CN102318752A - Nisin active protein feedstuff and preparation and application methods thereof - Google Patents
Nisin active protein feedstuff and preparation and application methods thereof Download PDFInfo
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Abstract
The invention discloses nisin active protein feedstuff and preparation and application methods thereof. An ideal eukaryotic protein high-level expression system of methylotrophs pichia pastoris is used, the black carp fish growth hormone genes are directionally cloned to a yeast integrative plasmid carrier, and efficiently expressed recombination fish growth hormone genetic engineering yeast is obtained through conversion, screening and inducible expression. Rice protein is used as fermentation culture materials for carrying out biological fermentation to prepare the nisin active protein feedstuff, then, the nisin active protein feedstuff is directly used or is mixed with common fish feedstuff to be used, when the production cost of the feedstuff is reduced, and the growth promotion and disease resistance improvement effects can be realized, so the fish culture benefits are increased.
Description
Technical field
The present invention relates to a kind of preparation method and application of bacterium peptide active protein feed, belong to the fishery cultivating field.
Background technology
Along with the development that aquatic products industry is produced, the essential protein feed of aquatic products more and more lacks.Global aquatic products industry desired protein was 2.3 hundred million tons in 1980, and nineteen ninety, this numeral surpassed 3.2 hundred million tons, and this numeral surpassed 600,000,000 tons in 2000, and reached about 1,300,000,000 tons to this numeral in 2006.Wherein the major protein source of aquatic feeds is rice protein, cotton dregs, the dish dregs of rice, vinasse etc., and therefore, the protein feed source of development of new improves efficiency of feed utilization and becomes present feed resource research focus with reduction Feed Manufacturing cost.
Yeast single cell protein has in remarkable advantages such as the yeast dry matter protein content up to 50% as feed; Contain abundant enzyme in the saccharomycete; Contain beta glucan (57.0%), manna oligosacchride (6.6%), glycoprotein (22.0%) and chitin isoreactivity composition; Both aquatic livestock was had somatotrophic effect, and can improve immunity of organism again, as far as aquatic livestock, yeast feed is a kind of desirable biological activity protein feed.Fast because of the yeast speed of production again, the cycle is short, is easy to characteristics such as genetic manipulation, and it is the desirable expressed receptor system of foreign gene.
Growth hormone (Growth hormone; GH) be a kind of single chain polypeptide hormone that comprises all vertebrate pituitary gland secretions of fish; Promote body growth to grow because it has, quicken the physiological function (Donaldson et al., 1979) of the synthetic and lipid degradation of albumen
[1], make it in the fish aquaculture industry, have great function, thereby obtain attention more and more widely.The mankind have utilized technique for gene engineering to synthesize the reorganization GH (rGH) of mammality and fish, and proof GH can be got into the activity (Hertz Y, 1991) of blood and maintenance promotion fish growth by the complete absorption of fish alimentary canal
[2]Xiao Dong etc., literary composition (Xiao Dong, 2000)
[3]Research shows that exogenous growth hormone just can decompose in 90%, 4 day in 24 hours and all disappears, can in the fish body, not accumulate in the fish body.Sun Xun etc. (Sun Xun, 1999)
[4], confirm that albonubes somatotropic hormone does not have any activity to mammiferous growth hormone receptor.Above result of study has been eliminated the doubt such as residual and safe of people to exogenous growth hormone.But the growth hormone of natural pig, ox, sheep, chicken, fish or utilize growth hormone that genetic engineering bacterium produces the people to the combination vigor of albonubes somatotropic hormone acceptor than low 150 times of the growth hormone of fish own.This results suggest changes the fish growth hormone genetic recombination over to and can be used as feed in the " Yishengsu " yeast and be widely used in during freshwater fish cultures, and this has crucial meaning to exploitation novel protein fish meal.
Summary of the invention
The black carp growth hormone gene that the objective of the invention is to recombinate directionally is incorporated in the Pichia pastoris and makes it to efficiently express; Utilize rice protein to carry out biofermentation for culture; Develop a kind of green, safety, efficient and contain the novel fodder of fish growth hormone and the methods for making and using same of this feed.
The objective of the invention is to realize in the following manner.
A kind of preparation method of bacterium peptide active protein feed:
(1) in fermentation tank, the zymotic fluid that contains following mass percent component ferments: derivant 0.5-1%, rice protein concentration 6-7%, fish growth hormone gene engineering microzyme seed liquor 5-6%, and all the other are water; The condition of fermentation is following: fermentation time 96h, fermentation temperature 28-30 ℃, pH6.0-7.5, rotating speed 300rpm, throughput 3L/min;
(2) mixture that fermentation is obtained carries out spray-drying and promptly gets;
Described fish growth hormone gene engineering microzyme is with the directed expression vector pPIC 3.5k that contains strong promoter AOX I, the construction of expression vector pPIC3.5K-bcGH of inserting of black carp growth hormone cDNA; Again its electric shock being transformed importing histidine defect type yeast GS115 bacterial strain obtains; With the seed liquor of the fish growth hormone gene engineering microzyme that obtains through the usefulness that obtains after the following activation culture process fermenting: the reorganization bcGH barms line of getting-80 ℃ of refrigerator preservations is transferred to the MD flat board; 30 ℃ of constant temperature are inverted and were cultivated 3-5 days; Connecing the dull and stereotyped seed of the well-grown MD of 1 ring shakes in the bottle to the 500mL that the 50mL seed culture medium is housed; Place 300rpm constant temperature shaking table, 30 ℃ of shaken cultivation bacterium liquid OD600 to 2-6, the time is to get final product in 10-24 hour.
Fermentation time 96h in the step (1), pH6.5, fish growth hormone gene engineering microzyme seed liquor 6%, 30 ℃ of fermentation temperatures, derivant concentration 1%, rice protein concentration 7%.
The described spray-dired condition of step (2) is following: EAT 110-130 ℃, and intake velocity 1.00-1.20m
3/ min, sample introduction speed 12.00ml/min.
The described spray-dired condition of step (2) is preferably following: 120 ℃ of EATs, intake velocity 1.10m
3/ min, sample introduction speed 10.00-14.00ml/min.
The construction method of described fish growth hormone gene engineering microzyme specifically may further comprise the steps:
1) according to the black carp GH gene order AF389238 design primer of registering in the GeneBank database,
Upstream primer P1:5 '-ATG GCT AGA GCA TTA GTG CT-3 ';
Downstream primer P2:5 '-CTA CAG GGT GCA GTT GGA AT-3 ';
2) the Trizol kit specification according to Invitrogen company extracts the total RNA of black carp pituitary; The ddH2O that handles according to synthetic black carp cDNA first chain: the RNA template 5 μ L (1 μ g/ μ L) of invitrogen company reverse transcription kit specification, 1 μ L oligo (dT), 18 primers (0.5 μ g/ μ L), DEPC supplies 12 μ L, 65 ℃ of water-bath 5min; The centrifugal 30S of 300rpm; Be placed on 1min on ice immediately; Add 5 * Reaction Buffer, 4 μ L, 40U/ μ LRNase inhibitor 1 μ L, 10mM/ μ L dNTP Mix 2 μ L, reverse transcriptase 1 μ L (1U/ μ L) more successively; The centrifugal 30S of 300rpm is placed on 42 ℃ of water-bath 60min immediately, 70 ℃ of cessation reaction 5min.
Black carp growth hormone gene cDNA fragment cloning, reaction system is following: the cDNA 2 μ L of above-mentioned acquisition (1 μ g/ μ L), 10 * PCR Buffer, 2.5 μ L, 25mmol MgCl
21.5 μ L, 10mM/ μ L primer P1, each 1 μ L of P2,10mM/ μ LdNTP 1 μ L, it is 20 μ L that the Taq archaeal dna polymerase 1 μ L of 5U/ μ L, ddH2O supply; The PCR reaction process is: 94 ℃ of preparatory sex change 3min, and 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 10min, 4 ℃ of 10min; Through the RT-PCR amplified reaction, the result sees Fig. 1, and is consistent with expected results.
3) according to step 2) the black carp growth hormone cDNA sequence and the expression vector pPIC 3.5K MCS that obtain; Design a pair of Auele Specific Primer that contains the restriction enzyme site of EcoR I and Not I; The black carp growth hormone gene of amplified band restriction enzyme site is so that construction of expression vector pPIC3.5K-bcGH.Primer sequence is following:
pPIC3.5K-GHF：5’GTC
GAATTCACCATGGCTAGAGCATTAGTG-3’，
pPIC3.5K-GHR：5’TAT
GCGGCCGCTTAATGATGATGATGATGATGCAGGGTGCAGTTGGAAT-3’。
Wherein, the sequence of underscore mark is respectively the restriction enzyme site of EcoR I and Not I, and primer is given birth to worker's biotechnology Co., Ltd by Shanghai and synthesized.
With step 2) synthetic cDNA is template, uses primer pPIC3.5K-GHF, pPIC3.5K-GHR carries out pcr amplification.The PCR system: 2 * PCR TaqMix, the 12.5 μ L of the 2X1mL specification that east Sheng biotechnology company provides, each 1 μ L of 10mM/ μ L primer pPIC3.5K-GHF and pPIC3.5K-GHR, (1 μ g/ μ L) cDNA 2 μ L add dd H
2O to 25 μ L;
The pcr amplification program: 94 ℃ of preparatory sex change 3min, 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 10min, 4 ℃ of 10min;
4) after the PCR product that step 3) is obtained reclaims purifying; With EcoR I and Not I double digestion PCR product; After the extracting of phenol chloroform, and carry out double digestion equally and be connected through the pPIC3.5K of alkaline phosphatase treatment carrier segments, change bacillus coli DH 5 alpha over to; Screening positive clone obtains recombinant plasmid pPIC3.5K-bcGH;
5) recombinant plasmid pPIC3.5K-bcGH electric shock is transformed importing histidine defect type yeast GS115 bacterial strain (available from precious bioengineering (Dalian) Co., Ltd), obtain gene engineering microzyme.
A kind of bacterium peptide active protein feed is the feed that is prepared from above-mentioned method.Can feed directly be raised fish or mixed feeding with fish feed.
The present invention utilizes this desirable eukaryotic protein high level expression system of methylotrophy type pichia yeast Pichia Pastoris; With black carp albonubes somatotropic hormone gene directed cloning to the yeast integrative plasmid carrier; Through conversion, screening, abduction delivering, obtained the recombinant fish growth hormone gene engineering microzyme that efficiently expresses.Through being that fermentation culture medium carries out biofermentation with the rice protein, preparation bacterium peptide active protein feed is then directly raised feed fish or is mixed feeding with fish feed.When reducing the Feed Manufacturing cost, realize growth promotion and improve disease-resistant effect, thereby increase the fish culture benefit.
Description of drawings
Fig. 1 is a RT-PCR amplified production electrophoretogram;
Lane M:DNA marker Lane 1-3:RT-PCR result
Fig. 2 is the EcoR I/Not I restriction enzyme digestion and electrophoresis figure of pPIC3.5K-bcGH;
1-6:pPIC3.5K-bcGH EcoR I and Not I enzyme cut M:DNA marker
Fig. 3 is: recombination yeast bacterium colony PCR figure;
1-4: recombination yeast PCR is M:DNA marker as a result
Fig. 4 is the expression product SDS-PAGE (12%) of pPIC 3.5k-bcGH recombinant yeast;
1 negative control, M:Marker, 2-7 induce 24 hours sampling culture medium PH to be respectively 4.5,5.5,6.0,6.5,7.0
Fig. 5 is the expression product SDS-PAGE (12%) of pPIC 3.5k-bcGH recombinant yeast;
1-6 and 7-12 induce 48 hours and the 72 hours culture medium PH that take a sample to be respectively: 4.5,5.5,6.0,6.5, and 7.0 M:Marker
Fig. 6 is the SDS-PAGE result of purifying bcGH;
1～7: the bcGH of purifying, M: albumen marker, 8: purifying protein not
Fig. 7 is the protein content calibration curve;
Fig. 8 is that fermentation time is to the influence of reorganization bcGH Yeast expression amount;
1, do not induce; 2, protein Marker; 3～7, induction time is respectively: 96h, 72h, 48h, 24h, 12h
Fig. 9 is that fermentation temperature is to the influence of reorganization bcGH Yeast expression amount;
1, protein Marker; 2～6, fermentation temperature is respectively: 32 ℃, 30 ℃, 28 ℃, 26 ℃, 24 ℃; 7, do not induce
Figure 10 is that the seed liquor inoculum concentration is to the influence of reorganization bcGH yeast abduction delivering;
1, do not induce; 2～6, inoculum concentration is respectively: 2%, 3%, 4%, 5%, 6%; 7, protein Marker
Figure 11 is that rice protein concentration is to the influence of reorganization bcGH yeast abduction delivering;
1, protein Marker; 2～6, rice protein concentration is respectively: 7%, 6%, 5%, 4%, 3%; 7, do not induce
Figure 12 is that methanol concentration is to the influence of reorganization bcGH yeast abduction delivering;
1, protein Marker; 2～6, methanol concentration is respectively: 2.5%, 2%, 1.5%, 1%, 0.5%; 7, do not induce
Figure 13 influences reorganization bcGH yeast abduction delivering for the pH value;
1, protein Marker; 2～6, the pH value is respectively: 8.0,7.5,7.0,6.5,6.0; 7, do not induce
The specific embodiment
Be intended to further specify the present invention below in conjunction with embodiment, and unrestricted the present invention.
1, the saccharomycetic structure of growth hormone gene engineering
According to black carp GH gene order (AF389238) the design primer of registering in the GeneBank database.Upstream primer P1:5 '-ATG GCT AGA GCA TTA GTG CT-3 '.Downstream primer P2:5 '-CTA CAG GGT GCA GTTGGAAT-3 '.
With reference to the Trizol kit specification of invitrogen company, extract the total RNA of black carp pituitary.DdH according to synthetic black carp cDNA first chain: the RNA template 5 μ L (1 μ g/ μ L) of invitrogen company reverse transcription kit specification, 1 μ L oligo (dT), 18 primers (0.5 μ g/ μ L), DEPC processing
2O supplies 12 μ L, 65 ℃ of water-bath 5min; The centrifugal 30S of 300rpm; Be placed on 1min on ice immediately; Add 5 * Reaction Buffer, 4 μ L, 40U/ μ L RNase inhibitor 1 μ L, 10mM/ μ L dNTP Mix 2 μ L, reverse transcriptase 1 μ L (1U/ μ L) more successively; The centrifugal 30S of 300rpm is placed on 42 ℃ of water-bath 60min immediately, 70 ℃ of cessation reaction 5min.Prepare black carp growth hormone gene cDNA fragment cloning again, reaction system is following: the cDNA 2 μ L (1 μ g/ μ L) that a last step obtains, 10 * PCR Buffer, 2.5 μ L, 25mmol MgCl
21.5 μ L, 10mM/ μ L primer P1, each 1 μ L of P2,10mM/ μ L dNTP 1 μ L, it is 20 μ L that the Taq archaeal dna polymerase 1 μ L of 5U/ μ L, ddH2O supply; The PCR reaction process is: 94 ℃ of preparatory sex change 3min, and 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 10min, 4 ℃ of 10min; The agarose gel electrophoresis result sees Fig. 1, and is consistent with expected results.
The PCR product is connected with pEASY-T1 cloning vector (available from precious bioengineering (Dalian) Co., Ltd) after gel reclaims the kit purifying.Reaction system is following: PCR product 1 μ l, and pEASY-T1 cloning vector 1 μ l, room temperature reaction 5min places cessation reaction on ice again.Change bacillus coli DH 5 alpha (available from precious bioengineering (Dalian) Co., Ltd) over to, picking white clone enlarged culture through bacterium colony PCR checking, is delivered the positive colony plasmid of verifying to Shanghai and is given birth to worker's sequencing analysis.Order-checking gained result consistent with the gene order that GeneBank uploads (Feng Hao, 2002)
[5]Explain and successfully cloned the black carp growth hormone gene.
The black carp growth hormone cDNA sequence and expression vector pPIC 3.5K (available from precious bioengineering (Dalian) Co., Ltd) MCS that obtain according to the front; Utilization Seqencer software designs a pair of special primer pPIC3.5K-GHF:5 '-GTCGAATTCACCATGGCTAGAGCATTAGTG-3 ', pPIC3.5K-GHR:5 '-TATGCGGCCGCTTAATGATGATGATGATGATGCAGGGTGCAGTTGGAAT-3 '.
CDNA with synthetic is a template, uses primer pPIC3.5K-GHF, and pPIC3.5K-GHR carries out pcr amplification.The PCR system: 2 * PCR TaqMix, the 12.5 μ L of the 2X1mL specification that east Sheng biotechnology company provides, each 1 μ L of 10mM/ μ L primer pPIC3.5K-GHF and pPIC3.5K-GHR, (1 μ g/ μ L) cDNA2 μ L adds dd H
2O to 25 μ L; The pcr amplification program: 94 ℃ of preparatory sex change 3min, 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 10min, 4 ℃ of 10min.The PCR product is after gel reclaims the kit purifying, with EcoR I and Not I double digestion PCR product, after the extracting of phenol chloroform; With carry out double digestion equally and be connected through the pPIC3.5K of alkaline phosphatase treatment carrier segments, change bacillus coli DH 5 alpha over to, screening positive clone; Extract plasmid; Bacterium colony PCR, EcoR I and Not I double digestion identify, are accredited as the positive further sequencing analysis of clone.With the positive recombinant plasmid called after pPIC3.5K-bcGH that obtains, the result sees Fig. 2 (the EcoR I of Lane 1-6:pPIC3.5K-bcGH and Not I enzyme are cut Lane M:DNA marker)
The competent preparation list of references of yeast (Frederick MA, 1995)
[6]In liquid nitrogen, take out Gs115 yeast competence; It is placed on ice to just melting immediately; Add linearizing pPIC3.5K-bcGH recombinant vector (5～10 μ g) and mix, change in the 0.1cm electricity revolving cup of precooling with Gs115 yeast (available from precious bioengineering (Dalian) Co., Ltd) competence; Place 5min on ice; Press voltage 600V, resistance 400 Ω, the condition of electric capacity 25 μ F shocks by electricity; The 1M sorbierite that adds the 1mL precooling immediately is applied to by (the every flat board of 200 μ L) on the MD flat board to cup immediately; Be inverted for 30 ℃ and cultivate 3～4d, to clone's (His+ transformant) generation.Carry out YPD flat board (concentration of G418 be respectively 1.0,2.0 and 3.0mg/ml) gradient screening multicopy to the pPIC3.5K-bcGH Pichia pastoris transformant on the MD flat board, cultivate 3～4d for 30 ℃.Screen positive transformant to well-grown high resistance transformant (more than the 2.0mg/ml) through the PCR method.The result sees Fig. 3 (Lane 1～4: recombination yeast PCR is Lane M:DNA marker as a result)
The positive yeast list of picking bacterium colony is seeded in the BMGY culture medium, and 30 ℃, 300r/min is cultured to OD600 and reaches 2～6; Centrifugal receipts bacterium, and be resuspended in BMMY culture medium (pH is 6.0), 30 ℃; 300r/min cultivated 4 days, and every 24h adds the methanol induction that final concentration is 1% (v/v).Collect thalline, carry out SDS-PAGE and analyze.The result sees that (Lane 1 negative control, LaneM:Marker, Lane 2～7 are induced sampling in 24 hours to Fig. 4, and medium pH is respectively 4.5,5.5,6.0,6.5,7.0.) and Fig. 5 (sampling in 48 hours, Lane M:Marker are induced in Lane1～6).
After inducing, centrifugal collection thalline is according to operation manual cracking and the digestion thalline of QIAGEN, with Ni Sepharose High Performance purifying protein.The sample of wash-out is packed into after molecular cut off is the bag filter of 3kD, puts into 1L0.9%NaCl solution, 4 ℃ of dialysis.Dialyzed sample is carried out SDS-PAGE electrophoresis and quantification of protein.With mouse-anti black carp growth hormone is one anti-, and goat-anti mouse lgG is two anti-, and chromogenic substrate is 3 ' 3 ' 5 ' 5 '-tetramethyl benzidine [TMB].Make negative control and blank with mice serum and PBST before the immunity respectively.Behind the abduction delivering 72h, bacterium liquid is through the cracking of liquid nitrogen, bead in the culture medium of pH6.5 for recombination yeast engineering bacteria Gsl15, and the histidine-tagged reorganization black carp growth hormone polypeptides of 6x is with in Ni-NTAMagnetic Agarose Bead absorption.Through after the affine absorption, only stay the fusion of the black carp growth hormone of expressing through the bacterial strain of inducing, see Fig. 6 in detail.Purified product dilutes 2000 times, 4000 times, 8000 times, 16000 times, 32000 times, 64000 times respectively, and one anti-dilutes 1000 times, 2000 times, 4000 times, 8000 times respectively, does negative control and blank simultaneously, and ELISA detects protein immunization originality.The result sees table 1.
The Elisa of table 1:bcGH analyzes
Because the absorbance of protein solution in the A450nm wave-length coverage is directly proportional with its content, the detection by quantitative of destination protein is standard protein with reference to ultraviolet absorption method with 1mg/mL BSA, is diluted to variable concentrations successively with 1X PBS then, and ELIASA is determined at A
450Absorbance under the nm is made into calibration curve and sees Fig. 7, records the A of the bc-GH albumen of nickel gel affinitive layer purification
450Nm mean value is 0.4122, and from regression equation y=0.7708x+0.068, can calculate the bc-GH expression is 893mg/L.
In the 10L of amount of fill 7L fermentation tank, under the 300rpm condition, research temperature, rice protein concentration, gene engineering microzyme seed liquor inoculum concentration, pH value, fermentation time, six single factors of derivant (being methyl alcohol) concentration are to the influence of the expression of albumen.Zymotic fluid consist of rice protein, the gene engineering microzyme seed liquor, derivant, all the other are water.And each component percentages of following appearance is mass percent.
The gene engineering microzyme seed culture method:
1) actication of culture: the reorganization bcGH barms line of getting-80 ℃ of refrigerator preservations is transferred to the MD flat board, and 30 ℃ of constant temperature are inverted and were cultivated 3-5 days, with the phenotype that detects the His+ transformant correct and vigor whether.
2) seed culture: connect the dull and stereotyped seed of the well-grown MD of 1 ring and shake in the bottle to the 500mL that the 50mL seed culture medium is housed; Repeat to do 6 shake-flask seeds; Place 300rpm constant temperature shaking table, 30 ℃ of shaken cultivation bacterium liquid OD600 to 2-6 (time is 10-24 hour) are as the seed liquor of fermentation usefulness.
According to the center combination design principle of Box-Behnken, more than six factors be independent variable, be index with the expression of destination protein, 6 factors, the five levels experiment (L of design optimization fermentation condition
25(5
6)), under 10L fermentation tank condition, explore the interaction during the fermentation of each factor, confirm its influence order and optimum technological condition for fermentation to index.
The single factor of fermentation condition is explored: I, in the 10L of amount of fill 7L fermentation tank; Under 30 ℃, the condition of pH6.5,0.5% derivant, 5% inoculum concentration, 5% rice protein, 300rpm; Explore the influence (12h, 24h, 48h, 72h, 96h) of fermentation time to exogenous protein expression, the result sees that Fig. 8 (Lane 1, does not induce; Lane 2: protein Marker; Lane 3～7: induction time is respectively 96h, 72h, 48h, 24h, 12h).II, in the 10L of amount of fill 7L fermentation tank; Under the condition of pH6.5,0.5% derivant, fermentation 72h, 5% inoculum concentration, 5% rice protein, 300rpm; Explore the influence (24 ℃, 26 ℃, 28 ℃, 30 ℃, 32 ℃) of cultivation temperature to exogenous protein expression, the result sees Fig. 9, and (Lane 1: protein Marker; Lane 2～6: fermentation temperature is respectively 32 ℃, 30 ℃, 28 ℃, 26 ℃, 24 ℃; Lane 7: do not induce).III, in the 10L of amount of fill 7L fermentation tank; Explore the influence (2%, 3%, 4%, 5%, 6%) of inoculum concentration to exogenous protein expression under the condition of 30 ℃ of temperature, pH6.5,0.5% derivant, fermentation 72h, 5% rice protein, 300rpm, the result sees Figure 10, and (Lane 1: do not induce; Lane 2～6: inoculum concentration is respectively 2%, 3%, 4%, 5%, 6%; Lane 7: protein Marker).
Single factor that fermentation medium is formed is explored: I, in the 10L of amount of fill 7L fermentation tank; Under the condition of 30 ℃, pH6.5,0.5% derivant, fermentation 72h, 5% inoculum concentration, 300rpm; Explore the influence (3%, 4%, 5%, 6%, 7%) of rice protein concentration to protein expression, the result sees Figure 11, and (Lane 1: protein Marker; Lane 2～6: rice protein concentration is respectively 7%, 6%, 5%, 4%, 3%; Lane 7: do not induce).II, in the 10L of amount of fill 7L fermentation tank; Under the condition of 30 ℃, pH6.5,5% rice protein, fermentation 72h, 5% inoculum concentration, 300rpm; Explore the influence (0.5%, 1%, 1.5%, 2%, 2.5%) of derivant concentration to protein expression, the result sees Figure 12, and (Lane 1: protein Marker; Lane 2～6: methanol concentration is respectively 2.5%, 2%, 1.5%, 1%, 0.5%; Lane 7: do not induce).III, in the 10L of amount of fill 7L fermentation tank; Under the condition of 30 ℃, 5% rice protein, 0.5% derivant, fermentation 72h, 5% inoculum concentration, 300rpm; Explore the influence (6.0,6.5,7.0,7.5,8.0) of medium pH value to protein expression, the result sees Figure 13, and (Lane 1: protein Marker; Lane 2～6:pH value is respectively 8.0,7.5,7.0,6.5,6.0; Lane 7: do not induce).
Center combination design principle according to Box-Behnken; With temperature, rice protein concentration, inoculum concentration, pH value, fermentation time, six factors of derivant is independent variable; Expression with destination protein is an index, 6 factors, the five levels experiment (L of design optimization fermentation condition
25(5
6)), explore the interaction during the fermentation of each factor, confirm suitable technological condition for fermentation.Protein expression figureofmerit under each factor level combination condition carries out range analysis, confirms optimum technological condition for fermentation.According to table 3 result of calculation, each factor extreme difference R value size relatively, can find out that each factor influences the primary and secondary order to reorganization bcGH yeast growth: C＞B＞E＞A＞D＞F, the optimum combination condition that ferments is A
4B
2C
5D
2E
5F
5, promptly fermentation temperature is 30 ℃, pH6.5, fermentation time 96h, derivant concentration 1%, inoculum concentration 6%, rice protein concentration 7%.
Table 2 bcGH engineering bacterium fermentation factor and level
Table 3 bcGH engineering bacterium fermentation orthogonal experiments
10L ferment tank result of the test
Under optimum fermentation combination condition, use the flame inocalation method that bcGH engineering bacteria seed liquid is inoculated in the 10L fermentation tank, verify its ferment effect in fermentation tank.Air mass flow is 3L/min, and mixing speed is 300r/min, in rice protein concentration 7%, and 30 ℃ of fermentation temperatures, pH6.5, inoculum concentration 6% is induced fermentation 96h under derivant concentration 1% condition.Fermentation ends, through SDS-PAGE electrophoresis detection and Bandscan software analysis, the bcGH expression is higher than shake flask fermentation, and the bcGH expression accounts for 32.39% of total protein.
In addition, also find at rice protein concentration 5-7%, fermentation temperature 28-30 ℃, pH6.0-7.5, inoculum concentration 5-6% induces fermentation 96h in the derivant concentration 0.5-1% condition and range, also can reach better effects.
The spray-drying research of growth hormone gene engineering saccharomycete liquid fermentation rice protein mixture
With the growth hormone gene Yeast engineering bacteria is index, adopts orthogonal test to inquire into the influence of spray-drying factors such as EAT, intake velocity and sample introduction speed to its survival rate.Explore the interaction of each factor in dry run, confirm that it is to the influence order of index and optimum drying condition combination.
Each factor of spray-drying is to the influence of bcGH engineering bacteria survival rate: I, with intake velocity, sample introduction speed stuck-at-.10m
3Under the condition of/min, 12.00ml/min, research is along with the influence (100 ℃, 110 ℃, 120 ℃, 130 ℃) of EAT difference to bcGH engineering bacteria survival rate, and the result sees table 4.Under II, the condition with EAT, 20 ℃ of sample introduction speed stuck-at-s, 12.00ml/min, the research intake velocity is to the influence (1.00m of bcGH engineering bacteria survival rate
3/ min, 1.10m
3/ min, 1.20m
3/ min, 1.30m
3/ min), the result sees table 5.III, with EAT, 20 ℃ of intake velocity stuck-at-s, 1.10m
3Under the condition of/min, research sample introduction speed is to the influence (8ml/min, 10ml/min, 12ml/min, 14ml/min) of bcGH engineering bacteria survival rate, and the result sees table 6.
Roughly confirmed spray-dired condition through single factor experiment; In order to make acquisition bcGH engineering bacteria survival rate reach the highest; Select EAT, intake velocity, 3 factors of sample introduction speed for use, adopt the positive method for designing of three factors, four levels to carry out the optimization test of spray-drying condition.Analyze the result of different spray-drying conditions according to plate count.And the engineering bacteria survival rate index under each factor level combination condition carried out range analysis, confirm best drying process condition.According to result of calculation shown in the table 8, relatively each factor extreme difference R value is big or small, and that can find out each factor bcGH expression influences primary and secondary in proper order: EAT＞sample introduction speed＞intake velocity, optimum fermentation combination condition is A
3B
2C
3, promptly EAT is 120 ℃, intake velocity 1.10m
3/ min, sample introduction speed 12.00ml/min.Find simultaneously, at EAT 110-130 ℃, intake velocity 1.00-1.20m
3/ min also can obtain better effects in the sample introduction speed 10.00-14.00ml/min condition and range.
Table 4 EAT is to the influence of bcGH engineering bacteria survival rate
Table 5 intake velocity is to the influence of bcGH engineering bacteria survival rate
Table 6 sample introduction speed is to the influence of bcGH engineering bacteria survival rate
Table 7 spray-drying orthogonal test factor level table
Table 8 spray-drying orthogonal experiments
Growth hormone gene engineering saccharomycete feed is to the physiological action of fish
With the grass carp about initial average weight 1g is experimental subjects; Feed respectively and add the daily ration of 3.0%, 6.0%, 9.0%, 12.0% and 15.0% engineering bacterium fermentation rice protein (being growth hormone gene engineering saccharomycete feed of the present invention) and common fermentation rice protein in the basal feed; Be labeled as A1, A2, A3, A4, A5 and B1, B2, B3, B4, B5 group respectively; Basal diet A0 is a control group, regulates protein level with glycine, and preparation waits the nitrogen daily ration.Each organizes feed formula and nutritional labeling is seen table 9.Each horizontal group is established 3 repetitions, raises 50 tail fishes in each aquarium, carries out the growth test of ingesting in 8 weeks by a definite date, the every group of corresponding feed of throwing something and feeding.Day feeding volume is the 3.0%-6.0% of fish body weight.About 27 ℃ of water temperatures, 24h blowing aeration, dissolved oxygen＞5mg/L, ammonia nitrogen＜0.1mg/L, pH 6.5-7.5.Inquire into and add of the influence of engineering bacterium fermentation rice protein in the feed the grass carp growth.
After the engineering bacterium fermentation rice protein that adds different proportion was raised grass carp 56d with the common fermentation rice protein, the average body weight average of the grass carp of test group was significantly higher than control group, and the A average body weight average of respectively organizing grass carp is higher than B, and each is organized.In addition, the rate of body weight gain of test group all is significantly higher than control group, and the rate of body weight gain of each group of A is higher than each group of B; The test group feed coefficient all significantly is lower than control group, and A respectively organize feed coefficient all be lower than B each the group; Survival rate test group and control group do not have significant difference (P＞0.05), and growth result is seen table 10.Table 10 shows, when adding the ratio of engineering bacterium fermentation rice protein 9%-15%, rate of body weight gain of grass carp and feed coefficient effect are better, but there was no significant difference (P＞0.05).Be that 12% effect is best with addition wherein, higher bcGH fermentation rice protein addition possibly cause fish body digestive organs, and over-burden, and influence is digested and assimilated and grown.This research shows that fully black carp growth hormone gene Yeast engineering bacteria fermentation rice protein has significantly promoted the grass carp growth.
Table 9 feed formula and nutritional labeling %
Table 10 engineering bacterium fermentation rice protein or common fermentation rice protein the influence of throwing something and feeding to grass carp growth and survival rate
The relative weight gain rate (%)=(end weight-initial weight)/initial weight * 100; Feed coefficient=total the amount of raising/(last initial weight that weighs) of throwing; Survival rate (%)=test is the fish tail number/last fish tail number of test * 100 just.
List of references:
[1]Donaldson，E.M.，U.H.M.Fagerlund，D.A.Higgs?and?J.R.Mc?Bride.Hormonal?enhancement?of?growth.In：Fish?Physiology(eds?W.S.Hoar，D.J.Randall?and?J.R.Brett)[J].Academic?Press，New?York，NY，1979，8，456-599.
[2]Hertz?Y，Tchelet?A，Madar?Z，et?al.Absorption?of?bioactive?human?growth?hormone?after?oral?administration?in?the?common?carp(Cyprinus?carpio)and?its?enhancement?by?deoxycholate[J].J?Comp?Physiol，1991，161B：159～163.
[3] Xiao Dong, Chen Songlin is sternly peaceful etc. the fish alimentary canal has the ability [J] that absorbs exogenous growth hormone. zoological research, 2000,21 (2): 103～107.
[4] Sun Xun, Zhu Shangquan. the structure of growth hormone and function [J]. foreign medical science physiology, pathology science and clinical fascicle, 1999,19 (1): 6～9.
[5] Feng Hao, Ceng Zhiqiang, Cheng Jia, Liu Shaojun, Liu Jun. the clone of black carp growth hormone cDNA and sequence analysis. laser biology journal [J], 2002,11 (6): 407～411.
[6]Frederick?MA，Roger?B，Robert?EK，et?al.Short?Protocols?in?Molecular?Biology，3rd?ed[J].John?Wiley?&?Sons，Inc，1995.
Claims (7)
1. the preparation method of a bacterium peptide active protein feed is characterized in that, may further comprise the steps:
(1) in fermentation tank, the zymotic fluid that contains following mass percent component ferments: derivant 0.5-1%, rice protein concentration 6-7%, fish growth hormone gene engineering microzyme seed liquor 5-6%, and all the other are water; The condition of fermentation is following: fermentation time 96h, fermentation temperature 28-30 ℃, pH6.0-7.5, rotating speed 300rpm, throughput 3L/min;
(2) mixture that fermentation is obtained carries out spray-drying and promptly gets;
Described fish growth hormone gene engineering microzyme is with the directed expression vector pPIC3.5k that contains strong promoter AOX I, the construction of expression vector pPIC3.5K-bcGH of inserting of black carp growth hormone cDNA; Again its electric shock being transformed importing histidine defect type yeast GS115 bacterial strain obtains; With the seed liquor of the fish growth hormone gene engineering microzyme that obtains through the usefulness that obtains after the following activation culture process fermenting: the reorganization bcGH barms line of getting-80 ℃ of refrigerator preservations is transferred to the MD flat board; 30 ℃ of constant temperature are inverted and were cultivated 3-5 days; Connecing the dull and stereotyped seed of the well-grown MD of 1 ring shakes in the bottle to the 500mL that the 50mL seed culture medium is housed; Place 300rpm constant temperature shaking table, 30 ℃ of shaken cultivation bacterium liquid OD600 to 2-6, the time is to get final product in 10-24 hour.
2. preparation method according to claim 1 is characterized in that, the construction method of described fish growth hormone gene engineering microzyme specifically may further comprise the steps:
1) according to the black carp GH gene order AF389238 design primer of registering in the GeneBank database,
Upstream primer P1:5 '-ATG GCT AGA GCA TTA GTG CT-3 ';
Downstream primer P2:5 '-CTA CAG GGT GCA GTT GGA AT-3 ';
2) extract the total RNA of black carp pituitary, carry out cDNA by RT-PCR and synthesize: cDNA first chain is synthetic: the ddH that the RNA template 5 μ L of 1 μ g/ μ L, 0.5 μ g/ μ L oligo (dT), 18 primers, 1 μ L, DEPC handle
2O supplies 12 μ L, 65 ℃ of water-bath 5min; The centrifugal 30S of 300rpm; Be placed on 1min on ice immediately; Add 5 * Reaction Buffer4 μ L, 40U/ μ L RNase inhibitor 1 μ L, 10mM dNTP Mix 2 μ L, 1U/ μ L reverse transcriptase 1 μ L more successively; The centrifugal 30S of 300rpm is placed on 42 ℃ of water-bath 60min immediately, 70 ℃ of cessation reaction 5min;
Prepare pcr amplification again, reaction system is following: 1 μ g/ μ L cDNA, 1 μ L, 10 * PCR Buffer2.5 μ L, 25mmol MgCl
21.5 μ L, 10mM/ μ L primer P1, each 1 μ L of P2,10mmol dNTP 1 μ L, the Taq archaeal dna polymerase 1 μ L of 5u/ μ L, ddH
2It is 20 μ L that O supplies; The PCR reaction process is: 94 ℃ of preparatory sex change 3min, and 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 10min, 4 ℃ of 10min;
3) according to step 2) black carp growth hormone cDNA sequence that obtains and the MCS of expression vector pPIC3.5K,
Design a pair of special primer that contains the restriction enzyme site of EcoR I and Not I,
pPIC3.5K-GHF：5’-GTCGAATTCACCATGGCTAGAGCATTAGTG-3’，
pPIC3.5K-GHR：5’-TATGCGGCCGCTTAATGATGATGATGATGATGCAGGGTGCAGTTGGAAT-3’；
The PCR system: 2 * PCR TaqMix, 12.5 μ L, each 1 μ L of 10mM/ μ L pPIC3.5K-GHF and pPIC3.5K-GHR, the cDNA 2 μ L of 1 μ g/ μ L add ddH
2O to 25 μ L;
The pcr amplification program: 94 ℃ of preparatory sex change 3min, 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 10min, 4 ℃ of 10min;
4) after the PCR product that step 3) is obtained reclaims purifying; With EcoR I and Not I double digestion PCR product; After the extracting of phenol chloroform, and carry out double digestion equally and be connected through the pPIC3.5K of alkaline phosphatase treatment carrier segments, change bacillus coli DH 5 alpha over to; Screening positive clone obtains recombinant plasmid pPIC3.5K-bcGH;
5) recombinant plasmid pPIC3.5K-bcGH electric shock is transformed importing histidine defect type yeast GS115 bacterial strain, obtain gene engineering microzyme.
3. preparation method according to claim 1 is characterized in that, fermentation time 96h in the step (1), pH6.5, fish growth hormone gene engineering microzyme seed liquor 6%, 30 ℃ of fermentation temperatures, derivant concentration 1%, rice protein concentration 7%.
4. preparation method according to claim 1 is characterized in that, the described spray-dired condition of step (2) is following: EAT 110-130 ℃, and intake velocity 1.00-1.20m
3/ min, sample introduction speed 12.00ml/min.
5. preparation method according to claim 4 is characterized in that, the described spray-dired condition of step (2) is following: 120 ℃ of EATs, intake velocity 110m
3/ min, sample introduction speed 10.00-14.00ml/min.
6. a bacterium peptide active protein feed is characterized in that, is in any feed that described method is prepared from of claim 1-5.
7. the application process of the described bacterium peptide of claim 6 active protein feed is characterized in that, feed is directly raised fish or mixed feeding with fish feed.
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Cited By (2)
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| CN103014058A (en) * | 2012-12-03 | 2013-04-03 | 广东现代农业集团研究院有限公司 | Method for industrially producing grouper growth hormone recombination gene protein |
| CN103478441A (en) * | 2013-07-29 | 2014-01-01 | 长沙学院 | Rice active bacterium peptide fish feed and application thereof |
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2011
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103014058A (en) * | 2012-12-03 | 2013-04-03 | 广东现代农业集团研究院有限公司 | Method for industrially producing grouper growth hormone recombination gene protein |
| CN103478441A (en) * | 2013-07-29 | 2014-01-01 | 长沙学院 | Rice active bacterium peptide fish feed and application thereof |
| CN103478441B (en) * | 2013-07-29 | 2016-03-30 | 长沙学院 | A kind of rice active bacterium peptide fish meal and application thereof |
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