CN101003809A - Method for preparing engineering bacterium for recombining plant alexin PD5 till its products - Google Patents
Method for preparing engineering bacterium for recombining plant alexin PD5 till its products Download PDFInfo
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Abstract
This invention relates to a method for preparing recombinant plant defensin PD5 with engineering bacteria. The method comprises: amplifying plant defensin PD5 gene, cloning the gene, transferring E. coli, transforming Pichia, expressing engineering bacteria product with a high efficiency, and purifying plant defensin PD5. The method has such advantages as high yield, simple purification process, and low cost. The obtained recombinant plant defensin PD5 can be used to treat crop diseases, perserve food and fruits, antisepticize cosmetics, and manufacture feed additives and medical products.
Description
[technical field]
The invention belongs to gene engineering technology field, relate in particular to the preparation method of the engineering bacteria of a kind of recombinant plant alexin PD5 until product.
[background technology]
Harmful microorganism is human being's production, life and a healthy important injurious factor always, on agricultural, can cause the disease of farm crop, in aquaculture, can cause animal ill, in foodstuffs industry, can produce and rot to go mouldy, and for human beings'health fearful killer especially.The human development in struggling against with these harmful microbes their means of many controls.Agricultural chemicals is in agricultural, and sanitas is on food, and the widespread use of microbiotic in animal, human medical treatment obtained great success; But because itself there is bad or unfavorable factor in these means, bring disadvantageous effect for the mankind and environment, as the pollution of agricultural chemicals to environment, sanitas is to the detrimentally affect of human health, and antibiotic side effect and the chemical sproof generation of microorganism etc., force the constantly new better control harmful microbe means of development of human needs.
Antibacterial peptide is the non-specific immunity factor that a class can be resisted multiple pathogenic bacterial infection, extensively is present in the various organisms, has that molecular weight is little, thermostability is high, characteristics such as good water solubility, sterilizing power are strong, has a broad antifungal spectrum.The antibacterial peptide of Fa Xianing is cecropin (cecropin) the earliest, is separated from cherish guppy sky silkworm chrysalis and is obtained by the rub Boman of university of this moral GoerTek of Sweden.From comprise various organisms such as microorganism, animal, plant and the mankind, separate, identify nearly 1500 antibacterial peptide genes or gene fragment at present.The antibacterial peptide in various sources there are differences at aspects such as antimicrobial spectrums.Plant alexin is molecule amount little (about 5kD is made up of 45-54 amino acid), be alkalescence, be rich in halfcystine, have the small peptide of complex three-dimensional pleated sheet structure, under the low concentration level, just can suppress a series of phytopathogens and other pathogenic bacterias, but nontoxic to people, animal, vegetable cell.
Plant alexin PD5 separates the polypeptide with anti-microbial activity that obtains from Queensland nut.The separated clone of its gene (secreting, expressing of plant alexin PD5 gene in pichia spp, Long Liangkun etc., 2006, the microbiology circular, 33 (5): 65～69), 150 Nucleotide of its nucleotide sequence total length, one section 50 amino acid whose polypeptide, i.e. plant alexin PD5 encode.The molecular weight 5627.35Da of plant alexin PD5, theoretical iso-electric point is 9.30.
From Queensland nut, separate the natural phant alexin PD5 that obtains, not only to phytopathogen such as Cuba point sickle spore bacterium (Fusarium oxysporum f.sp.cubense), cabbage black rot bacterium (Xanthomonacampestris) etc. has had strong inhibitory effects, and to intestinal bacteria (Escherichia coli), streptococcus aureus (Staphylococcus aureaus), Salmonellas (Salmonella sp.), the false unit cell people such as (Pseudomonas aeruginosa) of verdigris, the poultry pathogenic bacterium all have the obvious suppression effect, at crop disease control, the food fruit freshness preserving, fodder additives, aspects such as the pharmaceutical prod (secreting, expressing of plant alexin PD5 gene in pichia spp that have a wide range of applications, Long Liangkun etc., 2006, the microbiology circular, 33 (5): 65～69).But natural phant alexin PD5 is low at the plant materials intensive amount, the separation purification difficult, thereby causes production cost too high, is not easy to use of large-scale production.
[summary of the invention]
The present invention aims to provide the preparation method of the engineering bacteria of a kind of recombinant plant alexin PD5 until product, and this method cost is low, and the expression of polypeptides amount height of acquisition is easy to realize industrialization.
The engineering bacteria of a kind of recombinant plant alexin PD5 of the present invention is until the preparation method of product, and its operation steps is:
The amplification of A, plant alexin PD5 gene: in the presence of synthetic primer, be template,, obtain plant alexin PD5 gene DNA fragment by the pcr gene amplification with recombinant plasmid PTG/PD5;
B, gene clone and intestinal bacteria transform: with restriction enzyme XhoI and EcoR I respectively enzyme cut the gene DNA fragment and the expression vector pPIC9 plasmid of amplification acquisition, ligase enzyme is cut product and is converted into competent escherichia coli cell, through the screening of resistance substratum, obtain to contain the intestinal bacteria of recombinant expression vector pPIC-PD5;
C, pichia spp transform: extracting recombinant expression vector pPIC-PD5 plasmid from intestinal bacteria, cut this plasmid with restriction enzyme Bg/lII and SalI enzyme respectively, enzyme is cut the competent cell that product is converted into pichia spp, screen through selective medium, obtain the pichia spp of positive colony, i.e. recombinant plant alexin PD5 engineering bacteria;
D, with the engineering bacteria enlarged culturing, abduction delivering, plant alexin PD5 purifying.
The engineering bacteria of a kind of recombinant plant alexin PD5 of the present invention is until the preparation method of product, and more preferably operation steps is:
A. the amplification of plant alexin PD5 gene:
Synthetic contains the primer of restriction enzyme enzyme recognition site, introduces the encoding sequence of XhoI recognition site and signal peptide cutting site Kex2 in the synthetic upstream primer, introduces EcoR I site in the synthetic downstream primer, and the sequence of its synthetic primer is:
In the presence of this synthetic primer, be template with recombinant plasmid PTG/PD5, by the pcr gene amplification, obtain plant alexin PD5 gene DNA fragment.
The PCR reaction system is: 0.1 μ L plasmid DNA template, 2.0 μ L10 * reaction buffer, 0.4 μ L10mmol/L dNTPs, 1.0 μ L 5 μ mol/L primer P1,1.0 μ L 5 μ mol/L primer P2,0.2 μ L 5U/ μ LEx Taq enzyme replenishes distilled water to cumulative volume 20 μ L.Reaction parameter is: 94 ℃ of pre-sex change 4min, and 94 ℃ of sex change 30sec, 54 ℃ of annealing 1min, 72 ℃ are extended 30sec, and 30 circulations are extended 10min for back 72 ℃.Reaction product reclaims according to producer's explanation with DNA Gel Extraction Kit.
B. gene clone and intestinal bacteria transform
With restriction enzyme XhoI and EcoR I respectively enzyme cut gene DNA fragment and expression vector pPIC9 plasmid.Enzyme is cut product and connected according to following reaction system: the enzyme that adds about 200ng gene DNA fragment in the PCR pipe successively cuts back to close product, the enzyme of 50ng pPIC9 plasmid cuts back to close product, 5 μ L connect liquid, replenish ddH2O to cumulative volume 1 μ L, and 16 ℃ of following reactions are spent the night.
Getting 5 μ L connection product joins in the intestinal bacteria Top10 competent cell suspension of 100 μ L, Calcium Chloride Method transforms, coat LB substratum (containing penbritin), cultivate 16h for 37 ℃, get positive colony, to carry out gene sequencing behind the alkaline lysis method of extracting recombinant expression vector pPIC-PD5 plasmid, sequencing result (as shown in Figure 3) shows that plant alexin PD5 gene correctly is cloned in the expression vector.
C. pichia spp transforms: extracting recombinant expression vector pPIC-PD5 plasmid from intestinal bacteria, cut this plasmid with restriction enzyme BglII and SalI enzyme respectively.Getting enzyme cuts product 1 μ L and mixes with the competent cell of 50 μ L pichia spp GS115 or SMD1163, after carrying out the electricity conversion under the condition of voltage 1.5kV, electric capacity 25 μ F, resistance 200 Ω, electric shock time 10msec, coat MD and select substratum, cultivate 72h for 30 ℃, the pichia spp of picking positive colony, i.e. recombinant plant alexin PD5 engineering bacteria.
D. with the engineering bacteria enlarged culturing, abduction delivering, plant alexin PD5 purifying:
1, the enlarged culturing of engineering bacteria: picking efficiently expresses the single bacterium colony of engineering bacteria of recombinant plant alexin PD5, at YPD (1% yeast extract, 2% peptone, 2% glucose) or BMGY (1% yeast extract, 2% peptone, 100mM pH6.0 potassium phosphate buffer, 1.34% yeast nitrogen, 4 * 10
-5% vitamin H, 1% glycerine) vibrate cultivation in 30 ℃, 200rpm among the liquid nutrient medium 100mL.Work as OD
600During=2-6, the centrifugal collection thalline of 1500g.
2, the abduction delivering of engineering bacteria: will collect the thalline that obtains with 200～600mLMM (1.34% yeast nitrogen, 4 * 10
-5The % vitamin H, 0.5% methyl alcohol) or BMMY (1% yeast extract, 2% peptone, 100mM pH6.0 potassium phosphate buffer, 1.34% yeast nitrogen, 4 * 10
-5% vitamin H, 0.5% methyl alcohol) substratum is resuspended, makes OD
600=1, vibrate cultivation in 30 ℃, 200rpm, it is 0.5% that every 24h replenishes methyl alcohol to final concentration, coinduction is cultivated 96h.
3, purifying makes recombinant plant alexin PD5 albumen: with the centrifugal 5min of nutrient solution 10000g, collect supernatant liquor, carry out column chromatography with HiTrap CM Sepharose Fast Flow ion exchange column.With 10 times of volume buffer A (0.05M NH
4AC, pH5.4) balance cylinder, last sample is with buffer A liquid (NH
4Ac0.05M, pH 5.4, and 5ml 0.2ml/min) is washed till the registering instrument baseline and walks flatly, uses buffer B (0.05MNH again
4Ac, 1M NaCl pH 9.1,10ml, 1ml/min) gradient elution is collected the elutriant that penetrates peak and each component respectively, with Tricine-SDS-PAGE analyzing proteins wash-out situation.The elutriant that contains recombinant plant alexin PD5 protein ingredient is collected the elutriant that contains recombinant plant alexin PD5 protein ingredient with molecular sieve Sephadex G-25 desalination, and lyophilize promptly makes recombinant plant alexin PD5 albumen dry product.
Beneficial effect of the present invention is:
(1) as Fig. 2, the expression cassette of plant alexin PD5 gene in engineering bacteria is P
Aox1-S-pd5-TT (P
Aox1Be the promotor of alcohol oxidase gene aox1, S is the a-factor secretory signal sequence, and TT is the terminator of alcohol oxidase gene aox1), this expression system expression amount height, good stability, active high, be convenient to suitability for industrialized production.
(2) genetic engineering bacterium of the recombinant plant alexin PD5 that makes up by the genetic engineering technique method has improved the expression amount of recombinant plant alexin PD5 greatly.
(3) expression product is present in the expression supernatant, only needs centrifugal collection supernatant, just can obtain pure recombinant plant alexin PD5 through primary column chromatography and desalination, just can obtain pure recombinant plant alexin PD5 dry product through lyophilize again.
(4) produce recombinant plant alexin PD5 by the method for genetic engineering technique,, significantly reduce production costs than chemosynthesis or purification natural phant alexin PD5 method.
Thereby method output height of the present invention, purifying process is simple, and production cost is low.
The recombinant plant alexin PD5 that the present invention makes can be used for aspects such as crop disease control, food fruit freshness preserving, makeup be anticorrosion, can also be used to make fodder additives, pharmaceutical prod etc.
[description of drawings]
Fig. 1 is the design of graphics of recombinant expression vector pPIC-PD5;
Fig. 2 is the expression cassette synoptic diagram of plant alexin PD5 gene in engineering bacteria;
Fig. 3 is the sequencing result figure of recombinant expression vector pPIC-PD5;
Fig. 4 is that recombinant plant alexin PD5 is to the active figure of the inhibition of banana blight bacteria (Fusarium oxysporumf.sp.cubense);
Fig. 5 is that recombinant plant alexin PD5 is to the active figure of the inhibition of streptococcus aureus ATCC6538;
Fig. 6 is that recombinant plant alexin PD5 is to the active figure of the inhibition of intestinal bacteria ATCC8739.
[embodiment]
The engineering bacteria of embodiment 1 preparation recombinant plant alexin PD5 is until product
The amplification of A, plant alexin PD5 gene:
Synthetic contains the primer of restriction enzyme enzyme recognition site, introduces the encoding sequence of XhoI recognition site and signal peptide cutting site Kex2 in the synthetic upstream primer, introduces EcoR I site in the synthetic downstream primer, and the sequence of its synthetic primer is:
In the presence of this synthetic primer, be template with recombinant plasmid PTG/PD5 (Guangdong Microbes Inst DSMZ provides), by the pcr gene amplification, obtain plant alexin PD5 gene DNA fragment;
The PCR reaction system is: 0.1 μ L plasmid DNA template, 2.0 μ L10 * reaction buffer, 0.4 μ L10mmol/L dNTPs, 1.0 μ L 5 μ mol/L primer P1,1.0 μ L 5 μ mol/L primer P2,0.2 μ L 5U/ μ LEx Taq enzyme replenishes distilled water to cumulative volume 20 μ L.Reaction parameter is: 94 ℃ of pre-sex change 4min, and 94 ℃ of sex change 30sec, 54 ℃ of annealing 1min, 72 ℃ are extended 30Sec, and 30 circulations are extended 10min for back 72 ℃.Reaction product reclaims according to producer's explanation with DNA Gel Extraction Kit.
B, gene clone and intestinal bacteria transform:
With restriction enzyme XhoI (available from Invitrogen company) and EcoR I (available from Invitrogen company) according to producer's operation instructions respectively enzyme cut gene DNA fragment and expression vector pPIC9 plasmid (available from Invitrogen company).Enzyme is cut product and connected according to following reaction system: the enzyme that adds about 200ng gene DNA fragment in the PCR pipe successively cuts back to close product, and the enzyme of 50ng pPIC9 plasmid cuts back to close product, and 5 μ L connect liquid, replenishes ddH
2O is to cumulative volume 10 μ L, and 16 ℃ of following reactions are spent the night.
Getting 5 μ L connection product joins in the intestinal bacteria Top10 competent cell suspension of 100 μ L, Calcium Chloride Method transforms, coat LB substratum (containing penbritin), cultivate 16h for 37 ℃, get positive colony, to carry out gene sequencing behind the alkaline lysis method of extracting recombinant expression vector pPIC-PD5 plasmid, sequencing result (as shown in Figure 3) shows that plant alexin PD5 gene correctly is cloned in the expression vector.
C, pichia spp transform:
Extracting recombinant expression vector pPIC-PD5 plasmid from intestinal bacteria is cut this plasmid with restriction enzyme BglII and SalI according to producer's operation instructions enzyme respectively.Getting enzyme cuts product 1 μ L and mixes with the competent cell of 50 μ L pichia spp GS115 or SMD1163, after carrying out the electricity conversion under the condition of voltage 1.5kV, electric capacity 25 μ F, resistance 200 Ω, electric shock time 10msec, coat MD and select substratum, cultivate 72h for 30 ℃, the pichia spp of picking positive colony, i.e. recombinant plant alexin PD5 engineering bacteria.
D, with the engineering bacteria enlarged culturing, abduction delivering, plant alexin PD5 purifying.
1. the enlarged culturing of engineering bacteria
Picking efficiently expresses the single bacterium colony of engineering bacteria of recombinant plant alexin PD5, at YPD (1% yeast extract, 2% peptone, 2% glucose) or BMGY (1% yeast extract, 2% peptone, 100mM pH6.0 potassium phosphate buffer, 1.34% yeast nitrogen, 4 * 10
-5% vitamin H, 1% glycerine) vibrate cultivation in 30 ℃, 200rpm among the liquid nutrient medium 100mL.Work as OD
600During=2-6,1500g, the centrifugal collection thalline of 5min.
2. the abduction delivering of engineering bacteria
With the thalline that collect to obtain with 200～600mLMM (1.34% yeast nitrogen, 4 * 10
-5The % vitamin H, 0.5% methyl alcohol) or BMMY (1% yeast extract, 2% peptone, 100mM pH6.0 potassium phosphate buffer, 1.34% yeast nitrogen, 4 * 10
-5% vitamin H, 0.5% methyl alcohol) substratum is resuspended, makes OD
600=1, vibrate cultivation in 30 ℃, 200rpm, carry out abduction delivering, it is 0.5% that every 24h replenishes methyl alcohol to final concentration, coinduction is cultivated 96h.
3. purifying makes recombinant plant alexin PD5 albumen
With the nutrient solution of abduction delivering, the centrifugal 5min of 10000g collects supernatant liquor, carries out column chromatography with HiTrap CMSepharose Fast Flow ion exchange column.With 10 times of volume buffer A (0.05MNH
4AC, pH5.4) balance cylinder, last sample is with buffer A liquid (NH
4Ac 0.05M, pH5.4,5ml 0.2ml/min) is washed till the registering instrument baseline and walks flatly, uses buffer B (0.05M NH again
4Ac, 1M NaCl pH9.1,10ml, 1ml/min) gradient elution is collected the elutriant that penetrates peak and each component respectively, with Tricine-SDS-PAGE analyzing proteins wash-out situation.The elutriant that contains recombinant plant alexin PD5 protein ingredient is collected the elutriant that contains recombinant plant alexin PD5 protein ingredient with molecular sieve Sephadex G-25 desalination, and lyophilize promptly makes recombinant plant alexin PD5 albumen dry product.Every liter of nutrient solution can make pure product 133mg approximately.
Embodiment 2 recombinant plant alexin PD5 are to the restraining effect of banana blight bacteria (Fusarium oxysporum f.sp.cubense)
Banana blight bacteria (Fusarium oxysporum f.sp.cubense) (Guangdong Microbes Inst DSMZ provides) is cultivated 14d in 25 ℃ on the PDA flat board, treat that it produces spore, under aseptic condition, wash spore with sterilized water, filtered through gauze, collect spore, make bacteria suspension, bacteria suspension is joined among the PDA of thawing, make spore concentration and be about 1 * 10
5Contain the bacterium flat board, punch tool punching joins the recombinant plant alexin PD5 albumen of 20 μ l 133mg/L in the hole, cultivates 2d in 25 ℃, observe fungistatic effect, result's (seeing accompanying drawing 4) shows that recombinant plant alexin PD5 albumen has the activity of resisting banana vascular wilt bacterium.
With (ATCC6538) overnight incubation on the NA substratum of streptococcus aureus (Staphylococcus aureus subsp.Rosenbach), collect thalline, resuspended with sterilized water, this bacteria suspension is joined among the NA of thawing, make bacterial concentration and be about 1 * 10
6Cfu/ml contains the bacterium flat board, punch tool punching joins the recombinant plant alexin PD5 albumen of 20 μ l 133mg/L in the hole, cultivates 1d in 28 ℃, observe fungistatic effect, result's (seeing accompanying drawing 5) shows that recombinant plant alexin PD5 albumen has the activity of anti-streptococcus aureus.
Embodiment 4 recombinant plant alexin PD5 are to the restraining effect of intestinal bacteria (Escherichia coli)
With intestinal bacteria (Escherichia coli) (ATCC87 39) overnight incubation on the NA substratum, collect thalline, resuspended with sterilized water, this bacteria suspension is joined among the NA of thawing, make bacterial concentration and be about 1 * 10
6Cfu/ml contains the bacterium flat board, punch tool punching joins the recombinant plant alexin PD5 albumen of 20 μ l 133mg/L in the hole, cultivates 1d in 37 ℃, observe fungistatic effect, result's (seeing accompanying drawing 6) shows that recombinant plant alexin PD5 albumen has anticolibacillary activity.
Sequence table
＜110〉Guangdong Microbes Inst
＜120〉engineering bacteria of recombinant plant alexin PD5 is until the preparation method of product
<130>HB06003
<140>200610124282.1
<141>2006-12-18
<160>3
<170>PatentIn?version?3.3
<210>1
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉upstream primer
<400>1
ggccgctcga?gaaaagaggt?agaaagctgt?gtg 33
<210>2
<211>26
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉downstream primer
<400>2
ctgcaggaat?tcctattaat?gacaat 26
<210>3
<211>790
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(100)..(249)
＜223〉plant alexin PD5 gene
<400>3
ttgccatttt?ccaacagcac?aaataacggg?ttattgttta?taaatactac?tattgccagc 60
attgctgcta?aagaagaagg?ggtaagcttg?gacaagagag?gtagaaagct?gtgtgattct 120
ctgagccata?aattcagggg?tttgtgctta?agccacggga?actgtgctaa?tgtgtgtcgg 180
actgagggtt?ttcccggcgg?tcattgcaaa?gggttccggc?gtcgttgctt?ctgtgagaag 240
aattgtcatt?aataggaatt?cctgcagccc?gggggatctc?ccatgtctct?actggtggtg 300
gtgcttcttt?ggaattattg?gaaggtaagg?aattgccagg?tgttgctttc?ttatccgaaa 360
agaaataaat?tgaattgaat?tgaaatcgat?agatcaattt?ttttcttttc?tctttcccca 420
tcctttacgc?taaaataata?gtttatttta?ttttttgaat?attttttatt?tatatacgta 480
tatatagact?attatttatc?ttttaatgat?tattaagatt?tttattaaaa?aaaaattcgc 540
tcctctttta?atgcctttat?gcagtttttt?ttttcccatt?cgatatttct?atgttcgggg 600
ttcagcgtat?tttaagttta?ataactcgaa?aaattctgcg?ttccgttaaa?gctcctgcct 660
cgcgccgttt?ccggggatga?cgggtgaaaa?acctctgaca?ccatgcagct?acccggagaa 720
cggtcacagc?cttggtctgt?caacctgaat?gccggggagc?aaaacaaagc?ccgtcagggc 780
ggccaccggg 790
Claims (10)
1, the engineering bacteria of recombinant plant alexin PD5 is characterized in that until the preparation method of product its operation steps is:
The amplification of A, plant alexin PD5 gene: in the presence of synthetic primer, be template,, obtain plant alexin PD5 gene DNA fragment by the pcr gene amplification with recombinant plasmid PTG/PD5;
B, gene clone and intestinal bacteria Top10 transform: with restriction enzyme Xho I and EcoR I respectively enzyme cut the gene DNA fragment and the expression vector pPIC9 plasmid of amplification acquisition, ligase enzyme is cut product and is converted into intestinal bacteria Top10 competent cell, through the screening of resistance substratum, obtain to contain the intestinal bacteria Top10 of recombinant expression vector pPIC-PD5;
C, pichia spp transform: extracting recombinant expression plasmid pPIC-PD5 plasmid from intestinal bacteria Top10, cut this recombinant expression plasmid pPIC-PD5 with restriction enzyme Bg1II and Sa1I enzyme respectively, enzyme is cut the competent cell that product is converted into pichia spp GS115 and SMD1163, screen through selective medium, obtain the pichia spp GS115 and the SMD1163 of positive colony, i.e. recombinant plant alexin PD5 engineering bacteria;
D, with the engineering bacteria enlarged culturing, abduction delivering, plant alexin PD5 purifying.
2, the method for claim 1, it is characterized in that in the steps A, introduce the encoding sequence of XhoI recognition site and signal peptide cutting site Kex2 in the synthetic upstream primer, introduce the EcoRI recognition site in the synthetic downstream primer, the sequence of its synthetic primer is:
P1 5′ggc?cgc?tcga?gaa?aag?agg?tag?aaa?gct?gtg?tg?3′，
P2 5′ctg?cag?gaa?ttc?cta?tta?atg?aca?at?3′。
3, the method for claim 1 is characterized in that containing penbritin in the resistance substratum among the step B.
4, the method for claim 1 is characterized in that the selection substratum among the step C is a MD auxotroph substratum.
5, the method for claim 1 is characterized in that pichia spp GS115 and SMD1163 have inserted plant alexin PD5 gene in the chromogene group, and its expression cassette is P
Aox1-S-pd5-TT, wherein, P
Aox1Be the promotor of alcohol oxidase gene aox1, S is the a-factor secretory signal sequence, and TT is the terminator of alcohol oxidase gene aox1.
6, the method for claim 1 is characterized in that the enlarged culturing of engineering bacteria among the step D, and concrete steps are: engineering bacteria is vibrated cultivation in YPD or BMGY liquid nutrient medium, work as OD
600During=2-6, the centrifugal collection thalline of 1500g.
7, the method for claim 1 is characterized in that the abduction delivering of engineering bacteria among the step D, and concrete steps are: the thalline that enlarged culturing obtains is resuspended with MM or BMMY substratum, make OD
600=1, vibration is cultivated, and it is 0.5% that every 24h replenishes methyl alcohol to final concentration, cultivates 96h.
8, the method for claim 1, the purifying that it is characterized in that engineering bacteria among the step D, concrete steps are: the nutrient solution that centrifugal abduction delivering obtains, collect supernatant liquor, carry out column chromatography, collect recombinant plant alexin PD5 protein ingredient, lyophilize obtains recombinant plant alexin PD5 albumen.
9, method as claimed in claim 8, it is characterized in that 10, the centrifugal collection supernatant liquor of 000g, 5min, supernatant liquor carries out column chromatography with HiTrap CM Sepharose Fast Flow ion exchange column: earlier with distilled water pipe blow-through and cylinder, use 10 times of volume buffer A (0.05M NH4AC then, pH 5.4) the balance cylinder, last sample is with buffer A liquid (NH
4Ac 0.05M, pH 5.4, and 5ml 0.2ml/min) is washed till the registering instrument baseline and walks flatly, uses buffer B (0.05M NH again
4Ac, 1M NaCl pH 9.1,10ml, 1ml/min) gradient elution is collected the elutriant that penetrates peak and each component respectively, in Tricine-SDS-PAGE analyzing proteins wash-out situation; Contain the elutriant molecular sieve Sephadex G-25 desalination of recombinant plant alexin PD5 protein ingredient.
10,, it is characterized in that vibration culture condition wherein is: 30 ℃, 200rpm as claim 6 or 7 or 8 described methods.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586256A (en) * | 2012-01-16 | 2012-07-18 | 华南理工大学 | Method for expressing human beta-defensin 3 in yeast expression system |
| CN103893022A (en) * | 2014-04-04 | 2014-07-02 | 广州舒泰生物技术有限公司 | Application of antibacterial peptide as preservative in preparing cosmetics |
| CN108948163A (en) * | 2018-08-01 | 2018-12-07 | 四川百可馨生物制药有限公司 | Queensland nut plant alexin and its application |
-
2006
- 2006-12-18 CN CN 200610124282 patent/CN101003809A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586256A (en) * | 2012-01-16 | 2012-07-18 | 华南理工大学 | Method for expressing human beta-defensin 3 in yeast expression system |
| CN103893022A (en) * | 2014-04-04 | 2014-07-02 | 广州舒泰生物技术有限公司 | Application of antibacterial peptide as preservative in preparing cosmetics |
| CN108948163A (en) * | 2018-08-01 | 2018-12-07 | 四川百可馨生物制药有限公司 | Queensland nut plant alexin and its application |
| CN108948163B (en) * | 2018-08-01 | 2020-11-06 | 四川百可馨生物制药有限公司 | Macadamia nut plant defensin and application thereof |
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