CN104017848B - A kind of method that utilization Herba Polygoni hydropiperiss promote saccharomyces cerevisiae secreting, expressing human β defensin 3 - Google Patents
A kind of method that utilization Herba Polygoni hydropiperiss promote saccharomyces cerevisiae secreting, expressing human β defensin 3 Download PDFInfo
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- CN104017848B CN104017848B CN201410231528.XA CN201410231528A CN104017848B CN 104017848 B CN104017848 B CN 104017848B CN 201410231528 A CN201410231528 A CN 201410231528A CN 104017848 B CN104017848 B CN 104017848B
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- herba polygoni
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of method that utilization Herba Polygoni hydropiperiss promote saccharomyces cerevisiae secreting, expressing human β defensin 3.The method comprises the steps：The seed liquid of Prepare restructuring saccharomyces cerevisiae INVSC1/pY α hBD 3；Seed liquid is inoculated in Herba Polygoni hydropiperiss growth medium and is cultivated, collects thalline；Cultivate in thalline to be inoculated in Herba Polygoni hydropiperiss inducing culture, collect supernatant；Then bacteriostatic test is carried out, fungistatic effect is represented with the size of antibacterial circle diameter.The invention provides a kind of novel, low cost, the growth medium of hypersecretion amount and inducing culture, improve the ability of 3 secreting, expressing human β defensin 3s of recombinant Saccharomyces cerevisiae INVSC1/pY α hBD.Growth and protein excretion expression of the collocation of the carbon-nitrogen ratio in the growth of the present invention and inducing culture to saccharomyces cerevisiae is highly beneficial, hence it is evident that better than existing cultural method, be that more solid foundation is established in the industrialization and industrialization of recombinant Saccharomyces cerevisiae.
Description
Technical field
The invention belongs to microbe application field, and in particular to a kind of to promote saccharomyces cerevisiae secreting, expressing people β using Herba Polygoni hydropiperiss
The method of alexin 3.
Background technology
Alexin is the subfamily of maximum in antibacterial peptide family, is the biological class antagonism external source pathogen for producing in vivo
The defensive polypeptide active substance of pathogenic effects, is the important component part of organism innate immunity, with the group such as interferon, complement
Into the immune defense system of host.Research up to now finds that alexinic species has：Research of Mammalian Defensins, insecticide defence
Element and plant alexin；And according to the position of disulfide bond, the difference and precursor in connection is different with expression way, alexin is again
It is divided into α-alexin, 3 class of beta-alexin and θ-alexin.
Human β defensin 3 (human D-defensin-3, hBD-3) is calendar year 2001 Harder etc. in Psoriasis
The alexin D family bacteriostatic activity highest peptide found in tissue.The micromolecule polypeptide that hBD-3 is made up of 45 aminoacid,
Relative molecular mass is 5154.7, is made up of 1 α spiral and 3 antiparallel β-pleated sheet lamellas, while there is a short spiral
Ring is formed in amino terminal region.HBD-3 forms 3 disulfide bond by 6 cysteine residues, its connected mode be 2～4, l～
5th, 3～6.3 antiparallel β-pleated sheet lamellas are formed by 17～19,27～31,39～43 residues.
For other beta-defensins, the feature of human β defensin 3 is wider：(1) significant antibacterium, antifungal
And the activity of antiviral, and Mlc is lower than other members for having been observed by alexin family；(2) in connection first
Important function served as bridge is played in it and Acquired immune response；(3) induce professional antigen presenting cell to produce and activate, have
Prestige becomes vaccine adjuvant；(4) its antibacterial activity is non-salt ionic concentration sensitivity, is difficult to cause haemolysis.In abuse antimicrobial drug
Thing, bacterial resistance sex chromosome mosaicism increasingly serious today, human beta-defensin is because with efficient sterilizing, being not likely to produce resistance mutation, nothing
The malicious, suitability is wide, steady performance, is expected to replace traditional antibiotic, is developed into antibacterium of new generation, funguses, disease
The medicine of poison and anticancer, application and development have a extensive future.
But, human β defensin 3 is really applied to production practices, it is necessary to initially set up with low cost, efficient stable
Preparation technology, solves big batch metaplasia and produces problem.At present, alexin is mainly obtained by following 3 kinds of approach：(1) induction life
Isolate and purify after object secreting, expressing；(2) chemosynthesis；(3) build phylaxin gene engineered strain.But naturally occurring defence
Plain yield is very low, it is impossible to extracted from animal and plant body in a large number, and isolate and purify the component with antibacterial activity be undoubtedly one it is non-
Often loaded down with trivial details work；And chemosynthesis alexin there is also high cost, produce difficult weakness in enormous quantities, therefore, front 2 kinds of methods
The demand of alexin industrialized production cannot be met.In contrast, alexin is produced with engineering bacteria industrial fermentation, can be extensive
Production, its with short production cycle, low production cost and is not affected by external environment such as season and climate changes, thus exploration is appropriate
Alexin ways of regeneration becomes the focus of the area research.There are many experiments to carry out the alexin of separate sources recombinant expressed
Attempt, the expression system being related to includes E. coli system, Yeast system etc..
Huang Pengliang (Huang Pengliang, Zhao Yahua, Xu Shaopeng. people's beta-alexin 3 solubility expression and its biology in escherichia coli
The identification of activity. life science, 2005,9 (2)：129-133) etc. according to escherichia coli to arginine codon using inclined
People beta-alexin 3 is synthesized easily, the abduction delivering success in escherichia coli.Though coli expression system has certain simplification
And popularity, but still have some defects：1) Eukaryotic protein post-translational modification and the course of processing are lacked, disulfide bond cannot
It is properly formed, have impact on the correct folding of albumen, the antibacterial peptide activity after expression is affected；2) protein expressed is more to wrap
The presence of form containing body, alexinic purification and recovery, not only increase cost, and affect alexinic biological activity；3) miscellaneous egg
Bai Duo, purification step complexity etc..
Tang Yuqian etc. (Wu Hui, Chen Yi, Xiao Junmei, Tang Yuqian. the clone of human β defensin 3 gene and its in saccharomyces cerevisiae
Expression. modern food science and technology, 2012,28 (9)：1123-1127) the gene of human β defensin 3 is inserted in pY α carriers, and
The successful expression in saccharomyces cerevisiae INVSC1, the recombined human β defence of recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 secreting, expressings
Element 3 shows good bacteriostatic activity.
Herba Polygoni hydropiperiss, also known as Herba polygoni hydropiperiss, be Polygonaceae arsesmart Herba Polygoni hydropiperiss herb (Zhao Chunguang. wild Caulis et Folium Polygalae Tenuifoliae prevents and treats testudinate fish disease
Herba polygoni hydropiperiss. Southern Rural Area report, 2003,4-6).Polygonaceae plant is mainly distributed on north temperate zone, and Polygonum is that one of them is medicinal big
Category, is distributed in all parts of the country.Herba Polygoni hydropiperiss acrid in the mouth is sour, warm in nature, with the effect of intestinal stasis relieving removing dampness, dispelling wind for relieving itching, removing stasis to stop bleeding, removing toxic substances,
The growth of antibacterial can be suppressed to a certain extent.Auxin in Herba Polygoni hydropiperiss containing abundant yeast and needed for rhizopus, after air-drying
Herba Polygoni hydropiperiss hay powder contain nitrogen-free extract 41.7%, crude protein 15.5%, crude fat 2.6%, calcareous 4.03%, phosphorus 0.52%,
Also containing volatile oil, tannin, flavone, chromocor derivative, nepalin, persicarin, persicarin -7- methyl ethers, the glad element of Fructus rhamni (Rhamnus davurica Pall.) and Radix Hyperici Monogyni (Herba Hyperici Monogyni)
The effective ingredient such as glycoside (Li Jiashou, Chen Jingxian. process for making yellow rice wine. China Wine Industry Association yellow wine branch, 2004,59-62).
It follows that Herba Polygoni hydropiperiss play the role of to promote the breeding such as yeast (Gao Yongqiang, Xu great Xin. Herba polygoni hydropiperiss are to mechanized yellow rice wi ne yeast
Influence research. wine brewing, 2009,36 (6):67-68.).And saccharomyces cerevisiae, used as fungal expression system, expressing protein amount is relatively low,
Limit its application.
The content of the invention
In order to overcome the shortcoming of prior art and deficiency, the primary and foremost purpose of the present invention is to provide one kind to promote using Herba Polygoni hydropiperiss
The method of saccharomyces cerevisiae secreting, expressing human β defensin 3.The present invention is for the current recombinant Saccharomyces cerevisiae INVSC1/ for not yet optimizing
A kind of deficiency in pY α-hBD-3 growths and fermentation condition, there is provided novel, inexpensive, efficient culture medium, improves people β
The secretion yield of alexin 3.
Another object of the present invention is to provide the application of said method.
The purpose of the present invention is achieved through the following technical solutions：It is a kind of to promote saccharomyces cerevisiae secreting, expressing people β using Herba Polygoni hydropiperiss
The method of alexin 3, comprises the steps：
(1) recombinant Saccharomyces cerevisiae (Saccharomyces cerevisiae) INVSC1/pY α-hBD-3 strains are lined
On YPD culture medium flat plates, train and 2～3d are cultivated in 28～30 DEG C to growing single bacterium colony；The streak inoculation of picking single bacterium colony is cultivated in YPD
On base flat board, 1～2d is cultivated in 28～30 DEG C, then picking single bacterium colony is inoculated in 5～10mL YPD fluid mediums, 28～30
DEG C, 180～250r/min overnight incubations, obtain seed liquid；
(2) take seed liquid prepared by step (1) to be inoculated in Herba Polygoni hydropiperiss growth medium, 28～30 DEG C, 180～250r/
Min shaking cultures, cultivate 24～36h to OD600For 1～4 when stop growing culture, obtain culture fluid；
(3) the culture fluid low-speed centrifugal obtained in taking step (2), abandons supernatant, with twice of sterile water wash, low speed from
The heart, abandons supernatant, obtains recombinant Saccharomyces cerevisiae grown cultures thalline；
(4) the recombinant Saccharomyces cerevisiae grown cultures thalline obtained by step (3) is forwarded to into Herba Polygoni hydropiperiss inducing culture, 28～30
DEG C, 180～250r/min shaking culture, cultivate 48～72h, collect induction broth；
(5) induction broth for collecting step (4), low-speed centrifugal, abandon precipitation, collect supernatant, BCA albuminimetries
Determine the protein content of supernatant；
(6) bacterium solution is added in nutrient broth medium, make flat board containing bacterium, after flat board solidification, place cattle thereon
Tianjin cup, and the supernatant obtained by step (5) is added, 37 DEG C of 16～18h of culture observe the diameter of inhibition zone, with antibacterial circle diameter
Size represent fungistatic effect.
After above-mentioned culture terminates, the OD of recombinant Saccharomyces cerevisiae culture fluid after grown cultures is detected600Value reaches 10.0
More than, the protein content after inducing culture in culture supernatant reaches more than 80mg/mL, and supernatant carries out fungistatic effect detection
As a result show that antibacterial circle diameter reaches more than 2.5cm, illustrate that the cultural method can effectively obtain high-biomass, the weight of high expression
Group saccharomyces cerevisiae；With general uracil auxotrophy SC-U culture medium culturing recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-
3, the OD of culture fluid after grown cultures600It is worth for 4.0 or so, the protein content after inducing culture in culture supernatant is about
35mg/mL, the result of supernatant fungistatic effect detection show that antibacterial circle diameter is 1cm or so；Illustrate to contrast general SC-U trainings
Foster base, growth of the Herba Polygoni hydropiperiss growth medium to recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 have obvious facilitation, water
Knotweed inducing culture significantly improves the secreting, expressing of human β defensin 3, and bacteriostatic diameter increases more than 1.5cm；
The SC-U culture medium of described general uracil auxotrophy is made by the steps：20g/L glucoses,
6.7g/L YNB, each 0.1g/L of adenine, arginine, cysteine, leucine, lysine, threonine and tryptophan, Radix Asparagi
The each 0.05g/L of propylhomoserin, histidine, isoleucine, methionine, Phenylalanine, proline, serine, L-Tyrosine and L-Valine；
It is standby after 121 DEG C of autoclaving 20min；
YPD culture medium flat plates described in step (1) refer to glucose 20g/L, yeast extract 10g/L, peptone 20g/
L, 15～20g/L of agar；
YPD fluid mediums described in step (1) refer to glucose 20g/L, yeast extract 10g/L, peptone 20g/
L；
In above-mentioned steps (1), in order to obtain good seed activation effect, so inventor is to -80 DEG C of guarantors of conventional glycerol
The recombinant Saccharomyces cerevisiae strain deposited carries out flat board activation twice, then transfers liquid YPD medium shake-flask culture again overnight, so as to
Seed liquid needed for obtaining；Because having carried out flat board activation twice, it can be ensured that the removal completely of glycerol, strain are completely extensive
It is multiple, be conducive to the experiment in later stage；
Herba Polygoni hydropiperiss growth medium described in step (2) is preferably made by the steps：50～90g/L glucoses, 6.7g/
L YNB, each 0.1g/L of adenine, arginine, cysteine, leucine, lysine, threonine and tryptophan, aspartic acid,
The each 0.05g/L of histidine, isoleucine, methionine, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 20～
40g/L peptones, 20～60g/L Herba Polygoni hydropiperiss；It is standby after 121 DEG C of autoclaving 20min；
Seed liquid described in step (2) is inoculated in Herba Polygoni hydropiperiss growth medium, preferably by volume (1～3)：100
Ratio is inoculated with；
Herba Polygoni hydropiperiss inducing culture described in step (4) is preferably made by the steps：20～40g/L glucoses, 6.7g/
L YNB, each 0.1g/L of adenine, arginine, cysteine, leucine, lysine, threonine and tryptophan, aspartic acid,
The each 0.05g/L of histidine, isoleucine, methionine, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 30～
50g/L sucrose, 20～40g/L yeast extracts, 20～60g/L Herba Polygoni hydropiperiss；It is standby after 121 DEG C of autoclaving 20min；
Recombinant Saccharomyces cerevisiae grown cultures thalline described in step (4) is forwarded to Herba Polygoni hydropiperiss inducing culture, preferably often
100mL culture fluid is collected the recombinant Saccharomyces cerevisiae grown cultures thalline for obtaining and is forwarded to 100mL Herba Polygoni hydropiperiss inducing cultures；
Herba Polygoni hydropiperiss described in step (2), (4) preferably refer to Herba Polygoni hydropiperiss dry powder, and preparation method is as follows：Pluck in 6～July, growing height
Herba Polygoni hydropiperiss (Polygonum hydropiper) plant of 40～80cm is reached, cryogenic vacuum 12～16h of lyophilization after cleaning is used
After pulverizer is clayed into power, 100 mesh sieves are crossed standby；
Sterile water wash described in step (3) is preferably and adds 5～10mL sterilized water per 100mL culture fluid, low after shaking up
Speed centrifugation, abandons supernatant；
The condition of the low-speed centrifugal described in step (3), (5) is preferably 5000r/min room temperature centrifugation 10min；
Described room temperature is preferably 20～30 DEG C；
The concrete operations of the BCA albuminimetries described in step (5) are as follows：A：Used according to BCA protein quantifications test kit
Description prepares protein standard and working solution, after every 200 μ L BCA working solutions and the mixing of 20 μ L standard substance, 37 DEG C of placement 30min,
Its absorbance is surveyed after cooling under 562nm.With protein concentration as abscissa, light absorption value is that vertical coordinate draws standard curve；B：Take
The 100 μ L of supernatant of gained in step (4), add 1000 μ L BCA working solutions after appropriate dilution, mix, 37 DEG C of placement 30min,
Its absorbance is determined after cooling under 562nm (data of testing protein sample should not exceed standard curve range)；C：It is bent with standard
Line compares, and calculates the concentration of testing sample, is repeated 3 times and averages；
Bacterium solution described in step (6) is preferably escherichia coli (Escherichia coli) or staphylococcus aureuses
(Staphylococcus aureus) shaken cultivation in nutrient broth medium under the conditions of 37 DEG C, to mid-log phase, is stopped
The bacterium solution of gained is cultivated only；The final concentration of described escherichia coli or staphylococcus aureuses is preferably 104～105cfu/mL；
The thickness containing bacterium flat board described in step (6) is preferably 4～6mm；
The diameter of the Oxford cup described in step (6) is preferably 8mm, and sterilized process is needed before use；
The supernatant obtained by addition step (5) described in step (6), adds 200 μ L steps in being preferably each Oxford cup
(5) supernatant obtained by；
Described utilization Herba Polygoni hydropiperiss promote the method for saccharomyces cerevisiae secreting, expressing human β defensin 3 improving saccharomyces cerevisiae secretion
Application in expression human β defensin 3.
The present invention mechanism be：Auxin in Herba Polygoni hydropiperiss containing abundant yeast and needed for rhizopus, has promotion yeast
The effect of the breedings such as bacterium, therefore in recombinant Saccharomyces cerevisiae growth medium, add appropriate Herba Polygoni hydropiperiss which can be promoted to grow, induction training
Appropriate Herba Polygoni hydropiperiss are added also to promote the secreting, expressing of albumen etc. in saccharomyces cerevisiae body during supporting.
The present invention is had the following advantages relative to prior art and effect：
(1) it is of the invention it is important that in grown cultures and the inducing culture phase of recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3
Between add Herba Polygoni hydropiperiss, Herba Polygoni hydropiperiss to be conducive to the growth of yeast quasi-microorganism, can also promote the secreting, expressing of microorganism vivo protein etc.；And
The formula of grown cultures and inducing culture is improved respectively, the component and its consumption of its formula are that the present inventor passes through in a large number
Experiment and data analysiss it is preferred obtained by, the collocation of the carbon-nitrogen ratio in culture medium is defendd to the growth of recombinant Saccharomyces cerevisiae and people β
The expression of element 3 is all highly beneficial.
(2) 50～90g/L glucoses are added in the Herba Polygoni hydropiperiss growth medium of the inventive method as carbon source, 20～40g/L
Peptone as nitrogen source, relative to the carbon source and nitrogen source of adding other species and ratio, to recombinant Saccharomyces cerevisiae INVSC1/pY α-
The grown cultures facilitation of hBD-3 becomes apparent from.20～60g/L Herba Polygoni hydropiperiss, detection are added on the basis of preferred carbon nitrogen source ratio
The OD of culture fluid600Value reaches more than 10.0 in 36h, compares with general SC-U culture medium culturings, OD600Value double with
On.It is indicated above the growth that Herba Polygoni hydropiperiss growth medium has greatly facilitated recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3.
(3) in the Herba Polygoni hydropiperiss inducing culture of the inventive method add content be 30～50g/L sucrose as carbon source, 20～
40g/L yeast extracts as nitrogen source, relative to the carbon source and nitrogen source of adding other species and ratio, to recombinant Saccharomyces cerevisiae
The facilitation of INVSC1/pY α-hBD-3 high efficient expressions hBD-3 becomes apparent from.On the basis of this carbon nitrogen source ratio, add 20～
60g/L Herba Polygoni hydropiperiss, after culture 72h, in supernatant, protein content reaches more than 80mg/mL.Compare with general SC-U culture medium
Culture, in supernatant, protein content doubles the above.It is indicated above Herba Polygoni hydropiperiss inducing culture and greatly improves restructuring wine
Brewer yeast INVSC1/pY α-hBD-3 secrete the ability of human β defensin 3.
(4) the recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 of the inventive method culture, are compared and are cultivated with general SC-U
Base culture, antibacterial circle diameter increase more than 1.5cm, it was demonstrated that the Herba Polygoni hydropiperiss culture medium after improvement is increased than general SC-U culture medium
The secreting, expressing amount of human β defensin 3.In addition, because reporting that Herba Polygoni hydropiperiss also have certain sterilizing ability, therefore to unconverted hBD-3
The recombinant Saccharomyces cerevisiae INVSC1/pYES2 of gene is cultivated with the inventive method, and the supernatant containing Herba Polygoni hydropiperiss composition is without bright
Aobvious fungistatic effect, shows that the fungistatic effect that the supernatant that the inventive method culture is obtained is produced is mainly derived from human β defensin 3
Secretion.The general SC-U culture medium culturings of contrast, Herba Polygoni hydropiperiss culture medium promote the secretion of human β defensin 3, make the antibacterial work of supernatant
With significantly improving.
Description of the drawings
Fig. 1 is 1 recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 of embodiment in Herba Polygoni hydropiperiss growth medium (Herba Polygoni hydropiperiss containing 20g/L)
The fungistatic effect figure of the supernatant of gained after the middle culture 48h of middle culture 24h, Herba Polygoni hydropiperiss inducing culture (Herba Polygoni hydropiperiss containing 20g/L), wherein
1E-1：Containing 105Cfu/mL E. coli plates；1E-2：Containing 104Cfu/mL E. coli plates；1S-1：Containing 105Cfu/mL is golden yellow
Color staphylococcuses flat board；1S-2：Containing 104Cfu/mL staphylococcus aureuses flat boards.
Fig. 2 is 2 recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 of embodiment in Herba Polygoni hydropiperiss growth medium (Herba Polygoni hydropiperiss containing 40g/L)
The fungistatic effect figure of the supernatant of gained after the middle culture 60h of middle culture 30h, Herba Polygoni hydropiperiss inducing culture (Herba Polygoni hydropiperiss containing 40g/L), wherein
2E-1：Containing 104Cfu/mL E. coli plates；2E-2：Containing 105Cfu/mL E. coli plates；2S-1：Containing 104Cfu/mL is golden yellow
Color staphylococcuses flat board；2S-2：Containing 105Cfu/mL staphylococcus aureuses flat boards.
Fig. 3 is 3 recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 of embodiment in Herba Polygoni hydropiperiss growth medium (Herba Polygoni hydropiperiss containing 60g/L)
The fungistatic effect figure of the supernatant of gained after the middle culture 72h of middle culture 36h, Herba Polygoni hydropiperiss inducing culture (Herba Polygoni hydropiperiss containing 60g/L), wherein
3E-1：Containing 104Cfu/mL E. coli plates；3E-2：Containing 105Cfu/mL E. coli plates；3S-1：Containing 104Cfu/mL is golden yellow
Color staphylococcuses flat board；3S-2：Containing 105Cfu/mL staphylococcus aureuses flat boards.
Fig. 4 is that 4 recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 of embodiment cultivate 36h, SC-U training in SC-U culture medium
In foster base cultivate 72h after gained supernatant fungistatic effect figure, wherein 4E-1：Containing 104Cfu/mL E. coli plates；4E-
2：Containing 105Cfu/mL E. coli plates；4S-1：Containing 104Cfu/mL staphylococcus aureuses flat boards；4S-2：Containing 105cfu/mL
Staphylococcus aureuses flat board.
Fig. 5 is 5 recombinant Saccharomyces cerevisiae INVSC1/pYES2 of embodiment cultures in knotweed growth medium (Herba Polygoni hydropiperiss containing 40g/L)
The fungistatic effect figure of the supernatant of gained, wherein 5E-1 after the middle culture 72h of 36h, Herba Polygoni hydropiperiss inducing culture (Herba Polygoni hydropiperiss containing 40g/L)：
Containing 104Cfu/mL E. coli plates；5E-2：Containing 105Cfu/mL E. coli plates；5S-1：Containing 104Cfu/mL golden yellow Portugal
Grape coccus flat board；5S-2：Containing 105Cfu/mL staphylococcus aureuses flat boards.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition.
Strain used in embodiment is recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 and recombinant Saccharomyces cerevisiae INVSC1/
PYES2, is genetic engineering strain (Wu Hui, Chen Yi, Xiao Junmei, the Tang's language obtained by the present inventor is built using genetic engineering means
It is modest. the clone of 3 gene of people's beta-alexin and its expression in saccharomyces cerevisiae. modern food science and technology, 2012,28 (9)：1123-
1127)。
Embodiment 1
(1) preparation of seed liquid：
The recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 glycerol strains of preservation are lined in YPD culture medium flat plates, is put
Be placed in 30 DEG C of incubators 2～3d is cultivated to growing single bacterium colony；
YPD culture medium prescriptions used are as follows：Glucose 20g/L, yeast extract 10g/L, peptone 20g/L；Solid
Agar 15g/L is added in culture medium.
In above-mentioned single bacterium colony, the full single bacterium colony streak inoculation of picking is on YPD culture medium flat plates, in 30 DEG C of incubators
Middle culture 2d, then the full single bacterium colony of picking is inoculated in 5～10mL YPD fluid mediums, 30 DEG C, the training of 250r/min shaking flasks
Support overnight, obtain seed liquid.
(2) take the Herba Polygoni hydropiperiss growth medium after seed liquid 1mL prepared by step (1) is inoculated in 100mL sterilizings, 30 DEG C,
250r/min shaking cultures, cultivate 24h to OD600For 1～4 when stop growing culture, obtain culture fluid.
Herba Polygoni hydropiperiss grown cultures based formulas used are as follows：50g/L glucoses, 6.7g/L YNB, adenine, arginine, half
The each 0.1g/L of cystine, leucine, lysine, threonine and tryptophan, aspartic acid, histidine, isoleucine, first sulfur ammonia
The each 0.05g/L of acid, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 20g/L peptones, 20g/L Herba Polygoni hydropiperiss.
(3) the culture fluid low-speed centrifugal (5000rpm, 10min) in step (2) is taken, supernatant is abandoned, 10mL sterilized water is added
Twice of cleaning, low-speed centrifugal (5000rpm, 10min) abandons supernatant, obtains recombinant Saccharomyces cerevisiae grown cultures thalline.
(4) the recombinant Saccharomyces cerevisiae grown cultures thalline obtained by step (3) is forwarded to the Herba Polygoni hydropiperiss induction after 100mL sterilizings
Culture medium, 30 DEG C, 250r/min shaking cultures, low-speed centrifugal (5000rpm, 10min) after culture 48h are abandoned precipitation, collect supernatant
Liquid, BCA albuminimetries determine the protein content of supernatant.
Herba Polygoni hydropiperiss inducing culture based formulas used are as follows：20g/L glucoses, 6.7g/L YNB, adenine, arginine, half
The each 0.1g/L of cystine, leucine, lysine, threonine and tryptophan, aspartic acid, histidine, isoleucine, first sulfur ammonia
The each 0.05g/L of acid, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 30g/L sucrose, 20g/L yeast extracts,
20g/L Herba Polygoni hydropiperiss.
The concrete operations of BCA albuminimetries are as follows：A：Made according to BCA protein quantification test kits (being purchased from Takara companies)
Protein standard and working solution are prepared with description, after every 200 μ L BCA working solutions and the mixing of 20 μ L standard substance, 37 DEG C of placements
30min, surveys its absorbance under 562nm after cooling.With protein concentration as abscissa, light absorption value is that vertical coordinate draws standard song
Line；B：The 100 μ L of supernatant of gained in step (4) being taken, 1000 μ L BCA working solutions being added after appropriate dilution, is mixed, 37 DEG C put
Put 30min, after cooling, under 562nm, determine its absorbance (data of testing protein sample should not exceed standard curve range)；C：
Compare with standard curve, calculate the concentration of testing sample, be repeated 3 times and average.
(5) fungistatic effect inspection：Final concentration of 10 are added in nutrient broth medium4cfu/mL、105cfu/mL
Bacillus coli DH 5 alpha (Escherichia coli, E.coli DH5 α) (be purchased from Guangdong Province's Culture Collection,
ATCC69790) (it is purchased from Guangdong Province's Microbiological Culture Collection with staphylococcus aureuses (Staphylococcus aureus)
The heart, ATCC6538), flat board containing bacterium (thickness is 4mm) is made, after flat board solidification, the aseptic cattle of a diameter of 8mm is placed thereon
Tianjin cup, and the supernatant obtained by 200 μ L steps (4) is added, 16h is cultivated in 37 DEG C of incubators, the diameter of inhibition zone is observed, to press down
The size of bacterium loop diameter represents fungistatic effect.
After above-mentioned culture terminates, recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 culture fluid after grown cultures is detected
OD600Value reaches 10.3, and the protein content after inducing culture in culture supernatant reaches 85mg/mL, and supernatant carries out antibacterial effect
The result of fruit detection is respectively 10 as shown in figure 1, supernatant is added to concentration4cfu/mL、105Cfu/mL's is flat containing escherichia coli
In plate, antibacterial circle diameter is respectively 2.8cm, 2.5cm；Supernatant is added to concentration and is respectively 104cfu/mL、105Cfu/mL's contains
In staphylococcus aureuses flat board, antibacterial circle diameter is respectively 2.9cm, 2.5cm, shows a large amount of secretion people β in culture supernatant
Alexin 3, fungistatic effect is obvious.
Embodiment 2
(1) the seed liquid 1mL that prepared by Example 1- (1) be inoculated in 100mL sterilizing after Herba Polygoni hydropiperiss growth medium, 29
DEG C, 220r/min shaking culture, cultivate 30h to OD600For 1～4 when stop growing culture, obtain culture fluid.
Herba Polygoni hydropiperiss grown cultures based formulas used are as follows：70g/L glucoses, 6.7g/L YNB, adenine, arginine, half
The each 0.1g/L of cystine, leucine, lysine, threonine and tryptophan, aspartic acid, histidine, isoleucine, first sulfur ammonia
The each 0.05g/L of acid, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 30g/L peptones, 40g/L Herba Polygoni hydropiperiss.
(2) the culture fluid low-speed centrifugal (5000rpm, 10min) in step (1) is taken, supernatant is abandoned, 10mL sterilized water is added
Twice of cleaning, low-speed centrifugal (5000rpm, 10min) abandons supernatant, obtains recombinant Saccharomyces cerevisiae grown cultures thalline.
(3) the recombinant Saccharomyces cerevisiae grown cultures thalline obtained by step (2) is forwarded to the Herba Polygoni hydropiperiss induction after 100mL sterilizings
Culture medium, 29 DEG C, 220r/min shaking cultures, low-speed centrifugal (5000rpm, 10min) after culture 60h are abandoned precipitation, collect supernatant
Liquid, BCA albuminimetries determine the protein content of supernatant.
Herba Polygoni hydropiperiss inducing culture based formulas used are as follows：30g/L glucoses, 6.7g/L YNB, adenine, arginine, half
The each 0.1g/L of cystine, leucine, lysine, threonine and tryptophan, aspartic acid, histidine, isoleucine, first sulfur ammonia
The each 0.05g/L of acid, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 40g/L sucrose, 30g/L yeast extracts,
40g/L Herba Polygoni hydropiperiss.
The operational approach of BCA albuminimetries is consistent with embodiment 1- (4).
(4) fungistatic effect inspection：Final concentration of 10 are added in nutrient broth medium4cfu/mL、105cfu/mL
Bacillus coli DH 5 alpha (E.coli DH5 α) and staphylococcus aureuses (Staphylococcus aureus), make flat containing bacterium
Plate (thickness is 5mm), after flat board solidification, places the aseptic Oxford cup of a diameter of 8mm thereon, and adds 200 μ L steps (3)
The supernatant of gained, cultivates 17h in 37 DEG C of incubators, observe the diameter of inhibition zone, represent antibacterial with the size of antibacterial circle diameter
Effect.
After above-mentioned culture terminates, recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 culture fluid after grown cultures is detected
OD600Value reaches 11.5, and the protein content after inducing culture in culture supernatant reaches 98mg/mL, and supernatant carries out antibacterial effect
The result of fruit detection is respectively 10 as shown in Fig. 2 supernatant is added to concentration4cfu/mL、105Cfu/mL's is flat containing escherichia coli
In plate, antibacterial circle diameter is respectively 3.1cm, 3.0cm；Supernatant is added to concentration and is respectively 104cfu/mL、105Cfu/mL's contains
In staphylococcus aureuses flat board, antibacterial circle diameter is respectively 2.9cm, 2.7cm, shows a large amount of secretion people β in culture supernatant
Alexin 3, fungistatic effect is notable.
Embodiment 3
(1) the seed liquid 1mL that prepared by Example 1- (1) be inoculated in 100mL sterilizing after Herba Polygoni hydropiperiss growth medium, 28
DEG C, 180r/min shaking culture, cultivate 36h to OD600For 1～4 when stop growing culture, obtain culture fluid.
Herba Polygoni hydropiperiss grown cultures based formulas used are as follows：90g/L glucoses, 6.7g/L YNB, adenine, arginine, half
The each 0.1g/L of cystine, leucine, lysine, threonine and tryptophan, aspartic acid, histidine, isoleucine, first sulfur ammonia
The each 0.05g/L of acid, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 40g/L peptones, 60g/L Herba Polygoni hydropiperiss.
(2) the culture fluid low-speed centrifugal (5000rpm, 10min) in step (1) is taken, supernatant is abandoned, 10mL sterilized water is added
Twice of cleaning, low-speed centrifugal (5000rpm, 10min) abandons supernatant, obtains recombinant Saccharomyces cerevisiae grown cultures thalline.
(3) the recombinant Saccharomyces cerevisiae grown cultures thalline obtained by step (2) is forwarded to the Herba Polygoni hydropiperiss induction after 100mL sterilizings
Culture medium, 28 DEG C, 180r/min shaking cultures, low-speed centrifugal (5000rpm, 10min) after culture 72h are abandoned precipitation, collect supernatant
Liquid, BCA albuminimetries determine the protein content of supernatant.
Herba Polygoni hydropiperiss inducing culture based formulas used are as follows：40g/L glucoses, 6.7g/L YNB, adenine, arginine, half
The each 0.1g/L of cystine, leucine, lysine, threonine and tryptophan, aspartic acid, histidine, isoleucine, first sulfur ammonia
The each 0.05g/L of acid, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 50g/L sucrose, 40g/L yeast extracts,
60g/L Herba Polygoni hydropiperiss.
The operational approach of BCA albuminimetries is consistent with embodiment 1- (4).
(4) fungistatic effect inspection：Final concentration of 10 are added in nutrient broth medium4cfu/mL、105Cfu/mL's is big
Enterobacteria DH5 α (E.coli DH5 α) and staphylococcus aureuses (Staphylococcus aureus), make flat board containing bacterium
(thickness is 6mm), after flat board solidification, places the aseptic Oxford cup of a diameter of 8mm thereon, and adds 200 μ L steps (3) institutes
The supernatant for obtaining, cultivates 18h in 37 DEG C of incubators, observe the diameter of inhibition zone, represent antibacterial effect with the size of antibacterial circle diameter
Really.
After above-mentioned culture terminates, recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 culture fluid after grown cultures is detected
OD600Value reaches 10.5, and the protein content after inducing culture in culture supernatant reaches 88mg/mL, and supernatant carries out antibacterial effect
The result of fruit detection is respectively 10 as shown in figure 3, supernatant is added to concentration4cfu/mL、105Cfu/mL's is flat containing escherichia coli
In plate, antibacterial circle diameter is respectively 3.2cm, 3.1cm；Supernatant is added to concentration and is respectively 104cfu/mL、105Cfu/mL's contains
In staphylococcus aureuses flat board, antibacterial circle diameter is respectively 3.0cm, 2.8cm, shows a large amount of secretion people β in culture supernatant
Alexin 3, fungistatic effect is notable.
Embodiment 4
(1) the seed liquid 1mL that prepared by Example 1- (1) is inoculated in the SC-U culture medium after 100mL sterilizings, and 30
DEG C, 250r/min shaking culture, cultivate 36h to OD600For 1～4 when stop growing culture, obtain culture fluid.
SC-U culture medium prescriptions used are as follows：20g/L glucoses, 6.7g/L YNB, adenine, arginine, half Guang ammonia
The each 0.1g/L of acid, leucine, lysine, threonine and tryptophan, aspartic acid, histidine, isoleucine, methionine,
The each 0.05g/L of Phenylalanine, proline, serine, L-Tyrosine and L-Valine.
(2) the culture fluid low-speed centrifugal (5000rpm, 10min) in step (1) is taken, supernatant is abandoned, with sterile water wash two
Time, low-speed centrifugal (5000rpm, 10min) abandons supernatant, obtains recombinant Saccharomyces cerevisiae grown cultures thalline.
(3) the recombinant Saccharomyces cerevisiae grown cultures thalline obtained by step (2) is forwarded to the SC-U cultures after 100mL sterilizings
In base, 30 DEG C, 250r/min shaking cultures, low-speed centrifugal (5000rpm, 10min) after culture 72h are abandoned precipitation, collect supernatant
Liquid, BCA albuminimetries determine the protein content of supernatant.
The operational approach of BCA albuminimetries is consistent with embodiment 1- (4).
(4) fungistatic effect inspection：Final concentration of 10 are added in nutrient broth medium4cfu/mL、105cfu/mL
Bacillus coli DH 5 alpha (E.coli DH5 α) and staphylococcus aureuses (Staphylococcus aureus), make flat containing bacterium
Plate (thickness is 5mm), after flat board solidification, places the aseptic Oxford cup of a diameter of 8mm thereon, and adds 200 μ L steps (3)
The supernatant of gained, cultivates 18h in 37 DEG C of incubators, observe the diameter of inhibition zone, represent antibacterial with the size of antibacterial circle diameter
Effect.
After above-mentioned culture terminates, recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 culture fluid after grown cultures is detected
OD600It is worth for 4.2, the protein content after inducing culture in culture supernatant is 35.5mg/mL, supernatant carries out fungistatic effect
The result of detection is respectively 10 as shown in figure 4, supernatant is added to concentration4cfu/mL、105Cfu/mL containing E. coli plate
In, antibacterial circle diameter is respectively 1.2,1.1cm；Supernatant is added to concentration and is respectively 104cfu/mL、105Cfu/mL containing golden yellow
In color staphylococcuses flat board, antibacterial circle diameter is respectively 1.2cm, 0.8cm.Therefore, general SC-U culture medium, Herba Polygoni hydropiperiss life are compared
Growth of the long culture medium to recombinant Saccharomyces cerevisiae INVSC1/pY α-hBD-3 has obvious facilitation, Herba Polygoni hydropiperiss inducing culture
After culture 72h, in supernatant, the secretory volume of human β defensin 3 is more than the twice of general SC-U culture supernatants.
Embodiment 5
(1) preparation of seed liquid：
The recombinant Saccharomyces cerevisiae INVSC1/pYES2 glycerol strains of preservation are lined in YPD culture medium flat plates, is positioned over
3d is cultivated in 30 DEG C of incubators to growing single bacterium colony；
YPD culture medium prescriptions used are as follows：Glucose 20g/L, yeast extract 10g/L, peptone 20g/L；Solid
Agar 20g/L is added in culture medium.
In above-mentioned single bacterium colony, the full single bacterium colony streak inoculation of picking is on YPD culture medium flat plates, in 30 DEG C of incubators
Middle culture 2d, then the full single bacterium colony of picking is inoculated in YPD fluid mediums, 30 DEG C, 250r/min shake-flask culture overnight, obtain
To seed liquid.
(2) take seed liquid 1mL prepared by step (1) and be inoculated in 100mL Herba Polygoni hydropiperiss growth mediums, 30 DEG C, 250r/min
Shaking culture, cultivates 36h to OD600For 1～4 when stop growing culture, obtain culture fluid.
Herba Polygoni hydropiperiss grown cultures based formulas used are as follows：70g/L glucoses, 6.7g/L YNB, adenine, arginine, half
The each 0.1g/L of cystine, leucine, lysine, threonine and tryptophan, aspartic acid, histidine, isoleucine, first sulfur ammonia
The each 0.05g/L of acid, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 30g/L peptones, 40g/L Herba Polygoni hydropiperiss.
(3) the culture fluid low-speed centrifugal (5000rpm, 10min) in step (2) is taken, supernatant is abandoned, it is clear with 10mL sterilized water
Wash twice, low-speed centrifugal (5000rpm, 10min) abandons supernatant, obtains recombinant Saccharomyces cerevisiae grown cultures thalline.
(4) the recombinant Saccharomyces cerevisiae grown cultures thalline obtained by step (3) is forwarded to into the Herba Polygoni hydropiperiss inducing culture after sterilizing
Base, 30 DEG C, 250r/min shaking cultures, low-speed centrifugal (5000rpm, 10min) after culture 72h are abandoned precipitation, collect supernatant,
BCA albuminimetries determine the protein content of supernatant.
Herba Polygoni hydropiperiss inducing culture based formulas used are as follows：20g/L glucoses, 6.7g/L YNB, adenine, arginine, half
The each 0.1g/L of cystine, leucine, lysine, threonine and tryptophan, aspartic acid, histidine, isoleucine, first sulfur ammonia
The each 0.05g/L of acid, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 40g/L sucrose, 20g/L yeast extracts,
40g/L Herba Polygoni hydropiperiss.
The operational approach of BCA albuminimetries is consistent with embodiment 1- (4).
(5) fungistatic effect inspection：Final concentration of 10 are added in nutrient broth medium4cfu/mL、105cfu/mL
Bacillus coli DH 5 alpha (E.coli DH5 α) and staphylococcus aureuses (Staphylococcus aureus), make flat containing bacterium
Plate (thickness is 5mm), after flat board solidification, places the aseptic Oxford cup of a diameter of 8mm thereon, and adds 200 μ L steps (3)
The supernatant of gained, cultivates 18h in 37 DEG C of incubators, observe the diameter of inhibition zone, represent antibacterial with the size of antibacterial circle diameter
Effect.
After above-mentioned culture terminates, recombinant Saccharomyces cerevisiae INVSC1/pYES2 culture fluid after grown cultures is detected
OD600Value reaches 10.2, and the protein content after inducing culture in culture supernatant reaches 14.6mg/mL, and supernatant carries out antibacterial effect
As a result the result of fruit detection as shown in figure 5, show supernatant without obvious bacteriostasis, therefore embodiment 1- (5), embodiment 2-
(5), in embodiment 3- (5), the fungistatic effect of culture supernatant comes from the secretion of human β defensin 3, rather than the effect of Herba Polygoni hydropiperiss.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any spirit without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (8)
1. a kind of method that utilization Herba Polygoni hydropiperiss promote saccharomyces cerevisiae secreting, expressing human β defensin 3, it is characterised in that including following step
Suddenly：
(1) recombinant Saccharomyces cerevisiae (Saccharomyces cerevisiae) INVSC1/pY α-hBD-3 strains are lined into YPD
In culture medium flat plate, 2～3d are cultivated in 28～30 DEG C to growing single bacterium colony；The streak inoculation of picking single bacterium colony is flat in YPD culture medium
On plate, 1～2d are cultivated in 28～30 DEG C, then picking single bacterium colony is inoculated in YPD fluid mediums, 28～30 DEG C, 180～
250r/min overnight incubations, obtain seed liquid；
(2) take seed liquid prepared by step (1) to be inoculated in Herba Polygoni hydropiperiss growth medium, 28～30 DEG C, 180～250r/min shakes
Culture is shaken, 24～36h to OD is cultivated600For 1～4 when stop growing culture, obtain culture fluid；
(3) the culture fluid low-speed centrifugal obtained in taking step (2), abandons supernatant, and with twice of sterile water wash, low-speed centrifugal is abandoned
Supernatant, obtains recombinant Saccharomyces cerevisiae grown cultures thalline；
(4) the recombinant Saccharomyces cerevisiae grown cultures thalline obtained by step (3) is forwarded to into Herba Polygoni hydropiperiss inducing culture, 28～30 DEG C,
180～250r/min shaking cultures, cultivate 48～72h, collect induction broth；
Herba Polygoni hydropiperiss growth medium described in step (2) is made by the steps：50～90g/L glucoses, 6.7g/L YNB, gland
The each 0.1g/L of purine, arginine, cysteine, leucine, lysine, threonine and tryptophan, aspartic acid, histidine,
The each 0.05g/L of isoleucine, methionine, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 20～40g/L eggs
White peptone, 20～60g/L Herba Polygoni hydropiperiss；It is standby after 121 DEG C of autoclaving 20min；
Herba Polygoni hydropiperiss inducing culture described in step (4) is made by the steps：20～40g/L glucoses, 6.7g/L YNB, gland
The each 0.1g/L of purine, arginine, cysteine, leucine, lysine, threonine and tryptophan, aspartic acid, histidine,
The each 0.05g/L of isoleucine, methionine, Phenylalanine, proline, serine, L-Tyrosine and L-Valine, 30～50g/L sugarcanes
Sugar, 20～40g/L yeast extracts, 20～60g/L Herba Polygoni hydropiperiss；It is standby after 121 DEG C of autoclaving 20min.
2. the method that utilization Herba Polygoni hydropiperiss according to claim 1 promote saccharomyces cerevisiae secreting, expressing human β defensin 3, its feature
It is also to comprise the steps：
(5) induction broth for collecting step (4), low-speed centrifugal, abandon precipitation, collect supernatant, and BCA albuminimetries are determined
The protein content of supernatant；
(6) bacterium solution is added in nutrient broth medium, makes flat board containing bacterium, after flat board solidification, place Oxford cup thereon,
And add supernatant obtained by step (5), 37 DEG C of 16～18h of culture to observe the diameter of inhibition zone, with the size of antibacterial circle diameter
Represent fungistatic effect.
3. the method that utilization Herba Polygoni hydropiperiss according to claim 1 promote saccharomyces cerevisiae secreting, expressing human β defensin 3, its feature
It is：Seed liquid described in step (2) is inoculated in Herba Polygoni hydropiperiss growth medium, by volume (1～3)：100 ratio connects
Kind.
4. the method that utilization Herba Polygoni hydropiperiss according to claim 1 promote saccharomyces cerevisiae secreting, expressing human β defensin 3, its feature
It is：Recombinant Saccharomyces cerevisiae grown cultures thalline described in step (4) is forwarded to Herba Polygoni hydropiperiss inducing culture, is every 100mL trainings
Nutrient solution is collected the recombinant Saccharomyces cerevisiae grown cultures thalline for obtaining and is forwarded to 100mL Herba Polygoni hydropiperiss inducing cultures.
5. the method that utilization Herba Polygoni hydropiperiss according to claim 1 promote saccharomyces cerevisiae secreting, expressing human β defensin 3, its feature
It is：Herba Polygoni hydropiperiss described in step (2), (4) refer to Herba Polygoni hydropiperiss dry powder, and preparation method is as follows：Plucked in 6～July, 40～80cm high Herba Polygoni hydropiperiss
(Polygonum hydropiper) plant, cryogenic vacuum 12～16h of lyophilization after cleaning, after being clayed into power with pulverizer,
Cross 100 mesh sieves standby.
6. the method that utilization Herba Polygoni hydropiperiss according to claim 2 promote saccharomyces cerevisiae secreting, expressing human β defensin 3, its feature
It is：Bacterium solution described in step (6) is escherichia coli (Escherichia coli) or staphylococcus aureuses
The bacterium solution of (Staphylococcus aureus)；Described escherichia coli or final concentration of the 10 of staphylococcus aureuses4~
105cfu/mL。
7. the method that utilization Herba Polygoni hydropiperiss according to claim 2 promote saccharomyces cerevisiae secreting, expressing human β defensin 3, its feature
It is：
The thickness containing bacterium flat board described in step (6) is 4～6mm；
A diameter of 8mm of the Oxford cup described in step (6).
8. the utilization Herba Polygoni hydropiperiss described in any one of claim 1～7 promote the method for saccharomyces cerevisiae secreting, expressing human β defensin 3 to exist
Improve the application in saccharomyces cerevisiae secreting, expressing human β defensin 3.
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