CN105349578A - Chicken GM-CSF protein, and preparation method and application thereof - Google Patents
Chicken GM-CSF protein, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a preparation method for chicken GM-CSF protein. The preparation method comprises the following steps: a, synthesizing an optimized nucleotide sequence for encoding the chicken GM-CSF protein, wherein the nucleotide sequence is shown as SEQ ID No: 1; b, fabricating an expression vector by use of the SEQ ID No: 1, wherein the expression vector is pFastBac1-his-thrombin-GM-CSF; c, performing virus packaging on the expression vector obtained in the step b to obtain a virus stock; d, performing multiplication culture on the virus stock, infecting sf9 cells in the logarithmic phase, cultivating the infected sf9 cells for 24-72 h, collecting a supernatant in a culture medium, and performing centrifugation and chromatography treatment to obtain the chicken GM-CSF protein. The invention further provides the chicken GM-CSF protein prepared by the method and application of the chicken GM-CSF protein. The preparation method is simple, the protein expression level is high, magnification is easy to realize, and mass production is facilitated.
Description
Technical field
The present invention relates to genetically engineered field, be specifically related to a kind of chicken GM-CSF albumen and its preparation method and application.
Background technology
Cytokine is polypeptide or the albumen of the solubility that a class is secreted by active cells, by playing special biological activity with the acceptor interaction of cell surface.Cytokine has obvious immunological adjuvant effect, can the protective effect of enhanced virus vaccine, bacterial vaccine and parasite vaccine.Granulocyte-macrophage colony stimutaing factor (GM-CSF) under some antigen and Cytokine, stimulates by T cell, B cell, scavenger cell, endotheliocyte etc. the type cytokines with various biological activity produced.GM-CSF can not only hemopoietic cell proliferation, differentiation and maturation, and move from marrow to outer planet, and can also activated neutral granulocyte, scavenger cell and other monocytes, in cytokine network, occupy critical role, in immune response, there is vital role.GM-CSF, by promoting the proliferation and differentiation comprising the antigen presenting cell of dendritic cell, is attracted to injection site, enhancement antigen submission ability, thus reaches the effect strengthening immune effect.Therefore GM-CSF has very vast potential for future development as immunological adjuvant.
Dranoff etc. first using GM-CSF as immunological adjuvant, enhance the immune response of anti-tumor vaccine, be thus widely used as the immunological adjuvant of DNA vaccination, to overcome the defect of DNA vaccination immunogenicity difference.2004, Avery etc. screened the chicken GM-CSFcDNA with human GM-CSF homology from chicken cDNA library.Within 2008, Tan Bing has cloned the GM-CSF gene of Sichuan Mountain Dark-bone Chickens, and have expressed this albumen with coli expression carrier.Within 2011, Liu He Shan also utilizes coli expression carrier to have expressed GM-CSF gene.And utilize insect expression system to prepare the related basic research of chicken GM-CSF albumen, have no report both at home and abroad.Insect cell expression system has outstanding advantage, such as: expression level is high, be easy to amplify, the albumen produced with suitable posttranslational modification, and cell growth conditions is simple.Therefore potential advantages for development are had more compared with bacterium, yeast, mammalian cell expression system etc.
Infectious bursal disease (infectiousbursaldisease, IBD) be by infectious bursa of Fabricius virus (infectiousbursaldiseasevirus, IBDV) encroach on that the one that causes is acute, high degree in contact sexually transmitted disease, the fabricius bursa of main infringement chick and young chicken.This disease not only causes chicken death also to cause immunosuppression, is one of Infectious Diseases endangering world's aviculture at present.Along with the appearance of variant and highly virulent strain, the development speed of traditional vaccine has been difficult to the change of catching up with this disease.Although the recombinant vaccine prevention infectious bursal disease effect of single antigen VP2 is pretty good, also there is immune response strong not, immunogenicity is weak, the high relatively not defect of protection.
Summary of the invention
The object of the present invention is to provide the preparation method that a kind of protein expression level is high, be easy to amplify, be beneficial to the chicken GM-CSF albumen that scale operation needs, the application of the chicken GM-CSF albumen that the present invention also provides the method to obtain and this albumen.
For solving the problem, the technical solution adopted in the present invention is as follows:
A preparation method for chicken GM-CSF albumen, comprises the steps:
A. the nucleotide sequence of the coding chicken GM-CSF albumen of synthesis optimizing, the nucleotide sequence of the coding chicken GM-CSF albumen of described optimization is as shown in sequence SEQIDNo:1;
B. utilize the expression vector of sequence construct shown in SEQIDNo:1, described expression vector is: pFastBac1-his-thrombin-GM-CSF;
C. the expression vector obtained through b step is carried out virus packaging, obtain virus stocks;
D. the virus stocks obtained through step c is carried out amplification cultivation and (pass through amplification cultivation, virus titer can be improved), then collect the medium supernatant after amplification and infect the sf9 cell of logarithmic phase with the supernatant liquor collected, then metainfective sf9 cell 24-72h is cultivated in the medium, collect the supernatant in substratum, through centrifugal, Image processing, obtain chicken GM-CSF albumen.
In the present invention, preferred scheme is specially for synthesizing sequence shown in SEQIDNo:1 in described a step: carry out codon optimized with the gene order of insect cell preference codon to coding chicken GM-CSF albumen, to obtain final product; Described coding chicken GM-CSF albumen is the sequence being numbered NO:EU939770 that GeneBank announces.
In the present invention, preferred scheme is synthesized by chemical method for sequence shown in SEQIDNo:1 in described a step.
In the present invention, preferred scheme comprises the steps: for utilizing the expression vector of sequence construct shown in SEQIDNo:1 in described b step
1) by SEQIDNo:1 sequence clone on pUC57 vector plasmid, obtain pUC57-GM-CSF vector plasmid;
2) pUC57-GM-CSF vector plasmid is carried out pcr amplification, the primer pair wherein adopted in pcr amplification is:
Upstream primer 1:5 '-ATCACCTGGTGCCGCGCGGCAGCCTCGCTCAGCTCACTATCCTCCT-3 ';
Upstream primer 2:5 '-CGCGGATCCATGCATCACCATCACCATCACCTGGTGCCGCGCGGCAGCCTCG-3 ';
Downstream primer: 5 '-CCCAAGCTTTTAGATGCAATCTTTTTCTTCGGGC-3 ';
First with upstream primer 1 and downstream primer, first time pcr amplification is carried out to template pUC57-GM-CSF vector plasmid, again with upstream primer 2 and downstream primer to first time pcr amplification product carry out second time pcr amplification, described first time amplification adopts identical PCR reaction system and amplified reaction program with increasing for the second time:
In described pcr amplification, every 50 μ LPCR reaction systems comprise: 5 × Fastpfubuffer10 μ L, dNTPMix (2.5mM) 4 μ L, forward primer (10 μMs) 2 μ L, reverse primer (10 μMs) 2 μ L, Fastpfupolymerase1 μ L, template DNA (100ng/ μ L) 1 μ L, ddH
2o30 μ L;
Described pcr amplification reaction program is: 95 DEG C of 3min; 95 DEG C of 20s, 55 DEG C of 30s, 72 DEG C of 2min, 30 circulations; 72 DEG C of 5min;
3) by step 2) in amplification after product electrophoresis reclaim size be the band of 471bp, obtain goal gene; Wherein, the glue bed used by electrophoresis recovery is sepharose;
4) by pFastBac1 plasmid and step 3) in the goal gene collected carry out double digestion:
Endonuclease reaction is at 37 DEG C of water-bath 2h;
PFastBac1 plasmid enzyme restriction system is as follows: plasmid pFastBac11 μ g, 10 × Buffer2 μ l, 10 × BSA2 μ l, BamHI1 μ l, HindIII1 μ l, ddH2Oupto20 μ l;
It is as follows that goal gene enzyme cuts system: PCR reclaims product 14 μ l, 10 × Buffer2 μ l, 10 × BSA2 μ l, BamHI1 μ l, HindIII1 μ l;
Then, reclaim plasmid pFastBac1 enzyme respectively and cut large fragment and goal gene digestion products;
5) plasmid pFastBac1 enzyme step 4 be recovered to is cut large fragment and is connected with goal gene digestion products, and ligation, 22 DEG C of reactions 2 hours, obtains connecting product; Ligation system is as follows: goal gene digestion products 6 μ l, and pFastBac1 enzyme cuts large fragment 2 μ l, 10 × DNALigaseBuffer1 μ l, T4DNALigase1 μ l;
6) by step 5) the connection product that obtains and 100 μ lJM109 competence bacteriums mix after ice bath 30min, 42 DEG C of heat shock 45s, put immediately and place 2min on ice, add the 400 μ lLB substratum being preheated to room temperature, 37 DEG C of constant-temperature tables cultivate the centrifugal 1min of 1h, 4000rpm, discard 400 μ l culture supernatant, be spread evenly across on the LB flat board containing 100 μ g/mlAmpicillin resistances after remaining 100 μ l pipettor mixings, 37 DEG C of constant temperature culture carton upside down overnight incubation;
7) in step 6) the substratum after 37 DEG C of constant temperature culture carton upside down overnight incubation in picking 1 single colony inoculation in containing 5ml, in the LB nutrient solution of 100 μ g/mlAmpicillin resistances, 250rpm, 37 DEG C of constant-temperature table overnight incubation, with mini-scale plasmid extraction agent box extracting plasmid, utilize plasmid BamHI+HindIII to carry out double digestion qualification, then the correct recombinant clone of qualification is carried out sequence verification; Then, select correct pFastBac1-his-thrombin-GM-CSF vector plasmid and clone, for subsequent use.
In the present invention, preferred scheme is the expression vector in described step b is rhabdovirus expression vector.
In the present invention, preferred scheme is that the virus in described step c is packaged in sf9 insect cell and carries out.
In the present invention, preferred scheme is that described steps d specifically comprises:
Ι. the virus stocks obtained by step c is according to the ratio inoculation sf9 insect cell of MOI=0.05-0.1, and then cultivate 48h in the medium, collected by centrifugation substratum supernatant, carries out virus titer qualification;
II. by the step repeated infection sf9 cell in step I 3-4 time, improve virus titer, then by the ratio inoculation logarithmic phase sf9 insect cell of virus (through improving the recombinant virus after virus titer process) according to MOI=5-20, cultivate 24-72h in the medium, collected by centrifugation substratum supernatant;
III. get 1mLNi-NTA, with the BindingBuffer:(50mMTris (PH8.0) of 10 times of column volumes, 500mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine), cleaning balance pillar, flow velocity 2mL/min;
IV. by the supernatant liquor upper prop in step II, flow velocity is 1mL/min, collects and penetrates liquid;
The WashBuffer:(50mMTris (PH8.0) of V .10 times column volume, 500mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine, 20mM imidazoles) clean pillar, flow velocity 2mL/min;
VI .ElutionBuffer:(50mMTris, 300mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine, 75mM/250mM imidazoles, pH8.0) wash-out, flow velocity 1mL/min, collects elutriant, to obtain final product.
The present invention also provides the chicken GM-CSF obtained by aforesaid method albumen; This albumen may be used for preparing vaccine, as the preparation of infectious bursal disease DNA vaccination.
Compared with prior art, tool of the present invention has the following advantages:
1, the present invention is not limited to the method that traditional traditional tissue extraction DNA or RNA obtains goal gene, and can adopt the method for chemosynthesis chicken GM-CSF gene, gene chemical synthesis, without the need to template, does not thus limit by gene source, more economical, easy, time saving and energy saving;
2, the present invention is using baculovirus as expression vector, prepares chicken GM-CSF using insect cell as bio-reactor, compared with bacterium, yeast, eukaryotic cell expression system, because sf9 cell growth conditions is simple, and can without CO
2, serum-free, 27 DEG C of growths, therefore have and save the advantage that cost, output are high, can prepare on a large scale;
3, the chicken GM-CSF albumen that prepared by the present invention can be used as immunological adjuvant to be added in IBDVDNA vaccine, has security good, can improve the advantage of vaccine protection ratio.
Below in conjunction with the drawings and the specific embodiments, the present invention is described in detail.
Accompanying drawing explanation
Fig. 1 is the step 7 of the step b in embodiment 1) in double digestion qualification result figure;
Fig. 2 is the cell growth state figure in the substratum after the metainfective SF9 cell cultures 48h in the step Ι of steps d in embodiment 1;
Fig. 3 is the elutriant SDS-PAGE detected result figure of the steps d in embodiment 1;
Wherein, in Fig. 1, M is Trans2Kplusmarker; 1-5 is pFastBac1-his-thrombin-GM-CSFBamHI+HindIII double digestion qualification result;
In Fig. 3, M: albumen marker; 1#, 2# are chicken GM-CSF-his albumen.
Embodiment
A preparation method for chicken GM-CSF albumen, comprises the steps:
A. the nucleotide sequence of the coding chicken GM-CSF albumen of synthesis optimizing, the nucleotide sequence of the coding chicken GM-CSF albumen of described optimization is as shown in sequence SEQIDNo:1;
SEQIDNo:1：atgctcgctcagctcactatcctcctcgctctgggtgtcctctgctcccctgcccctactaccacctactcctgctgctacaaggtgtacaccatcctggaggagatcacctcccacctcgaatccaccgctgctactgccggtctcagcagcgtccccatggacatccgcgacaagacctgcctgcgtaataacctgaagaccttcattgagtccctgaagactaacggtaccgaggaggaatccggtatcgtctttcagctcaaccgtgtgcacgagtgtgagcgcctgtttagcaacatcactcccaccccccaagtccctgacaaggagtgtcgtactgcccaagtcagccgcgagaagttcgaagaagctctgaagacttttttcatctatctgtccgacgtcctgcccgaagaaaaagattgcatctaa；
B. utilize the expression vector of sequence construct shown in SEQIDNo:1, described expression vector is: pFastBac1-his-thrombin-GM-CSF;
C. the expression vector obtained through b step is carried out virus packaging, obtain virus stocks;
D. the virus stocks obtained through step c is carried out amplification cultivation, then collect the medium supernatant after amplification and infect the sf9 cell of logarithmic phase with the supernatant liquor collected, then metainfective sf9 cell 24-72h is cultivated in the medium, collect the supernatant in substratum, through centrifugal, Image processing, obtain chicken GM-CSF albumen.
In the present invention, preferred scheme is specially for synthesizing sequence shown in SEQIDNo:1 in described a step: carry out codon optimized with the gene order of insect cell preference codon to coding chicken GM-CSF albumen, to obtain final product; Described coding chicken GM-CSF albumen is the sequence being numbered NO:EU939770 that GeneBank announces.
In the present invention, preferred scheme is synthesized by chemical method for sequence shown in SEQIDNo:1 in described a step.
In the present invention, preferred scheme comprises the steps: for utilizing the expression vector of sequence construct shown in SEQIDNo:1 in described b step
1) by SEQIDNo:1 sequence clone on pUC57 vector plasmid, obtain pUC57-GM-CSF vector plasmid;
2) pUC57-GM-CSF vector plasmid is carried out pcr amplification, the primer pair wherein adopted in pcr amplification is:
Upstream primer 1:5 '-ATCACCTGGTGCCGCGCGGCAGCCTCGCTCAGCTCACTATCCTCCT-3 ';
Upstream primer 2:5 '-CGCGGATCCATGCATCACCATCACCATCACCTGGTGCCGCGCGGCAGCCTCG-3 ';
Downstream primer: 5 '-CCCAAGCTTTTAGATGCAATCTTTTTCTTCGGGC-3 ';
First with upstream primer 1 and downstream primer, first time pcr amplification is carried out to template pUC57-GM-CSF vector plasmid, again with upstream primer 2 and downstream primer to first time pcr amplification product carry out second time pcr amplification, described first time amplification adopts identical PCR reaction system and amplified reaction program with increasing for the second time:
In described pcr amplification, every 50 μ LPCR reaction systems comprise: 5 × Fastpfubuffer10 μ L, dNTPMix (2.5mM) 4 μ L, forward primer (10 μMs) 2 μ L, reverse primer (10 μMs) 2 μ L, Fastpfupolymerase1 μ L, template DNA (100ng/ μ L) 1 μ L, ddH
2o30 μ L;
Described pcr amplification reaction program is: 95 DEG C of 3min; 95 DEG C of 20s, 55 DEG C of 30s, 72 DEG C of 2min, 30 circulations; 72 DEG C of 5min;
3) by step 2) in amplification after product electrophoresis reclaim size be the band of 471bp, obtain goal gene; Wherein, the glue bed used by electrophoresis recovery is sepharose;
4) by pFastBac1 plasmid and step 3) in the goal gene collected carry out double digestion:
Endonuclease reaction is at 37 DEG C of water-bath 2h;
PFastBac1 plasmid enzyme restriction system is as follows: plasmid pFastBac11 μ g, 10 × Buffer2 μ l, 10 × BSA2 μ l, BamHI1 μ l, HindIII1 μ l, ddH2Oupto20 μ l;
It is as follows that goal gene enzyme cuts system: PCR reclaims product 14 μ l, 10 × Buffer2 μ l, 10 × BSA2 μ l, BamHI1 μ l, HindIII1 μ l;
Then, reclaim plasmid pFastBac1 enzyme respectively and cut large fragment and goal gene digestion products;
5) plasmid pFastBac1 enzyme step 4 be recovered to is cut large fragment and is connected with goal gene digestion products, and ligation, 22 DEG C of reactions 2 hours, obtains connecting product; Ligation system is as follows: goal gene digestion products 6 μ l, and pFastBac1 enzyme cuts large fragment 2 μ l, 10 × DNALigaseBuffer1 μ l, T4DNALigase1 μ l;
6) by step 5) the connection product that obtains and 100 μ lJM109 competence bacteriums mix after ice bath 30min, 42 DEG C of heat shock 45s, put immediately and place 2min on ice, add the 400 μ lLB substratum being preheated to room temperature, 37 DEG C of constant-temperature tables cultivate the centrifugal 1min of 1h, 4000rpm, discard 400 μ l culture supernatant, be spread evenly across on the LB flat board containing 100 μ g/mlAmpicillin resistances after remaining 100 μ l pipettor mixings, 37 DEG C of constant temperature culture carton upside down overnight incubation;
7) in step 6) the substratum after 37 DEG C of constant temperature culture carton upside down overnight incubation in picking 1 single colony inoculation in containing 5ml, in the LB nutrient solution of 100 μ g/mlAmpicillin resistances, 250rpm, 37 DEG C of constant-temperature table overnight incubation, with mini-scale plasmid extraction agent box extracting plasmid, utilize plasmid BamHI+HindIII to carry out double digestion qualification, then the correct recombinant clone of qualification is carried out sequence verification; Then, select correct pFastBac1-his-thrombin-GM-CSF vector plasmid and clone, for subsequent use.
In the present invention, preferred scheme is the expression vector in described step b is rhabdovirus expression vector.
In the present invention, preferred scheme is that the virus in described step c is packaged in sf9 insect cell and carries out.
In the present invention, preferred scheme is that described steps d specifically comprises:
Ι. the virus stocks obtained by step c is according to the ratio inoculation sf9 insect cell of MOI=0.05-0.1, and then cultivate 48h in the medium, collected by centrifugation substratum supernatant, carries out virus titer qualification;
II. by the step repeated infection sf9 cell in step I 3-4 time, improve virus titer, then by the ratio inoculation logarithmic phase sf9 insect cell of high titre recombinant virus according to MOI=5-20, cultivate 24-72h in the medium, collected by centrifugation substratum supernatant;
III. get 1mLNi-NTA, with the BindingBuffer:(50mMTris (PH8.0) of 10 times of column volumes, 500mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine), cleaning balance pillar, flow velocity 2mL/min;
IV. by the supernatant liquor upper prop in step II, flow velocity is 1mL/min, collects and penetrates liquid;
The WashBuffer:(50mMTris (PH8.0) of V .10 times column volume, 500mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine, 20mM imidazoles) clean pillar, flow velocity 2mL/min;
VI .ElutionBuffer:(50mMTris, 300mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine, 75mM/250mM imidazoles, pH8.0) wash-out, flow velocity 1mL/min, collects elutriant, to obtain final product.
The present invention also provides the chicken GM-CSF obtained by aforesaid method albumen; This albumen may be used for preparing vaccine, as the preparation of infectious bursal disease DNA vaccination.
Embodiment 1
A preparation method for chicken GM-CSF albumen, comprises the steps:
A. the nucleotide sequence of the coding chicken GM-CSF albumen of synthesis optimizing, the nucleotide sequence of the coding chicken GM-CSF albumen of described optimization is as shown in sequence SEQIDNo:1;
Synthesize sequence shown in SEQIDNo:1 in described a step to be specially: carry out codon optimized with the gene order of insect cell preference codon to coding chicken GM-CSF albumen, to obtain final product; Described coding chicken GM-CSF albumen is the sequence being numbered NO:EU939770 that GeneBank announces; Or by sequence shown in chemical process synthesis SEQIDNo:1;
B. utilize the expression vector of sequence construct shown in SEQIDNo:1, described expression vector is: pFastBac1-his-thrombin-GM-CSF; Described expression vector is rhabdovirus expression vector; Specifically comprise the steps:
1) by SEQIDNo:1 sequence clone on pUC57 vector plasmid, obtain pUC57-GM-CSF vector plasmid;
2) pUC57-GM-CSF vector plasmid is carried out pcr amplification, the primer pair wherein adopted in pcr amplification is:
Upstream primer 1:5 '-ATCACCTGGTGCCGCGCGGCAGCCTCGCTCAGCTCACTATCCTCCT-3 ';
Upstream primer 2:5 '-CGCGGATCCATGCATCACCATCACCATCACCTGGTGCCGCGCGGCAGCCTCG-3 ';
Downstream primer: 5 '-CCCAAGCTTTTAGATGCAATCTTTTTCTTCGGGC-3 ';
First with upstream primer 1 and downstream primer, first time pcr amplification is carried out to template pUC57-GM-CSF vector plasmid, again with upstream primer 2 and downstream primer to first time pcr amplification product carry out second time pcr amplification, described first time amplification adopts identical PCR reaction system and amplified reaction program with increasing for the second time:
In described pcr amplification, every 50 μ LPCR reaction systems comprise: 5 × Fastpfubuffer10 μ L, dNTPMix (2.5mM) 4 μ L, forward primer (10 μMs) 2 μ L, reverse primer (10 μMs) 2 μ L, Fastpfupolymerase1 μ L, template DNA (100ng/ μ L) 1 μ L, ddH
2o30 μ L;
Described pcr amplification reaction program is: 95 DEG C of 3min; 95 DEG C of 20s, 55 DEG C of 30s, 72 DEG C of 2min, 30 circulations; 72 DEG C of 5min;
3) by step 2) in amplification after product electrophoresis reclaim size be the band of 471bp, obtain goal gene; Wherein, the glue bed used by electrophoresis recovery is sepharose;
4) by pFastBac1 plasmid and step 3) in the goal gene collected carry out double digestion:
Endonuclease reaction is at 37 DEG C of water-bath 2h;
PFastBac1 plasmid enzyme restriction system is as follows: plasmid pFastBac11 μ g, 10 × Buffer2 μ l, 10 × BSA2 μ l, BamHI1 μ l, HindIII1 μ l, ddH2Oupto20 μ l;
It is as follows that goal gene enzyme cuts system: PCR reclaims product 14 μ l, 10 × Buffer2 μ l, 10 × BSA2 μ l, BamHI1 μ l, HindIII1 μ l;
Then, reclaim plasmid pFastBac1 enzyme respectively and cut large fragment and goal gene digestion products;
5) plasmid pFastBac1 enzyme step 4 be recovered to is cut large fragment and is connected with goal gene digestion products, and ligation, 22 DEG C of reactions 2 hours, obtains connecting product; Ligation system is as follows: goal gene digestion products 6 μ l, and pFastBac1 enzyme cuts large fragment 2 μ l, 10 × DNALigaseBuffer1 μ l, T4DNALigase1 μ l;
6) by step 5) the connection product that obtains and 100 μ lJM109 competence bacteriums mix after ice bath 30min, 42 DEG C of heat shock 45s, put immediately and place 2min on ice, add the 400 μ lLB substratum being preheated to room temperature, 37 DEG C of constant-temperature tables cultivate the centrifugal 1min of 1h, 4000rpm, discard 400 μ l culture supernatant, be spread evenly across on the LB flat board containing 100 μ g/mlAmpicillin resistances after remaining 100 μ l pipettor mixings, 37 DEG C of constant temperature culture carton upside down overnight incubation;
7) in step 6) the substratum after 37 DEG C of constant temperature culture carton upside down overnight incubation in picking 1 single colony inoculation in containing 5ml, in the LB nutrient solution of 100 μ g/mlAmpicillin resistances, 250rpm, 37 DEG C of constant-temperature table overnight incubation, with mini-scale plasmid extraction agent box extracting plasmid, utilize plasmid BamHI+HindIII to carry out double digestion qualification (as shown in Figure 1), then the correct recombinant clone of qualification is carried out sequence verification; Then, select correct pFastBac1-his-thrombin-GM-CSF vector plasmid and clone, for subsequent use;
C. the expression vector obtained through b step is carried out virus packaging, obtain virus stocks; Virus is packaged in sf9 insect cell carries out; Specifically comprise the steps:
A inoculates 8 × 10 in six orifice plates
5individual cell/2ml/ hole.Wherein adding FBS in SF-II 900SFM substratum makes its final concentration be 2%.Cultivate 1h, make cell attachment for 27 DEG C.
B prepares pFastbac1-his-thrombin-GM-CSF and CellfectinReagent mixture
A). with 100ulSF-II 900SFM substratum dilution 1ugBacmid.
B). before use CellfectinReagent is inverted 5-10 time, makes it fully mix, get the dilution of 6ulCellfectinReagent 100ulSF-II 900SFM substratum.
C). above-mentioned two kinds of diluents are merged (cumulative volume is about 210ul), mixes gently, incubated at room 15-45min.
During C prepares Bacmid and CellfectinReagent mixture, the substratum in six orifice plates is sopped up, wash once with 2mlSF-II 900SFM substratum, remove substratum.
The SF-II 900SFM substratum that D adds 800ul in the pipe of the mixture containing 210ul mixes gently, is added in each hole.
Cell in six orifice plates is hatched 5h by E in 27 DEG C of thermostat containers.
F removes mixture mixed solution, and every hole adds 2ml perfect medium (SF-II 900SFM substratum, containing dual anti-, 10%FBS)
G in 27 DEG C of humidified incubator, hatch 48h or until virus infection sign appears in cell.
H is when cell occurs infecting sign, and transferred to by upper strata substratum (about 2ml) in aseptic EP pipe with cover, the centrifugal 5min of 500g is to remove cell and large fragment.With the membrane filtration of the low 0.2um of protein binding rate.
Supernatant liquor containing virus is transferred in another aseptic EP pipe with cover by I, and the viral liquid lucifuge obtained is placed in 4 DEG C of refrigerators;
The virus stocks obtained through step c is carried out amplification cultivation by d, then the substratum supernatant after amplification is collected also and infect the sf9 cell of logarithmic phase with the supernatant liquor collected, then metainfective sf9 cell 24-72h is cultivated in the medium, collect the supernatant in substratum, through centrifugal, Image processing, obtain chicken GM-CSF albumen; Specifically comprise:
Ι. the virus stocks obtained by step c, according to the ratio inoculation sf9 insect cell of MOI=0.05-0.1, then cultivates 48h in the medium; Collected by centrifugation substratum supernatant, carries out virus titer qualification; Particularly, the same day is infected, at 25cm
25 × 10 are inoculated in Tissue Culture Flask
6individual cell.Wherein adding FBS in SF-II 900SFM substratum makes its final concentration be 2%.Cultivate 1h, make cell attachment, add appropriate viral MOI=0.05 for 27 DEG C, cultivate 48h (as shown in Figure 2) for 27 DEG C;
II. by the step repeated infection sf9 cell in step I 3-4 time, improve virus titer, then by the ratio inoculation logarithmic phase sf9 insect cell of high titre recombinant virus according to MOI=5-20, cultivate 24-72h in the medium, collected by centrifugation substratum supernatant;
III. get 1mLNi-NTA, with the BindingBuffer:(50mMTris (PH8.0) of 10 times of column volumes, 500mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine), cleaning balance pillar, flow velocity 2mL/min;
IV. by the supernatant liquor upper prop in step II, flow velocity is 1mL/min, collects and penetrates liquid;
The WashBuffer:(50mMTris (PH8.0) of V .10 times column volume, 500mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine, 20mM imidazoles) clean pillar, flow velocity 2mL/min;
VI .ElutionBuffer:(50mMTris, 300mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine, 75mM/250mM imidazoles, pH8.0) wash-out, flow velocity 1mL/min, collects elutriant, to obtain final product.
Detected by above-mentioned elutriant SDS-PAGE, detected result refers to Fig. 3.
Experimentation on animals
100 14 day age SPF chicken be divided into 5 groups at random, often organize 20, experimental group is GM-CSF albumen 300ug+1 plumage part IBDVVP2DNA vaccine that the present invention obtains; control group 1 is 1 plumage part IBDVVP2DNA vaccine, and control group 2 is VP2 empty carrier group, and control group 3 is PBS group; 14 day age, head exempted from; after 2 weeks, two exempt from, and observe dead and fabricius bursa pathology after attacking strong malicious BC6-85,96h after 7 days; calculate protection ratio; separately establish negative control group, injection PBS, but do not attack poison.Experimental result shows, the protection ratio of IBDVDNA vaccine can be brought up to 77% by 64% by chicken GM-CSF albumen
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.
Claims (10)
1. a preparation method for chicken GM-CSF albumen, is characterized in that comprising the steps:
A. the nucleotide sequence of the coding chicken GM-CSF albumen of synthesis optimizing, the nucleotide sequence of the coding chicken GM-CSF albumen of described optimization is as shown in sequence SEQIDNo:1;
B. utilize the expression vector of sequence construct shown in SEQIDNo:1, described expression vector is: pFastBac1-his-thrombin-GM-CSF;
C. the expression vector obtained through b step is carried out virus packaging, obtain virus stocks;
D. the virus stocks obtained through step c is carried out amplification cultivation, then collect the medium supernatant after amplification and infect the sf9 cell of logarithmic phase with the supernatant liquor collected, then metainfective sf9 cell 24-72h is cultivated in the medium, collect the supernatant in substratum, through centrifugal, Image processing, obtain chicken GM-CSF albumen.
2. the preparation method of chicken GM-CSF albumen according to claim 1, it is characterized in that synthesizing sequence shown in SEQIDNo:1 in described a step is specially: carry out codon optimized with the gene order of insect cell preference codon to coding chicken GM-CSF albumen, to obtain final product; Described coding chicken GM-CSF albumen is the sequence being numbered NO:EU939770 that GeneBank announces.
3. the preparation method of chicken GM-CSF albumen according to claim 1, is characterized in that: in described a step, shown in SEQIDNo:1, sequence is synthesized by chemical method.
4. the preparation method of chicken GM-CSF albumen according to claim 1, is characterized in that utilizing the expression vector of sequence construct shown in SEQIDNo:1 to comprise the steps: in described b step
1) by SEQIDNo:1 sequence clone on pUC57 vector plasmid, obtain pUC57-GM-CSF vector plasmid;
2) pUC57-GM-CSF vector plasmid is carried out pcr amplification, the primer pair wherein adopted in pcr amplification is:
Upstream primer 1:5 '-ATCACCTGGTGCCGCGCGGCAGCCTCGCTCAGCTCACTATCCTCCT-3 ';
Upstream primer 2:5 '-CGCGGATCCATGCATCACCATCACCATCACCTGGTGCCGCGCGGCAGCCTCG-3 ';
Downstream primer: 5 '-CCCAAGCTTTTAGATGCAATCTTTTTCTTCGGGC-3 ';
First with upstream primer 1 and downstream primer, first time pcr amplification is carried out to template pUC57-GM-CSF vector plasmid, again with upstream primer 2 and downstream primer to first time pcr amplification product carry out second time pcr amplification, described first time amplification adopts identical PCR reaction system and amplified reaction program with increasing for the second time:
In pcr amplification, every 50 μ LPCR reaction systems comprise: 5 × Fastpfubuffer10 μ L, dNTPMix (2.5mM) 4 μ L, forward primer (10 μMs) 2 μ L, reverse primer (10 μMs) 2 μ L, Fastpfupolymerase1 μ L, template DNA (100ng/ μ L) 1 μ L, ddH
2o30 μ L;
Pcr amplification reaction program is: 95 DEG C of 3min; 95 DEG C of 20s, 55 DEG C of 30s, 72 DEG C of 2min, 30 circulations; 72 DEG C of 5min;
3) by step 2) in amplification after product electrophoresis reclaim size be the band of 471bp, obtain goal gene; Wherein, the glue bed used by electrophoresis recovery is sepharose;
4) by pFastBac1 plasmid and step 3) in the goal gene collected carry out double digestion:
Endonuclease reaction is at 37 DEG C of water-bath 2h;
PFastBac1 plasmid enzyme restriction system is as follows: plasmid pFastBac11 μ g, 10 × Buffer2 μ l, 10 × BSA2 μ l, BamHI1 μ l, HindIII1 μ l, ddH2Oupto20 μ l;
It is as follows that goal gene enzyme cuts system: PCR reclaims product 14 μ l, 10 × Buffer2 μ l, 10 × BSA2 μ l, BamHI1 μ l, HindIII1 μ l;
Then, reclaim plasmid pFastBac1 enzyme respectively and cut large fragment and goal gene digestion products;
5) plasmid pFastBac1 enzyme step 4 be recovered to is cut large fragment and is connected with goal gene digestion products, and ligation, 22 DEG C of reactions 2 hours, obtains connecting product; Ligation system is as follows: goal gene digestion products 6 μ l, and pFastBac1 enzyme cuts large fragment 2 μ l, 10 × DNALigaseBuffer1 μ l, T4DNALigase1 μ l;
6) by step 5) the connection product that obtains and 100 μ lJM109 competence bacteriums mix after ice bath 30min, 42 DEG C of heat shock 45s, put immediately and place 2min on ice, add the 400 μ lLB substratum being preheated to room temperature, 37 DEG C of constant-temperature tables cultivate the centrifugal 1min of 1h, 4000rpm, discard 400 μ l culture supernatant, be spread evenly across on the LB flat board containing 100 μ g/mlAmpicillin resistances after remaining 100 μ l pipettor mixings, 37 DEG C of constant temperature culture carton upside down overnight incubation;
7) in step 6) the substratum after 37 DEG C of constant temperature culture carton upside down overnight incubation in picking 1 single colony inoculation in containing 5ml, in the LB nutrient solution of 100 μ g/mlAmpicillin resistances, 250rpm, 37 DEG C of constant-temperature table overnight incubation, with mini-scale plasmid extraction agent box extracting plasmid, utilize plasmid BamHI+HindIII to carry out double digestion qualification, then the correct recombinant clone of qualification is carried out sequence verification; Then, select correct pFastBac1-his-thrombin-GM-CSF vector plasmid and clone, for subsequent use.
5. the preparation method of chicken GM-CSF albumen according to claim 1, is characterized in that: the expression vector in described step b is rhabdovirus expression vector.
6. the preparation method of chicken GM-CSF albumen according to claim 1, is characterized in that: the virus in described step c is packaged in sf9 insect cell carries out.
7. the preparation method of chicken GM-CSF albumen according to claim 1, is characterized in that described steps d specifically comprises:
Ι. the virus stocks obtained by step c is according to the ratio inoculation sf9 insect cell of MOI=0.05-0.1, and then cultivate 48h in the medium, collected by centrifugation substratum supernatant, carries out virus titer qualification;
II. by the step repeated infection sf9 cell in step I 3-4 time, improve virus titer, then just virus, according to the ratio inoculation logarithmic phase sf9 insect cell of MOI=5-20, cultivates 24-72h, collected by centrifugation substratum supernatant in the medium;
III. get 1mLNi-NTA, with the BindingBuffer:(50mMTris (PH8.0) of 10 times of column volumes, 500mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine), cleaning balance pillar, flow velocity 2mL/min;
IV. by the supernatant liquor upper prop in step II, flow velocity is 1mL/min, collects and penetrates liquid;
The WashBuffer:(50mMTris (PH8.0) of V .10 times column volume, 500mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine, 20mM imidazoles) clean pillar, flow velocity 2mL/min;
VI .ElutionBuffer:(50mMTris, 300mMNaCl, 0.1%TritonX-100,1mMDTT, 5% glycerine, 75mM/250mM imidazoles, pH8.0) wash-out, flow velocity 1mL/min, collects elutriant, to obtain final product.
8. the chicken GM-CSF albumen obtained according to the method for any one of claim 1-7.
9. chicken GM-CSF albumen according to claim 8, it is preparing the application in vaccine.
10. chicken GM-CSF albumen according to claim 8, it is preparing the application in infectious bursal disease DNA vaccination.
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Cited By (4)
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| CN106177993A (en) * | 2016-07-06 | 2016-12-07 | 华南农业大学 | Infectious bursal disease virus DNA vaccination and construction method thereof |
| CN107188951A (en) * | 2017-05-16 | 2017-09-22 | 浙江海隆生物科技有限公司 | The preparation method of pig GM csf proteins and its parenteral solution and application |
| WO2017181920A1 (en) * | 2016-04-18 | 2017-10-26 | 江苏恒瑞医药股份有限公司 | Method for preparing recombinant human granulocyte colony-stimulating factor |
| CN110272478A (en) * | 2019-04-18 | 2019-09-24 | 肇庆大华农生物药品有限公司 | One breeder GM-CSF albumen, its monoclonal antibody, preparation method and application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017181920A1 (en) * | 2016-04-18 | 2017-10-26 | 江苏恒瑞医药股份有限公司 | Method for preparing recombinant human granulocyte colony-stimulating factor |
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| CN107188951A (en) * | 2017-05-16 | 2017-09-22 | 浙江海隆生物科技有限公司 | The preparation method of pig GM csf proteins and its parenteral solution and application |
| CN110272478A (en) * | 2019-04-18 | 2019-09-24 | 肇庆大华农生物药品有限公司 | One breeder GM-CSF albumen, its monoclonal antibody, preparation method and application |
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