CN1544629A - Method for preparing recombinant goose interleukin-2 protein and its application - Google Patents
Method for preparing recombinant goose interleukin-2 protein and its application Download PDFInfo
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Abstract
The invention discloses a recombinant goose interleukin-2(gsIL-2) protein preparing method and its use. The DNA fraction of RT-PCR amplified Zhedong white goose interleukin-2, prokaryotic expression carrier pBAD/HisB, and eukaryotic expression carriers pMET alpha B and pMETB compose expression particles pBAD/HisB/gsIL-2, pMET alpha B/gsIL-2 and pMETA/gsIL-2, which are converted to Escherichia coli LMG194, microzyme PMAD11 and PMAD16, respectively, then inducing expression. After inducing, making ultrasonic wave cracking and deposit elimination by centrifugation on Escherichia coli and microzyme PMAD16 to obtain crude products of gsIL-2 protein, PMAD11 expressed gsIL-2 is mainly separated in the culture medium, centrifugating and taking supernatant to obtain gsIL-2 protein crude products. Using Ni-NTAArgarrose protein purifying system, thus able to faster obtain gsIL-2 pure product, and the crude and pure products can act as immune assistant and disease-curing drugs. It applied genetic engineering technique to prepare recombinant protein gsIL-2, and has simple technical flow, low production cost, good stability, high bioactivity and other characters.
Description
Technical field
The present invention relates to biological technical field, relate in particular to the proteic preparation method and its usage of a kind of reorganization goose interleukin II.
Background technology
1.Schauenstein found at first that chicken splenic lymphocyte secretion is a kind of and had an active cytokine of the mammalian interleukin of being similar to element-2 sample Deng (1982), subsequently, the using sulfated ammonium precipitation of Schnetlzler etc. (1983), the bicarbonate of ammonia resuspension, Sephadex G-100 gel-filtration obtains two classes and has active isolate, and its molecular weight lays respectively between 9～11.5KDa and the 19.5～21.5Kda.After this Vainio etc. use and obtained molecular weight with quadrat method is the molecule of 13KDa, and they estimate that with isoelectrofocusing the PI of chicken IL-2 is 5.9.Frederickson etc. (1987) use Phenyl-sepharose, anionresin and superose-12 and cross methods such as post, isolate two classes and have active material, the one molecular weight is 14KDa, its two molecular weight is about 26～30KDa, the latter is stronger than the former activity, and both all have low wetting ability.Myers (1992) reported a kind of from the chicken splenic lymphocyte nutrient solution that ConA stimulates " five-step approach " of purifying chicken IL-2, i.e. (1) gel-filtration tentatively concentrates the chicken splenic lymphocyte suspension that ConA stimulates; (2) ammonium sulfate precipitation; (3) with Sephadex G-100 chromatography; (4) (high performanceliquid chromatography is HPLC) with (5) Phenyl-sepharose chromatography for the high back voltage liquid chromatography (LC).Purified IL-2 albumen carries out the SDS-PAGE electrophoresis under reductive condition, the albumen that obtains has two peak values, and molecular weight is about 36～39KDa and 17.5～25KDa, with the albumen alkanisation reduction on peak, use Sephadex G-100 chromatography again, can produce low-molecular-weight peak.
Sundick etc. (1997) obtain chicken IL-2 gene first by the method in construction cDNA library from chicken spleen, Choi etc. (1998) are cloned into prokaryotic expression carrier PUC18 and carrier for expression of eukaryon pcDNA3 to the chicken IL-2 gene that contains long 430 Nucleotide of signal sequence respectively, express in intestinal bacteria and CHO-K1 cell respectively, two kinds of albumen that obtain all have the lymphopoietic biologic activity of the chicken T of promotion.Stepaniak etc. (1999) compare deep research to chicken IL-2 vivoexpression, the chicken IL-2 gene of removing leading peptide is expressed in intestinal bacteria Pgex-2t system, the albumen that obtains has the proteic biologic activity of natural chicken IL-2, but expression efficiency is low only to be 63 μ g/L, and mostly be non-solubility albumen, be unfavorable for proteic recovery and purifying.Be head it off, they have adopted pET32a
+Efficient expression system is brought up to 4mg/L with expressing quantity, and the overwhelming majority is a soluble proteins.Simultaneously, this scholar complete encoder block that will contain leading chicken IL-2 gene is cloned into carrier for expression of eukaryon pCR3.1
+In, in the kidney fibroblast, express, found that this system not only can stably express chicken IL-2 albumen, and go up cleer and peaceful purified albumen and all have good biologic activity, thereby proved that chicken IL-2 is similar with Mammals IL-2, its activity does not also rely on leader sequence.
2. chicken IL-2 precursor protein is made up of 143 amino acid, and sophisticated albumen is made up of 121 amino acid.It and Mammals IL-2 have similar biologic activity, as promoting bone-marrow-derived lymphocyte and the lymphocytic propagation of T, strengthen the functions such as killing activity of NK cell, play a significant role in immunity of organism.Research based on chicken IL-2, we utilize the increased dna fragmentation of East Zhejiang province white goose IL-2 of RT-PCR technology from splenic lymphocyte, and realized that efficiently expressing in protokaryon and eukaryotic cell, expression product have the biologic activity of stimulation goose spleen lymphocyte proliferation.
Summary of the invention
The purpose of this invention is to provide the proteic preparation method and its usage of a kind of reorganization goose interleukin II.
The step of reorganization goose interleukin II protein preparation method is as follows:
1) dna fragmentation that in the white goose monokaryon lymphocyte of East Zhejiang province, clones goose interleukin II (gsIL-2) with contain P
BADThe prokaryotic expression carrier pBAD/His B of promotor is set up into expression vector pBAD/HisB/gsIL-2; Perhaps above-mentioned gsIL-2DNA fragment with contain P
ADGIThe carrier for expression of eukaryon pMET α B of promotor is set up into secreted expression carrier pMET α B/gsIL-2; Perhaps above-mentioned gsIL-2 dna fragmentation with contain P
ADGIThe carrier for expression of eukaryon pMETB of promotor is set up into type expression vector pMET B/gsIL-2 in the born of the same parents;
2) use RM as basic medium, by fermenting and amplifying colibacillus engineering LMG194;
3) use BMDY as basic medium,, add nutrient substances such as nitrogenous source, carbon source at the expression different time by fermenting and amplifying Yeast engineering bacteria PMAD11 and PMAD16;
4) intestinal bacteria LMG194 and yeast PMAD16 induces after ultrasonic treatment, the centrifugal precipitation of going are the gsIL-2 raw product, the gsIL-2 that PMAD11 expresses mainly secretes in substratum, is the gsIL-2 raw product through the centrifuging and taking supernatant.Can obtain the pure product of gsIL-2 with Ni-NTA Argarose protein purification system;
5) after raw product or pure product add stablizer such as glycine and solubility promoter, make finished product after sterile filtration, packing and the freeze-drying, be stored in-20 ℃ the cold storage environment.
Reorganization gsIL-2 albumen is used for enhance immunity function and immunological adjuvant; Be used for the treatment of the palmipeds disease;
Advantage of the present invention: the gsIL-2 albumen of (1) escherichia coli expression mainly exists in the genetic engineering bacterium with the form of fusion rotein, through ultrasonic wave target protein is discharged, do not need other processing just can obtain the gsIL-2 crude protein, and protein stability is good, biologic activity is higher; (2) select secretor type Yeast expression carrier pMET α B for use, expressed gsIL-2 albumen major part is secreted in the substratum, and not only without any need for processing just can obtain the gsIL-2 raw product, and target protein has satisfactory stability and biologic activity; (3) select nonsecreting type Yeast expression carrier pMET B for use, the gsIL-2 albumen of expression mainly exists in the engineering bacteria with the form of fusion rotein, just can obtain the gsIL-2 crude protein through ultrasonication, and crude protein has than advantages of higher stability and biologic activity; (4) used Ni-NTA Agarose protein purification system is simple to operate, the purity of protein height that obtains.(5) reorganization gsIL-2 is remarkable as immunological adjuvant and disease therapeuticing medicine effect.
Embodiment
1 goose interleukin-2 gene clone stimulates goose spleen monocyte with canavailn (ConA), the Trizol single stage method is extracted total RNA, by RT-PCR method amplification with cloned East Zhejiang province white goose interleukin II cDNA fragment, be connected to sequencing vector pGEM-T Easy Vector, measure the gsIL-2 dna sequence dna.
The pcr amplification product subclone of the colibacillary structure of 2 recombinant expressed goose inactivated interleukin-2 genetic engineerings goose interleukin II is in a new expression vector pBAD/His B, be built into gsIL-2 prokaryotic expression carrier pBAD/His B/gsIL-2, it can be expressed in intestinal bacteria LMG194, make gsIL-2 albumen account for 9～10% of bacterial protein, expression rate does not reduce through going down to posterity more than 100 times.
The saccharomycetic structure of 3 recombinant expressed goose inactivated interleukin-2 genetic engineerings with the pcr amplification product subclone of goose interleukin II in novel carrier for expression of eukaryon pMET α B and pMET B, be built into gsIL-2 expression vector pMET α B/gsIL-2 and pMET B/gsIL-2 respectively, with BMDY as basic medium, with methyl alcohol as inductor, by optimizing dissolved oxygen, concussion speed, processing parameters such as fermentation pH value, can make the gsIL-2 gene obtain expressing in P.methanolica expression strain PMAD11 and PMAD16, expression amount accounts for 20～25% and 10～12% of bacterial protein respectively., expression rate does not reduce through going down to posterity more than 100 times.
The preparation and the purifying process of 4 reorganization goose interleukin II albumen raw product.
The preparation of the raw product of being set up and purifying process flow process are simple, good stability, active high.This technology has following characteristics: the gsIL-2 albumen of (1) escherichia coli expression mainly exists in the engineering bacteria with the form of fusion rotein, through ultrasonic wave target protein is discharged, do not need other processing just can obtain the gsIL-2 crude protein, and its biologic activity is higher; (2) select secretor type Yeast expression carrier pMET α B for use, expressed gsIL-2 albumen major part is secreted in the substratum, and not only without any need for processing just can obtain the thick product of gsIL-2 albumen, and target protein has good biologic activity; (3) select nonsecreting type Yeast expression carrier PMET B for use, the gsIL-2 albumen of expression mainly exists in the engineering bacteria with the form of fusion rotein, just can obtain the gsIL-2 crude protein through ultrasonication, and crude protein has than higher biologic activity; (4) used Ni-NTA Agarose protein purification system is simple to operate, the purity of protein height that obtains.
5 raw product or pure product add solubility promoters such as phosphoric acid buffer, with the degerming of microvoid membrane filtration, can be used as immunological adjuvant and disease therapeuticing medicine.
The important biomolecule of IL-2 is learned function and is promoted the lymphocytic propagation of T exactly, we use the goose spleen T lymphocyte that stimulates through ConA as target cell, with mtt assay the proteic activity of reorganization gsIL-2 is detected, confirm that expression product has higher biologic activity.
The present invention uses recombinant DNA technology, adopts the reverse transcription PCR method to obtain a kind of reorganization gsIL-2 albumen.Through downstream purification, the product under the pilot scale has reached the specification of quality of clinical application.
The design of embodiment 1. Oligonucleolide primers is with synthetic
According to the duck of registering among the GenBank (AF294323), chicken (AF017645) and turkey (AJ007463) IL-2 nucleotide sequence, design 1 pair of RT-PCR primer:
Upstream primer, 5 '-AATACTAGCACAGAGACAACCAG-3 '
Downstream primer, 5 '-TTACTGAAATTTATTAAATATCATCTA-3 '
The lymphocytic separation of embodiment 2. spleen monokaryons
1 monthly age of aseptic collection East Zhejiang province white goose spleen, shred and place no Ca
2+, Mg
2+(717mmol/L K among the ionic PBS
2HPO
4, 283mmol/L KH
2PO
4PH7.2), 4 ℃ of centrifugal 10min of 300 * g, get 4 ℃ of centrifugal 30min of supernatant 500 * g and collect lymphocyte, wash 2 times with PBS then, with RPMI1640 nutrient solution (not containing calf serum) washing 1 time, behind trypan blue (0.1%) the dyeing viable count, cell is made into 1 * 10 again with RPMI1640 grown cultures liquid (containing 10% calf serum, the penicillin of 100IU/ml and the Streptomycin sulphate of 100 μ g/ml)
7The cell suspension of/ml.Add 10mg/L final concentration ConA in the cell suspension, the packing Tissue Culture Plate, 5% CO2gas incubator is cultivated 16h for 40 ℃ and is collected lymphocyte culture Trizol reagent single stage method extraction cell total rna.
Embodiment 3.RT-PCR amplifying target genes fragment
Goose IL-2 cDNA first chain is synthesized in First Strand cDNA Synthesis Kit reverse transcription; PCR is carried out in transcription product 30 circulations under 95 ℃ of 1min, 47 ℃ of 45s, 72 ℃ of 2min environment, and last 72 ℃ are extended 10min.The PCR product is through 1% agarose gel electrophoresis analysis.
The structure and the sequencing of embodiment 4. sequencing vectors
Reclaim purpose PCR product fragment with PCR Purification test kit purifying, utilize T-A clone strategy to be directly connected in pGEM-T Easy carrier, connect product Transformed E .coli TOP10 competent cell, at the dull and stereotyped enterprising row filter of the LB that contains penbritin, X-gal and IPTG.PCR and enzyme are cut the evaluation positive colony.Select 5 positive colonies to check order.East Zhejiang province white goose interleukin-2 gene Nucleotide and derivation aminoacid sequence see Table 1.
1 AATACTAGCACAGAGACAACCAGAACACTGACAAGATGTGCAAAGTACTGATCTTCAGCTGCCTTTCGG 69
2 M C K V L I F S C L S 69
1 TAGTAATGCTTATGACTACAGCTTATGGAGCACCTCTATCAGAGAAAAATGACACTCTTACAACTTTAA 138
2 V V M L M T T A Y G A P L S E K N D T L T T L 138
1 TAAAAGATTTAGAAAAGCTGGGAACAAGCATGAAGAAGATTAATCTTGAGCTCTACACACCAAATGAGA 207
2 I K D L E K L G T S M K K I N L E L Y T P N N 207
1 AACAGGAGTGCAGCTGGCAAACTCTACAATGTTACTTGAAAGAAATAGTCACCTTGGAGAATGAAATTG 276
2 K Q E C S W Q T L Q C Y L K E I V T L N E N I 276
1 AAGATGAGGATGAAATTGAAGATGAGAACGTATTTAGTGTTAGGAATATTGAAAAGAATCTCCAAAAAC 345
2 E D E D E I E D E N V F S V R N I E K N L Q K 345
1 TTACGGACCTAATTCCCCCAGGAACGGGATGCAAGATCTGTGAAGCTAATGACAAGAAGGAGTTTCCTG 414
2 L T D L I P P G T G C K I C E A N D K K E F P 414
1 AATTTCACCGAGAGCTGACCAACTTTCTGAGATCTATGCTAAAATAAGCAGATGACCATTTATATTTTT 483
2 E F H R E L T N F L R S M L K - 483
1 ACTGCTACGTTATTTATTTAAGTATTTAATTACAAATAATTTATATATTTTACCCTGGGGCTAACTAAC 552
1 TTGCTGTCCATTTTGGGACCACTGTATGCTCTTAGTGTGGGTGATAACGTGTCTGTTCTAAGATCACAT 621
1 TTGATCCTTTCTGTAAGCCCTACGGGCTCAAAATGTATGTTGGAAAACTAATTGGTTCTCACTTTGTCA 690
1 GTAAACTTAAAATGACAACTATTTATTGAAAGAATTGTTAAAAGTTACTTGTAGATGATATTTAATAAA 759
1 TTTCAGTAA 768
Table 1. East Zhejiang province white goose interleukin-2 gene Nucleotide and derivation aminoacid sequence (SEQ No.1)
Embodiment 5.pBAD/His B/gsIL-2 expression vector establishment, screening and evaluation
According to East Zhejiang province white goose interleukin II sequence of having cloned, synthetic upstream and downstream primer: the UP1 (5 ' GC at maturation protein sequence two ends
GAATTCGCACCTCTATCAGAGAA3 '), 5 ' end contains the EcoRI restriction enzyme site; DOWN1 (5 ' GC
AAGCTTTTATTTTAGCATAGATCT 3 '), 5 ' end contains the HandIII restriction enzyme site; Being template with pGEM-T-Easy/gsIL-2 under 95 ℃ of 1min, 47 ℃ of 45s, 72 ℃ of 2min environment, PCR is carried out in 30 circulations, extends 10min at last.After PCR product electrophoresis reclaims, behind EcoRI, HandIII double digestion, recovery purpose fragment is also cloned in the EcoRI+HandIII enzyme of expression plasmid pBAD/His B and is cut in the window, make up recombinant expression plasmid pBAD/His B/gsIL-2, transform host bacterium TOP10, extract recombinant plasmid and carry out PCR, double digestion evaluation and sequencing.
The fermentative production (intestinal bacteria) of embodiment 6. reorganization gsIL-2
Recombinant plasmid through identifying changes LMG194 host bacterium over to, the picking mono-clonal is inoculated in the RM substratum that contains penbritin and glucose, 37 ℃ of overnight incubation, in 1: 100 ratio culture is inoculated in the RM substratum, the 250rpm concussion was cultivated about 2 hours, make OD600 ≈ 0.5, added the L (+) of 20% concentration-pectinose inducing culture 3 hours in 1: 100 ratio, centrifugal collection thalline.Thalline is added an amount of 1 * SDS sample-loading buffer, adopt 15% SDS-PAGE to identify, the Bradford method is measured the product expression amount can reach 9～10%.
Embodiment 7.pMET α B/gsIL-2 and pMETB/gsIL-2 expression vector establishment, screening and evaluation
According to East Zhejiang province white goose interleukin II sequence, at the synthetic upstream and downstream primer in its maturation protein sequence two ends: upstream primer with example 5 in UP1; Downstream primer DOWN2 (5 ' GC
GGATCCTATTTTAGCATAGATCT3 '), 5 ' end contains the BamHI restriction enzyme site.With pGEM-T-Easy/gsIL-2 is that template is carried out PCR, and amplification condition is with example 5.After PCR product electrophoresis reclaims, behind EcoRI, BamHI double digestion, recovery purpose fragment is also cloned respectively in the EcoRI+BamHI enzyme in expression plasmid pMET α B and pMETB strong promoter downstream and is cut in the window, make up recombinant expression plasmid, transform host bacterium TOP10, extract recombinant plasmid and carry out PCR, double digestion evaluation and sequencing.
Reported method such as employing Choi prepare PMAD11 and PMAD16 competent cell, get 100 μ lPMAD11 and PMAD16 yeast competent cell and 2 μ g respectively and cut completely through the KpnI enzyme that pMET α B/gsIL-2 and pMETB/gsIL-2 mix, use Bio-Rad Gene Pluser at 375V/cm, 25 μ F, transform under the condition of 250 Ω, the YPAD that adds the 1ml room temperature then, hatch 1h for 28～30 ℃, the centrifugal 3min of 1500 * g room temperature collects thalline, again be suspended from 100 μ l, 1 * YNB, bacterium liquid is coated MD to be selected on the flat board, cultivated 3～4 days for 28～30 ℃, observe the growing state of transformant.White colony on the random screening MD flat board extracts pastoris genomic dna with Genomic DNA mini-prepkit and carries out the pcr amplification evaluation.
The embodiment 8. reorganization proteic fermentative production of gsIL-2 (yeast)
Picking PCR is accredited as male PMAD11 and PMAD16 clone and is inoculated in the 20mL BMDY substratum respectively, and 28～30 ℃, 16～18h to OD is cultivated in the 250r/min concussion
600=2～10.Room temperature, the centrifugal 5min of 1500 * g collects thalline and is resuspended in the 10mL BMMY substratum.28～30 ℃, 250r/min continues concussion and cultivated 4 days, adding 5% methyl alcohol in per 24 hours, to make the methyl alcohol final concentration be 0.5%, the centrifugal collection respectively gone up cleer and peaceful precipitation, the SDS-PAGE electrophoresis carries out gsIL-2 and expresses evaluation, it is 20～25% that the Bradford method is measured the expression amount of gsIL-2 in PMAD11, and the expression amount in PMAD16 is 10～12%.
The preservation of embodiment 9. recombination engineering colon bacillus and stability
Engineering bacteria adds 30% glycerine ,-70 ℃ of preservations.Short-term is preserved bacterial classification can use the LB flat board.Be to check the stability of engineering bacteria, with original strain, 50 generations and 100 generation bacterium respectively the extracting plasmid carry out sequencing.The three is identical as a result.Simultaneously 3 generation bacterial classification amplification cultivation thing expression level suitable, prove that the engineering bacteria among the present invention is very stable.
Saccharomycetic preservation of embodiment 10. recombination engineerings and stability
Engineering bacteria adds 30% glycerine ,-70 ℃ of preservations.Short-term is preserved bacterial classification can use the YPAD flat board.Be to check the stability of engineering bacteria, with original strain, 50 generations and 100 generation bacterium respectively the extracting plasmid carry out sequencing.The three is identical as a result.Simultaneously 3 generation bacterial classification amplification cultivation thing expression level suitable, prove that the engineering bacteria among the present invention is very stable.
The preparation and the separation and purification of embodiment 11. reorganization gsIL-2 crude protein
The gsIL-2 albumen of expressing through intestinal bacteria LMG194 mainly exists with the form of fusion rotein, thalline is behind ultrasonic disruption, can obtain gsIL-2 albumen raw product, further the Ni-NTAAgarose protein purification system with QIAGEN company can obtain the pure product of gsIL-2 albumen faster.
The gsIL-2 albumen that contains the P.methanolica expression strain PMAD11 expression of pMET α B/gsIL-2 recombinant vectors is mainly secreted in substratum, can obtain gsIL-2 albumen raw product through the centrifuging and taking supernatant, the gsIL-2 albumen that contains the P.methanolica expression strain PMAD16 expression of pMETB/gsIL-2 recombinant vectors mainly exists in the yeast cell, gsIL-2 albumen raw product can be obtained through ultrasonic treatment, the centrifugal precipitation of going, the pure product of gsIL-2 albumen can be obtained faster with the Ni-NTA Agarose protein purification system of QIAGEN company
The proteic evaluation of embodiment 12. reorganization gsIL-2
The HPLC purity checking is equal more than 98%, mtt assay is measured the biologic activity of raw product and pure product, intestinal bacteria and P.methanolica zymic expression product not only have the activity that stimulates the T lymphocyte growth, and active activity apparently higher than ConA inductive T lymphocyte supernatant liquor.
Embodiment 13. reorganization gsIL-2 albumen physico-chemical property and stability
Reorganization gsIL-2 albumen is more stable to heat, and 56 ℃ 1 hour, 37 ℃ 12 hours or 70 ℃ were handled still retentive activity in 15 minutes, can preserve more than 1 year for 4 ℃, and its activity keeps stablizing in pH2～9 scopes.Reorganization gsIL-2 albumen is to the proteolytic enzyme sensitivity, and is insensitive to DNA enzyme, RNA enzyme.
Embodiment 14. reorganization gsIL-2 albumen are as immunological adjuvant and disease therapeuticing medicine.
Can make goose salmonella typhi organ intrusion rate reduce by 49%～58% with reorganization gsIL-2 albumen, in addition, inject the goose embryo, can significantly reduce the Salmonellas organ intrusion rate of incubation period goose embryo with reorganization gsIL-2 albumen.Aspect parasite,, behind reorganization gsIL-2 albumen subcutaneous injection goose in 1 age in week, can obviously reduce the output of egg capsule behind (p＜0.05) coccidium infection in the day before yesterday or metainfective the 1st day that goose infects coccidia.After 30 age in days geese infect gosling plague; the Effective Vate of Protection of intramuscular injection anti gosling plague serum is 78.2%; the protection ratio that intramuscular injection reorganization gsIL-2 albumen adds anti gosling plague serum is up to 91.5%, and separately intramuscular injection anti gosling plague serum and gosling plague vaccine protection ratio be 64.8% and 2.7%.
Claims (7)
1. reorganization goose interleukin II proteic preparation method is characterized in that its step is as follows:
1) dna fragmentation that in the white goose monokaryon lymphocyte of East Zhejiang province, clones goose interleukin II (gsIL-2) with contain P
BADThe prokaryotic expression carrier pBAD/His B of promotor is set up into expression vector pBAD/HisB/gsIL-2; Perhaps above-mentioned gsIL-2DNA fragment with contain P
AUG1The carrier for expression of eukaryon pMET α B of promotor is set up into secreted expression carrier pMET α B/gsIL-2; Perhaps above-mentioned gsIL-2 dna fragmentation with contain P
AUG1The carrier for expression of eukaryon pMETB of promotor is set up into type expression vector pMET B/gsIL-2 in the born of the same parents;
2) use RM as basic medium, by fermenting and amplifying colibacillus engineering LMG194;
3) use BMDY as basic medium,, add nutrient substances such as nitrogenous source, carbon source at the expression different time by fermenting and amplifying Yeast engineering bacteria PMAD11 and PMAD16;
4) intestinal bacteria LMG194 and yeast PMAD16 induces after ultrasonic treatment, the centrifugal precipitation of going are the gsIL-2 raw product, the gsIL-2 that PMAD11 expresses mainly secretes in substratum, is the gsIL-2 raw product through the centrifuging and taking supernatant.Can obtain the pure product of gsIL-2 with Ni-NTA Argarose protein purification system;
5) after raw product or pure product add stablizer such as glycine and solubility promoter, make finished product after sterile filtration, packing and the freeze-drying, be stored in-20 ℃ the cold storage environment.
2. the proteic preparation method of a kind of reorganization goose interleukin II according to claim 1,, it is characterized in that said gsIL-2 dna fragmentation and contain P
BADThe prokaryotic expression carrier pBAD/His B of promotor is set up into expression vector pBAD/His B/gsIL-2, through electric transformed into escherichia coli LMG194 is the gsIL-2 gene engineering colibacillus, behind L (+)-pectinose abduction delivering, product exists in the engineering bacteria with the form of fusion rotein.
3. the proteic preparation method of a kind of reorganization goose interleukin II according to claim 1 is characterized in that above-mentioned said gsIL-2 dna fragmentation and contains P
AUG1The carrier for expression of eukaryon pMET α B of promotor is set up into secreted expression carrier pMET α B/gsIL-2, and PMAD11 is the gsIL-2 gene engineering microzyme through electric shock transformed yeast bacterium, and methanol induction is expressed, and product is present in the engineering bacteria substratum with the secretory product form; PMET α B/gsIL-2 electricity transformed yeast bacterium PMAD16, methanol induction is expressed, and product mainly is present in the engineering bacteria.
4. the proteic preparation method of a kind of reorganization goose interleukin II according to claim 1, it is characterized in that said by fermenting and amplifying colibacillus engineering LMG194, with RM as basic medium, be by broken coli somatic, with Ni-NTA Argarose protein purification system purified product.
5. the proteic preparation method of a kind of reorganization goose interleukin II according to claim 1, it is characterized in that said by fermenting and amplifying Yeast engineering bacteria PMAD16, with BMDY as basic medium, expressing nutrient substances such as different time adding nitrogenous source, carbon source, be by broken Yeast engineering bacteria PMAD16 thalline, carry out purifying with Ni-NTA Argarose protein purification system.
6. the proteic preparation method of a kind of reorganization goose interleukin II according to claim 1, it is characterized in that said by fermenting and amplifying Yeast engineering bacteria PMAD11, with BMDY as basic medium, expressing nutrient substances such as different time adding nitrogenous source, carbon source, Yeast engineering bacteria PMAD11 secretory product carries out purifying with the Ni-NTAArgarose protein purification system.
7. the proteic purposes of reorganization goose interleukin II is characterized in that it is used to improve vaccine immunity as immunological adjuvant and renders a service; As the aquatic bird medicine have antiviral, antibiotic and parasiticide active.
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| US8632995B2 (en) | 2006-07-27 | 2014-01-21 | Wyeth Llc | High-cell density fed-batch fermentation process for producing recombinant protein |
| CN104178494A (en) * | 2013-05-23 | 2014-12-03 | 上海华新生物高技术有限公司 | Preparation process and application of human interleukin 2 (IL-2) |
| CN109091671A (en) * | 2018-09-26 | 2018-12-28 | 中国农业科学院北京畜牧兽医研究所 | Goose specificity oligodeoxynucleotide immunologic adjuvant and its application |
| CN114369152A (en) * | 2022-01-24 | 2022-04-19 | 扬州大学 | Recombinant chicken interleukin-9 protein and preparation and application of antibody thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US8632995B2 (en) | 2006-07-27 | 2014-01-21 | Wyeth Llc | High-cell density fed-batch fermentation process for producing recombinant protein |
| US9279000B2 (en) | 2006-07-27 | 2016-03-08 | Wyeth Llc | High-cell density fed-batch fermentation process for producing recombinant protein |
| CN105463045A (en) * | 2006-07-27 | 2016-04-06 | 惠氏公司 | High-cell density fed-batch fermentation methods for producing recombinant protein |
| US10301663B2 (en) | 2006-07-27 | 2019-05-28 | Wyeth, Llc | High-cell density fed-batch fermentation process for producing recombinant protein |
| CN104178494A (en) * | 2013-05-23 | 2014-12-03 | 上海华新生物高技术有限公司 | Preparation process and application of human interleukin 2 (IL-2) |
| CN109091671A (en) * | 2018-09-26 | 2018-12-28 | 中国农业科学院北京畜牧兽医研究所 | Goose specificity oligodeoxynucleotide immunologic adjuvant and its application |
| CN114369152A (en) * | 2022-01-24 | 2022-04-19 | 扬州大学 | Recombinant chicken interleukin-9 protein and preparation and application of antibody thereof |
| CN114369152B (en) * | 2022-01-24 | 2023-11-21 | 扬州大学 | Preparation and application of recombinant chicken interleukin-9 protein and antibody thereof |
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