CN107216379A - Cynoglossus semilaevis IGF I proteins and its vivoexpression preparation method and application - Google Patents
Cynoglossus semilaevis IGF I proteins and its vivoexpression preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of Cynoglossus semilaevis IGF I proteins and its vivoexpression preparation method and application, the nucleotides sequence of described albumen is classified as SEQ IDNO:1；Amino acid sequence is SEQ IDNO:3.The present invention also provides a kind of vivoexpression preparation method of Cynoglossus semilaevis IGF I proteins, and it includes mature peptide sequence alterations, recombinant expression carrier structure, recombinant expression carrier induced expression and recombinant protein purification；The present invention utilizes the ripe peptide sequences of Cynoglossus semilaevis IGF I transformed according to yeast codons Preference and construction of eukaryotic expression vector recombinant expression carrier, and IGF I high efficient expression is successfully realized in Yeast engineering bacteria, the Cynoglossus semilaevis IGF I recombinant proteins with bioactivity are obtained, there is obvious growth-promoting effect after being applied in the form of feed addictive.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, and in particular to a kind of Cynoglossus semilaevis IGF-I albumen and its external
Express preparation method and application.
Background technology
Growth factor-insulin-like growth factor axis (GH/IGF-I axis) plays pass in fish growth growth course
The physiological regulating control effect of key.IGF-I is the Downstream regulatory factor of growth axis, is more guarded in evolution, and it gives birth in bony fish
Length, reproduction, metabolism, osmotic pressure regulation, behavior, ingest and be immunized in terms of all have important physiological function.In order to promote
IGF-I is applied in fish culture production, and scientific research and cultivation practitioner are wished to IGF-I mature peptides Expression product in vitro,
Obtain after IGF-I protein product as functional growing metabolism or immunomodulator, be used in modes such as feed addictives special
Developed with mixed feed.There are some researches show restructuring IGF-I albumen passes through injection, leaching as growth promotion or the metabolic regulation factor
Bubble or the mode of feed addition can effectively facilitate the growth metabolism of cultured fishes, and fish are especially homologous to foreign recombinant proteins
Recombinant protein to absorb fast, metabolism fast, will not produce in vivo accumulative thus safe and reliable.So, utilize gene engineering expression
Means obtain restructuring fish insulin-like growth factor and for cultured fishes adjusting and controlling growth, have in culture fishery important
Application value.At present, perch, Rofe are successfully realized in prokaryotic expression system (Escherichia coli) using genetic engineering means
The IGF-I in-vitro recombination expressions of the fish such as fish, grass carp, rainbow trout, turbot, obtain the IGF-I recombinant proteins of bioactivity,
Laid a good foundation for applications of the fish IGF-I in breeding production.But, prokaryotic expression system is in the presence of lacking that many is difficult to overcome
Point：Such as prokaryotic hosts bacterium may produce endotoxin in itself, and overexpression may result in non-physiological reaction；Prokaryotic expression system
The destination protein of generation is often expressed with inclusion bodies, causes product purification difficult；Prokaryotic expression system post translational processing is modified
System imperfection, the bioactivity of expression product is relatively low.To overcome the shortcomings of prokaryotic expression carrier, researchers develop eucaryon
Expression system, its advantage is to include target DNA and eukaryotic gene regulating and controlling sequence substantially without homology, it is to avoid the non-specificity of gene
Activation suppresses；Compared with prokaryotic expression, inducible gene expression efficiency is significantly improved；Strict controlling gene expression, the egg of acquisition
White product has bioactivity, without complex process such as separation, denaturation, renaturation.At present, commonly used in genetic engineering research
Eukaryotic expression system has yeast expression system, insect cell expression system and mammalian cell expression system.Cynoglossus semilaevis is
The important sea-farming tuna fisheries of China, it has also become one of species leading greatly of China's left-eyed flounder aquaculture industry three.Half sliding-tongue
Sole has 2-3 times slow compared with raun of obvious growth differences sex dimorphism, i.e. the milter speed of growth, is unfavorable for the lasting hair of industry
Exhibition.If developing corresponding biological products using the gene outcome for possessing growth metabolism adjusting function, realize sliding to cultivation half
Tongue sole growth differences and the artificial regulatory of metabolism, it will bring great economic benefit for aquaculture industry development, have a extensive future.
At present, it has therefore proved that IGF-I grows with metabolic process in Cynoglossus semilaevis, and there is important physiological regulating control to act on, but due to still
Without ripe IGF-I protein products, thus not yet applied in breeding production.In addition, also having no Cynoglossus semilaevis in the market
The special functional growth metabolism regulation and control specialist additive of cultivation.
The content of the invention
In order to solve above-mentioned technical barrier, promote applications of the IGF-I in cynoglossus semilaevis cultivation production, the present invention provides one
The external eukaryotic expression for planting Cynoglossus semilaevis IGF-I albumen is prepared and application process, is transformed using according to yeast codons Preference
With construction of eukaryotic expression vector after the ripe peptide sequences of Cynoglossus semilaevis IGF-I crossed, addition restriction enzyme site and 6 × His sequence labels
Recombinant expression carrier, and IGF-I high efficient expression is successfully realized in Yeast engineering bacteria, obtain with bioactivity
Cynoglossus semilaevis IGF-I recombinant proteins, have obvious growth-promoting effect after being applied in the form of feed addictive.
The present invention is achieved through the following technical solutions：
A kind of Cynoglossus semilaevis IGF-I albumen, the nucleotides sequence of described albumen is classified as SEQ IDNO:1；
A kind of Cynoglossus semilaevis IGF-I albumen, the amino acid sequence of described albumen is SEQ IDNO:3；
The present invention provides a kind of vivoexpression preparation method of Cynoglossus semilaevis IGF-I albumen, and it includes ripe peptide sequence and changed
Make, recombinant expression carrier is built, recombinant expression carrier induced expression and recombinant protein purification；
Described mature peptide sequence alterations, in order to carry out high efficient expression, to the nucleotides sequence of Cynoglossus semilaevis IGF-I mature peptides
Arrange SEQ IDNO:1 is transformed according to the codon preference of saccharomycete, obtains the nucleosides of the suitable high efficient expression in saccharomycete
Acid sequence SEQ IDNO:2；
Described recombinant expression carrier is built, in purpose nucleotide sequence SEQ IDNO:Restriction enzyme site and His are added on 2
Recombinated after × 6 labels with carrier for expression of eukaryon pPICZaA, composition can express amino acid sequence be SEQ IDNO:3 half cunning
The recombinant expression carrier pPICZaA-IGF-I of tongue sole quasi-insulin growthing factor I (IGF-I) polypeptide.
Further, addition restriction enzyme site refers to add XhoI restriction enzyme sites in purpose nucleotide sequence upstream, adds in downstream
Plus XbaI enzyme cutting site, and introduce pichia pastoris protein enzyme Ste13 restriction enzyme sites in purpose nucleotide sequence upstream；
Further, the addition label of His × 6 refers to make an addition to purpose nucleotide sequence downstream, is easy to destination protein after expression
Ni-sepharose purification.
Described recombinant expression carrier induced expression, recombinant expression carrier is linearized, electricity is transduceed into chemoreception
The expression bacterial strain Pichia pastoris X33 of state, form recombinant strains pPICZaA-IGF-I-Pichia pastoris
X33, the positive colony for selecting high efficient expression is cultivated, and recombinantly expresses bacterium secreting, expressing destination protein using methanol induction.
Further, described recombinant expression carrier induced expression specific method is the recombinant expression carrier for taking linearisation
PPICZaA-IGF-I adds the expression bacterial strain Pichia pastoris X33 in Competent, is placed in electric revolving cup, ice
It is upper place 6 minutes after, converted using electroporation, while add sorbierite, the liquid after electricity is turned be transferred in centrifuge tube 30 DEG C it is quiet
Only 1.5h, is then transferred in YPD culture mediums and cultivates 2h, breeding condition：30 DEG C, 200rpm shakes, then, is coated with YPD flat boards, training
Support；
The positive colony of flat board culture is selected, with BMGY medium cultures, then 30 DEG C of 200rpm collect thalline and utilize
When BMMY is cultivated to OD values to 1.0, methanol induction recombinant strains secreting, expressing destination protein is added.
Further, the methanol induction concentration is 0.5%；
Further, the condition of the induction is：Temperature is 30 DEG C, and pH is 5.0, and induction time is 36h.
Described recombinant protein purification, refers to the Cynoglossus semilaevis IGF-I of culture recombinantly expressing saccharomycete supernatant with sulphur
Be redissolved after sour ammonium precipitation, supernatant after micro-filtrate membrane filtration through Filter column hanging column, then with i.e. available after elution
The destination protein of purifying.The purpose of purifying protein is mainly the measuring and calculating scientific research such as destination protein yield and biological activity determination,
Yeast expression bacterium solution can directly be admixed without purifying during production application and application is fed in feed.
Further, ammonium sulfate concentrations are 350mg/L；
Further, micro-filtration membrane aperture is 0.45 μm.
The beneficial effect of the present invention compared with prior art：
(1) nucleotide sequence for the Cynoglossus semilaevis IGF-I mature peptides that the present invention is used, according to the close of expression yeast host bacterium
After the transformation of numeral Preference, the plasmid after being recombinated with carrier for expression of eukaryon imports table after Pichia pastoris X33 saccharomycete
Up to more efficient.
(2) the carrier for expression of eukaryon pPICZaA that the present invention is used belongs to secreted expression carrier, and destination protein is directly secreted
In extracellular, it is easy to isolate and purify, is a kind of carrier for expression of eukaryon being most widely used at present.Meanwhile, pPICZaA expression vectors
Genetic background it is clear, possess perfect expression control system and natural posttranslational modification system, destination protein expression efficiency
It is higher also closer to native protein, with stable natural biology activity, greatly save production technology and manpower and materials.
(3) present invention is expressed Cynoglossus semilaevis IGF-I recombinant eukaryotic expression plasmids in yeast, can be by yeast
The extracellular quantization secretion production of recombinant protein is quickly bred and realizes, meanwhile, on the basis of optimal inductive condition is obtained, also
Pilot scale culture is carried out using the industrialized production such as fermentation tank equipment, so that obtain high yield and substantially reduce production cost,
With higher economy.Yeast is widely used in feed and food service industry, the IGF-I recombinant proteins that the present invention is obtained and yeast table
It vacuum dried can be waited up to bacterium mixed liquor after processing, aquatic feeds directly applied to as additive, with green, safety, life
State, the advantage of environmental protection.
Brief description of the drawings
Expression of Fig. 1 Cynoglossus semilaevis IGF-I recombinant proteins under condition of different pH：1、pH3.0；2、pH4.0；3、pH5.0；
4、pH6.0；5、pH7.0；
The influence that Fig. 2 Cynoglossus semilaevis IGF-I recombinant proteins are bred to human breast cancer cell；
The influence that Fig. 3 Cynoglossus semilaevis IGF-I recombinant proteins are expressed liver IGF-I mRNA and IGF-II mRNA.
Embodiment
Technical scheme is further explained below by embodiment combination accompanying drawing, but the protection of the present invention
Scope formal is not limited by embodiment is any.
Embodiment
A kind of Cynoglossus semilaevis IGF-I albumen, the nucleotides sequence of described albumen is classified as SEQ IDNO:1；
The amino acid sequence of described albumen is SEQ IDNO:3；Its vivoexpression preparation method, it includes mature peptide sequence
Row transformation, recombinant expression carrier structure, recombinant expression carrier induced expression and recombinant protein purification.Comprise the following steps that：
1st, mature peptide sequence alterations and synthesis
By sequence alignment analysis, the ripe peptide sequence of Cynoglossus semilaevis IGF-I genes, total length 204bp, by 68 ammonia are obtained
Base acid composition, molecular weight about 7.5kD, including 4 functional domains of B, C, A, D.In order to carry out high efficient expression, core is carried out to mature peptide
Nucleotide sequence (SEQ IDNO:1) transform, by artificial synthesized mode obtain adapt to saccharomycete high efficient expression IGF-I into
The nucleotide sequence fragment SEQ IDNO of ripe peptide:2, its amino acid sequence segments is SEQ IDNO:3.
2nd, recombinant expression carrier is built
In purpose nucleotide sequence SEQ IDNO:XhoI restriction enzyme sites are added in 2 upstreams, in downstream addition XbaI enzyme cutting position
Point, while introduce pichia pastoris protein enzyme Ste13 restriction enzyme sites in upstream, and after the label of sequence downstream His × 6 with eucaryon table
Recombinated up to carrier pPICZaA, composition can express amino acid sequence be SEQ IDNO:3 Cynoglossus semilaevis insulin-like growth
The recombinant expression carrier pPICZaA-IGF-I of factor I (IGF-I) polypeptide.
3rd, recombinant expression carrier induced expression
Prepare the Pichia pastoris X33 bacterial strains in Competent.The recombination expression for taking 10ml to linearize is carried
Body pPICZaA-IGF-I adds the expression bacterial strain Pichia pastoris X33 that 80ml is in Competent, is placed in electric revolving cup
It is interior, after placing 6 minutes on ice, converted using electroporation, while adding 1ml 1M sorbierites, the liquid after electricity is turned is transferred to centrifugation
30 DEG C of static 1.5h in pipe, are then transferred in YPD culture mediums and cultivate 2h, breeding condition：30 DEG C, 200rpm shakes.Then, it is coated with
YPD flat boards, culture.
The positive colony of flat board culture is selected, with BMGY medium cultures (30 DEG C, 200rpm), thalline is then collected and utilizes
When BMMY is cultivated to OD values to 1.0, methanol induction recombinant strains secreting, expressing destination protein is added.Experiment determines most preferably to lure
Sliver part is：Methanol concentration is 0.5%, and temperature is 30 DEG C, and pH is 5.0, and induction time is 36h.
4th, recombinant protein purification
After methanol induction expression 36h, the supernatant of Yeast expression bacterium solution of culture is taken with 350mg/L ammonium sulfate precipitations (4
DEG C), then by 0.45 μm of micro-filtrate membrane filtration after being dissolved with elution buffer.Filtered fluid filters hanging column by His labels adsorption column,
Albumen wash-out is got off with eluent again, you can the destination protein purified.Destination protein with Tricine-SDS-PAGE and
Western blot method validations confirm.Destination protein is obtained with BCA methods and Tricine-SDS-PAGE gel image analysis method
Ultimate density.
5th, biological activity determination
Human breast cancer cell and Cynoglossus semilaevis liver cell are acted on the Cynoglossus semilaevis IGF-I albumen of the purifying of acquisition,
Test whether the restructuring destination protein obtained has bioactivity.As a result show, the Cynoglossus semilaevis IGF-I recombinant proteins of acquisition can
Significantly human breast cancer cell is stimulated to breed (Fig. 2), while can also significantly regulate and control the isogenic expression (figure of liver IGF-I, IGF-II
3), show that the Cynoglossus semilaevis IGF-I recombinant proteins obtained have obvious bioactivity.
6th, the application of Cynoglossus semilaevis IGF-I recombinant proteins
The external eukaryotic expression preparation method of Cynoglossus semilaevis IGF-I albumen that the present invention is provided can successfully realize Cynoglossus semilaevis
High efficient expression of the IGF-I mature peptides in yeast, suitably fermentation etc. industrialized production, can prepare with scale.By what is isolated and purified
Cynoglossus semilaevis IGF-I recombinant proteins, aqua is preserved into deionized water, available for scientific experiment research.For example, according to 2.5 μ g/
Kg and 25 μ g/kg IGF-I recombinant protein dosage, is injected intraperitoneally Cynoglossus semilaevis juvenile fish, once every two weeks, the body in 45d respectively
Increase again and 35.75% and 50.91% is respectively increased compared with control group, growth-promoting effect is clearly.It is being spray-dried, is being freezed
Deng processing after can obtain the yeast powder product of Cynoglossus semilaevis IGF-I recombinant proteins, be directly applicable for feed addictive and use.
It is added to 0.1% ratio in feed, feeds Cynoglossus semilaevis juvenile fish 60d, feeds group experiment fish body weight growth rate compared with control group
Experiment fish is high by 30.7%, and growth-promoting effect is obvious.
SEQUENCE LISTING
<110>Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
<120>Cynoglossus semilaevis IGF-I albumen and its vivoexpression preparation method and application
<130> wu
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 204
<212> DNA
<213> Cynoglossus semilaevis
<400> 1
ggcccggaga ccctgtgcgg ggcggagctg gtcgacacgc tgcagtttgt gtgtggagag 60
agaggctttt atttcagcaa accaacwggc tatggcccta actcacggcg gtctcgtggc 120
atcgtggacg agtgctgctt ccaaagctgt gagctgcggc gcctggagat gtactgcgcg 180
ccagccaaga ctggcaaagc agct 204
<210> 2
<211> 204
<212> DNA
<213> Cynoglossus semilaevis
<400> 2
ggtccagaga ctctgtgtgg tgctgagctg gttgacactc tgcaattcgt ttgtggtgag 60
agaggtttct acttcagtaa gccaactggt tacggtccaa actctcgtcg ttctcgtggt 120
attgttgacg agtgttgttt ccaaagttgt gagctgcgtc gtctggagat gtactgtgct 180
ccagccaaga ctggtaaggc tgct 204
<210> 3
<211> 68
<212> PRT
<213> Cynoglossus semilaevis
<400> 3
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Thr Leu Gln Phe
1 5 10 15
Val Cys Gly Glu Arg Gly Phe Tyr Phe Ser Lys Pro Thr Gly Tyr Gly
20 25 30
Pro Asn Ser Arg Arg Ser Arg Gly Ile Val Asp Glu Cys Cys Phe Gln
35 40 45
Ser Cys Glu Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Ala Lys Thr
50 55 60
Gly Lys Ala Ala
65
Claims (10)
1. a kind of Cynoglossus semilaevis IGF-I albumen, it is characterised in that the nucleotides sequence of the Cynoglossus semilaevis IGF-I albumen is classified as SEQ
ID NO:1。
2. a kind of Cynoglossus semilaevis IGF-I albumen described in claim 1, it is characterised in that the Cynoglossus semilaevis IGF-I albumen
Amino acid sequence is SEQ ID NO:3.
3. the vivoexpression preparation method of Cynoglossus semilaevis IGF-I albumen described in claim 1, it is characterised in that it includes mature peptide
Sequence alterations, recombinant expression carrier structure, recombinant expression carrier induced expression and recombinant protein purification；
Described mature peptide sequence alterations, to the nucleotide sequence SEQ ID NO of Cynoglossus semilaevis IGF-I mature peptides:1 according to yeast
The codon preference of bacterium is transformed, and obtains the nucleotide sequence SEQ ID NO of the suitable high efficient expression in saccharomycete:2；
Described recombinant expression carrier is built, in purpose nucleotide sequence SEQ ID NO:Restriction enzyme site and His × 6 are added on 2
Recombinated after label with carrier for expression of eukaryon pPICZaA, composition can express amino acid sequence be SEQ ID NO:3 half sliding-tongue
The recombinant expression carrier pPICZaA-IGF-I of sole quasi-insulin growthing factor I polypeptide；
Described recombinant expression carrier induced expression, recombinant expression carrier is linearized, and electricity is transduceed into Competent
Bacterial strain Pichia pastoris X33 are expressed, recombinant strains pPICZaA-IGF-I-Pichia pastoris are formed
X33, the positive colony for selecting high efficient expression is cultivated, and recombinantly expresses bacterium secreting, expressing destination protein using methanol induction.
4. method according to claim 3, it is characterised in that added described in described recombinant expression carrier construction step
Restriction enzyme site refers to add XhoI restriction enzyme sites in purpose nucleotide sequence upstream, in downstream addition XbaI enzyme cutting site, and
Purpose nucleotide sequence upstream introduces pichia pastoris protein enzyme Ste13 restriction enzyme sites.
5. method according to claim 3, it is characterised in that added in described recombinant expression carrier construction step His ×
6 labels refer to make an addition to purpose nucleotide sequence downstream.
6. method according to claim 3, it is characterised in that described recombinant expression carrier induced expression specific method is
The recombinant expression carrier pPICZaA-IGF-I of linearisation is taken to add the expression bacterial strain Pichia in Competent
Pastoris X33, are placed in electric revolving cup, after placing 6 minutes on ice, are converted using electroporation, while sorbierite is added, by electricity
Liquid after turning is transferred to 30 DEG C of static 1.5h in centrifuge tube, is then transferred in YPD culture mediums and cultivates 2h, breeding condition：30 DEG C,
200rpm shakes, then, is coated with YPD flat boards, culture；
The positive colony of flat board culture is selected, with BMGY medium cultures, then 30 DEG C of 200rpm are collected thalline and trained using BMMY
When supporting to OD values to 1.0, methanol induction recombinant strains secreting, expressing destination protein is added.
7. method according to claim 6, it is characterised in that the methanol induction concentration is 0.5%.
8. method according to claim 6, it is characterised in that the condition of the induction is：Temperature is 30 DEG C, and pH is 5.0,
Induction time is 36h.
9. method according to claim 3, it is characterised in that described recombinant protein purification, refers to half sliding-tongue of culture
Sole IGF-I recombinantly expresses saccharomycete supernatant to be redissolved after ammonium sulfate precipitation, and supernatant is after micro-filtrate membrane filtration through filtering
Post hanging column, then the destination protein can be purified after elution.
10. method according to claim 9, it is characterised in that described ammonium sulfate concentrations are 350mg/L, micro-filtration membrane aperture
For 0.45 μm.
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| CN109320601A (en) * | 2018-10-18 | 2019-02-12 | 天津林达生物科技有限公司 | Recombinant IGF-1 protein and high expression and its use in promoting cell proliferation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101466398A (en) * | 2006-06-09 | 2009-06-24 | 诺瓦提斯公司 | Stabilized insulin-like growth factor polypeptides |
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2018
- 2018-06-01 CN CN201810553738.9A patent/CN108752459A/en active Pending
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| CN101466398A (en) * | 2006-06-09 | 2009-06-24 | 诺瓦提斯公司 | Stabilized insulin-like growth factor polypeptides |
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| 刘芝亮: "半滑舌鳎生长轴关键基因的重组表达及对生长与生殖的调控机制研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109320601A (en) * | 2018-10-18 | 2019-02-12 | 天津林达生物科技有限公司 | Recombinant IGF-1 protein and high expression and its use in promoting cell proliferation |
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| Publication number | Publication date |
|---|---|
| CN108752459A (en) | 2018-11-06 |
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