CN1552857A - Brewer's yeast recombined hepatitis B virus surface antigen stream plus addition femrentation - Google Patents
Brewer's yeast recombined hepatitis B virus surface antigen stream plus addition femrentation Download PDFInfo
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Abstract
A hepatitis B surface antigen is produced with beer yeast engineering bacteria. The process is carried out by: 1) fermenting seeds of the beer yeast engineering bacteria, 2) fermenting by the said seeds, and 3) separating and purifying fermented mixture to obtain hepatitis B surface antigen, specially, flow adding adenine, adenosine, adenosine monophosphate, adenosine diphosphate or adenosine triphosphate at the rate of 14mg/L.h by weight into its culture medium in the said step 2. It achieves in higher expression level of recombined HBsAg.
Description
Technical field
The present invention relates to the microbial fermentation field.Particularly, the present invention relates to utilize the zymotechnique of production of superficial antigen of hepatitis B from yeasts.
Background technology
Crowd's sum that the whole world is influenced by hepatitis B reaches 2,000,000,000 people, and it is hepatitis B virus surface antigen (HBsAg) carrier that 3.5 hundred million people are arranged.Infect hepatitis B and cause lifelong carrier state; The serious sequela of development is liver cirrhosis and primary hepatocyte hepatocarcinoma.Whole world hepatitis B causes death toll every year is up to the million people.
China belongs to the hepatitis B hotspot, is world hepatitis big country, has that the people infected hepatitis B more than 600,000,000, and the HBsAg carrying rate reaches about 10%.Wherein have 1,200 ten thousand chronic hepatitis patient to lose or partly lose work capacity, struggle in slight illness all day, and suffer from not having good medicine, seeking medical advice produces little effect, and therefore many families go into poverty.There are every year nearly 1,000,000 people to suffer from acute hepatitis B, have 250,000 people to die from relevant liver cancer of hepatitis B and liver cirrhosis approximately.
Hepatitis B virus is found by DaneShi that in 1970 know that by electron microscopic examination a large amount of hepatitis B viruses (HBV) particle is arranged among the patients serum who has infected hepatitis B virus, diameter 42-47nm is called the DaneShi particle, and its serum-concentration can be up to 10
11/ ml, so patient blood has extremely strong infectivity.Also can detect the sphere that a large amount of diameters are 22nm and subvirus coat protein (HBsAg) particle of fibrous type in addition in the patient blood; They are not with viral DNA, no infectivity, and serum-concentration can be up to 10
13/ ml.
The control hepatitis B mainly relies on prevention; Hepatitis B vaccine is the effective weapon of prevention hepatitis B.By separation and purification HBsAg subviral particle from the hepatitis B patients'blood, succeeded in developing first-generation blood source polypeptide hepatitis B vaccine.Used the simplest eukaryotic cell yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) afterwards as recipient cell, people have successfully made up DNA recombinant HBsAg engineering strain again, and the HBsAg that it is expressed assembles on the endoplasmic reticulum of yeast cell and becomes subviral particle; Thereby become the fabulous raw material of development s-generation genetically modified polypeptide hepatitis B vaccine.
U.S. FDAs in 1986 have been ratified the yeast saccharomyces cerevisiae recombinant hepatitis B vaccine of Merck company first.Another yeast saccharomyces cerevisiae recombinant hepatitis B vaccine of Smith Kline Beecham company in 1989 also obtains the approval of FDA.The several years whole world is succeeded in developing several yeast and Chinese hamster ovary celI recombinant hepatitis B vaccine in succession subsequently.Up to the present, existing in the world billions of crowd has inoculated the yeast saccharomyces cerevisiae recombinant hepatitis B vaccine, and its validity and security have obtained abundant affirmation.After children's vaccination, the immune protective rate of hepatitis B and hepatitis D is reached 98%; And it also is first effective anti-cancer vaccine in the world, can effectively prevent primary hepatocyte hepatocarcinoma.Initial immunity is injected three pins and can be protected more than 5 years, and later per 5 years booster shots one pins can be protected all the life.
At present, adopt yeast saccharomyces cerevisiae (Saccharomyces cerevisiae yeast) engineering bacteria to produce the HBsAg of product reorganization next life in the world mostly.The content of this respect for example, has just been described in following document:
United States Patent (USP) 5,013,650 disclose a kind of fed-batch fermentation technology of utilizing yeast saccharomyces cerevisiae to produce recombinant HBsAg, in order to increase the output of foreign protein.The recipient bacterium of used beer yeast engineering bacteria strain is that leucine is auxotrophic; And comprise reorganization 2 μ plasmids.These 2 μ plasmids of recombinating comprise the gene of the hepatitis B surface antigen of encoding, 5 ' end and galactase (GAL4) gene promoter and the glyceraldehyde 3-phosphate dehydro-genase promotor (GAPDH) of this gene can be operatively connected, and 3 ' end and ethanol dehydrogenase I terminator can be operatively connected; Described reorganization 2 μ plasmids also comprise the leucic gene of coding, and the 5 ' end and the 3 ' end of this gene can be operatively connected with promotor and terminator respectively.The fed-batch fermentation processing requirement that this patent is described: stream adds glucose in the substratum that does not contain semi-lactosi, makes cell concn grow to maximum value; By methods such as centrifugal, filtration or dialysis, replace original substratum as the substratum of carbon source again, induce the expression of HBsAg with this with semi-lactosi.And this patent describes is 16 liters of pilot processes, make glucose concn be lower than 0.01% (W/V) and be difficult to realize that the essentially no production application of the technology of this patent is worth at hundreds of replacement mediums fully that rise in the production technique.
United States Patent (USP) 6,232,111 also disclose a kind of batch fermentation technology of utilizing yeast saccharomyces cerevisiae to produce recombinant HBsAg, in order to increase the productive rate of expression of recombinant yeast HBsAg.Used beer yeast engineering bacteria is that leucine is auxotrophic, and comprises reorganization 2 μ plasmids.These 2 μ plasmids of recombinating comprise the gene with the coding hepatitis B surface antigen, and the 5 ' end and the 3 ' end of this gene can be operatively connected with glyceraldehyde 3-phosphate dehydro-genase promotor and ethanol dehydrogenase I terminator respectively; Described reorganization 2 μ plasmids also comprise the leucic gene of coding, and the 5 ' end and the 3 ' end of this gene can be operatively connected with promotor and terminator respectively.This technology comprises the steps:
A) provide a certain amount of yeast extract as test;
B) measure the concentration of VITAMIN B4 in this yeast extract;
C) if the VITAMIN B4 of measuring restrains dry weights less than 0.06 gram/42, the concentration of then adjusting VITAMIN B4 is that 0.06 gram/42 grams are to 0.10 gram/42 grams.
This patent is by adjusting the differences between batches of VITAMIN B4 component in the basic medium yeast extract under the condition that is in carbon source concentration restriction, the difference when adjusting cell growth in the batch fermentation between the biomass of phase.
A subject matter that exists in present batch fermentation technology is, is disposable feeding intake because the substratum of batch fermentation contains unartificial yeast leaching powder, crude soya bean peptone and three kinds of components of glucose, can't regulate in the production process.Wherein special quality with yeast extract powder has the greatest impact to recombinant HBsAg productivity; Even give a long price for imported raw material,, cause different fermentations batch recombinant HBsAg productivity instability because there are big differences between batches in yeast extract powder.Though cell concn is very high sometimes, and the productivity of recombinant HBsAg is very low.The 2nd, in the batch fermentation, be about 15-20 hour during the growth of cell mutually; And when 20-25 hour subsequently antigen presentation phase because glucose exhausted, cell stops growing, and causes final cell concentration low excessively, also is unfavorable for improving the recombinant HBsAg expression level.
In sum, be necessary existing processes is improved, thereby improve the productive rate that yeast saccharomyces cerevisiae is produced recombinant HBsAg.
Summary of the invention
Contain in the method for beer yeast engineering bacteria production hepatitis B surface antigen of exogenous plasmid of non-inducible promoter in utilization, the inventor finds that the productivity of recombinant HBsAg is subjected to multifactorial influence, and the growth velocity of cereuisiae fermentum is a main factor in the fermenting process of wherein growing.The inventor finds, when the recombinant Saccharomyces cerevisiae that utilizes non-inducible promoter carries out the production of HBsAg in batches, though expressing, the growth of cell and HBsAg carry out simultaneously, but still can be when producing fermentation stage and be divided into two phase: one is phase during with the main growth of being grown to of cell, this time continue about 10-20 hour from producing fermentation beginning back mutually; Another phase for based on the expression of HBsAg expression the time, this time when above-mentioned growth afterwards lasting mutually 20-50 hour or longer time.
The inventor is through intensive research, find no matter be when growth mutually or when expressing phase, the growth limitation factor of yeast cell mainly is a VITAMIN B4, thereby phase fed batch fermentation production technique when having designed new expression finally, the recombinant HBsAg productivity of this technology unit of making fermentation volume was improving several times significantly on original level.Add benefit by low-speed flow and go into VITAMIN B4, increased biomass on the one hand, also guaranteed the retention of recombinant plasmid in the engineering bacteria on the other hand, thereby improved the output of recombinant HBsAg.In flow feeding technology of the present invention, adapt with the stream rate of acceleration of VITAMIN B4, low speed is mended into materials such as carbon source, nitrogenous sources simultaneously, and the suitableeest growth velocity that can also better control yeast cell is between 0.01-0.03/hr.
Particularly, the invention provides a kind of method of utilizing beer yeast engineering bacteria production hepatitis B surface antigen (HBsAg), wherein said cereuisiae fermentum engineering recipient bacterium is ade (VITAMIN B4) auxotroph cell, its thalline pinkiness; Described cereuisiae fermentum engineering F-strain still is that leucine is auxotrophic, and described beer yeast engineering bacteria comprises reorganization 2 μ plasmids but do not contain 2 natural μ plasmids; Described reorganization 2 μ plasmids comprise the gene with the coding hepatitis B surface antigen, and the 5 ' end and the 3 ' end of this gene can be operatively connected with glyceraldehyde 3-phosphate dehydro-genase promotor and ethanol dehydrogenase terminator respectively with respectively; Described reorganization 2 μ plasmids also comprise the leucic gene of coding, and the 5 ' end and the 3 ' end of this gene can be operatively connected with promotor and terminator respectively.This method comprises the steps:
1) the seed culture step of described beer yeast engineering bacteria;
2) seed that utilizes step 1) to make is produced the step of fermentation; With
3) from step 2) the fermenting mixture of gained separation and purification go out the step of hepatitis B surface antigen;
The method is characterized in that, above-mentioned in step 2) in the weight of VITAMIN B4, add VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) or adenosine triphosphate with the speed stream in substratum that is lower than the 14mg/ liters per hour, doubling time of yeast cell is controlled to be 23.0-35.0 hour.Sometimes VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) or adenosine triphosphate are referred to as the material that contains VITAMIN B4 hereinafter.
In the method for the invention, preferably in the weight of VITAMIN B4, add VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) or adenosine triphosphate with the data rate stream that is lower than 10 mg/litre hour.More preferably in the weight of VITAMIN B4, add VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) or adenosine triphosphate with the data rate stream that is lower than 6 mg/litre hour.Most preferably in the weight of VITAMIN B4, add VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) or adenosine triphosphate with the data rate stream that is lower than 2 mg/litre hour.
In the method for the invention, wherein VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) or adenosine triphosphate can add in the substratum with the successive fed-batch mode.Also can be added in the substratum with discrete mode stream.
In order to prolong time of producing fermentation and the expression amount that further improves hepatitis b surface antigen protein, in above-mentioned steps 2) in except stream adds the material that contains VITAMIN B4, can also add additional yeast extract powder with the speed stream in fermenting mixture that is lower than 1.0 grams per liters hour, add with the speed stream in fermenting mixture that is lower than 0.8 grams per liter hour and replenish soy peptone or add supplementary carbon source, doubling time of yeast cell is controlled to be 23.0-35.0 hour with the speed stream in fermenting mixture that is lower than 3.0 grams per liters hour.
In the present invention, described non-inducible promoter includes, but not limited to the glyceraldehyde 3-phosphate dehydro-genase promotor.The described terminator that can be operatively connected with hbsag gene includes, but not limited to ethanol dehydrogenase I terminator.
In the method for the invention, can be in step 2) beginning back several hrs just adds material, yeast extract powder, soy peptone and/or the carbon source that contains VITAMIN B4.General preferred production certainly when fermentation begins back about 12 hours added, more preferably add described yeast extract powder, soy peptone or carbon source in back about 15 hours, because this moment, nitrogenous source, carbon source and the some other nutritive ingredient that influences the yeast saccharomyces cerevisiae growth in basic medium was consumed almost in production fermentation beginning.
In the method for the invention, can the material that contain VITAMIN B4 be added in the yeast fermentation mixture various fed-batch modes.Can in batches the substance flow that contains VITAMIN B4 be added in the fermenting mixture.For example, add first kind of material such as VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) and/or adenosine triphosphate that contains VITAMIN B4 in very first time section with first rate, and then contain any or several mixtures in the material of VITAMIN B4 such as VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP), the adenosine triphosphate to add with identical or different second kind of first kind of material that contains VITAMIN B4 with the identical or different speed of first rate in second time period.Certainly, the first rate and second speed should be lower than 14 mg/litre hour.Also can with constant speed the material that contains VITAMIN B4 be added in the fermention medium in a continuous manner, can also with the speed that changes the material that contains VITAMIN B4 be added in the fermention medium in a continuous manner.Consider from the angle of control convenient for production, preferably the mode that adds with the constant rate of speed Continuous Flow.
In the method for the invention, can various fed-batch modes be added in the yeast fermentation mixture by yeast extract powder, soy peptone or carbon source.Can in batches yeast extract powder, soy peptone or carbon source be added in the fermenting mixture.For example, add yeast extract powder with first rate in very first time section, and then second time period to add yeast extract powder with the identical or different speed of first rate.Certainly, the first rate and second speed should be lower than 1.0 grams per liters hour.Equally, soy peptone or carbon source also can be added in the fermenting mixture by this way, just first and second of soy peptone should be added rate-controlling and be no more than the level of 0.8 gram soy peptone/rise fermenting mixture hour, first and second the rate-controlling of adding of carbon source is being lower than the level of 3.0 gram carbon sources/rise fermenting mixture hour.The mode that also can Continuous Flow adds is added yeast extract powder, soy peptone or carbon source in the fermenting mixture.And the mode that continuous flow adds is preferred, because can make the yeast of producing fermentation that an environment that had not only helped growing but also helped exogenous gene expression is arranged like this.The continuous flow rate of acceleration of yeast extract powder most preferably Be Controlled is lower than in the scope of 0.5 grams per liter hour.The continuous flow rate of acceleration Be Controlled of soy peptone is lower than in the scope of 0.38 grams per liter hour.The continuous flow rate of acceleration of carbon source hour is incremented to 3.0 grams per liters hour in time since 0.1 grams per liter, preferably hour is incremented to 2.2 grams per liters hour in time since 1.1 grams per liters.
In the method for the invention described carbon source not being had too much restriction, so long as can be got final product as the required carbon source of growth by used saccharomyces cerevisiae engineered yeast, for example can be glucose, sucrose, lactose, fructose, maltose, glycerine, or the like.But it is preferred being easy to by the monose that yeast utilizes.
In the used saccharomyces cerevisiae engineered yeast of the present invention in the contained reorganization 2 μ plasmids, preferably the promotor that can be operatively connected with the leucine gene has disappearance at 3 ' end, makes that the leucine expression level of beer yeast engineering bacteria of the leucine auxotrophy type that contains single copy recombinant plasmid is about 5% of a wild-type cereuisiae fermentum.So, also need not in substratum, to add in addition the leucine of external source, only utilize the selective pressure of substratum, the required engineering bacteria that contains the 2 μ plasmids of recombinating is selected in the seed culture stage.
Described in the method for the invention beer yeast engineering bacteria can be haploid strains, also can be the diploid bacterial strain.Described in the method for the invention beer yeast engineering bacteria can be haploid strains, also can be the diploid bacterial strain.Be preferably haploid strains.F-strain as the beer yeast engineering bacteria that is used for the inventive method includes, but not limited to cereuisiae fermentum 2150-2-3 (MATa, adel, leu2-04, cir
0) (referring to, for example, by a genetically engineered drug-basis and the clinical book write by Wu Wutong etc. that the People's Health Publisher published in March, 1996,150-151 page or leaf), PF161 (MATa, adel, leu2-04, cir
0) (referring to, for example, Valenzuela et.al., 1985, Biotechnology 3:317-320) and PF403 (MATa/ α, adel, leu2-04, cir
0) (referring to, for example, Cheryl A.Schulman et.al., Journal of Biotechnology, 21 (1991): 109-126).
The cereuisiae fermentum 2150-2-3 (MATa, adel, leu2-04, the cir that have plasmid pHBS56-GAP347/33
0).In the contained plasmid pHBS56-GAP347/33 of this bacterial strain, include LEU (leucine) gene, 3 of the promotor of this LEU gene ' end has lacked the 29bp base sequence, causes its leucine expression level to descend, and the leucine expression level of single copy plasmid has only 5% of wild strain.Therefore at the growth fermentation stage, used substratum is not contain leucicly, under the selective pressure of LEU, only comprises plasmid copy number and could survive in no leucic substratum greater than 20 cell.
The present invention preferably leaches powder and soy peptone adding the unartificial yeast that phase Continuous Flow when expressing when containing the VITAMIN B4 material adds lower concentration, thereby limit wherein contained leucine concentration, make the relative leucine auxotrophy type of the leucine prototroph cell cell that has 20 above plasmid copies have growth vigor; Thereby realized that when fermentative production finishes leucine prototroph cell retention reaches or near 100%.
Not with 2 natural μ plasmids, contain 2 μ plasmids of recombinant HBsAg in the transformant in the yeast saccharomyces cerevisiae recombinant HBsAg engineering strain recipient bacterium of the present invention.But the toxicity of its pair cell is aggravated with copy number; Using artificial synthetic yeast nitrogen base (YNB) substratum that elects though can make leucine prototroph cell retention reach 100%, makes cell copy toxic tolerance to height and reduces, and finally causes the average copy number of cell can only maintain about 20.The unartificial yeast that the present invention adopts Continuous Flow to add lower concentration is leached powder, and the various cytokines that wherein contain help improving cell height is copied toxic tolerance; Can make the average copy number of 2 μ plasmids of recombinant HBsAg in the cell increase to (the natural 2 μ plasmid copy numbers that brewing yeast cell has can reach more than 300) more than 100; Thereby advantageously improved the weight ratio productivity (HBsAg[μ g]/wet cell weight [g]) of surface antigen.
The promotor of employed in embodiments of the present invention yeast saccharomyces cerevisiae recombinant HBsAg engineering strain expression cassette is glyceraldehyde 3-phosphate dehydro-genase (GAPDH), and it is high-intensity continuous expression type promotor; The content of its RNA reaches 5% of cell total rna.Because GAPDH is the characteristic of high-intensity continuous expression promotor, adopt to prolong and produce phase cycle when expressing in the fermentation, increase the expression level (HBsAg[μ g]/rise fermenting mixture) of recombinant HBsAg; Produce fermentation period by original 40 hours, progressively extend to 52 hours, 64 hours, until 72 hours, the volume ratio productivity of hepatitis B surface antigen(HBsAg) prolonged in time, was linear direct ratio and rose.
For detecting the growing state of yeast saccharomyces cerevisiae, can utilize technology known in the art to detect CO in the fermentation tail gas in the feed supplement whole process
2And O
2Volumetric molar concentration, keep respiratory quotient (RQ)≤1; At this moment carbon source does not accumulate ethanol through the oxidative pathway metabolism; It is excessive that otherwise stream adds carbon source concentration, respiratory quotient (RQ)〉1, then carbon source accumulates ethanol through the metabolism of glycolysis approach, unfavorable cell growth and expression.The gas analyzer that can adopt Perkin-Elmer to produce, for example MAG 1200 type analysis instrument are to CO in the waste gas of reactor eliminating
2And O
2Level is analyzed, and is used to calculate the RQ value.
The beer yeast engineering bacteria of Shi Yonging is cereuisiae fermentum 2150-2-3 (MATa, adel, the leu2-04 cir that has plasmid pHBS56-GAP347/33 in an embodiment of the present invention
0), its building process sees also U.S. Pat SN 4,880,734 and USSN 5,013,650.In the contained plasmid pHBS56-GAP347/33 of this bacterial strain, include LEU (leucine) gene, 3 of the promotor of this LEU gene ' end has lacked the 29bp base sequence, causes its leucine expression level to descend, and the leucine expression level of single copy plasmid has only 5% of wild strain.Therefore at the growth fermentation stage, used substratum is not contain leucicly, under the selective pressure of LEU, only comprises plasmid copy number and could survive in no leucic substratum greater than 20 cell.Foreign gene plasmid pHBS56-GAP347/33 inserts exogenous gene expression framework (by the GAPDH promotor, HBsAgadw2 gene, three parts such as the compound terminator composition of viral terminator sequence and ADH1 terminator sequence) in 2 μ plasmids; Insert yeast saccharomyces cerevisiae LEU gene expression construct in addition, as the selective marker of transformant.
The fermentation technology of yeast saccharomyces cerevisiae recombinant HBsAg engineering strain is: yeast is stored bacterial classification inoculation cultivate to substratum, by four growth phases, progressively enlarge the cell cultures amount.Last harvested cell, filtering and concentrating through broken damping fluid washing, is concentrated into 35% (by weight/volume), and packing is freezing, and to put-60 ℃ of preservations standby.
Description of drawings:
Fig. 1 is 800 liters of production tank fermentation technology schemas in the existing production technique.
Fig. 2 is according to 800 liters of production tank fermentation technology schemas in the preferred production technique of the present invention.
Fig. 3 shows that the material that contains VITAMIN B4 is the figure of an important factor of restriction saccharomyces cerevisiae engineered yeast growth.Wherein, X-coordinate is represented time of taking a sample, and numeral 1 is illustrated in from fermentation beginning sampling in the time of back 31 hours, and numeral 2 is illustrated in from fermentation beginning sampling in the time of back 38 hours, numeral 3 is illustrated in from fermentation beginning sampling in the time of back 48 hours, and numeral 4 is illustrated in from fermentation beginning sampling in the time of back 53 hours; Ordinate zou represent the fermentation culture sample that records in 660nm optical density value (OD
660).Rhombus among Fig. 3 is represented control group; The experimental group that glucose is added in the square expression; Trilateral represents to add the experimental group of yeast extract powder; Multiplication sign represents to add the experimental group of lower concentration VITAMIN B4; The rice word table shows the experimental group of adding the high density VITAMIN B4.
The description of embodiment
The applicant is below in conjunction with accompanying drawing, and the solution of the present invention is further described in the mode of embodiment.But scope of the present invention is not subjected to the restriction of content of the embodiment of these illustrative.The saccharomyces cerevisiae engineered yeast strain of adopting in the embodiment of the invention is 2150-2-3 (MATa, adel, leu2-04, cir
0, contain plasmid pHBS56-GAP347/33), obtain by transfer of technology from U.S. Merck company.The building process of this project bacterial strain can be referring to United States Patent (USP) 4,880, and 734 and United States Patent (USP) 5,013,650.
In the following example, if no special instructions, all percentage ratio is weightmeasurement ratio percentage ratio.
According to the various substratum of formulated shown in the following table 1, wherein minimum medium consists of yeast extract powder 1.65g/53ml, soy peptone 1.925g/53ml, 25% glucose 3.75ml.The method of its preparation is: take by weighing DIFCO company and produce yeast extract powder 1.65g, Sheffield company produces soy peptone 1.925g, adds water dissolution 49.25ml constant volume (yeast powder of increase, VITAMIN B4 add in this step).121 ℃, sterilization in 20 minutes.After the cooling, add 20 minutes 25% glucose 3.75ml of 121 ℃ of sterilizations (glucose of increase adds in this step).
With identical inoculum size, with recombinant HBsAg engineering strain 2150-2-3 (MATa, adel, leu2-04, cir
0) in the substratum of various prescriptions shown in the following table 1, shaking in the bottle and cultivating, the OD value of 660nm is surveyed in samplings in 31,38,48,53 hours, and data are summarised in the following table 1:
Table 1
| Sample time | Minimum medium (53 milliliters) | Minimum medium (53 milliliters)+25% glucose (2.2ml) | Minimum medium (53 milliliters)+yeast extract powder 0.66g | Minimum medium (53 milliliters)+VITAMIN B4 8mg | Minimum medium (53 milliliters)+VITAMIN B4 18mg |
| ?31hr(1) | ????11.55 | ????11.27 | ????12.95 | ????14.525 | ????17.125 |
| ?38hr(2) | ????12.54 | ????11.7 | ????14.4 | ????16.38 | ????18.9 |
| ?48hr(3) | ????12.24 | ????12.692 | ????15.64 | ????19.24 | ????20.8 |
| ?53hr(4) | ????13.25 | ????13.26 | ????16.55 | ????19.58 | ????22.45 |
The OD value of the biomass in the last table 1 is changed line in time, and the result as shown in Figure 3.This Fig. 3 shows the content positive correlation of thalli growth amount and VITAMIN B4, and is whether irrelevant with the increase of glucose, but the dependency certain with being added with of yeast extract powder, this may be the cause that also contains VITAMIN B4 in the yeast extract powder.
Basically according to document Cheryl A.Schulman et.al., Journal ofBiotechnology, 21 (1991): 109-126) the described method of " Cell growth " part is carried out the seed culture step.Particularly, the bacterial classification of storing is thawed, be inoculated in 250 ml shake flasks that contain 50 milliliters of 5XLEU selective mediums, cultivated 18 hours in 28 ℃ of rotating speeds with 350rpm, on average be transferred to two then and respectively contain 2 liters of 500 milliliters of 5XLEU-selective mediums shake in the bottle, cultivated 18 hours in 28 ℃ of rotating speeds again with 350rpm.The composition of described 5XLEU-selective medium is with document Cheryl A.Schulman et.al., JournalofBiotechnology, 21 (1991): 109-126) described.
30 liters of fermentor tank continuous feedings are cultivated and adopted the YEPD substratum to carry out as follows: weighing import unartificial yeast is leached powder (DIFCO company product, the commodity article No. is 0127-01) 371.2 grams (content in fermented liquid is 32 grams per liters), the import crude soya bean peptone (product of Sheffield company, the commodity article No. is 59022) 417.6 grams (content in fermented liquid is 36 grams per liters), add water to 9.9 liters, 121 ℃, sterilization in 30 minutes, after the cooling, add 700 milliliters of 25% the glucose solutions of sterilizing, with the 9N phosphoric acid solution accent pH to 6.0 that sterilize.Access is according to 1 liter in the sophisticated seed of cultivation mentioned above.This whole production fermentation culture stage all maintains 28 ± 1 ℃, with the 300rpm rotating speed, cultivates with 6 liters/minute air flow.Be cultured to 14 hours, the beginning feed supplement.The BT00-100M peristaltic pump that the feed supplement device adopts Chinese Hebei province Baoding Lange company to produce is done transmission power, 1.6mm silicone tube do pipeline and mend into the mixing solutions that contains glucose, yeast extract powder, soy peptone and VITAMIN B4, it is 0.26 grams per liter hour that the benefit of yeast extract powder is gone into speed, it is 0.26 grams per liter hour that the benefit of soy peptone is gone into speed, it is 1.6 mg/litre hour that the benefit of VITAMIN B4 is gone into speed, and it is 1.2 grams per liters hour that the benefit of glucose is gone into speed.Be cultured to 40 hours and finish fermentation, 0.37 kilogram of results wet cell.Of Cheryl A.Schulman (1991, ibid, and literary composition is described), the method for utilizing defective type substratum and perfect medium cultivation counting to compare is measured, and recording leucine prototroph cell retention is 100%; Of Cheryl A.Schulman (1991, ibid, and literary composition is described), to measure according to the quantitative electrophoresis method, the average foreign gene plasmid copy number that records cell is about 100.With the fragmentation twice under the condition of 1000 crust of APV2000 minitype high voltage refiner, with Abbott Laboratories' enzyme immunoassay test kit, according to the indication of manufacturer, the content that records every liter of fermented liquid hepatitis B surface antigen (HBsAg) is 30.3mg.
The culturing step of seed is with embodiment 2.
Produce fermentation step according to following manner, the rotating speed of the source of wherein used material, feed supplement device, leavening temperature, air flow, fermentor tank, measure leucine prototroph cell retention method, measure cell average foreign gene plasmid copy number method and to measure the method for HBsAg content in every liter of fermented liquid all described identical with embodiment 2.
30 liters of fermentor tank continuous feedings are cultivated: the import unartificial yeast is leached powder 32 grams per liters, and import crude soya bean peptone 36 grams per liters add water to 9.9 liters, 121 ℃, sterilization in 30 minutes is after the cooling, add 700 milliliters of 25% the glucose solutions of sterilizing, with the 9N phosphoric acid solution accent pH to 6.0 that sterilize.Insert 1 liter in sophisticated seed, be cultured to 14 hours, the beginning feed supplement.Mend the mixing solutions of going into to contain glucose, yeast extract powder, soy peptone and VITAMIN B4, it is 0.40 grams per liter hour that the benefit of yeast extract powder is gone into speed, it is 0.20 grams per liter hour that the benefit of soy peptone is gone into speed, it is 2.0 mg/litre hour that the benefit of VITAMIN B4 is gone into speed, and it is 1.2 grams per liters hour that the benefit of glucose is gone into speed.Be cultured to 52 hours and finish fermentation, 0.36 kilogram of results wet cell.Measuring leucine prototroph cell retention is 100%; The average foreign gene plasmid copy number of cell is about 100.With the fragmentation twice under the condition of 1000 crust of APV2000 minitype high voltage refiner, the HBsAg content that records every liter of fermented liquid with import Abbott Laboratories enzyme-linked immunosorbent assay is 54.78mg.
The culturing step of seed is with embodiment 2.
Produce fermentation step according to following manner, the rotating speed of the source of wherein used material, feed supplement device, leavening temperature, air flow, fermentor tank, measure leucine prototroph cell retention method, measure cell average foreign gene plasmid copy number method and to measure the method for HBsAg content in every liter of fermented liquid all described identical with embodiment 2.
30 liters of fermentor tank continuous feedings are cultivated: the import unartificial yeast is leached powder 32 grams per liters, and import crude soya bean peptone 36 grams per liters add water to 9.9 liters, 121 ℃, sterilization in 30 minutes is after the cooling, add 700 milliliters of 25% the glucose solutions of sterilizing, with the 9N phosphoric acid solution accent pH to 6.0 that sterilize.Insert 1 liter in the seed cultivated with the feed supplement mode, be cultured to 14-15 hour, begin feed supplement.Mend the mixing solutions of going into to contain glucose, yeast extract powder, soy peptone and adenosine triphosphate, it is 0.50 grams per liter hour that the benefit of yeast extract powder is gone into speed, it is 0.30 grams per liter hour that the benefit of soy peptone is gone into speed, it is 3.0 mg/litre hour that the benefit of adenosine triphosphate is gone into speed (by the cubage of VITAMIN B4), and the benefit of glucose is gone into speed, and to be 1.2 grams per liters hour hour increase progressively to 1.6 grams per liters.Be cultured to 64 hours and finish fermentation, 0.5 kilogram of results wet cell.Measuring leucine prototroph cell retention is 100%; The average foreign gene plasmid copy number of cell is about 100.With the fragmentation twice under the condition of 1000 crust of APV2000 minitype high voltage refiner, the HBsAg content that records every liter of fermented liquid with enzyme-linked immunosorbent assay is 109.29mg.
The culturing step of seed is with embodiment 2.
Produce fermentation step according to following manner, the rotating speed of the source of wherein used material, feed supplement device, leavening temperature, air flow, fermentor tank, measure leucine prototroph cell retention method, measure cell average foreign gene plasmid copy number method and to measure the method for HBsAg content in every liter of fermented liquid all described identical with embodiment 2.
30 liters of fermentor tank continuous feedings are cultivated: the import unartificial yeast is leached powder 32 grams per liters, and import crude soya bean peptone 36 grams per liters add water to 9.9 liters, 121 ℃, sterilization in 30 minutes is after the cooling, add 700 milliliters of 25% the glucose solutions of sterilizing, with the 9N phosphoric acid solution accent pH to 6.0 that sterilize.Insert 1 liter in sophisticated seed, be cultured to 14 hours, the beginning feed supplement.Two BT00-100M peristaltic pumps that the feed supplement device adopts Lange company to produce are done transmission power, and the silicone tube of 1.6mm is done pipeline.A benefit is gone into glucose solution, and speed hour increases gradually from 1.2 grams per liters hour to 2.4 grams per liters; Another benefit goes into to contain the mixing solutions of yeast extract powder, soy peptone and adenosine triphosphate, it is 0.55 grams per liter hour that the benefit of yeast extract powder is gone into speed, it is 0.35 grams per liter hour that the benefit of soy peptone is gone into speed, it is 4.0 mg/litre hour that the benefit of adenosine triphosphate is gone into speed (by the cubage of VITAMIN B4), and the benefit of glucose is gone into speed for hour increasing progressively from 1.2 grams per liters hour to 2.4 grams per liters.Be cultured to 72 hours and finish fermentation, 0.57 kilogram of results wet cell.Measuring leucine prototroph cell retention is 100%; The average foreign gene plasmid copy number of cell is about 100.With the fragmentation twice under the condition of 1000 crust of APV2000 minitype high voltage refiner, the HBsAg content that records every liter of fermented liquid with import Abbott Laboratories enzyme-linked immunosorbent assay is 149.04mg.
Embodiment 6
The culturing step of seed is with embodiment 2.
Produce fermentation step according to following manner, the rotating speed of the source of wherein used material, feed supplement device, leavening temperature, air flow, fermentor tank, measure leucine prototroph cell retention method, measure cell average foreign gene plasmid copy number method and to measure the method for HBsAg content in every liter of fermented liquid all described identical with embodiment 2.
30 liters of fermentor tank continuous feedings are cultivated: the import unartificial yeast is leached powder 32 grams per liters, and import crude soya bean peptone 36 grams per liters add water to 9.9 liters, 121 ℃, sterilization in 30 minutes is after the cooling, add 700 milliliters of 25% the glucose solutions of sterilizing, with the 9N phosphoric acid solution accent pH to 6.0 that sterilize.Insert 1 liter in sophisticated seed, be cultured to 14-15 hour, the beginning feed supplement.It is 14.0 mg/litre hour that the benefit of VITAMIN B4 is gone into speed, and it is 1.2 grams per liters hour that the benefit of glucose is gone into speed.Add VITAMIN B4 and continue 15 hours.Glucose rate in tempo continues to add.Be cultured to 40 hours and finish fermentation, 0.36 kilogram of results wet cell.Measuring leucine prototroph cell retention is 100%; The average foreign gene plasmid copy number of cell is about 100.With the fragmentation twice under the condition of 1000 crust of APV2000 minitype high voltage refiner, the HBsAg content that records every liter of fermented liquid with import Abbott Laboratories enzyme-linked immunosorbent assay is 32mg.
Embodiment 7
The culturing step of seed is with embodiment 2.
Then 2 liters seed is transferred in 70 liters of fermentor tanks that contain 50 liters of YEPD substratum.70 liters of fermentor tank batch fermentations are cultivated: the import unartificial yeast is leached 1.6 kilograms in powder, and 1.8 kilograms of import crude soya bean peptones add water to 45 liters, 121 ℃, sterilization in 30 minutes is after the cooling, add 3 liters of 25% the glucose solutions of sterilizing, with the 9N phosphoric acid solution accent pH to 5.0 that sterilize.Insert 2 liters in sophisticated seed.This whole production fermentation culture stage all maintains 28 ± 1 ℃, with the 350rpm rotating speed, cultivates with 25 liters/minute air flow, cultivates 18 hours.
Produce fermentation step according to following manner, the rotating speed of the source of wherein used material, feed supplement device, leavening temperature, air flow, fermentor tank, measure leucine prototroph cell retention method, measure cell average foreign gene plasmid copy number method and to measure the method for HBsAg content in every liter of fermented liquid all described identical with embodiment 2.
800 liters of fermentor tank continuous feedings are cultivated: the import unartificial yeast is leached 14 kilograms in powder, and 19.25 kilograms of import crude soya bean peptones add water to 400 liters, 121 ℃, sterilization in 30 minutes is after the cooling, add 35 liters of 25% the glucose solutions of sterilizing, with the 9N phosphoric acid solution accent pH to 6.0 that sterilize.Insert 50 liters in sophisticated seed.This whole production fermentation culture stage all maintains 28 ± 1 ℃, with the 200rpm rotating speed, cultivates with 200 liters/minute air flow.Be cultured to 15 hours, the beginning feed supplement.The 7549-60 peristaltic pump that the feed supplement device adopts Millipore company to produce is done transmission power, and the silicone tube of 3.5mm is done pipeline, and one tunnel feed supplement is mended into glucose solution, and speed hour increases gradually from 1.1 grams per liters hour to 2.2 grams per liters; Mend into the solution that contains yeast extract powder, soy peptone and VITAMIN B4 on another road, and it is 0.32 grams per liter hour that the benefit of yeast extract powder is gone into speed, and it is 0.22 grams per liter hour that the benefit of soy peptone is gone into speed, and it is 1.8 mg/litre hour that the benefit of VITAMIN B4 is gone into speed.Be cultured to 64 hours and finish fermentation, the results wet cell weighs 38.5 kilograms.Measuring leucine prototroph cell retention is 100%; The average foreign gene plasmid copy number of cell is about 100.Detected glucose concn in the one time fermentation liquid in per 5 hours, until fermentation ends, glucose concn all is lower than 0.05 (g/L) after the 15th hour.With the fragmentation twice under the condition of 1000 crust of APV2000 minitype high voltage refiner, the HBsAg content that records every liter of fermented liquid with import Abbott Laboratories enzyme-linked immunosorbent assay is 138mg.
Comparative Examples 1
The culturing step of seed is with embodiment 2.
Produce fermentation step according to following manner, the rotating speed of the source of wherein used material, feed supplement device, leavening temperature, air flow, fermentor tank, measure leucine prototroph cell retention method, measure cell average foreign gene plasmid copy number method and to measure the method for HBsAg content in every liter of fermented liquid all described identical with embodiment 2.
30 liters of former batch fermentation technologies of fermentor tank: the import unartificial yeast is leached powder 32 grams per liters, and import crude soya bean peptone 36 grams per liters add water to 9.9 liters, 121 ℃, sterilization in 30 minutes is after the cooling, add 700 milliliters of 25% the glucose solutions of sterilizing, with the 9N phosphoric acid solution accent pH to 6.0 that sterilize.Insert 1 liter in sophisticated seed, cultivated 40 hours.0.18 kilogram of results wet cell.With the fragmentation twice under the condition of 1000 crust of APV2000 minitype high voltage refiner, three batches of content that record every liter of fermented liquid hepatitis B surface antigen (HBsAg) with import Abbott Laboratories enzyme-linked immunosorbent assay are up to 6.35mg.
Comparative Examples 2
The culturing step of seed is with embodiment 2.
Then 2 liters seed is transferred in 70 liters of fermentor tanks that contain 50 liters of YEPD substratum.70 liters of fermentor tank batch fermentations are cultivated: the import unartificial yeast is leached 1.6 kilograms in powder, and 1.8 kilograms of import crude soya bean peptones add water to 45 liters, 121 ℃, sterilization in 30 minutes is after the cooling, add 3 liters of 25% the glucose solutions of sterilizing, with the 9N phosphoric acid solution accent pH to 5.0 that sterilize.Insert 2 liters in sophisticated seed.This whole production fermentation culture stage all maintains 28 ± 1 ℃, with the 350rpm rotating speed, cultivates with 25 liters/minute air flow, cultivates 18 hours.
Carry out 800 liters of fermentor tanks according to following manner and produce fermentation steps, the source of wherein used material, measure leucine prototroph cell retention method, measure cell average foreign gene plasmid copy number method and to measure the method for HBsAg content in every liter of fermented liquid all described identical with embodiment 2.
800 liters of fermentor tank batch fermentations are cultivated: the import unartificial yeast is leached 14 kilograms in powder, and 19.25 kilograms of import crude soya bean peptones add water to 530 liters, 121 ℃, sterilization in 30 minutes is after the cooling, add 35 liters of 25% the glucose solutions of sterilizing, with the 9N phosphoric acid solution accent pH to 5.0 that sterilize.Insert 50 liters in sophisticated seed.This whole production fermentation culture stage all maintains 28+1 ℃, with the 200rpm rotating speed, cultivates with 200 liters/minute air flow.Be cultured to 40 hours and finish fermentation, the results wet cell weighs 9 kilograms.Measuring leucine prototroph cell retention is 100%; The average foreign gene plasmid copy number of cell is about 100.With the fragmentation twice under the condition of 1000 crust of APV2000 minitype high voltage refiner, the HBsAg content that records every liter of fermented liquid with import Abbott Laboratories enzyme-linked immunosorbent assay is 14mg.
The test example
Adopt Spectronic 20 (Bausch and Lomb), inventor's convection current to add before additional yeast extract powder, soy peptone and the glucose and the yeast cell A during fermentation ends
660Under absorbance value measure, the result is as shown in table 2 below:
Table 2
Claims (18)
1. method of utilizing beer yeast engineering bacteria production hepatitis B surface antigen, the recipient bacterium of wherein said beer yeast engineering bacteria strain is that VITAMIN B4, leucine are auxotrophic; Described beer yeast engineering bacteria comprises reorganization 2 μ plasmids but does not contain 2 natural μ plasmids; Described reorganization 2 μ plasmids comprise the gene of the hepatitis B surface antigen of encoding, and the 5 ' end and the 3 ' end of this gene can be operatively connected with glyceraldehyde 3-phosphate dehydro-genase promotor and ethanol dehydrogenase terminator respectively; Described reorganization 2 μ plasmids also comprise the leucic gene of coding, and the 5 ' end and the 3 ' end of this gene can be operatively connected with promotor and terminator respectively; This method comprises the steps:
1) the seed fermentation step of described beer yeast engineering bacteria;
2) seed that utilizes step 1) to make carries out the step of fermentation culture; With
3) from step 2) the fermenting mixture of gained separation and purification go out the step of hepatitis B surface antigen;
The method is characterized in that, above-mentioned in step 2) in the weight of VITAMIN B4, add VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) or adenosine triphosphate with the speed stream in substratum that is lower than the 14mg/ liters per hour.
2. the described method of claim 1 wherein in the weight of VITAMIN B4, adds VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) or adenosine triphosphate with the data rate stream that is lower than 10 mg/litre hour.
3. the described method of claim 2 wherein in the weight of VITAMIN B4, adds VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) or adenosine triphosphate with the data rate stream that is lower than 6 mg/litre hour.
4. the described method of claim 3 wherein in the weight of VITAMIN B4, adds VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) or adenosine triphosphate with the data rate stream that is lower than 2 mg/litre hour.
5. each described method among the claim 1-4, wherein VITAMIN B4, adenosine, adenylic acid, adenosine diphosphate (ADP) or adenosine triphosphate add in the substratum with the successive fed-batch mode.
6. the described method of claim 5 is wherein in above-mentioned steps 2) in also add yeast extract powder, add carbon source with the soybean protein peptone or with the speed stream in fermenting mixture that is lower than 3.0 grams per liters hour with the speed that is lower than 0.8 grams per liter hour stream in fermenting mixture with the speed stream in fermenting mixture that is lower than 1.0 grams per liters hour.
7. the described method of claim 6, wherein above-mentioned steps 2) in cultivate beginning described stream add operation of beginning after 10 hours from fermentation.
8. the described method of claim 7, wherein said yeast extract powder adds in the fermenting mixture in the mode that continuous flow adds.
9. the described method of claim 8, wherein said continuous flow rate of acceleration are controlled in the scope that is lower than 0.50 grams per liter hour.
10. the described method of claim 7, wherein said soy peptone adds in the fermenting mixture in the mode that continuous flow adds.
11. the described method of claim 10, wherein said continuous flow rate of acceleration are controlled in the scope that is lower than 0.38 grams per liter hour.
12. the described method of claim 7, wherein said carbon source is added in the fermenting mixture in the mode that continuous flow adds.
13. the described method of claim 12, wherein said continuous flow rate of acceleration increase progressively for a short time from 1.1 grams per liters hour-2.4 grams per liters in time.
14. the described method of claim 13, wherein said carbon source are glucose, sucrose, lactose, fructose, maltose or glycerine.
15. the described method of claim 15, wherein stating the promotor that can be operatively connected with hbsag gene in the 2 μ plasmids is the glyceraldehyde 3-phosphate dehydro-genase promotor, and the terminator that can be operatively connected with hbsag gene is an ethanol dehydrogenase I terminator.
16. the described method of claim 15, the promotor that can be operatively connected with the leucine gene in the wherein said 2 μ plasmids has disappearance at 3 ' end, and the feasible leucine expression level that contains the beer yeast engineering bacteria of the leucine auxotrophy type that singly copies recombinant plasmid is 5% of a wild-type cereuisiae fermentum.
17. the described method of claim 16, wherein said beer yeast engineering bacteria are haploid strains.
18. the described method of claim 17, wherein said beer yeast engineering bacteria are cereuisiae fermentum 2150-2-3 (MATa, ade1, leu2-04, the cir that has plasmid pHBS56-GAP347/33
0).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103305574A (en) * | 2013-07-16 | 2013-09-18 | 深圳康泰生物制品股份有限公司 | Recombinant saccharomyces cerevisiae fermentation medium expressing HBsAg (epatitis B surface antigen), preparation method and fermentation process therefor |
| CN105985150A (en) * | 2015-01-28 | 2016-10-05 | 浙江泛亚生物医药股份有限公司 | Isaria cicadae coremium liquid culture medium |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305574A (en) * | 2013-07-16 | 2013-09-18 | 深圳康泰生物制品股份有限公司 | Recombinant saccharomyces cerevisiae fermentation medium expressing HBsAg (epatitis B surface antigen), preparation method and fermentation process therefor |
| CN103305574B (en) * | 2013-07-16 | 2016-01-27 | 深圳康泰生物制品股份有限公司 | The recombinant Saccharomyces cerevisiae bacteria fermentation culture medium of HBsAg expression and compound method thereof and zymotechnique |
| CN105985150A (en) * | 2015-01-28 | 2016-10-05 | 浙江泛亚生物医药股份有限公司 | Isaria cicadae coremium liquid culture medium |
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