CN108752459A - Cynoglossus semilaevis IGF-I albumen and its vivoexpression preparation method and application - Google Patents
Cynoglossus semilaevis IGF-I albumen 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 albumen and its vivoexpression preparation method and application, the nucleotides sequence of the albumen to be classified as SEQ ID NO:2；Amino acid sequence is SEQ ID NO:3.The present invention also provides a kind of vivoexpression preparation methods of Cynoglossus semilaevis IGF-I albumen, it includes mature peptide sequence alterations, recombinant expression carrier structure, recombinant expression carrier induced expression and recombinant protein purification；The present invention utilizes the Cynoglossus semilaevis IGF-I maturations peptide sequence being transformed according to yeast codons Preference and construction of eukaryotic expression vector recombinant expression carrier, and the high efficient expression of IGF-I is successfully realized in Yeast engineering bacteria, biologically active Cynoglossus semilaevis IGF-I recombinant proteins are obtained, there is apparent 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 of key acts on.IGF-I is the Downstream regulatory factor of growth axis, more conservative in evolution, is given birth in bony fish
Length, reproduction, metabolism, osmotic pressure adjusting, behavior, ingesting and be immunized etc. all has important physiological function.In order to promote
IGF-I is applied in fish culture production, and scientific research and cultivation practitioner wish to IGF-I mature peptides Expression product in vitro,
It is used as functional growing metabolism or immunomodulator after obtaining the protein product of IGF-I, for special in a manner of feed addictive etc.
It is developed with mixed feed.Have studies have shown that recombination IGF-I albumen is as growth promotion or the metabolic regulation factor, by injecting, soaking
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 absorb that fast, metabolism is fast, will not generate in vivo accumulative thus safe and reliable.So utilizing gene engineering expression
Means obtain recombination fish insulin-like growth factor and are used for cultured fishes adjusting and controlling growth, have in culture fishery important
Application value.Currently, genetic engineering means has been utilized to be successfully realized perch, Rofe in prokaryotic expression system (Escherichia coli)
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,
It lays a good foundation for applications of the fish IGF-I in breeding production.But prokaryotic expression system is lacked in the presence of what many was difficult to overcome
Point：If prokaryotic hosts bacterium itself may will produce endotoxin, 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 is not perfect, and the bioactivity of expression product is relatively low.To overcome the shortcomings of that prokaryotic expression carrier, researchers develop eukaryon
Expression system, its advantage is that avoiding the non-specificity of gene substantially without homology with eukaryotic gene regulating and controlling sequence including target DNA
It activates or inhibits；Compared with prokaryotic expression, inducible gene expression efficiency significantly improves；Stringent controlling gene expression, the egg of acquisition
White product has bioactivity, without complex process such as separation, denaturation, renaturation.Currently, common 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 in China, it has also become one of type leading greatly of China's left-eyed flounder aquaculture industry three.Half sliding-tongue
Sole has apparent growth differences gender dimorphism, i.e. 2-3 times slow compared with raun of the milter speed of growth, is unfavorable for the lasting hair of industry
Exhibition.As can developing corresponding biological products using the gene outcome for having growth metabolism adjusting function, realize sliding to cultivation half
The artificial regulatory of tongue sole growth differences and metabolism, it will bring great economic benefit for aquaculture industry development, have a extensive future.
At present, it has therefore proved that IGF-I has important physiological regulating control effect in Cynoglossus semilaevis growth and development and metabolic process, but due to still
Without ripe IGF-I protein products, thus not yet applied in breeding production.In addition, also having no Cynoglossus semilaevis currently on the market
Cultivate dedicated functional growth metabolism regulation and control specialist additive.
Invention content
In order to solve above-mentioned technical barrier, applications of the IGF-I in cynoglossus semilaevis cultivation production, the present invention is promoted to provide one
The external eukaryotic expression of kind Cynoglossus semilaevis IGF-I albumen prepares and application process, is transformed using according to yeast codons Preference
The Cynoglossus semilaevis IGF-I maturation peptide sequences crossed, add after restriction enzyme site and 6 × His sequence labels with construction of eukaryotic expression vector
Recombinant expression carrier, and it is successfully realized in Yeast engineering bacteria the high efficient expression of IGF-I, it obtains biologically active
Cynoglossus semilaevis IGF-I recombinant proteins have apparent growth-promoting effect after being applied in the form of feed addictive.
The invention is realized by the following technical scheme：
The nucleotides sequence of a kind of Cynoglossus semilaevis IGF-I albumen, the albumen is classified as SEQ IDNO:2；
The amino acid sequence of a kind of Cynoglossus semilaevis IGF-I albumen, the albumen is SEQ IDNO:3；
The present invention provides a kind of vivoexpression preparation method of Cynoglossus semilaevis IGF-I albumen, it includes that ripe peptide sequence changes
Make, recombinant expression carrier structure, recombinant expression carrier induced expression and recombinant protein purification；
The 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 suitable for the nucleosides of the high efficient expression in saccharomycete
Acid sequence SEQ IDNO:2；
The recombinant expression carrier structure, in purpose nucleotide sequence SEQ IDNO:Restriction enzyme site and His are added on 2
Recombinated with carrier for expression of eukaryon pPICZaA after × 6 labels, 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 adding XhoI restriction enzyme sites in purpose nucleotide sequence upstream, is added in downstream
Add XbaI enzyme cutting site, and pichia pastoris protein enzyme Ste13 restriction enzyme sites are introduced in purpose nucleotide sequence upstream；
Further, the addition labels of His × 6 refer to making an addition to purpose nucleotide sequence downstream, convenient for destination protein after expression
Ni-sepharose purification.
The 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 are cultivated, and recombinantly express bacterium secreting, expressing destination protein using methanol induction.
Further, the recombinant expression carrier induced expression specific method is the recombinant expression carrier for taking linearisation
The expression bacterial strain Pichia pastoris X33 in Competent are added in pPICZaA-IGF-I, are placed in electric revolving cup, ice
It is upper place 6 minutes after, converted using electroporation, while sorbierite is added, the liquid after electricity is turned be transferred in centrifuge tube 30 DEG C it is quiet
Then only 1.5h is transferred in YPD culture mediums and cultivates 2h, breeding condition：30 DEG C, 200rpm shakes, and then, is coated with YPD tablets, training
It supports；
The positive colony for selecting tablet culture, 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 a concentration of 0.5%；
Further, the condition of the induction is：Temperature is 30 DEG C, pH 5.0, induction time 36h.
The recombinant protein purification refers to that the Cynoglossus semilaevis IGF-I that will be cultivated recombinantly expresses saccharomycete supernatant with sulphur
Sour ammonia-sinking is redissolved after forming sediment, supernatant after micro-filtrate membrane filtration through Filter column hanging column, then can be obtained after elution
The destination protein of purifying.The purpose of purifying protein mainly calculates the scientific researches such as destination protein yield and biological activity determination,
Yeast expression bacterium solution can directly be admixed and feed application in feed without purifying during production application.
Further, ammonium sulfate concentrations 350mg/L；
Further, micro-filtration membrane aperture is 0.45 μm.
The advantageous effect of the present invention compared with prior art：
(1) nucleotide sequence for the Cynoglossus semilaevis IGF-I mature peptides that the present invention uses, 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
2 times or more is improved up to efficiency.
(2) the carrier for expression of eukaryon pPICZaA that the present invention uses belongs to secreted expression carrier, and destination protein is directly secreted
It is a kind of carrier for expression of eukaryon being most widely used at present convenient for isolating and purifying in extracellular.Meanwhile pPICZaA expression vectors
Genetic background it is clear, have perfect expression control system and natural posttranslational modification system, destination protein expression efficiency
Higher also closer to native protein, has stable natural biology activity, production technology and manpower and materials is greatly saved.
(3) present invention expresses Cynoglossus semilaevis IGF-I recombinant eukaryotic expression plasmids in yeast, can be by yeast
Quickly breed and realize the extracellular quantization secretion production of recombinant protein, meanwhile, on the basis of obtaining optimum inductive condition, also
Pilot scale culture is carried out using the industrialized productions equipment such as fermentation tank, production cost is substantially reduced to obtain high yield,
With higher economy.Yeast is widely used in feed and food service industry, the IGF-I recombinant proteins that the present invention obtains and yeast table
After vacuum dried can waiting processing up to bacterium mixed liquor, aquatic feeds are directly applied to as additive, there is green, safety, life
State, the advantage of environmental protection.
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；
(SEQ IDNO are not transformed for Fig. 2 Cynoglossus semilaevis:1) and transformation after (SEQ IDNO:2) recombination of IGF-I sequences expression
Albumen：1, blank control；2,SEQ IDNO:The recombination IGF-I albumen of 1 sequence expression；3,SEQ IDNO:The weight of 2 sequences expression
Group IGF-I albumen；
The influence that Fig. 3 Cynoglossus semilaevis IGF-I recombinant proteins are proliferated human breast cancer cell；
The influence that Fig. 4 Cynoglossus semilaevis IGF-I recombinant proteins express liver IGF-I mRNA and IGF-II mRNA.
Specific implementation mode
Technical scheme of the present invention is further explained below by embodiment combination attached drawing, but the protection of the present invention
Range is not limited in any form by embodiment.
Embodiment
The nucleotides sequence of a kind of Cynoglossus semilaevis IGF-I albumen, the albumen is classified as SEQ IDNO:2；
The amino acid sequence of the 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.It is as follows：
1, mature peptide sequence alterations and synthesis
By sequence alignment analysis, Cynoglossus semilaevis IGF-I gene maturation peptide sequences, overall length 204bp, by 68 ammonia are obtained
Base acid forms, 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) be transformed, by artificial synthesized mode obtain adapt to saccharomycete high efficient expression IGF-I at
The nucleotide sequence fragment SEQ IDNO of ripe peptide:2, amino acid sequence segments are SEQ IDNO:3.
The expression effect of after ripening peptide sequence is transformed for detection, with SEQ IDNO:1 sequence is control, equally manually to close
SEQ IDNO are obtained at mode:1 segment, it is synchronous to carry out recombinant protein expression experiment.
2, 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, at the same upstream introduce pichia pastoris protein enzyme Ste13 restriction enzyme sites, and after the labels of sequence downstream His × 6 with eukaryon 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.
Check experiment group, in nucleotide sequence SEQ IDNO:XhoI restriction enzyme sites are added in 1 upstream, and XbaI is added in downstream
Restriction enzyme site, at the same upstream introduce pichia pastoris protein enzyme Ste13 restriction enzyme sites, and after the labels of sequence downstream His × 6 with
Carrier for expression of eukaryon pPICZaA is recombinated, composition can express amino acid sequence be SEQ IDNO:3 Cynoglossus semilaevis class pancreas islet
The recombinant expression carrier pPICZaA-IGF-I-C of plain growth factor I (IGF-I) polypeptide.Following tests operating procedure is identical.
3, recombinant expression carrier induced expression
Prepare the Pichia pastoris X33 bacterial strains in Competent.The recombinant expression that 10ml is linearized is taken to carry
The expression bacterial strain Pichia pastoris X33 that 80ml is in Competent are added in body pPICZaA-IGF-I, are placed in electric revolving cup
It is interior, it after placing 6 minutes on ice, is converted using electroporation, while 1ml 1M sorbierites are added, 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 tablets, culture.
The positive colony for selecting tablet culture is then collected thalline and is utilized with BMGY medium cultures (30 DEG C, 200rpm)
When BMMY is cultivated to OD values to 1.0, methanol induction recombinant strains secreting, expressing destination protein is added.Experiment determination most preferably lures
Conducting bar part is：Methanol concentration is 0.5%, and temperature is 30 DEG C, pH 5.0, induction time 36h.
4, recombinant protein purification
After methanol induction expresses 36h, take the supernatant of the Yeast expression bacterium solution of culture 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 label adsorption columns,
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 methods
Ultimate density.
Protein reconstitution expression of results shows, it is engineered after Cynoglossus semilaevis IGF-I maturations peptide sequence in Pichia pastoris X33 bacterium
High efficient expression (Fig. 1, Fig. 2) is obtained in strain, expression yield reaches 3.95g/L, and not modified IGF-I polypeptide sequences expression is produced
Amount is only 1.14g/L (Fig. 2), and expression efficiency promotes 2.46 times, it is seen that improved Cynoglossus semilaevis IGF-I maturations peptide sequence is more not
Modified IGF-I maturations peptide sequence has efficient expression in yeast.
5, 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.The result shows that the Cynoglossus semilaevis IGF-I recombinant proteins of acquisition can
Significantly stimulation human breast cancer cell is proliferated (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 apparent bioactivity.
6, the application of Cynoglossus semilaevis IGF-I recombinant proteins
The outer eukaryotic expression preparation method of Cynoglossus semilaevis IGF-I proteosomes provided by the invention can successfully realize Cynoglossus semilaevis
High efficient expression of the IGF-I mature peptides in yeast can prepare with scale suitable for industrialized productions such as fermentations.By what is isolated and purified
Cynoglossus semilaevis IGF-I recombinant proteins are preserved with deionized water into aqua, can be used for scientific experiment research.For example, according to 2.5 μ g/
Cynoglossus semilaevis juvenile fish is injected intraperitoneally, once every two weeks, in 45d inner bodies in the IGF-I recombinant protein dosage of kg and 25 μ g/kg 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 lyophilized
Deng the yeast powder product that can get Cynoglossus semilaevis IGF-I recombinant proteins after processing, it is directly applicable for feed addictive use.
It is added in feed with 0.1% ratio, 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 apparent.
Sequence table
<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> SIPOSequenceListing 1.0
<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
IDNO:2。
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 IDNO: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；
The mature peptide sequence alterations, to the nucleotide sequence SEQ IDNO of Cynoglossus semilaevis IGF-I mature peptides:1 according to yeast
The codon preference of bacterium is transformed, and is obtained suitable for the nucleotide sequence SEQ IDNO of the high efficient expression in saccharomycete:2；
The recombinant expression carrier structure, in purpose nucleotide sequence SEQ IDNO:Restriction enzyme site is added on 2 and His × 6 is marked
Recombinated with carrier for expression of eukaryon pPICZaA after label, composition can express amino acid sequence be SEQ IDNO:3 Cynoglossus semilaevis
The recombinant expression carrier pPICZaA-IGF-I of quasi-insulin growthing factor I polypeptide；
The 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 are cultivated, and recombinantly express bacterium secreting, expressing destination protein using methanol induction.
4. according to the method described in claim 3, it is characterized in that being added described in the recombinant expression carrier construction step
Restriction enzyme site refers to adding 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. according to the method described in claim 3, it is characterized in that in the recombinant expression carrier construction step add His ×
6 labels refer to making an addition to purpose nucleotide sequence downstream.
6. according to the method described in claim 3, it is characterized in that the recombinant expression carrier induced expression specific method is
Take the recombinant expression carrier pPICZaA-IGF-I of linearisation that the expression bacterial strain Pichia in Competent is added
Pastoris X33 are placed in electric revolving cup, after placing 6 minutes on ice, are converted using electroporation, while sorbierite is added, will be electric
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, and then, is coated with YPD tablets, culture；
The positive colony for selecting tablet culture, with BMGY medium cultures, then 30 DEG C of 200rpm are collected thalline and are trained using BMMY
When supporting to OD values to 1.0, methanol induction recombinant strains secreting, expressing destination protein is added.
7. according to the method described in claim 6, it is characterized in that the methanol induction a concentration of 0.5%.
8. according to the method described in claim 6, it is characterized in that the condition of the induction is：Temperature is 30 DEG C, pH 5.0,
Induction time is 36h.
9. according to the method described in claim 3, it is characterized in that the recombinant protein purification, refers to half sliding-tongue that will be cultivated
Sole IGF-I recombinantly expresses saccharomycete supernatant to be redissolved after ammonium sulfate precipitation, and supernatant is after micro-filtrate membrane filtration through filtering
Column hanging column, then can be obtained the destination protein of purifying after elution.
10. according to the method described in claim 9, it is characterized in that the ammonium sulfate concentrations are 350mg/L, micro-filtration membrane aperture
It is 0.45 μm.
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| CN201710648921.2A CN107216379A (en) | 2017-08-01 | 2017-08-01 | Cynoglossus semilaevis IGF I proteins and its vivoexpression preparation method and application |
| CN2017106489212 | 2017-08-01 |
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| CN201810553738.9A Pending CN108752459A (en) | 2017-08-01 | 2018-06-01 | Cynoglossus semilaevis IGF-I albumen and its vivoexpression preparation method and application |
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| YANGYUAN LI等: "Expression of insulin-like growth factor-1 of orange-spotted grouper(Epinephelus coioides) in yeast Pichia pastoris", 《PROTEIN EXPRESSION AND PURIFICATION》 * |
| 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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