenic material rapid conversion host cell, can be screened the Host Strains that can obtain to transform allogenic material by conditioned medium subsequently.
Further explaination by the following examples.
In following embodiment, method therefor is ordinary method if no special instructions, and concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3
rdEdition, 2001, NY, Cold Spring Harbor).
The approach that obtains of the various biomaterials that are described in embodiment only is to provide approach that a kind of experiment obtains to reach concrete disclosed purpose, should not become the restriction to biological material source of the present invention.In fact, the source of the present invention's biomaterial used is widely, any keep on the right side of the law and the moral ethics biomaterial that can obtain can be replaced and use according to the prompting in embodiment.
Embodiment implements under take technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, and embodiment will help to understand the present invention, but protection scope of the present invention is not limited to following embodiment.
In the present embodiment, allogenic material is take plasmid pET28b-Tat-EGFP as example, and host cell is take e. coli bl21 (DE3) as example.Reference as shown in Figure 1, is carried out following operation and is changed allogenic material over to host cell:
1) purifying, recovery and pre-treatment e. coli host cell
(1) purifying host cell: with LB solid medium (formula: Tryptones 10g/L, yeast extract paste 5g/L, NaCl10g/L, agar powder 15g/L), at 37 ℃ of underscore culture purified e. coli host cell BL21 (DE3), obtain the e. coli host cell mono-clonal;
(2) recovery host cell: picking e. coli host cell mono-clonal is inoculated in 5mL LB liquid nutrient medium (formula: Tryptones 10g/L, yeast extract paste 5g/L, NaCl10g/L), 37 ℃, 220rpm recovery spend the night (12-16 hour);
(3) pre-treatment host cell: the e. coli host cell through recovery is inoculated in the fresh LB liquid nutrient medium of 100mL (filling a prescription identical with step (2)) according to the ratio of volume ratio 1:100, and 37 ℃, 220rpm shake bacterium 2～3 hours to OD
600=0.6～1.0, to draw 500 μ L bacterium liquid and divide and be filled in 1.5mL EP pipe ,-80 ℃ of refrigerator precoolings are more than 12～24 hours.
2) vacuum lyophilization e. coli host cell
with vacuum freeze drier (available from Beijing development in science and technology company limited of Song Yuan Huaxing) be chilled in advance temperature reach-54 ℃ and stable after, the pretreated e. coli host cell of step 1) is taken out and is placed in rapidly vacuum freeze drier from-80 ℃ of cryogenic refrigerators, close intake valve and open vacuum pump after installing lid,-54 ℃ of cold condition are set, vacuum lyophilization host cell 12～24 hours or more than, close vacuum pump after to be dried and take out the intestinal bacteria of through Freeze Drying Technique, processing, the intestinal bacteria of the method preparation are " competence e. coli bl21 (DE3) " (having possessed the ability that allogenic material transforms of accepting), can use immediately or-20 ℃ save backup, perhaps also can be under 0-4 ℃ of condition storage and transport.
Below investigate " competence e. coli bl21 (DE3) " that obtain and whether possess the ability of accepting to change over to allogenic material.During with the allogenic material transformed host cell, continue following steps:
3) preparation intends transforming the aqueous solution of allogenic material
At first adopt ultraviolet spectrophotometer to measure pre-inversion plasmid pET28b-Tat-EGFP(construction process: 1. the cDNA fragment that amplifies EGFP by conventional PCR method; Restriction enzyme respectively enzyme cut goal gene EGFP, carrier pET28b-Tat, use the T4DNA ligase enzyme to connect after agarose gel electrophoresis reclaims.Reference (Wu Y, Ren C, Gao Y, Hou B, Chen T, Zhang C.A novel method for promoting heterologous protein expression in Escherichia coli by fusion with the HIV-1TAT core domain.Amino Acids.2010Aug; 39 (3): 811-20) or the building process of Chinese patent literature 201010119397.8 " utilizing TAT core peptide section to promote method and the dedicated expression vector therefor thereof of foreign protein solubility expression ") concentration, and use ddH
2O is diluted to 1ng/ μ l with it, respectively 5 μ l, 10 μ l, 25 μ l, 50 μ l, 100 μ l plasmid pET28b-Tat-EGFP is added to 500 μ l ddH
2Mix the aqueous solution of making different concns in O, hatched 30～60 minutes in 37 ℃ of thermostat water baths.
4) to above-mentioned steps 2) e. coli host cell (" competence e. coli bl21 (DE3) ") processed of the process vacuum lyophilization that obtains carries out fast rehydrating, obtains to have transformed the e. coli host cell of allogenic material with the conditioned medium screening:
(1) " competence e. coli bl21 (DE3) " that fast rehydrating: with step 2) obtains adds rapidly the aqueous solution that contains plasmid pET28b-Tat-EGFP of the step 3) preparation of processing through 37 ℃ of insulations, host cell equal-volume (500 μ l) before this aqueous solution and lyophilize,, in 37 ℃ (or 42 ℃) insulation 30 minutes, be placed in 37 ℃ of thermostat container 220rpm shaking culture 45～60 minutes after mixing gently;
(2) screening: after the lyophilize of learning from else's experience, rehydration is processed the e. coli host cell bacterium liquid 100 μ l of (with the aqueous solution of the listed different plasmid concentrations of step 3)), be applied to respectively the LB solid medium (formula: Tryptones 10g/L that conditioned medium namely contains kantlex, yeast extract paste 5g/L, NaCl10g/L, agar powder 15g/L, the microbiotic kantlex of final concentration 200 μ g/mL) in, 37 ℃ of lower constant temperature culture 12～16 hours, select the mono-clonal that grows out, obtain transforming the e. coli host cell of allogenic material, i.e. " Tat-EGFP transformed bacteria ".
The ability that is transformed by allogenic material pET28b-Tat-EGFP by observing mono-clonal quantity judgement competence e. coli bl21 (DE3) in experimentation, found that under 37 ℃ of insulations and 42 ℃ of different temperature condition, plasmid pET28b-Tat-EGFP increases along with the increase of plasmid concentration for the inversion quantity of competence e. coli bl21 (DE3), but there is no notable difference both, under Fig. 2 A(37 ℃ condition) and Fig. 2 B(42 ℃ condition under) shown in, therefore 37 ℃ of conditions of follow-up direct employing are carried out rehydration to allogenic material and are transformed.
Checking to the Tat-EGFP transformed bacteria continues following operation:
5) the e. coli host cell Tat-EGFP transformed bacteria that transforms allogenic material being carried out biochemical indicator detects
(1) the Tat-EGFP transformed bacteria is carried out the dynamic monitoring of growth curve
Above-mentioned 300 μ l " Tat-EGFP transformed bacteria " are adopted and contain kantlex (Kana according to the ratio of volume ratio 1:10
+, final concentration 200 μ g/mL) the LB liquid nutrient medium dilute and be inoculated into microbial growth curve monitoring microwell plate (350 μ l/ hole), real-time dynamic monitoring microbial growth state.
Monitoring result as shown in Figure 3.Fig. 3 first classifies normal bacteria as, does not use the pET28b-Tat-EGFP plasmid to transform, therefore when conditioned medium screens without any clonal growth out; Secondary series to the four row are three groups of parallel repetitions and contrast, and are " the Tat-EGFP transformed bacteria " of the plasmid that contains pET28b-Tat-EGFP, have all grown mono-clonal, illustrate that competent cell preparation method of the present invention is successfully.As seen: the intestinal bacteria (" Tat-EGFP transformed bacteria ") that transformed allogenic material can grow in the substratum of Kana resistance, and its growth curve is good; Observe and show for the bacterium colony that is grown in the solid culture primary surface, the intestinal bacteria colonial morphology that has transformed allogenic material is normal.Therefore showing step 2) " competence e. coli bl21 (DE3) " (e. coli host cell that vacuum lyophilization obtains) inherited character of obtaining do not change, can be used as allogenic material and transform the host cell that uses.
(2) the colibacillary exogenous protein expression that transforms allogenic material is detected
For whether further confirmation has transformed foreign protein in the intestinal bacteria of allogenic material can be according to expection imagination normal expression, it is " Tat-EGFP transformed bacteria " to be inoculated into 5mL contain the LB liquid nutrient medium of Kana (final concentration 200 μ g/mL) and spend the night (12～16 hours) of recovering that this step is detected; Next day, be inoculated into 150mL according to the ratio of volume ratio 1:100 and contain the LB liquid nutrient medium of Kana (final concentration 200 μ g/mL), and 37 ℃, 220rpm shake bacterium to OD
600=0.6-1.0; Then add inductor IPTG (final concentration 1mM) and in 30 ℃, 220rpm abduction delivering 6 hours, respectively at 0 hour, 2 hours, 4 hours and sampling in 6 hours, prepare protein sample, adopt 15%SDS-PAGE and immunoblot assay to detect foreign protein (TAT-EGFP) expression.
2 hours and 6 hours detected results are seen Fig. 4 A, Fig. 4 B, therefrom can find out, TAT-EGFP albumen (as shown by arrows in FIG.) by exogenous plasmid coding obtains to express, and shows that " competence e. coli bl21 (DE3) " can normal expression contains the coded TAT-EGFP albumen of exogenous plasmid pET28b-Tat-EGFP of TAT-EGFP gene.As seen allogenic material (this detect in the encoding gene of TAT-EGFP albumen) changing over to the intestinal bacteria competence host cell of vacuum lyophilization preparation not only, and these allogenic materials have also obtained desirable expression (this detect in take TAT-EGFP albumen as example) after changing over to, illustrate that the prepared competence host cell of the present invention can be used as the host cell of genetic expression.
As shown in Figure 5, allogenic material with plasmid pUC19(available from THERMO FISHER, 0.5 μ g/ μ l) be example, host cell is take bacillus coli DH 5 alpha as example.Following steps are identical with embodiment 1, except the special part that indicates, but essentially identical statement cross-reference.
1) purifying, recovery and pre-treatment e. coli host cell
(1) purifying host cell: with LB solid medium (formula: Tryptones 10g/L, yeast extract paste 5g/L, NaCl10g/L, agar powder 15g/L), at 37 ℃ of underscore culture purified e. coli host cells (DH5 α), obtain e. coli host cell DH5 alpha monoclonal;
(2) recovery host cell: picking DH5 alpha monoclonal is inoculated in 5mL LB liquid nutrient medium (formula: Tryptones 10g/L, yeast extract paste 5g/L, NaCl10g/L), 37 ℃, 220rpm recovery spend the night (12-16 hour);
(3) pre-treatment host cell: the DH5 α bacterium liquid through recovery is inoculated in the fresh LB liquid nutrient medium of 100mL according to the ratio of 1:100, and 37 ℃, 220rpm shake bacterium 3～4 hours to OD
600=0.6～1.0, be distributed into the 50ml centrifuge tube, place cooled on ice 10min, 4000rpm4 ℃ is centrifugal 10 minutes.The thalline that every 50ml bacterium liquid is collected is resuspended in the 2ml lyophilized vaccine, and the frozen-dried protective agent prescription is as follows: contain by weight gelatin 1%, and N.F,USP MANNITOL 7%, all the other are water.Divide and install in the 1.5mlEP pipe by the 200 every pipes of μ l ,-80 ℃ of refrigerator precoolings are more than 12～24 hours.
2) vacuum lyophilization e. coli host cell
with vacuum freeze drier (available from Beijing development in science and technology company limited of Song Yuan Huaxing) be chilled in advance temperature reach-54 ℃ and stable after, e. coli host cell is taken out and is placed in rapidly vacuum freeze drier from-80 ℃ of cryogenic refrigerators, close intake valve and open vacuum pump after installing lid,-54 ℃ of cold condition are set, vacuum lyophilization host cell 12～24 hours or more than, close vacuum pump after to be dried and take out the intestinal bacteria of through Freeze Drying Technique, processing, the intestinal bacteria of the method preparation are " competence bacillus coli DH 5 alpha ", possessed the ability that allogenic material transforms of accepting, can use immediately or-20 ℃ save backup, perhaps also can be under 0-4 ℃ of condition storage and transport.
3) preparation intends transforming the aqueous solution of allogenic material
With pre-inversion plasmid pUC19(available from THERMO FISHER, 0.5 μ g/ μ l) be added to 500 μ l LB liquid nutrient mediums (formula: Tryptones 10g/L, yeast extract paste 5g/L, NaCl10g/L) mix in and make the solution that concentration is 2pg/ μ l, be incubated 30～60 minutes in 37 ℃ of thermostat water baths.
4) the competence bacillus coli DH 5 alpha is carried out fast rehydrating, obtains to have transformed the e. coli host cell of allogenic material with the conditioned medium screening:
(1) e. coli host cell through vacuum lyophilization is carried out fast rehydrating: the competence bacillus coli DH 5 alpha is added rapidly the LB liquid nutrient medium that contains plasmid pUC19 of processing through 37 ℃ of insulations, mix gently and be placed in 37 ℃ of thermostat containers the 220rpm shaking culture 45～60 minutes;
(2) obtain to have transformed the e. coli host cell of allogenic material with the screening of conditioned medium pressure: the competent cell DH5 α bacterium liquid 100 μ l that after the lyophilize of learning from else's experience, rehydration is processed, be applied to the LB solid medium (formula: Tryptones 10g/L that conditioned medium namely contains penbritin, yeast extract paste 5g/L, NaCl10g/L, agar powder 15g/L, the microbiotic penbritin of final concentration 100 μ g/mL) in, grow mono-clonal 37 ℃ of lower constant temperature culture after 12～16 hours, obtain transforming the host cell of allogenic material, i.e. " pUC19 transformed bacteria ", show referring to Fig. 5, show that plasmid pUC19 successfully is transferred in " competence bacillus coli DH 5 alpha ".
Allogenic material with plasmid pUC19(available from THERMO FISHER, 0.5 μ g/ μ l) be example, host cell is take Pichia pastoris GS115 as example.Following steps are identical with embodiment 2 with embodiment 1, except the special part that indicates, but essentially identical statement cross-reference.
1) purifying, recovery and pre-treatment e. coli host cell
(1) purifying host cell: with YPD solid medium (formula: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, agar powder 20g/L),, 30 ℃ of underscore culture purified Pichia pastoris GS115s two days, obtain the yeast cell mono-clonal;
(2) recovery host cell: picking GS115 mono-clonal is inoculated in 5mL YPD liquid nutrient medium (formula: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L), 30 ℃, 220rpm recovery spend the night (12-16 hour);
(3) pre-treatment host cell: the GS115 bacterium liquid through recovery is inoculated in the fresh YPD liquid nutrient medium of 100mL according to the ratio of volume ratio 1:100, and 30 ℃, 220rpm shake bacterium 24～28 hours to OD
600=0.8～1.0, to draw 500 μ L bacterium liquid and divide and be filled in 1.5mL EP pipe ,-80 ℃ of refrigerator precoolings are more than 12～24 hours.
2) vacuum lyophilization Pichia pastoris GS115 host cell
with vacuum freeze drier (available from Beijing development in science and technology company limited of Song Yuan Huaxing) be chilled in advance temperature reach-54 ℃ and stable after, e. coli host cell is taken out and is placed in rapidly vacuum freeze drier from-80 ℃ of cryogenic refrigerators, close intake valve and open vacuum pump after installing lid,-54 ℃ of cold condition are set, vacuum lyophilization host cell 12～24 hours or more than, close vacuum pump after to be dried and take out the Pichia pastoris GS115 of processing through Freeze Drying Technique, the Pichia pastoris GS115 of the method preparation is " competence yeast ", possessed the ability that allogenic material transforms of accepting, can use immediately or-20 ℃ save backup, perhaps also can be under 0-4 ℃ of condition storage and transport.
3) preparation intends transforming the aqueous solution of allogenic material
With pre-inversion plasmid pUC19(available from THERMO FISHER, 0.5 μ g/ μ l) be added to 500 μ l ddH
2Mix in O and make the aqueous solution that concentration is 1ng/ μ l, be incubated 30～60 minutes in 37 ℃ of thermostat water baths.
4) the Pichia pastoris GS115 competent cell (" competence yeast ") of processing through vacuum lyophilization is carried out fast rehydrating, obtains to have transformed the yeast host cell of allogenic material with the conditioned medium screening:
(1) yeast host cell through vacuum lyophilization is carried out fast rehydrating: yeast host cell is added rapidly (with the host cell equal-volume before lyophilize) aqueous solution that contains plasmid pUC19 of processing through 37 ℃ of insulations,, in 37 ℃ of insulations 30 minutes, then be placed in 30 ℃ of thermostat container 220rpm shaking culture 1～4 hour after mixing gently;
(2) obtain to have transformed the yeast host cell of allogenic material with the conditioned medium screening: the yeast host cell bacterium liquid 100 μ l that after the lyophilize of learning from else's experience, rehydration is processed, (formula is: yeast extract paste 10g/L to be applied to respectively the YPD solid medium that conditioned medium namely contains penbritin, peptone 20g/L, glucose 20g/L, agar powder 20g/L, the microbiotic penbritin of final concentration 100 μ g/mL) in, grow mono-clonal 30 ℃ of lower constant temperature culture after 2～3 days, obtain transforming the host cell of allogenic material, i.e. " pUC19 yeast conversion bacterium ".Show referring to Fig. 6, show that plasmid pUC19 successfully changes in " competence yeast ", prepared the yeast competent cell and realized changing over to of allogenic material pUC19.
Claims (14)
1. the preparation method of a competence host cell comprises the following steps:
1) purifying, recovery and pre-treatment host cell;
2) process host cell with Vacuum Freezing ﹠ Drying Technology, obtain the competence host cell.
2. method according to claim 1, it is characterized in that: described host cell is any one protokaryon or eukaryotic host cell, comprises bacterial cell, fungal cell, vegetable cell and zooblast etc., is for example intestinal bacteria and yeast.
3. described method according to claim 1 and 2, it is characterized in that: the described purifying host cell of step 1) refers to former host cell streak culture at the solid culture primary surface, obtains mono-clonal; The recovery host cell refers to the host cell of purifying is cultivated certain hour again, makes it from stationary state, reenter growth conditions; The pre-treatment host cell refers to the host cell enlarged culturing of recovery to respective concentration.
4. method according to claim 3, it is characterized in that: the process of step 1) purifying, recovery and pre-treatment host cell is as follows:
(1) purifying host cell: get original host cell sectional streak with transfering loop and be inoculated in the solid medium that is applicable to cultivate host cell, cultivate under optimum conditions, obtain the host cell mono-clonal; For colibacillary be the LB solid medium, its formula is for Tryptones 10g/L, yeast extract paste 5g/L, NaCl10g/L, agar powder 15g/L; For yeast be the YPD solid medium, its formula is for yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, agar powder 20g/L; Being suitable for colibacillary culture condition is 37 ℃, 12～16h; The culture condition that is suitable for yeast is 30 ℃, 48h;
(2) recovery host cell: picking host cell mono-clonal is inoculated in the growth medium that 5mL is applicable to host cell, (37 ℃ of intestinal bacteria under optimal temperature; 30 ℃ of yeast culture) 220rpm recovery spend the night (12～16 hours); Be suitable for the colibacillary LB substratum that is, its formula is Tryptones 10g/L, yeast extract paste 5g/L, NaCl10g/L; What be suitable for yeast is the YPD substratum, and its formula is yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L;
(3) pre-treatment host cell: the ratio of the host cell through recovering according to volume ratio 1:100 is inoculated in the described growth medium of the fresh step of 100mL (2) (37 ℃ of intestinal bacteria under optimal temperature; 30 ℃ of yeast culture) 220rpm shakes bacterium to OD
600=0.6～1.0; Draw 500 μ l bacterium liquid and divide and be filled in 1.5mL EP pipe ,-80 ℃ of refrigerator precoolings are more than 12～24 hours.
According to claim 1 to 4 arbitrary described method, it is characterized in that: step 2) described Vacuum Freezing ﹠ Drying Technology processing, be after step 1) is processed host cell with vacuum freeze drier precooling 30～120 minutes until temperature reach-54 ℃ and be stabilized in this temperature after, then vacuum lyophilization host cell 12～24 hours or more than.
6. the competence host cell for preparing of the arbitrary described method of claim 1 to 5, and described competence host cell still possesses biological activity at 0-4 ℃ of temperature.
7. method that allogenic material is changed over to host cell, the competence host cell that utilizes the arbitrary described method of claim 1 to 5 to prepare, further comprising the steps:
3) prepare the aqueous solution of allogenic material to be transformed;
4) with the described aqueous solution to step 2) the competence host cell that obtains carries out fast rehydrating, obtains transforming the host cell bacterium liquid of allogenic material;
Change allogenic material over to host cell with this.
8. method according to claim 7, is characterized in that, allogenic material used comprises the biologically active substances such as nucleic acid (thymus nucleic acid (DNA), Yeast Nucleic Acid (RNA)), protein, oligopeptides, amino acid.
9. according to claim 7 or 8 described methods, is characterized in that, the allogenic material aqueous solution described in described step 3) uses ddH
2O, the allogenic material aqueous solution for preparing is incubated 30～60 minutes in 37 ℃ of water-baths.
10. according to claim 7 or 8 or 9 described methods, it is characterized in that, being operating as of step 4) fast rehydrating: with step 2) competence host cell adds rapidly the aqueous solution of the allogenic material of step 3) preparation, mixes gently and is placed on (37 ℃ of intestinal bacteria under optimal temperature; 30 ℃ of yeast culture) 220rpm shaking culture 45～60 minutes in thermostat container; Host cell equal-volume before the consumption of the allogenic material aqueous solution and lyophilize.
11. a method that generates the allogenic material transformed bacteria, is characterized in that, with the host cell that transforms allogenic material that the arbitrary described method of conditioned medium screening claim 7 to 10 obtains, the mono-clonal that obtains is the allogenic material transformed bacteria.
12. described method, is characterized in that according to claim 11, it is the LB solid medium that described conditioned medium is fit to colibacillary, formula is: Tryptones 10g/L, yeast extract paste 5g/L, NaCl10g/L, agar powder 15g/L, the microbiotic that is suitable for allogenic material of final concentration 100-200 μ g/mL; What be fit to yeast is the YPD solid medium, and filling a prescription is: yeast extract paste 10g/L, and peptone 20g/L, glucose 20g/L, agar powder 20g/L, final concentration 100-200 μ g/mL is suitable for the microbiotic of allogenic material.
13. according to claim 11 or 12 described methods, is characterized in that, described screening process is: will transform the host cell bacterium liquid inoculation of allogenic material or be applied to conditioned medium, (37 ℃ of intestinal bacteria under optimal temperature; 30 ℃ of yeast culture) constant temperature culture grew mono-clonal after 12～16 hours, namely obtained the allogenic material transformed bacteria.
14. the allogenic material transformed bacteria that the arbitrary described method of claim 11 to 13 prepares.
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| JP2001512310A (en) * | 1997-02-12 | 2001-08-21 | ライフ テクノロジーズ，インコーポレイテッド | Method for freeze-drying competent cells |
| US20020081565A1 (en) * | 2000-10-30 | 2002-06-27 | Sigma-Aldrich Co. | Process for producing freeze dried competent cells and use thereof in cloning |
| CN101264062A (en) * | 2007-03-12 | 2008-09-17 | 袁红杰 | Production technology for preparing freeze-drying genetic engineering bacterium competence cell and protective agent formula |
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