CN106520807A - Thymosin [alpha]1-porcine interferon [alpha] fusion protein gene and preparation method of recombinant protein of fusion protein gene - Google Patents
Thymosin [alpha]1-porcine interferon [alpha] fusion protein gene and preparation method of recombinant protein of fusion protein gene Download PDFInfo
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Abstract
The invention discloses a thymosin [alpha]1-porcine interferon [alpha] fusion protein gene and a preparation method of recombinant protein of the fusion protein gene. A nucleotide sequence of the reconstructed thymosin [alpha]1-porcine interferon [alpha] fusion protein gene provided by the invention is shown as SEQ ID NO.1. Meanwhile, the invention discloses a method for preparing the recombinant protein expressed by the gene and for conducting activity detection, wherein specifically, the method comprises such steps as reconstructing the thymosin [alpha]1-porcine interferon [alpha] fusion protein gene, cloning the gene and promoting secretory expression of the gene in pichia pastoris, preparing the recombinant protein and controlling fermentation culture conditions, improving such operating methods as the activity detection method and the like. The method provided by the invention is simple and easy to implement and is relatively low in cost; the efficient and stable secretory expression of the thymosin [alpha]1-porcine interferon [alpha] fusion protein in the pichia pastoris is achieved; and the expressed recombinant fusion protein is high in anti-viral activity and immune cell regulating activity levels; therefore, the invention lays a foundation for preparing green and no-residue biological products having functions of treating porcine viral diseases and immune regulation.
Description
Technical field
The present invention relates to a kind of transformation of thymosin α1-porcine interferon alpha antigen-4 fusion protein gene, further relates to containing the gene
The side of stably excreting expression and Prepare restructuring thymosin α1-pig interferon alpha fusion protein in the structure of expression vector, Pichia sp.
Method, and the antiviral activity and immunoregulation effect of recombinant thymin alpha-1-pig interferon alpha fusion protein, the invention belongs to dynamic
Thing genetic engineering and technical field of animal virology.
Background technology
Thymosin is the general name of the polypeptide hormone produced by thymic epithelial cell, also known as thymosin
(thymopeptide).The thymosin of application is the peptide material obtained from extraction purification in calf or porcine thymus mostly at present,
Its biological activity is mainly inducing T cell differentiation and maturation and regulation and control immunologic balance.It is mainly used in autoimmunity in people's doctor's clinic
Disease, such as rheumatoid arthritiss, lupus erythematosus, glomerulonephritiies and myasthenia graviss etc., are also used for malignant tumor, viral
Hepatitis and antibiotic are unable to the auxiliary treatment of the diseases such as the infection of effective control, there is no clinical practice in veterinary.
Thymosin (Thymosin, T) is made up of the amino acid residue of more than 25, in the various active components of thymosin
In, its active highest.Different according to isoelectric point, IP, thymosin can be divided into α, β and γ family.With thymosin α1 (T α 1) in α families
Most study, T α 1 are made up of 28 amino acid residues, are initially that Goldstein organizes BSA from calf thymuss
A kind of polypeptide of separating-purifying in (Thymosin Fraction 5, TF5), molecular weight are 3.1KDa, and isoelectric point, IP is 4.2
[Goldstein AL,etal.Thymosin alpha 1:isolation and sequence analysis of an
immunologycally active thymicpolypetide[J].ProcNatlAcadSci USA,1997,74(2):
725-729；Low TL,et al.The chemistry and biology of thymosinⅡ.Amino acid
sequence analysis of thymosin alpha 1and polypeptide beta l[J].J BiolChem,
1979,254(3):987-995.].The peptide sequence of thymosin α1 highly conserved [Goldstein AL, et between different animals
al.From lab to bedside:emerging clinical applicationsof thymosin alpha
1.Expert OpinBiol Ther,2009；9(5):593-608.].
Nineteen fifty-seven Issacs and Lindenmann are separated to a kind of life from the chick-embryo cell culture fluid of influenza infection
Active substances, because its interference is homologous and heterologus virus are replicated, thus are named as interferon (Interferon, IFN)
[Isaacs,A,et al.Virus interference.I.The interferon.Proc.R.Soc.London
Ser.1957.B 147:258-267.].Interferon-ALPHA (IFN-α) belongs to I type interferon, major function for " disease-resistant poison cell because
Son ".Thank Potiria pectinifera (Mukller et Tro Sehel), Wu Dan etc. and cloned pig IFN-α gene, construct prokaryotic expression carrier, and successfully express pig IFN-α；
Ge Li etc. has cloned pig IFN-α, constructs carrier for expression of eukaryon, and successfully expresses pig IFN-α；Cao Ruibing etc. is cloned and is changed
Pig IFN-α gene has been made, the high efficient expression in prokaryotic system has been realized；Yao Qingxia etc. is obtained in yeast expression system life
The restructuring porcine interferon alpha (Porcine Interferona, PoIFN-a) of thing activity simultaneously suppresses FMDV, PRV, PRRSV to which
Activity studied.Interferon is a kind of non-specific broad-spectrum antiviral biological preparation, can be used to treat many viral
Disease.Poplar emperor's teacher etc. studies discovery leukocyte interferon of pig and inoculates, and can substantially reduce pigletss sickness rate, and with 7 days
Age pigletss proceed by protective inoculation best results.Zheng Yongbo etc. carries out clinical trial, result of the test with leukocyte interferon of pig
Show, if directly treated with interferon, the cure rate to infected animal can be improved, if with interferon coordinate antibiotic and
Antiviral drugs are treated to infected animal, can more significantly improve the cure rate to infected animal.Zhang Quanjun etc. is by test
Find with clinical expanding test, leukocyte interferon of pig to suckling pig and some viral diarrheas of ablactational baby pig or virus with it is thin
The diarrhoea that bacterium mixed infection causes, has good preventive and therapeutic action.
There is the disclosed (application of patent application of two escherichia coli expression pig interferon-thymosin α1 fusion proteins at present
Number be respectively：CN200910217774.9 and CN201410063007.8), the two patent applications use prokaryotic expression system
To express GST- porcine interferon alphas-thymosin α1 fusion protein, the destination protein of expression is inclusion body, needs through inclusion body
Degeneration, renaturation, dialysis, cross column purification, proteolytic cleavage and remove GST and destination protein repurity (number of patent application is
CN200910217774.9, is shown in its description page 11；Number of patent application CN201410063007.8, is shown in its description the 3rd
Page), well-known to those skilled in the art to be, there is many uncertain factors in the degeneration of inclusion body and renaturation step, whole pure
Change process is extremely complex and is difficult to quality control, can greatly improve the production cost of destination protein.
The present invention is thymosin α1-porcine interferon alpha (T α 1-PoIFN α) fusion egg with Pichia sp. efficient secretory expression
In vain, in shaking flask, the expression of restructuring destination protein can reach 210mg/L, the expression of restructuring destination protein in fermentation tank
1100mg/L can be reached, expressed T α 1-PoIFN alpha fusion proteins are secretions, and in culture in the form of solvable
In supernatant, it is only necessary to which, through Purification by filtration, production cost is very low.So far, to have no both at home and abroad and come high with Pichia sp.
The report of effect secreting, expressing T α 1-PoIFN alpha fusion proteins.In sum it may be concluded that the present invention is red to finish both at home and abroad
The reported first of yeast efficient secretory expression T α 1-PoIFN alpha fusion proteins, the restructuring T α 1- of efficient secretory expression of the present invention
PoIFN alpha fusion proteins have very high antiviral activity and adjust immunologic cellular activity.Efficient stable secreting, expressing of the present invention
Restructuring T α 1-PoIFN alpha fusion proteins are to provide the green noresidue life with treatment porcine viral diseases and immunoloregulation function
Tetramune is laid a good foundation.
The content of the invention
It is an object of the invention to provide a kind of thymosin α1-porcine interferon alpha (T α 1-PoIFN α) fusion egg of synthetic
White gene；
It is a further object of the present invention to provide the DNA restructuring of the T α 1-PoIFN alpha fusion protein genes containing above-mentioned synthetic
Carrier and host cell；
Another object of the present invention is to provide a kind of method for preparing T α 1-PoIFN alpha fusion proteins and Activity determination.
In order to achieve the above object, present invention employs technical scheme below：
A kind of thymosin α1-porcine interferon alpha (the T α 1-PoIFN α) antigen-4 fusion protein gene of the present invention, it is characterised in that described
Gene nucleotide sequence as shown in SEQ ID NO.1.
A kind of T α 1-PoIFN alpha fusion protein genes of the present invention are not change thymosin α1 and the natural ammonia of porcine interferon alpha
On the premise of base acid sequence, to carrying out changing after T α 1-PoIFN alpha fusion proteins genes carry out DNA analysis and RNA structure predictions
Make what is obtained, which can make the stably excreting expression in Pichia sp. of T α 1-PoIFN alpha fusion proteins.
Present invention also offers a kind of recombinant expression carrier comprising described T α 1-PoIFN alpha fusion protein genes.
Preferably, described recombinant expression carrier is methanol adjustment type recombinant yeast expression vector, it is furthermore preferred that described
Recombinant vector be by the T α 1-PoIFN alpha fusion proteins gene clonings shown in SEQ ID NO.1 in Yeast expression carrier pPIC9K
Obtain, the recombinant expression carrier contains EcoRI and Not I restriction enzyme sites, a strong promoter AOX1 promoter and
Individual G418 resistances select site.
Further, present invention also offers a kind of host cell, the host cell is comprising shown in SEQ ID NO.1
Nucleotide sequence host cell, or be comprising the recombinant expressed load containing the nucleotide sequence shown in SEQ ID NO.1
The host cell of body.
Preferably, described host cell is eukaryotic cells.It is furthermore preferred that described host cell is methanol nutrition
Type recombinant yeast cell GS115.
Further, present invention also offers described T α 1-PoIFN alpha fusion proteins genes are preparing T α 1-PoIFN α
Application in fusion protein.
A kind of method for preparing T α 1-PoIFN alpha fusion proteins that the present invention is provided, it is characterised in that be by SEQ ID
Then the recombinant expression carrier for obtaining is converted place to expression vector by the T α 1-PoIFN alpha fusion proteins gene clonings shown in NO.1
Main bacterium, carries out the abduction delivering of recombiant protein, collects purification and obtains final product.
In the present invention, it is preferred to, described method is comprised the following steps：
(1) synthetic T α 1-PoIFN alpha fusion protein genes, the nucleotide sequence such as SEQ ID NO.1 of described gene
It is shown；
(2) by the T α 1-PoIFN alpha fusion proteins gene clonings of synthesis in Yeast expression carrier pPIC9K, electricity conversion
It is after GS115 competent cells, with MD plates and G418+YPD plate screenings, big in shaking flask Small Amount or fermentation tank after activation culture
Amount is expressed with methanol induction and carries out steriling test；
(3) sterile collection culture supernatant, after purification Jing filtrations or Prepare restructuring T α 1-PoIFN alpha fusion proteins, whole process
In carry out albumen steriling test；
(4) immune detection is carried out to T α 1-PoIFN alpha fusion proteins with monoclonal antibody；
(5) antiviral activity of detection restructuring T α 1-PoIFN alpha fusion proteins and regulation immunologic cellular activity.
In the present invention, it is preferred to, EcoRI restricted enzyme position is added in 5 ' ends of the gene described in step (1)
Point, adds Not I restriction endonuclease sites at its 3 ' end.
In the present invention, it is preferred to, the described condition expressed with methanol induction in a small amount in shaking flask is to add at interval of 24h
It is Jia 5～10 μ L100% in every milliliter of culture fluid to enter 100% methanol to final concentration of 0.5～1% (v/v) of methanol, i.e. addition
Methanol, carries out inducing culture, 26～30 DEG C of 250～300r/min, 96～120h of concussion and cultivate, harvests culture supernatant, Jing filter or
Prepare restructuring T α 1-PoIFN alpha fusion proteins after purification.
In the present invention, it is preferred to, it is described in fermentation tank with the step of methanol induction great expression and condition is：1)
Glycerol culture expands thalline：Fermentation tank parameter setting is respectively 600～1000r/min of mixing speed, 26～28 DEG C of temperature, control
Mode is P-I-D, maintains dissolved oxygen value (DO) 30% to 40%；2) methanol induction expression：Exist according to 8～12mL/L ratios
PTM1 culture medium is added in methanol, after mixing, is added in fermentation tank with the speed of 2.4～3.6mL/h/L/ Preliminary fermentation liquid and is lured
Expression is led, so that yeast adapts to the environment with methanol as sole carbon source, the speed for subsequently adding methanol is upgraded to 4.8～7.2mL/h/
L Preliminary fermentation liquid, finally improves the speed for adding methanol to 10～12mL/h/L/ Preliminary fermentation liquid, after starting abduction delivering, often
It takes expression supernatant is used for analysis of protein, after 72～96h of abduction delivering, terminates fermentation；Culture supernatant is harvested, Jing is filtered or purification
Prepare restructuring T α 1-PoIFN alpha fusion proteins afterwards.
Specifically, the method comprising the steps of：
(1) the synthetic T α 1-PoIFN alpha fusion proteins gene T α 1- on the premise of natural acid sequence is not changed
PoIFN α, the nucleotide sequence of the T α 1-PoIFN alpha fusion protein genes of the synthetic is as shown in SEQ ID NO.1.Then exist
Its 5 ' end adds EcoR I restriction endonuclease sites, adds Not I restriction endonuclease sites at its 3 ' end；By the people
The gene cloning of work synthesis obtains pUC-T α 1-PoIFN α into pUC57.
(2) by the plasmid pUC-T α 1-PoIFN α and pPIC9K comprising T α 1-PoIFN alpha fusion protein genes with EcoR I and
Not I double digestions, glue reclaim T α 1-PoIFN α and pPIC9K fragments after sepharose electrophoresis, connection T α 1-PoIFN α and pPIC9K are obtained
To Expression vector pPIC9K-T α 1-PoIFN α.
(3) the electricity conversion of Pichia sp.
With Sal I or Sac I linearisation pPIC9K-T α 1-PoIFN α, after mixing with GS115 competent cells, use
BioRad Gene Pulser electricity conversions, electric converted product are coated with MD flat boards, are switched to different dense after single bacterium colony is grown
In degree G418+YPD resistant panels, 26～30 DEG C of 2～3d of incubation.
(4) abduction delivering of albumen
From picking individual colonies in resistance plate activated after inducing culture, detect supernatant in destination protein expression,
Supernatant is centrifuged 3min with 12000g, and taking supernatant carries out purification, carries out SDS-PAGE detections with purifying protein.
(5) immunology detection of restructuring T α 1-PoIFN alpha fusion proteins
Supernatant to harvesting is centrifuged 3min with 12000g, and taking supernatant carries out SDS-PAGE, is passed through with the monoclonal antibody of PoIFN α
The reactionogenicity of Western-blot testing goal albumen.
(6) Activity determination of restructuring T α 1-PoIFN alpha fusion proteins
Supernatant to harvesting is centrifuged 3min with 12000g, then takes supernatant filtration, detection filter after in supernatant PoIFN α it is anti-
The regulation immunologic cellular activity of virus activity and T α 1.
When the shaking flask for T α 1-PoIFN alpha fusion proteins of recombinating is prepared in a small amount, comprise the following steps：
(1) single bacterium colony with G418 resistances grown on G418+YPD flat boards is chosen with sterilizing toothpick, the BMGY in 3mL is chosen
Enter line activating culture in fluid medium, cultivation temperature is 26～30 DEG C, the 250 turns/min shaken overnights in shaking table, to OD600
=2～6, cell is in exponential phase；
(2) the culture bacterium solution of step (1) is centrifuged into 3min under 1500g and room temperature condition and collects precipitation, be resuspended in 5mL's
In BMMY, control dissolved oxygen amount and prevent pollution, continue the shaken cultivation in shaking table in the test tube of 30mL；
(3) 100% methanol is added to final concentration of 1% (v/v) at interval of 24h, i.e., during addition is every milliliter of culture fluid
Plus 10 μ L100% methanol, inducing culture is carried out, 26～30 DEG C of 250r/min 96～120h of concussion and cultivate harvest culture supernatant, Jing
Filtration or after purification Prepare restructuring capsid protein.
In a large amount of preparations for T α 1-PoIFN alpha fusion proteins of recombinating, present invention employs 5L fermentation tanks and fermented
Expression, step are 1) glycerol culture amplification thalline：Fermentation tank parameter setting is respectively 600～1000r/min of mixing speed, temperature
26～28 DEG C, control mode is P-I-D, maintains dissolved oxygen value (DO) 30% to 40%；2) methanol induction expression：According to 8
～12mL/L ratios add PTM1 culture medium in methyl alcohol, after mixing, are added with the speed of 2.4～3.6mL/h/L/ Preliminary fermentation liquid
Enter in fermentation tank abduction delivering, maintain this low rate methanol to add 2～3h, so that yeast is adapted to methanol as sole carbon source
Environment, the speed for subsequently adding methanol are upgraded to 4.8～7.2mL/h/L Preliminary fermentation liquid, maintain 2h with this speed, finally improve and mend
Plus the speed of methanol is to 10～12mL/h/L/ Preliminary fermentation liquid, while detecting DO values and broth temperature and whether judging methanol
Excessive, after starting abduction delivering, taking expression supernatant daily is used for analysis of protein.After 72～96h of abduction delivering, terminate fermentation；Receive
Obtain culture supernatant, after purification Jing filtrations or Prepare restructuring T α 1-PoIFN alpha fusion proteins.
Compared to prior art, the invention has the beneficial effects as follows：
(1) present invention is that both at home and abroad relevant T α 1-PoIFN alpha fusion proteins efficient secretory expression have Gao Sheng in yeast
The reported first of thing activity.
(2) the T α 1-PoIFN alpha fusion proteins of efficient stable secreting, expressing of the present invention have treatment pig virus disease to provide
The green noresidue biological product of disease and immunoloregulation function are laid a good foundation.
(3) the inventive method is simple, and cost is relatively low.
Description of the drawings
SDS-PAGE results of the Fig. 1 for the T α 1-PoIFN alpha fusion proteins of the present invention of Pichia anomala expression；
M is pre-dyed albumen Marker, and 1 is T α 1-PoIFN alpha fusion protein fermentation expression supernatants, and 2 merge for T α 1-PoIFN α
Albumen shaking flask expresses supernatant；
Of the present invention T α 1-PoIFN alpha fusion protein Western-blot results of the Fig. 2 for Pichia anomala expression；
M is pre-dyed albumen Marker, and 1 is T α 1-PoIFN alpha fusion protein fermentation expression supernatants, and 2 merge for T α 1-PoIFN α
Albumen shaking flask expresses supernatant；
Antiviral activity testing results of the Fig. 3 for supernatant after the T α 1-PoIFN alpha fusion proteins filtration of Pichia anomala expression.
10-7、10-8Represent respectively and supernatant after the filtration comprising recombiant protein of expression is done into 10-7With 10-8Make after diluting again
MDBK cells are used, is then infected with VSV；Negative is the MDBK cells that blank is uninfected by VSV, without pathological changes, right as feminine gender
According to；Positive is the MDBK cells for infecting VSV, almost 100% pathological changes, used as positive control；10-7、10-8, Negative and
The shooting time of Positive pictures is almost consistent.
Specific embodiment
Below by specific embodiment and combine accompanying drawing the present invention will be further described, it should be understood that these realities
The purpose that example is only used for illustration is applied, protection scope of the present invention is in no way intended to limit.Those of ordinary skill in the art understand, in the present invention
Many changes can be carried out in spirit and scope set by claim to which, is changed, or even equivalent change, but fall within this
In the protection domain of invention.
Synthesis of the embodiment 1 through the T α 1-PoIFN alpha fusion protein genes of transformation
Transform after DNA analysis and RNA structure predictions are carried out to T α 1-PoIFN alpha fusion proteins genes, do not changing day
So synthetic T α 1-PoIFN alpha fusion protein genes on the premise of aminoacid sequence, are named as T α 1-PoIFN α, the artificial conjunction
Into T α 1-PoIFN alpha fusion protein genes nucleotide sequence as shown in SEQ ID NO.1.
It is prepared by a small amount of of embodiment 2T α 1-PoIFN alpha fusion proteins
1st, the structure of T α 1-PoIFN alpha fusion proteins gene engineering microzyme kind
(1) material and method：
Pichia yeast Pichiapastoris GS115, pPIC9K expression plasmids are purchased from U.S. Invitrogen public
Department.Archaeal dna polymerase, restricted enzyme EcoR I, Not I, Sac I are purchased from TaKaRa companies, and T4 DNA ligases are purchased from NEB
Company.BMGY, BMMY, YPD culture medium, is shown in Invitrogen companies Pichia sp. workbook.Plasmid extraction test kit, PCR
Product QIAquick Gel Extraction Kit is purchased from Axgen companies.One resists for anti-pig interferon alpha monoclonal antibodies, is self-control, and two resist for rabbit-anti Mus
IgG-HRP antibody, purchased from Sigma companies；
(2) structure of Expression vector pPIC9K-T α 1-PoIFN α
5 ' the ends of a T α 1-PoIFN alpha fusion protein gene T α 1-PoIFN α that embodiment 1 is synthesized by () add EcoR I limits
Property restriction enzyme site processed, adds Not I restriction endonuclease sites rear clones at its 3 ' end and pUC-T α 1- is obtained into pUC57
PoIFNα；
B plasmid pUC-T α 1-PoIFN α and pPIC9K comprising T α 1-PoIFN alpha fusion protein genes are used EcoR by () respectively
I and Not I double digestions, distinguish glue reclaim T α 1-PoIFN α and pPIC9K purpose fragments, the T that will be reclaimed after agarose gel electrophoresiies
The purpose fragment connection of α 1-PoIFN α and pPIC9K obtains Expression vector pPIC9K-T α 1-PoIFN α；
(3) the electricity conversion of recombinant yeast pichia pastoris
Using the restricted enzyme Sac I from TaKaRa companies by recombiant plasmid pPIC9K-T α 1-PoIFN α lines
Property after, mix with Pichia pastoris GS115 competent cell, it is electroporated with BioRad Gene Pulser electroporations, conversion
Parameter is 1.5kV, 25uF, 200 Ω；The Sorbitol of l mL ice baths after electric shock, is added immediately, after incubation, transformed bacteria solution is coated with
On MD flat boards, 26～30 DEG C of incubation 3d, after the single bacterium colony on flat board grows, are distinguished dibbling successively containing antibiotic
On the G418+YPD flat boards of G418 250,500,1000,2000,3000mg/L, 26～30 DEG C incubate 3d, then by G418
Single bacterium colony on 2000mg/L G418+YPD flat boards is distinguished dibbling successively again and is put down in the G418+YPD of G418 2500,3000mg/L
On plate, 26～30 DEG C of incubation 3d；The ability of restructuring yeast strains opposing G418 is integrated with plasmid copy number and is directly proportional.
(4) abduction delivering of recombiant protein
Chosen with sterilizing toothpick grow on G418+YPD flat boards containing T α 1-PoIFN alpha fusion protein bases of the present invention
Because of the G418 resistance single bacterium colonies of T α 1-PoIFN α, choose and enter line activating culture in the BMGY fluid mediums of 3mL, cultivation temperature is
28 DEG C, 250r/min shaken overnights, to OD600 ≈ 6.0, cell is in exponential phase, the culture bacterium solution for obtaining in 1500g and
3min is centrifuged under room temperature condition and collects precipitation, be resuspended in the BMMY of 5mL, prevent pollution, in the test tube relaying persistent oscillation of 30mL
Culture, at interval of 24h add 100% methanol to final concentration of 1% (v/v) of methanol, i.e. addition be in every milliliter of culture medium plus
Enter 10 μ L100% methanol, carry out inducing culture 4d, 28 DEG C of 250r/min shaken cultivation 96h harvest supernatants, then with 12000g from
Heart 3min takes supernatant by SDS-PAGE electrophoresis detection T α 1-PoIFN alpha fusion proteins (see Fig. 1), as a result shows expressed weight
The size of histone is consistent with expection, and the expression of albumen can reach 210mg/L；Product is transferred to into cellulose nitrate after electrophoresis
On plain film, Western-blot identifications (see Fig. 2) are carried out, one resists for anti-pig interferon alpha monoclonal antibodies, and two resist for rabbit-anti Mus
LgG-HRP antibody, as a result showing to express the recombiant protein for obtaining can be with anti-pig interferon alpha monoclonal antibodies and rabbit-anti Mus
There is immunoreation in lgG-HRP antibody.
A large amount of preparations of embodiment 3T α 1-PoIFN alpha fusion proteins
1st, material：
Strain containing T α 1-PoIFN alpha fusion protein gene T α 1-PoIFN α：GS115(pPIC9K-Tα1-PoIFNα)
(prepared by embodiment 2)；
Instrument：Fermentation tank, electrophresis apparatuses；
Culture medium：The concrete configuration method of YPD, BMGY, BSM and PTM1 fermentation medium is shown in Invitrogen companies Bi Chi
Yeast workbook；
2nd, method：
The accumulation of 2.1 seed culture and yeast cells Biomass
By frozen engineering bacteria in YPD Agar lining out, 26 DEG C of cultures.2mm, picking monoclonal bacterium colony are grown to bacterium colony
It is added in 10mL YPD culture fluid (seed culture medium), 26 DEG C, 250r/min shaken cultivation 24h.Above-mentioned culture 1mL is connect
Plant in 200m L YPD culture fluid, 26 DEG C, 250r/min shaken cultivation 24h so as to A600 ≈ 10.Prepare 2L BSM cultures
Base, adds 5L fermentation tanks, 121 DEG C, 30min autoclaved mediums and fermentation tank.Treat that culture medium is cooled to room temperature in fermentation tank
When, the pH value of BSM culture medium is adjusted to required numerical value with ammonia, then add PTM1 culture medium and biotin stock solution.Will be upper
State 100mL YPD culture strains and add fermentation tank, start fermentor cultivation, this is glycerol culture amplification thalline for the first stage,
Fermentation tank parameter setting is respectively mixing speed 800r/min, and 26 DEG C of temperature, control mode are P-I-D, maintain dissolved oxygen value
(DO) 30% to 40%, pure oxygen is passed through if necessary.This stage at least samples 1 time daily, surveys A600 and wet cell weight, analyzes ferment
Female bacteria growing state, visually with Microscopic observation bacterium solution, and stays supernatant for analysis of protein.After about 24h, DO values rise to close
100%, according to PTM1 culture medium is added in ratio 50% glycerol after autoclaving of every liter of 12mL PTM1 culture medium, mix
After even, be added in fermentation tank with the speed of 18.2mL/h/L Preliminary fermentation liquid, 200g/L is reached to thalline weight in wet base.Stopping is added sweet
After oil, observation DO values are risen to after being close to 100%, continue to " glycerol is hungry " state 30min, proceed to methanol induction expression rank
Section.
2.2 methanol inductions are expressed
PTM1 culture medium is added in methyl alcohol according to 12mL/L ratios, after mixing, with the speed of 3.6mL/h/L Preliminary fermentation liquid
Rate is added to abduction delivering in fermentation tank, maintains this low rate methanol to add 2h, so that yeast is adapted to methanol as sole carbon source
Environment.The speed for adding methanol is upgraded to 7.2mL/h/L Preliminary fermentation liquid, maintains 2h with this speed.The speed of methanol is added in raising
Rate is to 11mL/h/L Preliminary fermentation liquid, while detecting DO values and broth temperature and judging whether excessively methanol (stop mending methanol sight
DO value changes are surveyed, if after stopping mending methanol, DO values ascensional range in the 1min is more than 10%, illustrate that carbon source is limited, otherwise illustrates first
Alcohol excess), if carbon source is limited, accelerates to mend the speed of methanol, the speed for mending methanol should be slowed down if methanol is excessive, until suitable
Speed.After starting abduction delivering, sample 1 time every 12h, survey A600 and wet cell weight, analyze Yeast Growth state, naked eyes
With Microscopic observation bacterium solution, and take expression supernatant be used for analysis of protein.After abduction delivering 72h, terminate fermentation, after testing in fermentation tank
The expression of middle restructuring destination protein can reach 1100mg/L.Detected by SDS-PAGE electrophoresis (see Fig. 1) and Western-
T α 1-PoIFN alpha fusion proteins in blot (see Fig. 2) identification expression supernatants.
3rd, a large amount of preparations of restructuring T α 1-PoIFN alpha fusion proteins and Activity determination
By the fermentation supernatant for harvesting to filter after 12000g centrifugation 30min, after taking filtration respectively, supernatant carries out SDS-PAGE
With antiviral activity and regulation immunologic cellular activity detection (rosette).
1) antiviral activity test：Using MDBK cells/VSV detecting systems, few cells pathological changes (cytopathogenic
Effect, CPE) suppress method to determine the antiviral activity of recombiant protein.With 10 times (10 in 96 orifice plates-1、10-2、10-3、10-4、
10-5、10-6、10-7、10-8、10-9) doubling dilution expression filtration after supernatant (per 50 μ L of hole, if 8 repetitions), subsequently add per hole
Enter 100 μ LMDBK cell suspension (5 × 104Cell), if cell and virus control, 37 DEG C, 5%CO2 culture 24h are discarded in hole
Liquid, adds 200 μ LVSV suspension (200TCID per hole50/ 0.2mL), cell control well add 200 μ L culture medium, culture 36～
48h, the result of determination when the MDBK cells almost all in virus control wells occurs pathological changes, according to the lesion degree of cell, note
Protectiveness during record variable concentrations.The lesion degree of cell is divided into：Without obvious CPE, there is 25% or so CPE, have 50% left
Right CPE, there is 75% or so CPE, have 100% or so CPE.As a result calculate：Calculated according to Reed-Muench methods.Protection
Interferon amount in the recombiant protein dilution factor of 50%CPE is 1 antiviral activity unit (IU).
As a result show, after filtration, the antiviral activity of supernatant is 1.2 × 108IU/mL (see Fig. 3).
2) rosette：T α 1 have the activity for promoting cell surface receptor expression.Can be surveyed according to rosette
Determining fusion protein stimulates the activity of lymphocyte surface receptors expression, judges that the biology of thymosin α1 in fusion protein lives accordingly
Property.6, test tube is taken, wherein 3 each addition Hank's liquid 0.1mL are used as control.Other 3 respectively add sample solution 0.1mL to survey
Fixed tube, often the lymphocyte suspension 0.2mL of pipe plus de- E receptors, shakes up 37 DEG C of incubation 1h, adds sheep red blood cell (SRBC) suspension 0.2mL
500r/min is centrifuged 3min, is put into 4 DEG C of refrigerator overnights, and supernatant is abandoned in next day taking-up, often adds fixative 1 to drip in pipe, and jog stands
10min, adds dyeing liquor 2 to drip and shake up, starts counting up after standing 15min.Nattier blue in the visual field is that lymph is thin compared with maxicell
Born of the same parents, lymphocyte number (200) in counting number 16 block plaids of plate, counts E rosette forming cell (RFC)s number therein (knot altogether
Close the lymphocyte of more than 3 sheep red blood cell (SRBC)s) meter rosette forming rate, take each pipe meansigma methodss.Calculate T α's 1 according to formula
Activity：Sample activity=[(sample determination pipe average reading-control tube average reading)/100] × 100%, measurement result such as table 1
It is shown：
The T α 1 of table 1T α 1-PoIFN α recombination fusion proteins are active
| Rosetteses form average (%) | Active (%) | |
| Hank ' s liquid | 7 | - |
| Without recombiant protein lymphocyte control | 6 | - |
| Supernatant after filtration containing recombiant protein | 22 | 15 |
Result above shows that supernatant has obvious T α 1 active after the filtration comprising recombiant protein.
Sequence table
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture
<120>A kind of preparation method of thymosin α1-porcine interferon alpha antigen-4 fusion protein gene and its recombiant protein
<130> KLPI161371
<170>PatentIn 3.5
<210> 1
<211> 624
<212> DNA
<213> Tα1-PoIFNα
<400> 1
tctgatgctg ctgttgatac ttcttctgag attactacta aggatttgaa ggagaagaag 60
gaggttgttg aggaggctga gaacggttct ggtggtggtg gttctggtgg tggtggttct 120
ggttcttgtg atttgccaca aactcactct ttggctcaca ctagagcttt gagattgttg 180
gctcaaatga gaagaatttc tccattctct tgtttggatc acagaagaga tttcggttct 240
ccacacgagg ctttcggtgg taaccaagtt caaaaggctc aagctatggc tttggttcac 300
gagatgttgc aacaaacttt ccaattgttc tctactgagg gttctgctgc tgcttggaac 360
gagtctttgt tgcaccaatt ctacactggt ttggatcaac aattgagaga tttggaggct 420
tgtgttatgc aagaggctgg tttggagggt actccattgt tggaggagga ttctattaga 480
gctgttagaa agtacttcca cagattgact ttgtacttgc aagagaagtc ttactctcca 540
tgtgcttggg agattgttag agctgaggtt atgagatctt tctcttcttc tagaaacttg 600
caagatagat tgagaaagaa ggag 624
Claims (10)
1. a kind of thymosin α1-porcine interferon alpha antigen-4 fusion protein gene, it is characterised in that the nucleotide sequence of the gene such as SEQ
Shown in ID NO.1.
2. a kind of recombinant expression carrier of the thymosin α1 comprising described in claim 1-porcine interferon alpha antigen-4 fusion protein gene, excellent
Choosing, described recombinant expression carrier is methanol adjustment type recombinant yeast expression vector.
3. recombinant expression carrier as claimed in claim 2, it is characterised in that described recombinant expression carrier is by claim
Thymosin α1 described in 1-porcine interferon alpha antigen-4 fusion protein gene is obtained in being cloned into Yeast expression carrier pPIC9K, described heavy
Group expression vector contains EcoRI and Not I restriction enzyme sites, and a strong promoter AOX1 promoter and a G418 resistance are selected
Site.
4. a kind of host cell, it is characterised in that the host cell is comprising the nucleotide sequence shown in SEQ ID NO.1
Host cell, or be the host cell comprising the recombinant expression carrier described in Claims 2 or 3, it is preferred that described is thin
Born of the same parents are eukaryotic cell, it is furthermore preferred that described cell is methanotrophic recombinant yeast cell GS115.
5. the thymosin α1 described in claim 1-porcine interferon alpha antigen-4 fusion protein gene is in Prepare restructuring thymosin α1-pig interference
Application in plain alpha fusion protein.
6. a kind of method of Prepare restructuring thymosin α1-pig interferon alpha fusion protein, it is characterised in that be by claim 1 institute
The thymosin α1 stated-porcine interferon alpha antigen-4 fusion protein gene is cloned into expression vector, then converts the recombinant expression carrier for obtaining
Host Strains, the thymosin α1 recombinated-pig interferon alpha fusion protein abduction delivering are collected purification and are obtained final product.
7. method as claimed in claim 6, it is characterised in that comprise the following steps：
(1) synthetic thymosin α1-porcine interferon alpha antigen-4 fusion protein gene, the nucleotide sequence such as SEQ ID of described gene
Shown in NO.1；
(2) thymosin α1 of synthesis-porcine interferon alpha antigen-4 fusion protein gene is cloned in Yeast expression carrier pPIC9K, electricity turns
After changing GS115 competent cells, with MD plates and G418+YPD plate screenings, after activation culture in shaking flask Small Amount or fermentation tank
Expressed with methanol induction and carry out steriling test in a large number；
(3) sterile collection culture supernatant, Jing filtrations or after purification Prepare restructuring thymosin α1-pig interferon alpha fusion protein, entirely
During carry out albumen steriling test；
(4) immune detection is carried out to thymosin α1-pig interferon alpha fusion protein with monoclonal antibody；
(5) detect the antiviral activity of recombinant thymin alpha-1-pig interferon alpha fusion protein and adjust immunologic cellular activity.
8. method as claimed in claim 7, it is characterised in that add EcoRI limits in 5 ' ends of the gene described in step (1)
Property restriction enzyme site processed, adds Not I restriction endonuclease sites at its 3 ' end.
9. method as claimed in claim 7, it is characterised in that it is described in shaking flask with the condition of methanol induction expression in a small amount
It is to add 100% methanol to final concentration of 0.5～1% (v/v) of methanol at interval of 24h, i.e., during addition is every milliliter of culture fluid
Plus 5～10 μ L100% methanol, carry out inducing culture, 26～30 DEG C of 250～300r/min, 96～120h of concussion and cultivate harvest training
Foster supernatant, after purification Jing filtrations or Prepare restructuring thymosin α1-pig interferon alpha fusion protein.
10. method as claimed in claim 7, it is characterised in that it is described in fermentation tank with the step of methanol induction great expression
Rapid and condition is：1) glycerol culture amplification thalline：Fermentation tank parameter setting is respectively 600～1000r/min of mixing speed, temperature
26～28 DEG C, control mode is P-I-D, maintains dissolved oxygen value (DO) 30% to 40%；2) methanol induction expression：According to 8
～12mL/L ratios add PTM1 culture medium in methyl alcohol, after mixing, are added with the speed of 2.4～3.6mL/h/L/ Preliminary fermentation liquid
Enter in fermentation tank abduction delivering, so that yeast adapts to the environment with methanol as sole carbon source, subsequently add the speed liter of methanol
For 4.8～7.2mL/h/L Preliminary fermentation liquid, the speed for adding methanol is finally improved to 10～12mL/h/L/ Preliminary fermentation liquid, opened
After beginning abduction delivering, taking expression supernatant daily is used for analysis of protein, after 72～96h of abduction delivering, terminates fermentation；Harvest in culture
Clearly, Jing filtrations or after purification Prepare restructuring thymosin α1-pig interferon alpha fusion protein.
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| CN113234171A (en) * | 2021-04-30 | 2021-08-10 | 广州源博医药科技有限公司 | CaIFN-alpha & T alpha 1 fusion protein, vector, recombinant strain and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6319691B1 (en) * | 1999-06-15 | 2001-11-20 | Usa Universe Bioengineering, Inc. | Fusion proteins comprising IFN-alpha2b and TM-alpha1 |
| CN1611604A (en) * | 2003-12-23 | 2005-05-04 | 青岛宝依特生物技术研究所 | Swine alpha-interferon gene synthesis, expression vector establishment and product preparing method |
| CN1765932A (en) * | 2005-12-05 | 2006-05-03 | 吉林大学 | Fusion protein of extrasin alpha1 and interferon |
| CN103937828A (en) * | 2014-02-25 | 2014-07-23 | 南京洲邦生物科技有限公司 | Preparation method of fusion protein of porcine interferon-alpha 1 and thymosin-alpha 1 |
-
2016
- 2016-12-16 CN CN201611169628.XA patent/CN106520807B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6319691B1 (en) * | 1999-06-15 | 2001-11-20 | Usa Universe Bioengineering, Inc. | Fusion proteins comprising IFN-alpha2b and TM-alpha1 |
| CN1611604A (en) * | 2003-12-23 | 2005-05-04 | 青岛宝依特生物技术研究所 | Swine alpha-interferon gene synthesis, expression vector establishment and product preparing method |
| CN1765932A (en) * | 2005-12-05 | 2006-05-03 | 吉林大学 | Fusion protein of extrasin alpha1 and interferon |
| CN103937828A (en) * | 2014-02-25 | 2014-07-23 | 南京洲邦生物科技有限公司 | Preparation method of fusion protein of porcine interferon-alpha 1 and thymosin-alpha 1 |
Non-Patent Citations (6)
| Title |
|---|
| CHUN XIA ET AL.: "Cloning and expression of interferon-a/g from a domestic porcine breed and its effect on classical swine fever virus", 《VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY》 * |
| 刘先俊 等: "胸腺素α1 与复合α干扰素融合蛋白的表达及其生物学活性", 《生物工程学报》 * |
| 孙卫民 等: "《细胞因子研究方法学》", 31 July 1999, 人民卫生出版社 * |
| 王旻: "《生物工程》", 31 August 2015, 中国医药科技出版社 * |
| 祁岩超 等: "《细胞生长因子基础与临床》", 31 December 1994, 河南科学技术出版社 * |
| 龚大春 等: "《酵母生物学》", 31 October 2015, 中国水利水电出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113234171A (en) * | 2021-04-30 | 2021-08-10 | 广州源博医药科技有限公司 | CaIFN-alpha & T alpha 1 fusion protein, vector, recombinant strain and application thereof |
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