CN101717737A - Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof - Google Patents

Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof Download PDF

Info

Publication number
CN101717737A
CN101717737A CN200910273232A CN200910273232A CN101717737A CN 101717737 A CN101717737 A CN 101717737A CN 200910273232 A CN200910273232 A CN 200910273232A CN 200910273232 A CN200910273232 A CN 200910273232A CN 101717737 A CN101717737 A CN 101717737A
Authority
CN
China
Prior art keywords
buforin
ifn
expression
inf
pichia pastoris
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN200910273232A
Other languages
Chinese (zh)
Other versions
CN101717737B (en
Inventor
唐志如
冯泽猛
印遇龙
张友明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GUANGDONG HINABIOTECH CO., LTD.
Original Assignee
Institute of Subtropical Agriculture of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Subtropical Agriculture of CAS filed Critical Institute of Subtropical Agriculture of CAS
Priority to CN 200910273232 priority Critical patent/CN101717737B/en
Publication of CN101717737A publication Critical patent/CN101717737A/en
Application granted granted Critical
Publication of CN101717737B publication Critical patent/CN101717737B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses an antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris (Pichia pastoris FZM2009, CCTCC NO:M209259), and a preparation method and applications thereof. The method comprises the steps: A, the construction of expression cassette PAOX1-alpha Factor-IFN alpha-6xHis-bufforin II-Zeocinr; B the construction of expression engineering bacteria Pichia pastoris FZM 2009, wherein the engineering bacteria is resulted from the screening of antibiotics Zeocin and PCR identification; C, the preparation of fusion protein, which comprises the steps: 1) the recovery is implemented on a YPD plate containing 100mug/ml of Zeocin; 2) single clone is inoculated to a YPD-containing culture medium for vibration and culture; 3) 1 volume of culture is inoculated into 10 volumes of a fermentation culture medium for culture; 4) supernatant is obtained by centrifugation; 5) loading buffer with double volume is added into the supernatant, and electrophoresis detection is performed; 6) the supernatant is subject to a molecular sieve gel column to collect components; 7) the collected components are lyophilized to result in fusion protein; 8) buforin II and IFN alpha are obtained by the enzyme digestion of enterokinase on the fusion protein. The fusion protein buforin II and IFN alpha in fusion expression are applied to pig feeds. The method has great feasibility, simple and convenient operation, high expression, low cost and strong bioactivity.

Description

Antibacterial peptide buforin II and porcine INF-alpha fusion expression pichia pastoris and preparation method and application
Technical field
The present invention relates to biological technical field, more specifically relate to a kind of antibacterial peptide buforin II and porcine INF-alpha fusion expression Pichia yeast engineering, the preparation method who also relates to antibacterial peptide buforin II and INF-α simultaneously, antibacterial peptide buforin II and porcine INF-alpha can be widely used in the animal-feed, are a kind of novel antibacterial medicine after being further purified.
Background technology
Park had found buforin I first in the toad Bufo bufo gargarizans stomach-tissue of Asia in 1996.BuforinI and histone H 2The N-terminal of A has sequence homology, is isozyme Cb and Ca cracking H by pepsin C 2The Tyr of A 39-Ala 40Between peptide bond.Buforin II is by buforin I cracking under the effect of intracellular protein enzyme Lys-C, is that buforinI is from Thr 16To Lys 36The strong antibacterial peptide of totally 21 amino-acid residues, the anti-microbial activity of buforin II is strong than buforin I.Buforin II is the amphipathic antibacterial peptide of a kind of α spiral.The effect target of BuforinII is intracellular nucleic acid rather than cytolemma, enters cell under the situation of not damaging cytolemma rapidly, and is incorporated into macromole DNA and RNA in the born of the same parents with very strong avidity, makes bacterium death.Compare with other antibacterial peptides, buforin II has fungistatic effect preferably.Be used to research treatment septic shock and peritonitis, and obtained good effect.In addition the anti-microbial effect of buforinII with the situation of traditional microbiotic compatibility under be enhanced, also make buforin II at fodder additives, medicine and other fields has had wider application space.
Yet natural extract antibacterial peptide buforin II expense is high and yield poorly, and the chemosynthesis expense is also expensive, and utilizing biotechnology to carry out fermentative production buforin II is a relatively cheap preparation approach.Realized that at present buforin II is at expression in escherichia coli.Though it is short to utilize escherichia coli expression buforin II to have a cycle, cultivate and expend low the grade advantage is arranged, also exist subsequent purification cumbersome, shortcoming such as can not directly use.Pichia spp is one of now the most frequently used expression system, has alcohol oxidase AOX1 gene promoter (the strongest at present, one of promotor that Regulation Mechanism is the strictest).Its expression plasmid can be at the form stable integration of genomic specific site with single copy or multiple copied, and bacterial strain is easy to carry out high density fermentation, exogenous protein expression amount height.Pichia spp self exocrine protein seldom is easy to purifying if external secretion is expressed foreign protein in addition.
BuforinII has certain toxic action to fungi, and in order to reduce or remit the toxic action of antibacterial peptide to pichia spp, this research considers that catenation sequence is that the enteropeptidase enzyme is cut sequence buforinII and pig interferon α (INF-α) amalgamation and expression.The fusion rotein of expressing both can directly add feed to, and pig digestion back is cut to activated antibacterial peptide buforinII and porcine INF-alpha at its enteron aisle by the enteropeptidase enzyme, brings into play their biological effectiveness.Also available enteropeptidase vitro enzyme is cut fusion rotein, obtains the antibacterial peptide buforinII and the porcine INF-alpha of pharmaceutical grade, is used for making injection.Select the albumen reason of porcine INF-alpha as amalgamation and expression: INF-α is the natural immunity and the bridge that obtains immunity, can activate the cytotoxicity of NK cell and promote its propagation, regulates immunity of organism.And INF-α participates in directly but the invasion reaction of body antagonism virus especially as first kind Interferon, rabbit, and multiple virus diseases such as porcine epizootic diarrhea, rotavirus diarrhea, transmissible gastroenteritis are had the better prevention effect.Can be used as broad-spectrum antiviral drug and be used for the control of pig virus disease, have antivirus action extensive, residual little, cheap, pollution-free, have no drug resistance, characteristics such as drug residue free.
Summary of the invention
The object of the invention has been to provide the Pichia yeast engineering of a kind of antibacterial peptide buforinII and porcine INF-alpha fusion expression, the easy high density fermentation of this project bacterium, novel antibacterial peptide buforin II that produces and porcine INF-alpha fusion polypeptide be the expression amount height not only, and be easy to purifying, under the effect of body enteron aisle enteropeptidase, can produce biologic activity such as antibiotic, antiviral and adjusting immunity efficient, wide spectrum.
Another object of the present invention has been to provide the preparation method of a kind of antibacterial peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, this method is easy and simple to handle, with low cost, and this fusion polypeptide has the antibiotic and antiviral activity of high-efficiency broad spectrum after by enzymolysis.
A further object of the present invention is the preparation method who has been to provide a kind of fusion rotein buforin II and INF-α.Yeast pichia pasteris takes in Chinese Pharmacopoeia already, but is the hyoscine and the edible barms of generally acknowledging both at home and abroad.The AOX strong promoter of pichia spp is the strongest at present, one of promotor that Regulation Mechanism is the strictest, and inductor methyl alcohol cheapness, foreign gene is put in order into to the thalline genome, and is more stable, is difficult for taking place heredity and loses.Invention is used improved pichia spp as cultivating bacterium high density fermentation cultivation continuously, helps industrial production.Used fermention medium cheapness of while, culture condition is controlled easily, the purification step that the external secretion design simplifies greatly, because pichia spp self exocrine protein seldom, target protein can account for the 80-90% of total secretion Tot Prot, last culture just can obtain purer fusion rotein through chromatography again through the simple centrifugal nutrient solution supernatant that can obtain containing a large amount of fusion roteins.In a word, this preparation method has relatively cheap, simple to operate and advantage such as preparation amount is big.
Purpose in addition of the present invention is to be to provide a kind of fusion rotein buforin II and the application of INF-α in pig feed.Fusion rotein buforin II and the used cultivated material of INF-α, device is easy to get, and output is big, and production cost is low, and is antibacterial obvious with immune system usefulness, and drug residue free, and very big application potential is arranged in pig feed.Simultaneously, simple in pig feed application operating step because fusion rotein can be through the absorption of ingesting.Fusion rotein buforin II and INF-α can be resolved into INF-α and buforinII by enteropeptidase in the chitling road in addition, so both can suppress the harmful intestinal tract microbial growth, also can strengthen the resistivity of pig by regulating the immunity system of pig body to virus.
In order to achieve the above object, the present invention adopts following technical measures:
Express engineering bacteria Pichia pastoris FZM 2009Structure:
Material requested is:
Pichia spp Pichia pastoris KM71H and plasmid pPICZA α are available from invitrogen company; Intestinal bacteria GB2005 and YZ2005 are provided by Gene Bridge company; Protein purification reagent is available from MERK, and other conventional reagent are given birth to the worker available from Shanghai.
Intestinal bacteria GB2005, YZ2005 substratum are the LB substratum, and its moiety is: the 5g yeast extract; The 10g peptone; 5g sodium-chlor (NaCl); Add water 900ml, (NaOH) is adjusted to 7.0 with the pH value with 1M sodium hydroxide, is settled to 1L.The LB solid medium: adding agar powder in the LB liquid nutrient medium, to make its final concentration be 1.5% (m/v).
Adopt the homologous recombination method with expression cassette P AOX1-α Factor-IFN α-6 * His-bufforin II-Zeocin rWith the gene aox1 on the Pichia pastoris KM71H genome homologous recombination taking place, obtains the Pichia yeast engineering of a kind of amalgamation and expression antibacterial peptide buforin II and porcine INF-alpha, called after Pichia pastorisFZM 2009, the preservation of this bacterial strain, depositary institution: Chinese typical culture collection center, address: China. Wuhan. Wuhan University, postcode: 430072, preservation date: on November 11st, 2009, deposit number: CCTCCNO:M209259, classification name: Pichia yeast Pichia pastoris FZM 2009
The preparation method of the Pichia yeast engineering of a kind of amalgamation and expression antibacterial peptide buforin II and porcine INF-alpha the steps include:
A, expression cassette P AOX1-α Factor-IFN α-6 * His-bufforin II-Zeocin rStructure.Its detailed process is: at first with the synthetic IFN α nucleotide sequence of the artificial design of pichia spp preference codon, obtain being with homology arm PCR product IFN α-6 * His-bufforin II by the twice PCR amplification; Adopt the Red/ET homologous recombination technique that IFN α-6 * His-bufforin II is cloned on pPICZA α carrier or the plasmid then, use at last and obtain expression cassette P after Sac I enzyme is cut AOX1-α Factor-IFN α-6 * His-bufforin II-Zeocin r(specifically seeing accompanying drawing 1).
B, expression engineering bacteria Pichia pastoris FZM 001Structure.Obtain genome conformity expression cassette P is arranged AOX1-IFN α-6His-bufforin II-Zeocin rDetailed process be: adopt electroporation (1500V/cm) with expression cassette P after the linearizing AOX1-α Factor-IFN α-6 * His-bufforin II-Zeocin rExpression plasmid import among the Pichia pastoris KM71H.Obtain being integrated with on the genome expression cassette P through microbiotic Zeocin screening and PCR evaluation AOX1The engineering bacteria Pichia pastorisFZM of-IFN α-6His-bufforin II-Zeocin 2009(specifically seeing accompanying drawing 1).The preservation of this bacterial strain, depositary institution: Chinese typical culture collection center, address: China. Wuhan. Wuhan University, postcode: 430072, preservation date: on November 11st, 2009, deposit number: CCTCC NO:M209259, classification name: Pichia yeast Pichia pastorisFZM 2009
The preparation method of a kind of fusion rotein buforin II and INF-α the steps include:
A, material and solution preparation:
Pichia pastoris FZM provided by the present invention 2009
The engineering bacteria substratum is experiment fermentation optimization substratum, each composition quality mark %: glucose 4%, ammoniacal liquor 150 μ L, potassium primary phosphate (KH 2PO 4) 0.7%, magnesium sulfate heptahydrate (MgSO 4.7H 2O) 0.03%, green vitriol (FeSO 4.7H 2O) 0.05%, magnesium sulfate monohydrate (MnSO 4.H 2O) 0.05%, peptone 0.1%, pH5.5;
The PTM1 prescription is: copper sulfate (CuSO 4) 6g/L, Potassium Iodate (KI) 0.08g/L, magnesium sulfate monohydrate (MnSO 4H 2O) 3g/L, two molybdic acid hydrate sodium 0.2g/L, boric acid (H 3BO 3) 0.02g/L, zinc chloride (ZnCl 2) 20g/L, cobalt chloride 0.5g/L, green vitriol (FeSO 4.7H 2O) 65g/L, vitamin H (Biotin) 0.2g/L, sulfuric acid (H 2SO 4) 5mL/L.
The preparation process of B, fusion rotein IFN α-buforin II is:
1) recovery Pichia pastoris FZM on the YPD flat board that contains 100 μ g/ml zeocin 2009
2) choose mono-clonal in containing the YPD substratum from flat board, 17h is cultivated in 30 ℃ of joltings (240rpm);
3) volume is cultivated the Pichia pastoris FZM of 17h 2009, be inoculated in the long-pending optimization of the decaploid fermention medium, 30 ℃, 260rpm, 4d is cultivated in jolting; Every during this time 8h adds 0.15% ammoniacal liquor (v/v analytical pure ammoniacal liquor), and 24h adds 1% methyl alcohol (v/v analytical pure), 0.4%PTM1 (v/v);
4) the centrifugal 3min of 8000rpm (4 ℃) obtains supernatant;
5) get 10 μ l supernatants, carry out the SDS-PAGE electrophoresis detection;
6) supernatant liquor is crossed the molecular sieve gel column, collects respective components;
7) respective components of lyophilize collection obtains fusion rotein buforin II and INF-α;
8) cut acquisition buforin II and IFN α with the enteropeptidase enzyme.
A kind of fusion rotein buforin II and INF-α application process in pig starter feed are:
With lyophilize fusion rotein buforin II and INF-α, directly mix with additive, further be made into complete feed again, be applied in the pig starter feed, fusion rotein buforin II and the final addition of INF-α are to contain 80mg-100mg fusion rotein buforin II and INF-α in every kg daily ration.
This genetic engineering technique produces fusion rotein buforin II and INF-α has solved buforin II to strong this difficult point of host bacterium suicide effect, the novel fusion polypeptide of being produced is the expression amount height not only, also have efficient, broad-spectrum antimicrobial, antiviral and regulate biologic activity such as immunity after the digestion of enzymolysis or body enteron aisle, and produce fusion rotein buforin II with existing other genetic engineering technique and compare with INF-α and possess following characteristics:
1, use this expression system to produce novel antibacterial peptide buforin II and can overcome the toxic action of buforin II to the host bacterium, its reason is that this expression system is used pig interferon α as fusion rotein, has eliminated the suicide effect of buforin II to the host bacterium;
2, utilize the Red/ET homologous recombination technique that plasmid integration is gone into the engineering bacteria genome, heredity is more stable.
3, expression amount is than higher, and purifying easily, because pichia spp self exocrine protein is few, expressed proteins 60% is a target protein.
4, the substratum cheapness of fermentation use, purifying process is simple, makes the protein production cost reduce significantly.
Description of drawings
Fig. 1 is that antibacterial peptide buforin II and porcine INF-alpha fusion expression Pichia yeast engineering make up the whole process synoptic diagram
Wherein: P1: P2:
Figure G2009102732323D00052
P3:
Figure G2009102732323D00053
Embodiment
Embodiment 1:
The preparation method of the structure of the Pichia yeast engineering of antibacterial peptide buforin II and porcine INF-alpha fusion expression and antibacterial peptide buforin II.
1 material
1.1 bacterial classification and carrier
Bacterial classification and vector gene type and source see Table 1.
Table 1 bacterial classification and vector gene type and source
Figure G2009102732323D00061
1.2 toolenzyme
RNaseA, Proteinase K give birth to worker company available from Shanghai; Restriction enzyme is available from promega; 2 *PCR master Mix is available from sky, Beijing root biotech company.
1.3 test kit
In a small amount, middle amount and large quantity extracting plasmid test kit, yeast genes group reagent box is a QIAGEN company product.
1.4 substratum
LB liquid nutrient medium: yeast extract 5g; Peptone 10g; NaCl 5g; Add water and be settled to 1L.
The LB solid medium: adding final concentration in the LB liquid nutrient medium is the agar powder of 1.5% (m/v).
According to Fig. 1 the present invention is described in further detail below:
The preparation method of the Pichia yeast engineering of a kind of amalgamation and expression antibacterial peptide buforin II and porcine INF-alpha the steps include:
The acquisition of A, pig IFN α gene: the pig IFN α protein sequence (ID:CAA40477) according to the NCBI albumen database is announced, with reference to the pichia spp codon preference, design pig IFN α complete genome sequence, and synthetic by Shanghai invitrogen company.
The acquisition of B, IFN α-6 * His-bufforin II PCR product: classify template as with synthetic pig IFN α nucleotides sequence, with p1 (ATTGCTGCTA AAGAAGAAGG GGTATCTCTC GAGAAAAGAGAGGCTGA AGC TATGGCTCCA ACTTCTGCTT TCTT) and p2 (ATCCTTTCTCAACAATCTAT GAACTCTACC AACTGGAAAT TGCAAACCAG CTCTAGAAGATCTAGTCTTA TCATCATCAT CTTCCTTCTT TCTCAATCTA TCTTG) is primer, by 50 μ l reaction system (ddH 2O:39 μ l; DNTP (10mM): 1.0 μ l; 10 * pfu buffer:5.0 μ l; Oligoup (20pmol/ μ l): 1.5 μ l; Oligo down (20pmol/ μ l): 1.5 μ l; Template (212ng): 1.0 μ l; Pfupolymarase:1.0 μ l.) and response procedures (95 ℃ of pre-sex change, 3min; 10 circulations (95 ℃ of sex change, 45s; Anneal 63 ℃ 40s; Extend 72 ℃, 50s); 22 circulations (95 ℃ of sex change, 45s; Anneal 55 ℃ 40s; Extend 72 ℃, 50s) extend 72 ℃ at last, 10min) carry out pcr amplification and obtain IFN α-6 * His-bufforin II PCR product.
The acquisition of IFN α-6 * His-bufforin II PCR product of C, band homology arm: with IFN α-6 * His-bufforin II PCR product is template, with p1 and p3 (AACTCAATGATGATGATGATGATGGTCGAC GGCGCTATTC AGATCCTCTT CTGAGATGAG TTTTTGTTCCTTATCATCAT CATCCTTTCT CAACAATCTATGAAC) is primer, by 50 μ l reaction system (ddH 2O:39 μ l; DNTP (10mM): 1.0 μ l; 10 * pfu buffer:5.0 μ l; Oligo up (20pmol/ μ l): 1.5 μ l; Oligo down (20pmol/ μ l): 1.5 μ l; Template (212ng): 1.0 μ l; Pfu polymarase:1.0 μ l.) and response procedures (95 ℃ of pre-sex change, 3min; 10 circulations (95 ℃ of sex change, 45s; Anneal 63 ℃ 40sec; Extend 72 ℃, 50s); 22 circulations (95 ℃ of sex change, 45s; Anneal 55 ℃ 40s; Extend 72 ℃, 50s) extend 72 ℃ at last, 10min) carry out IFN α-6 * His-bufforin II PCR product that pcr amplification obtains the band homology arm.
D, expression cassette P AOX1-α Factor-IFN α-6 * His-bufforin II-Zeocin rAcquisition: the band homology arm IFN α-6 * His-bufforin II PCR product and pichia spp external secretion pPICZ Aa plasmid common-battery change among the intestinal bacteria E.coli YZ2005, utilize bacterium colony PCR and nucleotide sequencing to filter out plasmid pPICZA α-IFN α-6 * His-bufforin II then, use at last and obtain expression cassette P after Sac I enzyme is cut AOX1-α Factor-IFN α-6 * His-bufforin II-Zeocin r
The structure of E, engineering bacteria: adopt electroporation (1500V/cm) with linearizing expression cassette P AOX1-α Factor-IFN α-6 * His-bufforin II-Zeocin rImport among the Pichia pastoris KM71H.Obtain to be integrated with expression cassette P on the genome through the PCR evaluation AOX1-IFN α-6His-bufforin II-Zeocin rEngineering bacteria Pichia pastoris FZM 2009The preservation of this bacterial strain, depositary institution: Chinese typical culture collection center, address: China. Wuhan. Wuhan University, postcode: 430072, preservation date: on November 11st, 2009, deposit number: CCTCC NO:M209259, classification name: Pichia pastoris FZM 2009This strain morphology is learned characteristic: this bacterium bacterium colony is big and thick, moistening, and smooth surface is opaque, oyster white, and the edge is rounding very, provokes easily, and the bacterium colony quality is even, and the color of pros and cons and edge, central part is homogeneous very all.Cultural characters: this bacterium can well grow at solid-state or liquid YPD substratum, when cultivating in liquid YPD, if the big or incubation time of inoculum size can form obvious bacterium ball when long.Physiological property: Pichiapastoris FZM 2009Belong to methyl alcohol nutritional type yeast belong, can utilize methyl alcohol as carbon source, have the AOX strong promoter, be integrated with fusion rotein IFN α-bufforin II encoding gene on the genome, can a large amount secreting, expressing IFN α-bufforin II fusion rotein under methanol induction.Functional performance: Pichia pastoris FZM 2009High-density culture is produced fusion rotein buforin II and INF-α continuously, as fodder additives or medicine.
Embodiment 2:
A kind of engineering bacteria Pichia pastoris FZM 2009Preparation fusion rotein buforin II and INF-α method, its step is as follows:
A, material and solution preparation
Used engineering bacteria is Pichia pastoris FZM provided by the present invention 2009
The engineering bacteria substratum is experiment fermentation optimization substratum, each composition quality mark %: glucose 4%, ammoniacal liquor 150 μ L, KH 2PO 40.7%, MgSO 4.7H 2O, 0.03%FeSO 4.7H 2O, 0.05%MnSO 4.H 2O0.05%, 0.1%Peptone, pH5.5, culture condition are 30 ℃, and every 8h adds ammoniacal liquor one time, and 24h adds methyl alcohol one time, 0.4%PTM1 (v/v).
The PTM1 prescription is: copper sulfate (CuSO 4) 6g/L, Potassium Iodate (KI) 0.08g/L, magnesium sulfate monohydrate (MnSO 4H 2O) 3g/L, two molybdic acid hydrate sodium 0.2g/L, boric acid (H 3BO 3) 0.02g/L, zinc chloride (ZnCl 2) 20g/L, cobalt chloride 0.5g/L, green vitriol (FeSO 4.7H 2O) 65g/L, vitamin H (Biotin) 0.2g/L, sulfuric acid (H 2SO 4) 5mL/L.
The preparation method of B, a kind of fusion rotein buforin II and INF-α, its step is as follows:
1) recovery Pichia pastoris FZM on the YPD flat board that contains 100 μ g/ml Zeocin 2009
2) choose mono-clonal in containing the YPD substratum from the flat board of recovering, 30 ℃ of joltings (240rpm) were cultivated 17 hours;
3) volume is cultivated the Pichia pastoris FZM of 17h 2009, be inoculated in ten volumes and optimize in the fermention medium, 30 ℃, 260rpm, 4d is cultivated in jolting; Every during this time 8h adds 0.15% ammoniacal liquor (v/v analytical pure ammoniacal liquor), and 24h adds 1% methyl alcohol (v/v analytical pure) and 0.4%PTM1 (v/v);
4) the centrifugal 3min of 8000rpm (4 ℃) obtains supernatant;
5) get 10 μ l supernatants, carry out the SDS-PAGE electrophoresis detection;
6) supernatant liquor is crossed the molecular sieve gel column, collects respective components;
7) component is collected in lyophilize, obtains fusion rotein buforin II and INF-α;
8) cut acquisition buforin II and IFN α with the enteropeptidase enzyme, its ratio is 1: 1;
9) show that by external minimal inhibitory concentration test buforin II and IFN α (ratio is 1: 1) have fabulous anti-microbial property (the results are shown in Table 2).Show that by the weanling pig test every kg daily ration adds 80-100mg fusion rotein antibacterial peptide buforin II and INF-α can reduce diarrhea rate, also can improve weanling pig growth performance and immunologic function (the results are shown in Table 3,4).
A kind of fusion rotein buforin II and the application process of INF-α in feed are:
With exsiccant fusion rotein buforin II and INF-α, directly mix with additive, further be made into complete feed again, be applied in the pig starter feed, fusion rotein buforin II and the final addition of INF-α are to contain 80mg-100mg fusion rotein buforin II and INF-α in every kg feed.
Table 2 fusion rotein buforin II and INF-α are to the minimal inhibitory concentration of various pathogenic bacterias
Concentration unit: μ g/ml
Figure G2009102732323D00091
Table 3 fusion rotein buforin II and INF-α are to the influence of weanling pig growth performance
Figure G2009102732323D00092
Colleague's shoulder motes same letter is represented difference not remarkable (P＞0.05) in the table, and adjacent letters is represented significant difference (P＜0.05), and is alternate aLetter representation difference is (P＜0.01) extremely significantly.
Table 4 fusion rotein buforin II and INF-α are to the influence of weanling pig serum immune globulin concentration
Figure G2009102732323D00101
Colleague's shoulder motes same letter is represented difference not remarkable (P＞0.05) in the table, and adjacent letters is represented significant difference (P＜0.05), and alternate letter representation difference is (P＜0.01) extremely significantly.

Claims (4)

1. antibacterial peptide buforin II and porcine INF-alpha fusion expression pichia pastoris: it is characterized in that: Pichia yeast Pichia pastoris FZM 2009, CCTCC NO:M209259.
2. the preparation method of the Pichia yeast engineering of described a kind of amalgamation and expression antibacterial peptide buforin II of claim 1 and porcine INF-alpha the steps include:
A, expression cassette P AOX1-α Factor-IFN α-6 * His-bufforin II-Zeocin rStructure, at first, obtain being with homology arm PCR product IFN α-6 * His-bufforin II by the twice PCR amplification with synthetic pig interferon α (IFN α) nucleotide sequence of the artificial design of pichia spp preference codon; Next adopts the Red/ET homologous recombination technique that IFN α-6 * His-bufforin II is cloned on pPICZA α carrier or the plasmid; The 3rd is to use to obtain expression cassette P after Sac I enzyme is cut AOX1-α Factor-IFN α-6 * His-bufforin II-Zeocin r
B, expression engineering bacteria Pichia pastoris FZM 2009Structure, obtaining genome conformity has expression cassette 5 ' P AOX1The process of-IFN α-6His-bufforin II-Zeocin is: expression cassette P after adopting electroporation with linearizing AOX1-α Factor-IFN α-6 * His-bufforin II-Zeocin rExpression plasmid import among the Pichia pastorisKM71H, obtain being integrated with on the genome expression cassette 5 ' P through microbiotic Zeocin screening and PCR evaluation AOX1The engineering bacteria of-IFN α-6His-bufforin II-Zeocin.
3. the preparation method of described a kind of fusion rotein buforin II of claim 1 and INF-α the steps include:
A, material and solution preparation: each composition quality mark % of engineering bacteria substratum: glucose 4%, ammoniacal liquor 150 μ L, potassium primary phosphate 0.7%, magnesium sulfate heptahydrate 0.03%, green vitriol 0.05%, magnesium sulfate monohydrate 0.05%, peptone 0.1%, pH5.5;
The PTM1 prescription is: copper sulfate 6g/L, Potassium Iodate 0.08g/L, magnesium sulfate monohydrate 3g/L, two molybdic acid hydrate sodium 0.2g/L, boric acid 0.02g/L, zinc chloride 20g/L, cobalt chloride 0.5g/L, green vitriol 65g/L, vitamin H 0.2g/L, sulfuric acid 5mL/L;
The preparation process of B, antibacterial peptide fusion protein buforin II and INF-α is:
1) recovery Pichia pastoris FZM on the YPD flat board that contains 100 μ g/ml zeocin 2009
2) choose mono-clonal and be inoculated in and contain in the YPD substratum, 17h are cultivated in 30 ℃ of joltings;
3) volume is cultivated the Pichia pastoris FZM of 17h 2009, be inoculated in the ten volume fermention mediums, 30 ℃, 260rpm, 4d is cultivated in jolting; Every during this time 8h adds 0.15%v/v ammoniacal liquor, and 24h adds 1%v/v methyl alcohol and 0.4%v/v PTM1;
4) 8000rpm, 4 ℃ of centrifugal 3min obtain supernatant;
5) get 10 μ l supernatants, carry out the SDS-PAGE electrophoresis detection;
6) supernatant liquor is crossed the molecular sieve gel column, collects component;
7) component of lyophilize collection obtains fusion rotein buforin II and INF-α;
8) cut acquisition buforin II and IFN α with the enteropeptidase enzyme.
4. described a kind of fusion rotein buforin II of claim 3 and the application of IFN α in pig starter feed.
CN 200910273232 2009-12-11 2009-12-11 Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof Active CN101717737B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 200910273232 CN101717737B (en) 2009-12-11 2009-12-11 Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 200910273232 CN101717737B (en) 2009-12-11 2009-12-11 Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof

Publications (2)

Publication Number Publication Date
CN101717737A true CN101717737A (en) 2010-06-02
CN101717737B CN101717737B (en) 2012-05-30

Family

ID=42432376

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 200910273232 Active CN101717737B (en) 2009-12-11 2009-12-11 Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof

Country Status (1)

Country Link
CN (1) CN101717737B (en)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103923846A (en) * 2013-12-23 2014-07-16 华北制药集团新药研究开发有限责任公司 Pichia pastoris culturing medium
CN104017086A (en) * 2014-06-24 2014-09-03 任建廷 Fusion antimicrobial peptide and preparation method thereof
CN104017087A (en) * 2014-06-24 2014-09-03 任建廷 Pig-source antimicrobial peptide and preparation method thereof
CN104498377A (en) * 2015-01-13 2015-04-08 中国科学院亚热带农业生态研究所 Antioxidant polypeptide expression pichia pastoris engineering bacteria and application
CN105330748A (en) * 2014-08-14 2016-02-17 广东紫金正天药业有限公司 Human interferon IFN-gamma and LL-37 fusion protein
CN105330746A (en) * 2014-08-14 2016-02-17 广东紫金正天药业有限公司 Polyethylene glycol-modified human interferon and human antibacterial peptide fusion protein
CN105330747A (en) * 2014-08-14 2016-02-17 广东紫金正天药业有限公司 Human interferon IFN-alpha 2 alpha and LL-37 fusion protein
CN105713093A (en) * 2014-08-14 2016-06-29 广东紫金正天药业有限公司 Fusion protein of human interferon IFN-alpha 2b and LL-37
CN106854657A (en) * 2016-12-20 2017-06-16 广州格拉姆生物科技有限公司 It is a kind of assistant degradation protein and the prebiotic recombinant Saccharomyces cerevisiae of antibacterial peptide to be secreted
CN110256573A (en) * 2019-05-08 2019-09-20 吉林农业大学 A kind of heterozygous antibacterial peptide BM16R and its preparation method and application
CN110357971A (en) * 2019-08-21 2019-10-22 中国科学院微生物研究所 It is a kind of for promptly preventing the pig Interferon Alfacon-1 of African swine fever

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1158382C (en) * 2001-02-28 2004-07-21 华南农业大学 Preparation and application of antibiotic peptide pichia yeast
CN100558891C (en) * 2007-10-18 2009-11-11 中国科学院亚热带农业生态研究所 A kind of red/ET recombination method that is used for luminous light shaped bacillus

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103923846B (en) * 2013-12-23 2016-07-06 华北制药集团新药研究开发有限责任公司 A kind of pichia pastoris phaff culture medium
CN103923846A (en) * 2013-12-23 2014-07-16 华北制药集团新药研究开发有限责任公司 Pichia pastoris culturing medium
CN104017086A (en) * 2014-06-24 2014-09-03 任建廷 Fusion antimicrobial peptide and preparation method thereof
CN104017087A (en) * 2014-06-24 2014-09-03 任建廷 Pig-source antimicrobial peptide and preparation method thereof
CN105330748A (en) * 2014-08-14 2016-02-17 广东紫金正天药业有限公司 Human interferon IFN-gamma and LL-37 fusion protein
CN105330746A (en) * 2014-08-14 2016-02-17 广东紫金正天药业有限公司 Polyethylene glycol-modified human interferon and human antibacterial peptide fusion protein
CN105330747A (en) * 2014-08-14 2016-02-17 广东紫金正天药业有限公司 Human interferon IFN-alpha 2 alpha and LL-37 fusion protein
CN105713093A (en) * 2014-08-14 2016-06-29 广东紫金正天药业有限公司 Fusion protein of human interferon IFN-alpha 2b and LL-37
CN104498377A (en) * 2015-01-13 2015-04-08 中国科学院亚热带农业生态研究所 Antioxidant polypeptide expression pichia pastoris engineering bacteria and application
CN106854657A (en) * 2016-12-20 2017-06-16 广州格拉姆生物科技有限公司 It is a kind of assistant degradation protein and the prebiotic recombinant Saccharomyces cerevisiae of antibacterial peptide to be secreted
CN106854657B (en) * 2016-12-20 2018-05-15 广州格拉姆生物科技有限公司 A kind of energy assistant degradation protein simultaneously secretes the prebiotic recombinant Saccharomyces cerevisiae of antibacterial peptide
WO2018113429A1 (en) * 2016-12-20 2018-06-28 广州格拉姆生物科技有限公司 Probiotic recombinant saccharomyces cerevisiae for assisting protein degradation and antimicrobial peptide secretion
CN110256573A (en) * 2019-05-08 2019-09-20 吉林农业大学 A kind of heterozygous antibacterial peptide BM16R and its preparation method and application
CN110357971A (en) * 2019-08-21 2019-10-22 中国科学院微生物研究所 It is a kind of for promptly preventing the pig Interferon Alfacon-1 of African swine fever
CN110357971B (en) * 2019-08-21 2021-01-15 中国科学院微生物研究所 Pig compound interferon for emergency prevention of African swine fever

Also Published As

Publication number Publication date
CN101717737B (en) 2012-05-30

Similar Documents

Publication Publication Date Title
CN101717737B (en) Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof
CN106701606B (en) Genetic engineering candida utilis capable of degrading and utilizing kitchen waste and construction method thereof
CN101173004A (en) Insect antimicrobial peptide Thanatin and method for producing deletion mutant thereof
CN103103202A (en) Harpin coding gene (i) hrpZPsgS1(/i) or application of harpinZPsgS1 protein expressed by Harpin coding gene (i) hrpZPsgS1(/i)
CN101906165B (en) Expression product in series of two fish antibacterial peptide genes and expression method thereof
CN111004317B (en) Canine recombinant interferon alpha 7 and preparation method and application thereof
CN109295067A (en) A kind of the moral paddy insulin precursor-gene and its expression of codon optimization
CN101665798B (en) Method for preparing recombinant human serum albumin and interferon alpha fusion protein
CN102199201B (en) Recombinant fungal immunomodulatory protein gene in Ganoderma lucidum, protein coded whereby and application thereof
CN105063138A (en) Method for producing trametes versicolor immunomodulatory protein by virtue of pichia pastoris expression system
CN103602603A (en) Preparation method of bovine lactoferricin pichia pastoris engineering bacterium
CN100387704C (en) Lucid ganoderma fungus with high glycopeptide composite yield, its mutagen breeding method and use
CN101314762B (en) Yeast engineering bacterium for expression recombination of rainbow trout antimicrobial peptide Oncorhyncin II and preparation method thereof
CN105002103A (en) Fermentation method for recombinant G-CSF(15-75) polypeptide pichia pastoris
CN101525629B (en) Gene engineering preparation method of bioactive peptide containing human alpha defensin 5
CN102199202B (en) Gene of recombinant fungal immunomodulatory protein between ganodermas, protein coded thereby, and application thereof
CN106222175A (en) The optimized gene of channel catfish LEAP 2 mature peptide and the preparation method of recombiant protein thereof
CN103012578B (en) Recombinant porcine interleukin 2, and encoding gene and expression method thereof
CN101979572B (en) Preparation for South China Sea conus striatus toxin S4.3 and application
CN105400791A (en) Optimized gene of zebrafish defensin defbl3 and preparation method of recombinant protein thereof
CN105062906B (en) A kind of production method optimizing organophosphor hydrolytic enzyme Yeast engineering bacteria and its enzyme
CN103865865B (en) A kind of sea cucumber enteron aisle produces Sumizyme MP bacterial strain and application thereof
CN102168057B (en) Engineering bacteria expressing active peptides and method of preparing mixed polypeptide
CN105995216A (en) Lysozyme-based fish feed
CN102121025B (en) Multi-copy integrated expression vector and preparation method and application thereof in expression of bovine lactoferrin

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C53 Correction of patent for invention or patent application
CB03 Change of inventor or designer information

Inventor after: Tang Zhiru

Inventor after: Feng Zemeng

Inventor after: Yin Yulong

Inventor before: Tang Zhiru

Inventor before: Feng Zemeng

Inventor before: Yin Yulong

Inventor before: Zhang Youming

COR Change of bibliographic data

Free format text: CORRECT: INVENTOR; FROM: TANG ZHIRU FENG ZEMENG YIN YULONG ZHANG YOUMING TO: TANG ZHIRU FENG ZEMENGYIN YULONG

C14 Grant of patent or utility model
GR01 Patent grant
ASS Succession or assignment of patent right

Owner name: HINAPHARM PHARMACEUTICAL CO., LTD.

Free format text: FORMER OWNER: TROPICAL AGRICULTURAL ECOLOGICAL INST., CHINESE ACADEMY OF SCIENCES

Effective date: 20130712

C41 Transfer of patent application or patent right or utility model
COR Change of bibliographic data

Free format text: CORRECT: ADDRESS; FROM: 410125 CHANGSHA, HUNAN PROVINCE TO: 528515 FOSHAN, GUANGDONG PROVINCE

TR01 Transfer of patent right

Effective date of registration: 20130712

Address after: 528515 Guangdong province Foshan Gaoming Cangjiang Industrial Park West Sand Water District Heyuan Yang Road

Patentee after: Hinapharm Pharmaceutical Co., Ltd.

Address before: 410125 Hunan province Changsha Furong district Mapoling two Yuanda Road No. 1071

Patentee before: Institute of Subtropical Agriculture, Chinese Academy of Sciences

C56 Change in the name or address of the patentee

Owner name: GUANGDONG HINAPHARM BIOTECHNOLOGY CO., LTD.

Free format text: FORMER NAME: HINAPHARM PHARMACEUTICAL CO., LTD.

CP01 Change in the name or title of a patent holder

Address after: 528515 Guangdong province Foshan Gaoming Cangjiang Industrial Park West Sand Water District Heyuan Yang Road

Patentee after: GUANGDONG HINABIOTECH CO., LTD.

Address before: 528515 Guangdong province Foshan Gaoming Cangjiang Industrial Park West Sand Water District Heyuan Yang Road

Patentee before: Hinapharm Pharmaceutical Co., Ltd.