CN101050450A - Derivational expression method of glucose polycytidylate enzyme in expression system of lactobacillus casei - Google Patents
Derivational expression method of glucose polycytidylate enzyme in expression system of lactobacillus casei Download PDFInfo
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Abstract
This invention provides a method for induced expression of beta-glucosidase in Lactobacillus casei 393. The method comprises: expressing beta-glucosidase in Lactobacillus casei 393 with surface-expression vector pPG611.1 and secretive expression vector pPG612.1. The target protein is generated in host cells. The expressed proteins are combined with cell walls, and do not exist in the supernatant. This invention also optimizes the induction conditions. The expression system can be used to study antigen presentation.
Description
One, technical field
That the present invention relates to is a kind of preparation method of biomaterial, specifically
Two, background technology
In recent years, people are devoted to study novel oral vaccine to avoid traditional shortcoming that vaccinate was had always.A large amount of bacteriums, virus and parasitic pathogenic agent all enter body by mucomembranous surface, therefore are better than traditional vaccinate as a kind of immunization route oral vaccine; Reply though vaccinate can cause systemic immunity, can not effectively induce body to produce secretor type antibody sIgA; Oral vaccine not only use more convenient and cost not high, may produce the herd immunity effect for a lot of pathogenic agent oral immunities, when promptly only having several members to obtain immunity in colony inside, this immunizing power can be propagated between the inner member of colony, and these advantages are particularly useful for the not high country of industrialization degree.Therefore, research and the selection to oral vaccine antigen transmission live vector just becomes very important.
Ideal mucosa-immune live vector vaccine can promote between antigen and the immunity system effective contact, stimulate body fluid and cell immune response, produce secular immanoprotection action later on, and stable, nontoxicity in the single dose immunity.What study at most at present is to cause weak pathogenic bacteria carrier, because of it still may keep aggressive and toxicity they is restricted when being applied to children and immune deficiency person.Cause the safety and the environmental problem of weak microorganism widespread adoption at genetically engineered, having developed is the non-virulent gram-positive microorganism carrier of representative with the milk-acid bacteria.For centuries, lactobacillus is used to every field such as biological processing and food and feed be fresh-keeping, and they are considered to the food-grade microorganisms of a class safety, and other live bacterial vaccines carrier then is not classified as security classes; And some lactobacillus is a normal flora in the enteron aisle, can field planting in enteron aisle, and be the health-promoting factor of humans and animals, this point is that other live vector (attenuation intestinal bacteria, Salmonellas, virus) is incomparable; In addition, studies show that lactobacillus and the product thereof in the enteron aisle has antagonism to intestinal tract infections and some cancer, and very likely play the effect of anti-cholesterol by the cholesterol level in the reduction blood.Other possible effect of lactobacillus comprises: increase probiotics in the enteron aisle, reduce harmful bacterium; Increase the nutritive value of food, reduce lactose intolerance; Produce tumour necrosis factor, Interferon, rabbit, IgA antibody etc.A large amount of advantages makes lactobacillus become the most attractive candidate of live vector that oral vaccine antigen transmits.But select lactobacillus as expression system the most important thing is its safely, do not have intracellular toxin, the foreign protein of expression to need not purifying can directly to take together with thalline.Moreover, since lactobacillus can be in enteron aisle the field planting survival, the genetically engineered lactobacillus is through after oral, its expression be used for the treatment of or the albumen of immunization just can constantly be produced in enteron aisle and lasting playing a role.The lactobacillus expression vector has been successfully used to the expression of different foreign proteins, but nearly all not high as the efficient of host bacterium expressing protein with lactobacillus, therefore improves lactobacillus and expresses the focus that the ability of foreign protein has become research.
Three, summary of the invention
The object of the present invention is to provide the abduction delivering condition of a kind of definite β-Pu Taotanggansuanmei (gusA) in the lactobacterium casei expression system, for the expression of foreign gene in lactobacterium casei that realizes alternative gusA lays the foundation, determined that lactobacterium casei transmits the feasibility of carrier and expresses the method for glucuronidase with lactobacterium casei as oral vaccine.
The object of the present invention is achieved like this: with lactobacterium casei Lactobacillus casei393 is the host bacterium, expresses β-Pu Taotanggansuanmei by cell surface expression carrier pPG611.1 and cell exocrine expression vector pPG612.1.
With the D-wood sugar as inductor, picking pPG611.1/L..casei 393 and pPG612.1/L..casei 393, be inoculated in the MRS minimum medium that contains 2% wood sugar, in wood sugar, cultivate simultaneously not contain plasmid bacterium L.casei 393 and grow in the pPG611.1/L..casei 393 that contains in 2% glucose and cultivate in contrast, 37 ℃ of static overnight incubation are induced, and get an amount of bacterium liquid respectively after stopping cultivating and do electrophoretic analysis; Inductive condition is: pH value 6.0 to 7.0, replenish that D-wood sugar final concentration is 1%, concentration is 0.5mM in the lactose, 30 ℃ leave standstill that to be cultured to cell concentration be A
590OD=0.50.
The present invention determines that target protein all produces in cell, the target protein of cell surface expression is combined on the cell walls, and the target protein of secreting, expressing does not occur in supernatant, in fact still is combined on the thalline.The present invention uses the D-wood sugar to induce the expression of target protein as inductor; Simultaneously according to the growth characteristics of lactobacterium casei, explored the best abduction delivering condition of gusA from many aspects, efficiently expressing to the antigen transmission of this system laid a good foundation.
1. effective inductor
This experiment inductor of D-wood sugar as pPG611.1/L.casei 393 and pPG612.1/L.casei393, simultaneously in contrast with glucose, SDS-PAGE result shows that the D-wood sugar is induced and can express target protein, further analyze with Western-blot, the result shows that the gusA albumen of acquisition has antigenicity.
2. the target protein position of expressing
The result shows that lactobacterium casei pPG611.1/L.casei393 and pPG612.1/L.casei393 can express gusA under 2%D-wood sugar inductive condition.For the carrier pPG611.1/L.casei 393 of cell surface expression, find that through the SDS-PAGE electrophoresis detection target protein of its expression appears in the cellular lysate thing; Yet, with regard to secretion expression carrier pPG612.1/L.casei 393, by supernatant liquor being dialysed and after lyophilize handles, target protein does not appear through SDS-PAGE and Western-blot detection, in bacterial sediment, do not see obvious purpose band through the SDS-PAGE electrophoresis yet, and it is existing to find to have in the bacterial sediment purpose to take out of through the Western-blot detection, proves that thus secretion type expression host bacterium pPG612.1/L.casei 393 expressed proteins also are combined on the cell walls.
3. culture condition is to the influence of gusA expression amount
Because the target protein of the pPG612.1/L.casei 393 of secretion type expression is not secreted in the supernatant liquor through evidence, also in bacterial sediment, occur, the pPG611.1/L.casei 393 of its test method and cell surface expression is similar, therefore, this research is that example is explored from the following aspects with pPG611.1/L.casei 393 host bacterium only: the initial pH value of the kind of inductor, the concentration of inductor, induction time, culture temperature, substratum and the concentration of inhibitor glucose etc.Drawing the optimum expression condition by a large amount of tests is: with the basic MRS substratum of pH value between 6.0 to 7.0, replenish D-wood sugar final concentration and be 1% and lactose in concentration be 0.5mM, 30 ℃ leave standstill that to be cultured to cell concentration be A
590During the OD=0.50 left and right sides, its expression amount reaches peak value.
4. the cracking of lactobacillus
Because the cell walls of lactobacillus contains a large amount of peptidoglycans, so one of the difficult point of lactic acid bacillus genic engineering just is that it is difficult to cracking.Generally speaking, the cracking intestinal bacteria only need certain density SDS to get final product; Related data report, G+ bacterium such as genus bacillus, suis need be with 5～10mg/ml N,O-Diacetylmuramidase pre-treatment 1 hour, and the lactobacillus behind antibiotic-screening, almost do not have cracking with 10mg/ml N,O-Diacetylmuramidase processing a few hours.Yet, the lysate composition that adopts in this test: 10mM Tris-HCl, pH8.0, N,O-Diacetylmuramidase 10mg/ml.When cell concentration at A
590During the OD=0.5 left and right sides; after the precipitation of 1ml bacterium liquid added the abundant mixing of this kind lysate 100 μ l, 37 ℃ of effect 30～60minn got final product abundant cracking, therefore; therefrom extracting plasmid or foreign gene changed over to all become extremely easy more helps the development and the utilization of lactic acid bacillus genic engineering.
5. the development of lactic acid bacillus genic engineering has profound significance
Because the characteristic of the GRAS (generally regarded as safe) of lactobacillus, the proteic lactobacillus of expressive function can be by being settled in the people or the Mammals mucomembranous surface continues to play a role, thereby become a good expression system.This research is by with lactobacterium casei Lactobacillus casei393 being host bacterium expression label protein β-glucuronidasegene (gusA), determined the expression condition of new expression vector pPG611.1/L.casei 393 and pPG612.1/L.casei 393, successfully realize efficient, the stably express of gusA, proved the feasibility that this carrier system is expressed, and explore its best abduction delivering condition, for next step expression alien gene is laid a good foundation, also induce mucosa-immune to provide the foundation for the novel live vector vaccine of further developing security level.
Four, description of drawings
Fig. 1 and Fig. 2 are SDS-PAGE electrophoretic analysis and the Western-blot detections that lactobacterium casei pPG611.1/L.casei393 expresses gusA;
Among Fig. 1: 1. molecular weight standard albumen
2-3. wood sugar inductive lactobacterium casei pPG611.1/L.casei393
4. the not inductive lactobacterium casei pPG611.1/L.casei393 that cultivates of glucose
5. the empty bacterium L.casei393 of wood sugar inductive
Among Fig. 2: 1. wood sugar inductive lactobacterium casei L.casei 393
2. the not inductive lactobacterium casei pPG611.1/L.casei393 that cultivates of glucose
3. the lactobacterium casei pPG611.1/L.casei393 that cultivates of wood sugar
4. lactose-induced lactobacterium casei pPG611.1/L.casei393
Fig. 3 and Fig. 4 are the analyses that different concns wood sugar inductive lactobacterium casei pPG611.1/L.393 expresses gusA;
Fig. 5 is the SDS-PAGE electrophoretic analysis and the expression amount analysis of the different induction times of lactobacterium casei pPG611.1/L.393 with Fig. 6;
Among Fig. 5: 1. molecular weight standard albumen
2-9. cell concentration is A
590The OD value is followed successively by: 0.252; 0.257; 0.280; 0.310; 0.350; 0.410; 0.510; 0.631; 0.740 the time lactobacterium casei pPG611.1/L.casei393
Fig. 7 is the SDS-PAGE electrophoretic analysis of glucose content to the influence of gusA expression amount in lactobacterium casei pPG611.1/L.393;
1.1% wood sugar inductive lactobacterium casei pPG611.1/L.393 is as positive control
2. molecular weight standard albumen
3-8.pPG611.1/L.393 all be incubated among the MRS of glucose that 1% wood sugar contains different concns simultaneously, the glucose final concentration that it contained is followed successively by 0.01%; 0.1%; 0.5%; 1%; 2%; 3%
Fig. 8 is the influence of SDS-PAGE electrophoretic analysis culture temperature to gusA expression amount in empty carrier lactobacterium casei pPG611.1/L.casei 393;
1. the wood sugar inductive is in the lactobacterium casei pPG611.1/L.casei 393 of 28 ℃ of cultivations
2.30 the lactobacterium casei pPG611.1/L.casei 393 of ℃ cultivation
3.32 ℃ inductive lactobacterium casei pPG611.1/L.casei 393
4.37 ℃ wood sugar inductive lactobacterium casei pPG611.1/L.casei 393
5.40 ℃ wood sugar inductive lactobacterium casei pPG611.1/L.casei 393
6. the wood sugar inductive is in the lactobacterium casei pPG611.1/L.casei 393 of 42 ℃ of cultivations
7. molecular weight standard albumen
Fig. 9, the 10th, the different initial pH value of substratum are to the influence of gusA expression amount in lactobacterium casei pPG611.1/L.casei 393;
Among Fig. 9: 1.pH is 7.0 substratum inductive lactobacterium casei pPG611.1/L.casei 393
2.pH be 6.5 substratum inductive lactobacterium casei pPG611.1/L.casei 393
3.pH be 6.2 substratum inductive lactobacterium casei pPG611.1/L.casei 393
4.pH be 6.0 substratum inductive lactobacterium casei pPG611.1/L.casei 393
5.pH be 5.5 substratum inductive lactobacterium casei pPG611.1/L.casei 393
6.pH be 5.0 substratum inductive lactobacterium casei pPG611.1/L.casei 393
7. molecular weight standard albumen
Figure 11 is that concussion is cultivated and the influence of static cultivation to gusA expression amount in pPG611.1/L.393;
1. molecular weight standard albumen
2. the lactobacterium casei pPG611.1/L.casei 393 of static cultivation
3.70rpm the lactobacterium casei pPG611.1/L.casei 393 that concussion is cultivated
4.186rpm the lactobacterium casei pPG611.1/L.casei 393 that concussion is cultivated
Figure 12 is that SDS-PAGE and the Western-blot of lactobacterium casei pPG612.1/L.casei 393 detects.
393 pairs of positive contrasts of 1.1% wood sugar inductive lactobacterium casei pPG611.1/L.casei
2. the supernatant liquor of 1% wood sugar inductive lactobacterium casei pPG612.1/L.casei 393 after concentrating 50 times through dialysing
3. the supernatant liquor of inductive lactobacterium casei pPG612.1/L.casei393 after lyophilize concentrates 50 times
4. the bacterial sediment of the lactobacterium casei pPG612.1/L.casei 393 of glucose cultivation in contrast
5. the empty bacterium L.casei of wood sugar inductive 393 bacterial sediments are as negative control
6.1% wood sugar inductive is in the bacterial sediment of the lactobacterium casei pPG612.1/L.casei393 of 30 ℃ of cultivations
Five, embodiment
For a more detailed description to the present invention for example below:
1 wood sugar induces gusA to express
With the D-wood sugar as inductor, picking pPG611.1/L..casei 393 and pPG612.1/L..casei 393, be inoculated in the MRS minimum medium that contains 2% wood sugar, in wood sugar, cultivate simultaneously not contain plasmid bacterium L.casei 393 and grow in the pPG611.1/L..casei 393 that contains in 2% glucose and cultivate in contrast, 37 ℃ of static overnight incubation are induced, and get an amount of bacterium liquid respectively after stopping cultivating and do electrophoretic analysis.
Determining of 2 best xylose concentrations
An amount of pPG611.1/L..casei 393 is connected in the MRS liquid nutrient medium that contains the different concns wood sugar, 37 ℃ of standing over night are cultivated, the D-xylose concentration is respectively 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, induce end to get bacterium liquid and do electrophoretic analysis, further expression product is carried out preliminary quantitative analysis, draw best induced concentration by densitometric scan.
Determining of 3 best induction times
To spend the night inductive 5ml bacterium liquid with the centrifugal 3min of 4000r/min, discard about 4ml supernatant liquor after, the bacterial sediment that suspends again joins 20ml and contains in the MRS liquid nutrient medium of 2%D-wood sugar and carry out enlarged culturing, gets bacterium liquid with spectrophotometric instrumentation A in appropriate time
590The OD value was measured once and was got bacterium liquid then and handles every one hour, be SDS-PAGE and analyze, and drew pPG611.1/L..casei 393 with this and expressed best induction time or the OD value of gusA.
4 glucose concn are to the influence of expression amount
PPG611.1/L..casei 393 is connected among the MRS of different concns glucose 37 ℃ and leaves standstill cultivation, the glucose final concentration is respectively 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, wood sugar inductive final concentration is 2%, gets respectively after stop cultivating that an amount of bacterium liquid is done electrophoretic analysis and densitometric scan carries out quantitative analysis to expression product.
Determining of 5 best abduction delivering temperature
Because the lactobacterium casei optimum growth temperature is 37 ℃, but be lower than 30 ℃ or still can grow when being higher than 40 ℃ in temperature.Therefore 2%D-wood sugar inductive pPG611.1/L..casei393 is left standstill respectively and be incubated at different temperature, spend the night and induce, the temperature when expression amount reaches maximum is determined in electrophoretic analysis.
Determining of 6 optimum medium initial pH value
PPG611.1/L.casei 393 is connected to respectively in the MRS liquid nutrient medium of 2%D-wood sugar of different initial pH value (pH5.0,5.5,6.0,6.2,6.5 and 7), 37 ℃ of standing over night are induced, and handle bacterium liquid electrophoretic analysis result.
7 different training methods are to the influence of expression amount
The single bacterium colony of pPG611.1/L..casei 393 is inserted respectively in the MRS liquid nutrient medium of 2%D-wood sugar, place 37 ℃ of shaking tables to leave standstill overnight incubation and electrophoretic analysis respectively with 186rpm or 70rpm concussion overnight incubation and 37 ℃.
The SDS-PAGE of 8 expression products and Western-blot analyze
Lactobacterium casei pPG611.1/L.casei393 is a cell surface expression.The bacterium liquid of getting incubated overnight carries out SDS-PAGE specimen preparation and analysis after N,O-Diacetylmuramidase is handled; Lactobacterium casei pPG612.1/L.casei393 is that express in the extracellular, can be in substratum with protein excretion, therefore get the bacterium liquid of incubated overnight centrifugal after, keep supernatant and carry out respectively carrying out the SDS-PAGE specimen preparation after 4 ℃ of dialysis and lyophilize concentrate with 50 times and analyzing; For preventing that lactobacterium casei pPG612.1/L.casei393 expressing protein still is combined on the thalline, so the centrifugal back of inductive bacterium liquid gained bacterial sediment is also carried out the SDS-PAGE electrophoretic analysis.The data of expression amount are analyzed gained by dual wavelength TLC scanner (Shimadzu CS-930) in this research.
9 experimental results
9.1 lactobacterium casei pPG611.1/L.casei393 expresses the condition of gusA gene
9.1.1 SDS-PAGE and Western-blot detected result that wood sugar induces gusA to express
Carry out electrophoretic analysis (Fig. 1,2) after the pPG611.1/L.casei393 bacterium liquid processing with wood sugar and glucose incubated overnight, empty bacterium L.casei393 is incubated at makes negative control in the wood sugar simultaneously.Electrophorogram shows only has D-wood sugar inductive pPG611.1/L.casei393 a tangible protein band to occur at the 75KD place, and this molecular weight is consistent with gusA gene expression product theoretical value.Electrophoresis result (Fig. 1) can prove tentatively that the reorganization bacterium is incubated in the 2%D-wood sugar and can expresses the gusA target protein thus, further comes testing goal albumen (Fig. 2) with Western blot, and the result shows that the gusA albumen of acquisition has antigenicity.
9.1.2 determining of best xylose concentration
Lactobacterium casei is incubated among the MRS that contains the different concns wood sugar, its electrophoresis result such as Fig. 3, can analyze expression amount trend from figure: when wood sugar content is lower than 0.5%, detect substantially less than the gusA that expresses by the gusA of different concns D-wood sugar inductive pPG611.1/L.casei393, by the densitometric scan analysis as can be known when wood sugar content is 1%, the expression amount of gusA is up to 12.8% of bacterial protein, and xylose concentration was greater than 1% o'clock, its expression amount does not increase along with the increase of wood sugar content, has a declining tendency on the contrary.Repeatedly induce its expression amount of electrophoretic analysis and wood sugar relation with contents such as Fig. 4
9.1.3 determining of best induction time
This bacterium poor growth in wood sugar, bacterium liquid just occur obviously muddy after suitably the about 6h of enlarged culturing is carried out in dilution, per hour get a bacterium liquid and measure A
590The OD value and the electrophoresis that keeps sample.Different A
590OD value electrophoresis result such as Fig. 5, by this figure as can be seen: work as A
590OD was less than 0.51 o'clock, and the expression amount of gusA in pPG611.1/L.393 is A with induction time
590The increase of OD and increasing, and when cell concentration continued to rise, its amount of expressing target protein no longer increased.Its maximum expression amount can reach 14% of bacterial protein by analysis, its expression amount trend such as Fig. 6.
9.1.4 glucose concn is to the influence of expression amount
Almost in all carriers, express the inhibition that β-glucuronidase all is subjected to glucose, even there is inductor to occur.For whether the expression of gusA in this experiment of check and analysis also is subjected to the inhibition and the inhibition degree of glucose, carried out the inhibition test of different concns glucose, result such as Fig. 7, the concentration of glucose has the obvious suppression effect to the expression of gusA in the substratum.When the glucose final concentration is 0.1% not influence the expression amount of goal gene when following substantially, and when its content reaches 0.5%, even the content of inductor D-wood sugar is constant in the substratum, the purpose band of its expression also only mays be seen indistinctly, illustrate that expression amount has begun to descend, reach at 2% o'clock to glucose concn, can't see the purpose band of expression substantially.The content that glucose is described is an important factor that influences expression amount.
9.1.5 determining of best abduction delivering temperature
Lactobacterium casei pPG611.1/L.393 is carried out electrophoretic analysis, result such as Fig. 8 respectively at the differing temps cultivation.This figure shows that temperature is to influence the important factor that gusA expresses, the bacterium liquid of 28 ℃ of cultivations also can be expressed gusA, its expression amount of 30 ℃ (expression amount account for bacterial protein 13.4%) and 32 ℃ (expression amount account for bacterial protein 12.5%) is obviously greater than 37 ℃ (expression amounts account for bacterial protein 11.2%), and along with temperature continue increase its expression amount and obviously descend, 40 ℃ almost naked eyes be difficult to see the purpose band, though also obviously descend than 30 ℃ at 42 ℃ expression amount but still can be observed tangible purpose band and cultivate, illustrate that thus temperature is the important factor that influences expression amount, this result goes back the basic temperature with the lactobacterium casei growth of temperature range that the illustration purpose gene expresses in pPG611.1/L.casei393 identical, wide temperature range help pPG611.1/L.casei393 as the live vector definite value of antigen transmission in different animal bodies.
9.1.6 determining of optimum medium initial pH value
Whether the optimal pH of lactobacterium casei growth is 5.5-6.2, but also can grow in pH value is sour environment 5.5 below, identical to the pH requirement of substratum in order to detect the reorganization bacterium of expressing gusA, with its cultivation in the MRS of the initial pH of difference inducing culture.Result's (seeing Fig. 9,10) finds that when it is grown in pH be that speed is considerably slower than in other several pH in 5.0 the substratum time.And inductive bacterium liquid in the different PH is carried out SDS-PAGE, and find that when pH value is lower than 5.5 the expression amount of gusA in pPG611.1/L.casei 393 descends, when being 5.0, the pH value can't see the target protein band of expression substantially.The pH value is between 6.0 and 7.0 the time, and its expression amount is more or less the same.Because lactobacillus can make the nutrient solution acidifying in process of growth, therefore if remain pH in the process 6.0 between 7.0 the time whole inducing, the ability of its expressing protein can be strengthened greatly.
9.1.7 different training methods are to the influence of expression amount
Because of lactobacterium casei is little oxygen bacterium of having a liking for, so static cultivation should more help its growth and express goal gene, detected result show that the concussion of carrying out 37 ℃ of different rotating speeds is cultivated and 37 ℃ leave standstill cultivation, the expression amount difference of gusA is little, from electrophoresis Figure 11, leave standstill as can be seen when expression amount is cultivated a little more than concussion when cultivating, illustrate that shaking culture or static cultivation are little to its expression amount influence.
9.2 lactobacterium casei pPG612.1/L.casei393 expresses the condition of gusA gene
Inductive secreting, expressing lactobacterium casei pPG612.1/L.casei 393 supernatant liquors are respectively after concentrated 50 times of dialysis and lyophilize, SDS-PAGE electrophoresis and Western-blot detect and target protein all do not occur, show that its target protein is not secreted in the substratum.And the bacterial sediment of pPG612.1/L.casei 393 is detected the back at about 72KD place appearance tangible purpose band (seeing Figure 12) through Western-blot, illustrate that thus the gusA target protein that lactobacterium casei pPG612.1/L.casei 393 expresses is not secreted in the supernatant, but be combined on the thalline.
Claims (2)
1, the derivational expression method of a kind of β-Pu Taotanggansuanmei in the lactobacterium casei expression system, it is characterized in that: with lactobacterium casei Lactobacillus casei 393 is the host bacterium, expresses β-Pu Taotanggansuanmei by cell surface expression carrier pPG611.1 and cell exocrine expression vector pPG612.1.
2, the derivational expression method of β-Pu Taotanggansuanmei according to claim 1 in the lactobacterium casei expression system, it is characterized in that: with the D-wood sugar as inductor, picking pPG611.1/L..casei 393 and pPG612.1/L..casei 393, be inoculated in the MRS minimum medium that contains 2% wood sugar, in wood sugar, cultivate simultaneously not contain plasmid bacterium L.casei 393 and grow in the pPG611.1/L..casei 393 that contains in 2% glucose and cultivate in contrast, 37 ℃ of static overnight incubation are induced, and get an amount of bacterium liquid respectively after stopping cultivating and do electrophoretic analysis; Inductive condition is: pH value 6.0 to 7.0, replenish that D-wood sugar final concentration is 1%, concentration is 0.5mM in the lactose, 30 ℃ leave standstill that to be cultured to cell concentration be A
590OD=0.50.
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| CN103698521A (en) * | 2013-12-17 | 2014-04-02 | 江南大学 | Double-antibody sandwich method for detecting beta-glucuronidase in escherichia coli cells of food |
| CN103698521B (en) * | 2013-12-17 | 2015-09-23 | 江南大学 | A kind of double antibody sandwich method detecting β-glucuronidase in Escherichia coli cell in food |
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