CN106520807B - A kind of preparation method of thymosin α1-porcine interferon alpha antigen-4 fusion protein gene and its recombinant protein - Google Patents
A kind of preparation method of thymosin α1-porcine interferon alpha antigen-4 fusion protein gene and its recombinant protein Download PDFInfo
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Abstract
The invention discloses the preparation methods of a kind of thymosin α1-porcine interferon alpha antigen-4 fusion protein gene and its recombinant protein.It is of the invention it is engineered after thymosin α1-porcine interferon alpha antigen-4 fusion protein gene nucleotide sequence as shown in SEQ ID NO.1, the present invention discloses the preparation of the recombinant protein of the gene expression and the methods of Activity determination, more particularly to thymosin α1-porcine interferon alpha antigen-4 fusion protein gene transformation, the improvement of the preparation of the clone of gene and its secreting, expressing, recombinant protein in Pichia pastoris and the operating methods such as fermentation culture conditions and activity test method.The invention is simple and feasible, cost is relatively low, realize thymosin α1-pig interferon alpha fusion protein efficient stable secreting, expressing in Pichia pastoris, the recombination fusion protein of expression has high-caliber antiviral activity and adjusts immunologic cellular activity, to provide there are the green noresidue biological products for the treatment of porcine viral diseases and immunoloregulation function to lay a good foundation.
Description
Technical field
The present invention relates to a kind of transformations of thymosin α1-porcine interferon alpha antigen-4 fusion protein gene, further relate to containing the gene
Stably excreting expression and preparation and reorganization thymosin α1-pig interferon alpha fusion protein side in the building of expression vector, Pichia pastoris
Method and recombinant thymin alpha-1-pig interferon alpha fusion protein antiviral activity and immunoregulation effect, the invention belongs to dynamic
Object genetic engineering and technical field of animal virology.
Background technique
Thymin is the general name of the polypeptide hormone generated by thymic epithelial cells, also known as thymic peptide
(thymopeptide).The thymic peptide applied at present is the peptide material that extraction purification obtains from calf or porcine thymus mostly,
Its bioactivity is mainly inducing T cell differentiation and maturation and regulation immunologic balance.Clinic, which is cured, in people is mainly used for autoimmune
Disease, such as rheumatoid arthritis, lupus erythematosus, glomerulonephritis and myasthenia gravis are also used for malignant tumour, viral
The adjuvant treatment of the diseases such as the infection that hepatitis and antibiotic not can be effectively controlled there is no clinical application in animal doctor.
Thymosin extrasin (Thymosin, T) is made of 25 or more amino acid residues, in the various active principles of thymic peptide
In, active highest.According to isoelectric point difference, thymosin extrasin can be divided into α, β and γ family.With thymosin α1 (T α 1) in α family
Most study, T α 1 are made of 28 amino acid residues, are initially Goldstein from calf thymus tissue fifth component
A kind of polypeptide of separating-purifying, molecular weight 3.1KDa, isoelectric point 4.2 in (Thymosin Fraction 5, TF5)
[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 polypeptide 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 solution of influenza infection
Active substances because it interferes the duplication of homologous and heterologus virus, 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 be " disease-resistant poison cell because
Son ".It thanks petrel, Wu Dan etc. and has 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 is realized；Yao Qingxia etc. is obtained in yeast expression system life
The active recombination porcine interferon alpha of object (Porcine Interferona, PoIFN-a) simultaneously inhibits FMDV, PRV, PRRSV to it
Activity studied.Interferon is a kind of non-specific broad-spectrum antiviral biological agent, can be used for treating many viral
Disease.The researchs discovery such as poplar emperor's teacher is inoculated with leukocyte interferon of pig, can substantially reduce sucking pig disease incidence, and with 7 days
It is best that age sucking pig starts to carry out protective inoculation effect.Zheng Yongbo etc. carries out clinical test, test result with leukocyte interferon of pig
Show if directly treated with interferon, the cure rate to infected animal can be improved, if with interferon cooperate antibiotic and
Antiviral drugs treats infected animal, can more significantly improve the cure rate to infected animal.Zhang Quanjun etc. passes through test
It is found with clinical expanding test, leukocyte interferon of pig is to suckling pig and the certain virus diarrheas of weanling pig or virus and carefully
The diarrhea that bacterium mixed infection causes, there is good preventive and therapeutic action.
There are two the patent applications of Bacillus coli expression pig interferon-thymosin α1 fusion protein to disclose (application at present
Number it is respectively as follows: CN200910217774.9 and CN201410063007.8), the two patent applications use prokaryotic expression system
GST- porcine interferon alpha-thymosin α1 fusion protein is expressed, the destination protein of expression is inclusion body, need by inclusion body
Denaturation, renaturation, dialysis, cross column purification, proteolytic cleavage except (number of patent application is for GST and destination protein repurity
CN200910217774.9 is shown in its specification page 11；Number of patent application CN201410063007.8 is shown in its specification the 3rd
Page), well-known to those skilled in the art to be, there are many uncertain factors for the denaturation of inclusion body and renaturation step, entire pure
Change process is extremely complex and is difficult to carry out quality control, can greatly improve the production cost of destination protein.
The present invention is to merge egg with Pichia pastoris efficient secretory expression thymosin α1-porcine interferon alpha (T α 1-PoIFN α)
White, the expression quantity of restructuring destination protein can achieve 210mg/L in shaking flask, in the fermenter the expression quantity of restructuring destination protein
It can achieve 1100mg/L, expressed T α 1-PoIFN alpha fusion protein is secretion, and is present in culture in the form of soluble
In supernatant, it is only necessary to pass through Purification by filtration, production cost is very low.So far, have no next high with Pichia pastoris both at home and abroad
Imitate the report of secreting, expressing T α 1-PoIFN alpha fusion protein.In summary it may be concluded that the present invention is red to finish both at home and abroad
The report for the first time of yeast efficient secretory expression T α 1-PoIFN alpha fusion protein, the recombination T α 1- of efficient secretory expression of the present invention
PoIFN alpha fusion protein has very high antiviral activity and adjusts immunologic cellular activity.Efficient stable secreting, expressing of the present invention
Recombination T α 1-PoIFN alpha fusion protein is to provide the green noresidue life with treatment porcine viral diseases and immunoloregulation function
Tetramune is laid a good foundation.
Summary of the invention
The object of the present invention is to provide a kind of artificial synthesized thymosin α1-porcine interferon alphas (T α 1-PoIFN α) to merge egg
White gene；
It is a further object of the present invention to provide the DNA recombinations containing above-mentioned artificial synthesized T α 1-PoIFN alpha fusion protein gene
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 protein and Activity determination.
In order to achieve the above object, the invention adopts the following technical scheme:
A kind of thymosin α1-porcine interferon alpha (T α 1-PoIFN α) antigen-4 fusion protein gene of the 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 gene of the invention is not change thymosin α1 and the natural ammonia of porcine interferon alpha
Under the premise of base acid sequence, change after carrying out DNA analysis and RNA structure prediction to progress T α 1-PoIFN alpha fusion protein gene
It makes, T α 1-PoIFN alpha fusion protein stably excreting in Pichia pastoris can be made to express.
The present invention also provides a kind of recombinant expression carriers comprising the T α 1-PoIFN alpha fusion protein gene.
Preferably, the recombinant expression carrier is methanol adjustment type recombinant yeast expression vector, it is furthermore preferred that described
Recombinant vector be by T α 1-PoIFN alpha fusion protein gene cloning shown in SEQ ID NO.1 into Yeast expression carrier pPIC9K
It obtains, the recombinant expression carrier contains EcoRI and Not I restriction enzyme site, a strong promoter AOX1 promoter and one
A G418 resistance selects site.
Further, the present invention also provides a kind of host cell, the host cell is comprising shown in SEQ ID NO.1
Nucleotide sequence host cell, or to include that recombinant expression containing nucleotide sequence shown in SEQ ID NO.1 carries
The host cell of body.
Preferably, the host cell is eukaryotic cells.It is furthermore preferred that the host cell is methanol nutrition
Type recombinant yeast cell GS115.
Further, the present invention also provides the T α 1-PoIFN alpha fusion protein genes in preparation T α 1-PoIFN α
Application in fusion protein.
A kind of method preparing T α 1-PoIFN alpha fusion protein provided by the invention, it is characterised in that be by SEQ ID
Then obtained recombinant expression carrier is converted place to expression vector by T α 1-PoIFN alpha fusion protein gene cloning shown in NO.1
Main bacterium, carries out the inducing expression of recombinant protein, and collection purifies to obtain the final product.
In the present invention, it is preferred to, the method the following steps are included:
(1) artificial synthesized T α 1-PoIFN alpha fusion protein gene, the nucleotide sequence of the gene such as SEQ ID NO.1
It is shown；
(2) by the T α 1-PoIFN alpha fusion protein gene cloning of synthesis into Yeast expression carrier pPIC9K, electrotransformation
It is big in shaking flask Small Amount or fermentor after activation culture with MD plate and G418+YPD plate screening after GS115 competent cell
Amount is expressed with methanol induction and carries out steriling test；
(3) sterile collection culture supernatant, through filtering or preparation and reorganization T α 1-PoIFN alpha fusion protein after purification, whole process
Middle progress albumen steriling test；
(4) immune detection is carried out to T α 1-PoIFN alpha fusion protein with monoclonal antibody；
(5) antiviral activity of detection recombination T α 1-PoIFN alpha fusion protein and adjusting immunologic cellular activity.
In the present invention, it is preferred to, 5 ' ends of the gene described in step (1) joined EcoRI restriction enzyme position
Point joined Not I restriction endonuclease sites at its 3 ' end.
In the present invention, it is preferred to, the condition expressed in a small amount in shaking flask with methanol induction be at interval of for 24 hours plus
Enter 100% methanol to methanol final concentration of 0.5~1% (v/v), i.e. additional amount is to add 5~10 μ L100% in every milliliter of culture solution
Methanol, carry out Fiber differentiation, 26~30 DEG C of 250~300r/min, 96~120h of shake culture, harvest culture supernatant, through filtering or
Preparation and reorganization T α 1-PoIFN alpha fusion protein after purification.
In the present invention, it is preferred to, it is described in the fermenter with the step of methanol induction great expression and condition are as follows: 1)
Glycerol culture expands thallus: fermentor parameter setting is respectively 600~1000r/min of mixing speed, 26~28 DEG C of temperature, is controlled
Mode is P-I-D, maintains dissolved oxygen value (DO) 30% to 40%；2) methanol induction is expressed: being existed according to 8~12mL/L ratio
PTM1 culture medium is added in methanol, after mixing, is added in fermentor and is lured with the rate of 2.4~3.6mL/h/L/ Preliminary fermentation liquid
Expression is led, so that yeast is adapted to using methanol as the environment of sole carbon source, the rate for then adding methanol is upgraded to 4.8~7.2mL/h/
L Preliminary fermentation liquid finally improves and adds the rate of methanol to 10~12mL/h/L/ Preliminary fermentation liquid, after starting inducing expression, often
It takes expression supernatant for analysis of protein, after 72~96h of inducing expression, terminates fermentation；Culture supernatant is harvested, is filtered or is purified
Preparation and reorganization T α 1-PoIFN alpha fusion protein afterwards.
Specifically, the described method comprises the following steps:
(1) artificial synthesized T α 1-PoIFN alpha fusion protein gene T α 1- under the premise of not changing natural acid sequence
PoIFN α, the nucleotide sequence of the artificial synthesized T α 1-PoIFN alpha fusion protein gene is as shown in SEQ ID NO.1.Then exist
Its 5 ' end joined EcoR I restriction endonuclease sites, joined 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 gene with EcoR I and
Not I double digestion, glue recycles T α 1-PoIFN α and pPIC9K segment after agarose electrophoresis, and connection T α 1-PoIFN α and pPIC9K are obtained
To Expression vector pPIC9K-T α 1-PoIFN α.
(3) electrotransformation of Pichia pastoris
PPIC9K-T α 1-PoIFN α is linearized with Sal I or Sac I, after being mixed with GS115 competent cell, is used
BioRad Gene Pulser electrotransformation, electrotransformation product are coated with MD plate, are switched to after growing single colonie different dense
It spends in G418+YPD resistant panel, 26~30 DEG C of 2~3d of incubation.
(4) inducing expression of albumen
From picking individual colonies in resistance plate activated after Fiber differentiation, detect supernatant in destination protein expression,
Supernatant is centrifuged 3min with 12000g, and supernatant is taken to be purified, and carries out SDS-PAGE detection with purifying protein.
(5) immunology detection of T α 1-PoIFN alpha fusion protein is recombinated
3min is centrifuged with 12000g to the supernatant of harvest, takes supernatant to carry 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 T α 1-PoIFN alpha fusion protein is recombinated
3min is centrifuged with 12000g to the supernatant of harvest, then supernatant is taken to filter, after detection filtering in supernatant PoIFN α it is anti-
The adjusting immunologic cellular activity of virus activity and T α 1.
When the shaking flask for recombinating T α 1-PoIFN alpha fusion protein is prepared in a small amount, comprising the following steps:
(1) single colonie with G418 resistance grown on G418+YPD plate is chosen with sterilizing toothpick, chooses the BMGY in 3mL
Activation culture is carried out in fluid nutrient medium, cultivation temperature is 26~30 DEG C, the 250 turns/min shaken overnight in shaking table, until OD600
=2~6, cell is in logarithmic growth phase；
(2) the culture bacterium solution of step (1) is centrifuged 3min in 1500g and under room temperature and collects precipitating, be resuspended in 5mL's
In BMMY, controls dissolved oxygen amount and prevent from polluting, continue the shaken cultivation in shaking table in the test tube of 30mL；
(3) at interval of 100% methanol to final concentration of 1% (v/v) is added for 24 hours, i.e. additional amount is in every milliliter of culture solution
Add 10 μ L100% methanol, carry out Fiber differentiation, 26~30 DEG C of 250r/min 96~120h of shake culture harvest culture supernatant, warp
Filtering or after purification preparation and reorganization capsid protein.
In a large amount of preparations for recombinating T α 1-PoIFN alpha fusion protein, present invention employs 5L fermentors to ferment
Expression, step 1) glycerol culture amplification thallus: fermentor 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 is expressed: according to 8
PTM1 culture medium is added in~12mL/L ratio in methyl alcohol, after mixing, is added with the rate of 2.4~3.6mL/h/L/ Preliminary fermentation liquid
Enter into fermentor inducing expression, this low rate methanol is maintained to add 2~3h, so that yeast is adapted to using methanol as sole carbon source
Environment, the rate for then adding methanol are upgraded to 4.8~7.2mL/h/L Preliminary fermentation liquid, maintain 2h with this rate, finally improve and mend
Add the rate of methanol to 10~12mL/h/L/ Preliminary fermentation liquid, while detecting DO value and broth temperature and whether judging methanol
It is excessive, after starting inducing expression, take expression supernatant for analysis of protein daily.After 72~96h of inducing expression, terminate fermentation；It receives
Culture supernatant is obtained, through filtering or preparation and reorganization T α 1-PoIFN alpha fusion protein after purification.
Compared to the prior art, the beneficial effects of the present invention are:
(1) present invention is that related T α 1-PoIFN alpha fusion protein efficient secretory expression and has Gao Sheng in yeast both at home and abroad
Object is active to be reported for the first time.
(2) the T α 1-PoIFN alpha fusion protein of efficient stable secreting, expressing of the present invention is to provide to have treatment pig virus disease
The green noresidue biological products of disease and immunoloregulation function are laid a good foundation.
(3) the method for the present invention is simple and easy, and cost is relatively low.
Detailed description of the invention
Fig. 1 is the SDS-PAGE result of the T α 1-PoIFN alpha fusion protein 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 supernatant, and 2 merge for T α 1-PoIFN α
Albumen shaking flask expresses supernatant；
Fig. 2 is the T α 1-PoIFN alpha fusion protein Western-blot result 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 supernatant, and 2 merge for T α 1-PoIFN α
Albumen shaking flask expresses supernatant；
Fig. 3 is the antiviral activity testing result of supernatant after the T α 1-PoIFN alpha fusion protein of Pichia anomala expression filters.
10-7、10-8Supernatant does 10 after respectively indicating the filtering comprising recombinant protein by expression-7With 10-8Make after diluting again
With MDBK cell, then infected with VSV；Negative is the MDBK cell that blank is uninfected by VSV, no lesion, as negative right
According to；Positive is the MDBK cell for infecting VSV, almost 100% lesion, as positive control；10-7、10-8, Negative and
The shooting time of Positive picture is almost consistent.
Specific embodiment
Below by specific embodiment and in conjunction with attached 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
Change that many changes, modifications, and even equivalents may be made in spirit and scope set by claim, but fall within this
In the protection scope of invention.
The synthesis of T α 1-PoIFN alpha fusion protein gene of the embodiment 1 by transformation
It is transformed after carrying out DNA analysis and RNA structure prediction to T α 1-PoIFN alpha fusion protein gene, is not changing day
Artificial synthesized T α 1-PoIFN alpha fusion protein gene under the premise of right amino acid sequence, is named as T α 1-PoIFN α, the artificial conjunction
At T α 1-PoIFN alpha fusion protein gene nucleotide sequence as shown in SEQ ID NO.1.
A small amount of preparations of embodiment 2T α 1-PoIFN alpha fusion protein
1, the building of T α 1-PoIFN alpha fusion protein gene engineering microzyme kind
(1) material and method:
Pichia yeast Pichiapastoris GS115, pPIC9K expression plasmid is purchased from U.S. Invitrogen public affairs
Department.Archaeal dna polymerase, restriction enzyme EcoR I, Not I, Sac I are purchased from TaKaRa company, and T4 DNA ligase is purchased from NEB
Company.BMGY, BMMY, YPD culture medium are shown in Invitrogen company Pichia pastoris operation manual.Plasmid extraction kit, PCR
Product QIAquick Gel Extraction Kit is purchased from Axgen company.Primary antibody is anti-pig interferon alpha monoclonal antibodies, and for self-control, secondary antibody is rabbit-anti mouse
IgG-HRP antibody is purchased from Sigma company；
(2) building of Expression vector pPIC9K-T α 1-PoIFN α
(a) EcoR I limit is added in 5 ' ends for the T α 1-PoIFN alpha fusion protein gene T α 1-PoIFN α for synthesizing embodiment 1
Property restriction enzyme site processed joined Not I restriction endonuclease sites rear clone at its 3 ' end and obtain pUC-T α 1- into pUC57
PoIFNα；
(b) plasmid pUC-T α 1-PoIFN α and pPIC9K comprising T α 1-PoIFN alpha fusion protein gene are used into EcoR respectively
I and Not I double digestion, glue recycles T α 1-PoIFN α and pPIC9K target fragment respectively after agarose gel electrophoresis, by the T of recycling
α 1-PoIFN α connects to obtain Expression vector pPIC9K-T α 1-PoIFN α with the target fragment of pPIC9K；
(3) electrotransformation of recombinant yeast pichia pastoris
Using the restriction enzyme Sac I from TaKaRa company by recombinant plasmid pPIC9K-T α 1-PoIFN α line
Property after, mixed with Pichia pastoris GS115 competent cell, it is electroporated with BioRad Gene Pulser electroporation, conversion
Parameter is 1.5kV, 25uF, 200 Ω；After electric shock, the sorbierite of l mL ice bath is added immediately, after incubation, transformed bacteria solution is coated with
In on MD plate, 26~30 DEG C of incubation 3d are distinguished successively dibbling containing antibiotic after the single colonie on plate is grown
G418 250,500,1000,2000, on the G418+YPD plate of 3000mg/L, 26~30 DEG C incubate 3d, then by G418
Successively G418+YPD of the dibbling in G418 2500,3000mg/L is flat respectively again for single colonie on 2000mg/L G418+YPD plate
On plate, 26~30 DEG C of incubation 3d；It is directly proportional that the ability that the restructuring yeast strains resist G418 is integrated with plasmid copy number.
(4) inducing expression of recombinant protein
Chosen with sterilizing toothpick grown on G418+YPD plate containing T α 1-PoIFN alpha fusion protein base of the present invention
Because of the G418 resistance single colonie of T α 1-PoIFN α, chooses and carry out activation culture in the BMGY fluid nutrient medium of 3mL, cultivation temperature is
28 DEG C, 250r/min shaken overnight, until OD600 ≈ 6.0, cell is in logarithmic growth phase, obtained culture bacterium solution in 1500g and
Centrifugation 3min collects precipitating under room temperature, is resuspended in the BMMY of 5mL, prevents from polluting, and relays persistent oscillation in the test tube of 30mL
Culture, at interval of 100% methanol is added for 24 hours to methanol final concentration of 1% (v/v), i.e. additional amount is to add in every milliliter of culture medium
Enter 10 μ L100% methanol, carry out Fiber differentiation 4d, 28 DEG C of 250r/min shaken cultivation 96h harvest supernatants, then with 12000g from
Heart 3min takes supernatant to pass through SDS-PAGE electrophoresis detection T α 1-PoIFN alpha fusion protein (see Fig. 1), the results showed that expressed weight
The size of histone and expection are consistent, and the expression quantity of albumen can achieve 210mg/L；Product is transferred to cellulose nitrate after electrophoresis
It on plain film, carries out Western-blot identification (see Fig. 2), primary antibody is anti-pig interferon alpha monoclonal antibodies, and secondary antibody is rabbit-anti mouse
LgG-HRP antibody, the results showed that the recombinant protein expressed can be with anti-pig interferon alpha monoclonal antibodies and rabbit-anti mouse
LgG-HRP antibody is immunoreacted.
A large amount of preparations of embodiment 3T α 1-PoIFN alpha fusion protein
1, material:
Strain containing T α 1-PoIFN alpha fusion protein gene T α 1-PoIFN α: GS115 (pPIC9K-T α 1-PoIFN α)
(preparation of embodiment 2)；
Instrument: fermentor, electrophoresis apparatus；
Culture medium: the concrete configuration method of YPD, BMGY, BSM and PTM1 fermentation medium is shown in Invitrogen company Bi Chi
Yeast operation manual；
2, method:
The accumulation of 2.1 seed cultures and yeast cells biomass
By the engineering bacteria frozen in YPD Agr lining out, 26 DEG C of cultures.2mm, picking monoclonal colonies are grown to bacterium colony
Be added in 10mL YPD culture solution (seed culture medium), 26 DEG C, 250r/min shaken cultivation for 24 hours.Above-mentioned culture 1mL is connect
Kind into 200m L YPD culture solution, 26 DEG C, 250r/min shaken cultivation for 24 hours, make its A600 ≈ 10.Prepare 2L BSM culture
5L fermentor, 121 DEG C, 30min autoclaved medium and fermentor is added in base.Room temperature is cooled to culture medium in fermentor
When, PTM1 culture medium and biotin stock solution is then added to required numerical value in the pH value for adjusting BSM culture medium with ammonium hydroxide.It will be upper
It states 100mL YPD culture strain and fermentor is added, start fermentation tank culture, this expands thallus for first stage, that is, glycerol culture,
Fermentor parameter setting is respectively mixing speed 800r/min, and 26 DEG C of temperature, control mode is P-I-D, maintains dissolved oxygen value
(DO) 30% to 40%, it is passed through pure oxygen when necessary.This stage at least samples 1 time daily, surveys A600 and wet cell weight, analyzes ferment
Female bacterium growth conditions and stay supernatant for analysis of protein visually with microscopic observation bacterium solution.About for 24 hours after DO value rise to it is close
100%, according to PTM1 culture medium is added in ratio 50% glycerol after autoclaving of every liter of 12mL PTM1 culture medium, mix
It after even, is added in fermentor with the rate of 18.2mL/h/L Preliminary fermentation liquid, until thallus weight in wet base reaches 200g/L.Stopping is added sweet
After oil, observation DO value is risen to close to after 100%, continues to " glycerol is hungry " state 30min, is transferred to methanol induction expression rank
Section.
The expression of 2.2 methanol inductions
PTM1 culture medium is added in methyl alcohol according to 12mL/L ratio, after mixing, with the speed of 3.6mL/h/L Preliminary fermentation liquid
Rate is added to inducing expression in fermentor, this low rate methanol is maintained to add 2h, so that yeast is adapted to using methanol as sole carbon source
Environment.The rate for adding methanol is upgraded to 7.2mL/h/L Preliminary fermentation liquid, maintains 2h with this rate.Improve the speed for adding methanol
Rate detects DO value and broth temperature and judges whether methanol is excessive and (stop mending methanol and see to 11mL/h/L Preliminary fermentation liquid
The variation of DO value is surveyed, if stopping after mending methanol, DO value ascensional range in 1min is greater than 10%, and it is limited to illustrate carbon source, otherwise illustrates first
Alcohol is excessive), if carbon source is limited, accelerate the rate for mending methanol, the rate for mending methanol should be slowed down if methanol is excessive, until suitable
Speed.It after starting inducing expression, is sampled 1 time every 12h, surveys A600 and wet cell weight, analyze Yeast Growth state, naked eyes
With microscopic observation bacterium solution, and take expression supernatant for analysis of protein.After inducing expression 72h, terminate fermentation, through detecting in fermentor
The expression quantity of middle restructuring destination protein can achieve 1100mg/L.Pass through SDS-PAGE electrophoresis (see Fig. 1) detection and Western-
T α 1-PoIFN alpha fusion protein in blot (see Fig. 2) identification expression supernatant.
3, a large amount of preparations and the Activity determination of T α 1-PoIFN alpha fusion protein are recombinated
It is filtered after the fermentation supernatant of harvest is centrifuged 30min with 12000g, supernatant after filtering is taken to carry out SDS-PAGE respectively
With antiviral activity and adjusting immunologic cellular activity detection (rosette).
1) antiviral activity is tested: using MDBK cell/VSV detection system, few cells lesion (cytopathogenic
Effect, CPE) inhibit method measurement recombinant protein antiviral activity.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 filtering after supernatant (every 50 μ L of hole, if 8 repetitions), subsequent every hole adds
Enter 100 μ LMDBK cell suspensions (5 × 104Cell), if cell and virus control, 37 DEG C, 5%CO2 culture for 24 hours, discard in hole
200 μ LVSV suspension (200TCID are added in liquid, every hole50/ 0.2mL), cell control well 200 μ L culture mediums of addition, culture 36~
48h is determined when lesion occurs for the MDBK cell almost all in virus control wells as a result, being remembered according to the lesion degree of cell
Record protectiveness when various concentration.The lesion degree of cell is divided into: no apparent CPE, have 25% or so CPE, have 50% a left side
The CPE on the right side, there is 75% or so CPE, have 100% or so CPE.As a result it calculates: being calculated according to Reed-Muench method.Protection
Interferon amount in the recombinant protein dilution of 50%CPE is 1 antiviral activity unit (IU).
The result shows that the antiviral activity of supernatant is 1.2 × 10 after filtering8IU/mL (see Fig. 3).
2) rosette: T α 1 has the activity for promoting cell surface receptor expression.It can be surveyed according to rosette
Determine the activity that fusion protein stimulation lymphocyte surface receptors are expressed, judges that the biology of thymosin α1 in fusion protein is living accordingly
Property.6, test tube are taken, wherein 3 each Hank's liquid 0.1mL that are added are as control.Other 3 respectively add sample solution 0.1mL to survey
Fixed pipe, every pipe add the lymphocyte suspension 0.2mL of de- E receptor, shake up 37 DEG C of incubation 1h, and sheep red blood cell (SRBC) suspension 0.2mL is added
500r/min is centrifuged 3min, is put into 4 DEG C of refrigerator overnights, and next day, which takes out, abandons supernatant, and fixer 1 is added in every pipe and drips, jog is stood
10min is added dyeing liquor 2 and drips and shake up, starts counting after standing 15min.It is nattier blue thin for lymph compared with maxicell in the visual field
Born of the same parents, lymphocyte number (200) in 16 block plaids of counting number plate, counts E rosette forming cell (RFC) number therein (knot altogether
Close the lymphocyte of 3 or more sheep red blood cell (SRBC)s) meter rosette forming rate, take each pipe average value.Calculate T α's 1 according to formula
Activity: sample activity=[(sample measures pipe average reading-control tube average reading)/100] × 100%, measurement result such as table 1
It is shown:
1 activity of T α of table 1T α 1-PoIFN α recombination fusion protein
| Rosettes form average (%) | Active (%) | |
| Hank ' s liquid | 7 | - |
| Without recombinant protein lymphocyte control | 6 | - |
| Supernatant after filtering containing recombinant protein | 22 | 15 |
The above result shows that supernatant has apparent 1 activity of T α after the filtering comprising recombinant protein.
Sequence table
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture
<120>preparation method of a kind of thymosin α1-porcine interferon alpha antigen-4 fusion protein gene and its recombinant 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 (9)
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 include thymosin α1 described in claim 1-porcine interferon alpha antigen-4 fusion protein gene recombinant expression carrier, institute
The recombinant expression carrier stated is methanol adjustment type recombinant yeast expression vector.
3. recombinant expression carrier as claimed in claim 2, which is characterized in that the recombinant expression carrier is by claim
Thymosin α1 described in 1-porcine interferon alpha antigen-4 fusion protein gene is cloned into obtained in Yeast expression carrier pPIC9K, described heavy
Group expression vector contains EcoRI and Not I restriction enzyme site, a strong promoter AOX1 promoter and a G418 resistance selection
Site.
4. a kind of host cell, which is characterized in that the host cell is to include nucleotide sequence shown in SEQ ID NO.1
Host cell, or be the host cell comprising recombinant expression carrier described in Claims 2 or 3, the cell is first
Alcohol auxotype recombinant yeast cell GS115.
5. thymosin α1 described in claim 1-porcine interferon alpha antigen-4 fusion protein gene is in preparation and reorganization thymosin α1-pig interference
Application in plain alpha fusion protein.
6. a kind of method of preparation and reorganization thymosin α1-pig interferon alpha fusion protein, which comprises the following steps:
(1) artificial synthesized thymosin α1-porcine interferon alpha antigen-4 fusion protein gene, the nucleotide sequence of the gene such as SEQ ID
Shown in NO.1；
(2) thymosin α1 of synthesis-porcine interferon alpha antigen-4 fusion protein gene is cloned into Yeast expression carrier pPIC9K, electricity turns
After changing GS115 competent cell, with MD plate and G418+YPD plate screening, activate after cultivating in shaking flask Small Amount or fermentor
It is largely expressed with methanol induction and carries out steriling test；
(3) sterile collection culture supernatant, through filtering or preparation and reorganization thymosin α1-pig interferon alpha fusion protein after purification, entirely
Albumen steriling test is carried out in the process；
(4) immune detection is carried out to thymosin α1-pig interferon alpha fusion protein with monoclonal antibody；
(5) detection recombinant thymin alpha-1-pig interferon alpha fusion protein antiviral activity and adjusting immunologic cellular activity.
7. method as claimed in claim 6, it is characterised in that 5 ' ends of the gene described in step (1) joined EcoRI limit
Property restriction enzyme site processed joined Not I restriction endonuclease sites at its 3 ' end.
8. method as claimed in claim 6, it is characterised in that the condition expressed in a small amount in shaking flask with methanol induction
For at interval of 100% methanol is added for 24 hours to methanol final concentration of 0.5~1% (v/v), i.e. additional amount is in every milliliter of culture solution
Add 5~10 μ L100% methanol, carries out Fiber differentiation, 26~30 DEG C of 250~300r/min, 96~120h of shake culture, harvest training
Supernatant is supported, through filtering or preparation and reorganization thymosin α1-pig interferon alpha fusion protein after purification.
9. method as claimed in claim 6, it is characterised in that described in the fermenter with the step of methanol induction great expression
Rapid and condition are as follows: 1) glycerol culture expands thallus: fermentor 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 is expressed: according to 8
PTM1 culture medium is added in~12mL/L ratio in methyl alcohol, after mixing, is added with the rate of 2.4~3.6mL/h/L/ Preliminary fermentation liquid
Enter into fermentor inducing expression, so that yeast adapts to then add the rate liter of methanol using methanol as the environment of sole carbon source
For 4.8~7.2mL/h/L Preliminary fermentation liquid, the rate for adding methanol is finally improved to 10~12mL/h/L/ Preliminary fermentation liquid, is opened
It after beginning inducing expression, takes expression supernatant for analysis of protein daily, after 72~96h of inducing expression, terminates fermentation；In harvest culture
Clearly, through filtering or preparation and reorganization thymosin α1-pig interferon alpha fusion protein after purification.
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