CN101870733A - Poultry IL-2 and newcastle disease virus HN gene recombination fusion protein and application thereof - Google Patents
Poultry IL-2 and newcastle disease virus HN gene recombination fusion protein and application thereof Download PDFInfo
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Abstract
The invention relates to a preparation method and application of fusion protein (chIL-2-HN fusion protein) of recombining poultry interleukin-2 (IL-2) and newcastle disease virus hemagglutinin-neuraminidase (HN), belonging to the technical field of biological engineering. The fusion protein is fused by poultry IL-2 protein and newcastle disease virus HN protein by flexible Linker peptide; a DNA sequence for coding the fusion protein is inserted into an expression vector pPICZ alpha A; saccharomycete X-33 is electrically converted to obtain a genetically engineered microorganism for efficiently expressing recombination chIL-2-HN fusion protein which is prepared by liquid culture and purification; the recombination fusion protein can serve as the novel genetic engineering vaccine and can serve as a novel immunologic adjuvant for newcastle disease common vaccine. The chIL-2-HN fusion protein of the invention has favourable safety and no toxic or side effect, which is verified by experiments of animal immunoassay. In addition, the chIL-2-HN fusion protein can effectively strengthen the level of organism cellular immunity and humoral immunity and has wide application prospect.
Description
Technical field
The present invention relates to relate to fowl leukocyte Jie element-2(Interleukin-2, IL-2) with the newcastle disease virus HN fusion rotein, and the dna sequence dna of this fusion rotein of coding and carrier and the host cell that contains this dna sequence dna, also relate to recombination fusion protein and application thereof, belong to the technical field that the genetically engineered in the biological-pharmacy is produced vaccine and immunological adjuvant.
Background technology
Newcastle disease (Newcastledisease, ND) be by Avian pneumo-encephalitis virus (Newcastlediseasevirus, NDV) cause bird a kind of with expiratory dyspnea, the yellow-green colour of having loose bowels just, nervous function disorder and mucous membrane, serous coat is hemorrhage is height contact, the acute septic transmissible disease of principal character.Classified as one of maximum category-A transmissible disease of animal harm by International Office of Epizootics, caused the tremendous economic loss to aviculture.Newcastle disease is the No.1 disease that threatens aviculture because the ND M ﹠ M is all very high, the control success or not of this disease has directly been determined the development trend of aviculture.
The HN albumen of NDV-HN genes encoding is being brought into play important effect in the NDV pathogenic course.HN albumen is hemagglutinin-neuraminic acid zymoprotein, is NDV another kind of bigger glycoprotein except that F glycoprotein, also is the main host protective antigen of virus.HN has HA and two kinds of activity of NA, and the one, the adherent cell surface contains sialic acceptor; Another kind is by NA catalytic pyrolysis sialic acid acceptor, and these two kinds of activity play the vital role of recognizing cells acceptor, mediation virus absorption onto cell film in the virus infection cell processes.HN albumen is to induce body to produce the main diseases toxalbumin of neutrality antibody; in the body anti-infectious immunity, play an important role; HN albumen with gene engineering expression has good immunogenicity; can produce the specific antibody of higher level behind the immunity chicken, strong virus attack is had good provide protection.
The conventional vaccine that uses mainly contains the attenuated vaccine and the oil emulsion inactivated vaccine of various different virulence in the newcastle disease immunoprophylaxis.But the denominator of traditional newcastle disease inactivated vaccine is to induce the intensive humoral immunoresponse(HI), and cell immune response is then weak relatively.In the anti-infectious immunity of newcastle disease, antibody response is essential for the infection of body opposing virus, but can not stop the propagation of virus.Thereby; in recent years along with the develop rapidly of Protocols in Molecular Biology; the development of NDV recombinant vaccine has become the focus of people research, but also there are some parts not fully up to expectations in recombinant vaccine aspect the organism immune response inducing, and the protection of animal experiment effect is not ideal enough.Therefore, the investigator attempts to strengthen from many aspects the immune effect of NDV vaccine, wherein uses cytokine as the molecular immune adjuvant, has caused investigator's concern.
Interleukin II (Interleukin-2, IL-2) be a kind of lymphokine by the T lymphocytic emiocytosis, can promote the propagation and the differentiation of T cell, B cell, and can strengthen the killing activity of monocyte and NK cell, in the immune response process, play important regulatory role.Discover that IL-2 can specificity induce the body cell immunity, non-specific action is in humoral immunity of organism, mix use with antigenic substance, can make body produce immunizing power in advance, reduce stress reaction, reducing the immunity infringement, prolong potent antibodies and hold time, is a kind of natural immunostimulant.
Summary of the invention
The object of the present invention is to provide a kind of chIL-2-HN fusion rotein with good immune effect.
The present invention also aims to provide a kind of dna sequence dna of this fusion rotein of encoding.
The present invention also aims to provide a kind of carrier and host cell that contains this dna sequence dna.
The present invention also aims to provide a kind of is the preparation method of the genetically engineered chIL-2-HN fusion rotein of host cell with the Pichia yeast.
In addition, the present invention also aims to provide the application of a kind of this fusion rotein in the newcastle disease control.
To achieve these goals, technical scheme of the present invention has adopted a kind of chIL-2-HN fusion rotein, comprises the fowl leukocyte plain 2(chIL-2 that is situated between) aminoacid sequence, Avian pneumo-encephalitis virus hemagglutinin-neuraminidase (HN albumen) aminoacid sequence and in be situated between flexible Linker peptide ammino acid sequence between plain 2 aminoacid sequences and the proteic aminoacid sequence of newcastle disease virus HN of fowl leukocyte; By the fowl leukocyte plain 2(chIL-2 that is situated between) gene and newcastle disease virus HN gene be connected in series by flexible Linker peptide (G-G-G-G-S) and form.
This albumen has the aminoacid sequence shown in the SEQIDNO.7.
Described flexible Linker peptide has the aminoacid sequence shown in the SEQIDNO.6.
Simultaneously, technical scheme of the present invention has also adopted a kind of separated DNA sequence, and coding has the fusion rotein that sequence is the aminoacid sequence shown in the SEQIDNO.7.
This dna sequence dna has the nucleotide sequence shown in the SEQIDNO.5.
Technical scheme of the present invention has also adopted a kind of recombinant yeast pichia pastoris carrier and host cell, and it contains the dna sequence dna with the nucleotide sequence shown in the SEQIDNO.5.
The application of recombination fusion protein in newcastle disease prevention and treatment.
Particularly: chIL-2-HN fusion rotein of the present invention and dna sequence dna thereof are as follows:
The chIL-2-HN antigen-4 fusion protein gene is by flexible Linker(G-G-G-G-S by fowl IL-2 gene and newcastle disease virus HN gene) gene is in series and is connected, and the coded aminoacid sequence of this series connection dna sequence dna is the chIL-2-HN fusion rotein.On the one hand, newcastle disease virus HN albumen can induce body to produce high-caliber specific antibody, and strong virus attack is had good provide protection.Simultaneously, interleukin II can promote the propagation and the differentiation of T cell, B cell, can specificity induce the body cell immunity.The two connects by flexible amino acid peptide, can effectively bring into play biological function separately when keeping space structure independently.
The present invention has also adopted a kind of construction process that can efficiently express the genetic engineering bacterium of chIL-2-HN fusion rotein:
Be the low chIL-2-HN fusion rotein of production cost, must make up a kind of genetic engineering bacterium that can produce the chIL-2-HN fusion rotein, the genetic engineering bacterium that the present invention selects for use is a pichia spp X-33 bacterium, the pichia spp X-33 bacterial strain of reorganization, be to pass through homologous recombination technique, the gene integration of chIL-2-HN fusion rotein in pichia spp X-33 bacterium genome, is therefore carried the gene of chIL-2-HN fusion rotein in the pichia spp X-33 strain gene group of reorganization.
The present invention has also adopted a kind of preparation method of soluble recombining chIL-2-HN fusion rotein:
Select pichia yeast expression system for use, in the BMMY substratum, the pichia spp X-33 bacterium of reorganization can efficiently express the chIL-2-HN fusion rotein under methanol induction; Collect the nutrient solution supernatant behind Ni post affinitive layer purification, collect and penetrate the peak, lyophilize can obtain the reorganization chIL-2-HN fusion rotein of based on very high purity.
The present invention chIL-2-HN fusion protein immunization chicken of will recombinating.By mensuration and the animal of IL-4 and IFN-γ in the NDV-HN antibody of immune chicken and antibody subtype, spleen lymphocyte proliferation, the serum are attacked poison protection experiment.Estimate the immunological characteristic of reorganization chIL-2-HN.
The preparation method of reorganization chIL-2-HN fusion rotein of the present invention, its production may further comprise the steps:
(a) gene order of acquisition coding chIL-2-HN fusion rotein;
1. obtain fowl IL-2 gene order:
The nucleotide sequence (number of including: AY029588), utilize DNAStar, 2 primer fragments of primerpremier software design F according to fowl IL-2 among the GenBank
1, F
2Amplification fowl IL-2 gene.Primer is synthetic by Dalian Bao Bio-Engineering Company.Primers F 15 ' end is introduced
KpnThe I restriction enzyme site, F25 ' end is introduced flexible joint.
Primer sequence is as follows:
F1:SEQIDNO.1；
5’-CGGGTACCATGTGCAAAGTACTGATCTTTGGC-3’
F2:SEQIDNO.2；
5’-GCTGCCGCCGCCGCCTTTTTGCAGATATCTCAC-3’
With NDV-I is that vaccine is done 50 times of dilutions, is inoculated in instar chicken embryo on the 10th by allantoic cavity, and 0.2mL/ piece, 37 ℃ are continued hatching.48-72h takes out not dead chicken embryo in the inoculation back, gets the aseptic grinding of spleen, and carries out extraction and the amplification of total mRNA by the requirement of test kit immediately.Total mRNA is a template with chicken embryo spleen, RT-PCR amplification chicken IL-2 gene.By the requirement of RT-PCR test kit, in 50 μ L reaction systems, add 10 * buffer5 μ L respectively, MgCl210 μ L, dNTP mixture 5 μ L, RNA enzyme inhibitors 1 μ L, ThermoScript II 1 μ L, Taq enzyme 1 μ L, each 2 μ L of upstream and downstream primers F 1 and F2, template 5 μ L, no RNA enzyme water 18 μ L.50 ℃ of 30min reverse transcriptions, 94 ℃ of 2min deactivation ThermoScript II.Adopt touchdown PCR to increase, parameter is: 94 ℃ of 50s, and 58 ℃ of 45s, 72 ℃ of 100s circulate 6 times, 94 ℃ of 50s, 56 ℃ of 50s, 72 ℃ of 100s circulate 27 times, and 72 ℃ are extended 10min.(EthidiumBromide after EB) 1% agarose gel electrophoresis is identified, downcuts the purpose band to amplification PCR products, reclaims by glue recovery test kit (Dalian TaKaRa company) operation instruction subsequently and is fowl IL-2 gene fragment through containing ethidium bromide;
2. obtain the newcastle disease virus HN gene
The nucleotide sequence (number of including: GU573799.1), utilize DNAStar, 2 primer fragments of primerpremier software design F according to newcastle disease virus HN gene among the GenBank
3, F
4Amplification newcastle disease virus HN gene, primers F 35 ' end is introduced flexible joint, and F45 ' holds introducing
NotThe I restriction enzyme site.Primer sequence is as follows:
F3:SEQ.ID.NO.3；
5’-GGCGGCGGCGGCAGCATGGACCGTGTAGTTAGC-3’
F4:SEQ.ID.NO.4；
5’-TAGCGGCCGCAACTCTATCATCCTTGAGGATCTCAAC-3’
Frozen NDV strain after melting, 37 ℃ of water-baths is inoculated the non-immune chicken embryos of 9～10 ages in days (each embryo 0.2mL), 37 ℃ of hatchings by the allantoic cavity approach.Chicken embryo dead in the 24h discards, and the allantoic fluid of the dead chicken embryo of aseptic collection 24～48h (having red corpuscle or yolk to make allantoic fluid become muddy chicken blastochyle discards) behind multigelation 3 times, 8000r/min4 ℃ centrifugal 15min, is got supernatant liquor, and 20 ℃ of preservations are stand-by.The extraction of viral RNA is extracted the test kit explanation according to the precious biotechnology RNA of company limited in Dalian and is operated.Total RNA4 μ L with extraction is a template, adds MgCl
23 μ L10 * reverse transcription buffer2.5 μ L, dNTPMixture (10mmol/L) 2.5 μ L, RnaseInhititor0.5 μ L (20U), RT enzyme 0.5 μ L, Taq enzyme 0.5 μ L, primers F 31 μ L, primers F 41 μ L, sterilization dH
2O9.5 μ L, total system 25 μ L.Carry out the RT-PCR reaction by following reaction conditions: 50 ℃ of 40s, 94 ℃ of 2min, 94 ℃ of 60s, 55 ℃ of 60s, 72 ℃ 2min11 circulation; 94 ℃ of 60s, 53 ℃ of 60s, 72 ℃ 2min12 circulation; 94 ℃ of 60s, 51 ℃ of 60s, 72 ℃ 2min10 circulation; 72 ℃ of 10min.The PCR product downcuts the purpose band after containing the evaluation of ethidium bromide 1% agarose gel electrophoresis, reclaim by glue recovery test kit operation instruction subsequently and be the newcastle disease virus HN gene;
The gene order of 3. overlapping pcr amplification chIL-2-HN fusion rotein
With above-mentioned fowl IL-2 gene and the newcastle disease virus HN gene that 1. 2. obtains is template, utilizes F1 and F4 primer, by overlapping PCR method amplification chIL-2-HN fusion gene.
The PCR reaction conditions: 94 ℃ of pre-sex change 2min enter PCR circulation: 94 ℃ of 30s, and annealing temperature is from 55 ℃, every circulation 1min, totally 30 circulations; 72 ℃ are extended 10min, and the PCR product downcuts the purpose band after containing the evaluation of ethidium bromide 1% agarose gel electrophoresis, reclaim by glue recovery test kit operation instruction subsequently and are the chIL-2-HN fusion gene;
(b) gene order of the chIL-2-HN fusion rotein that step (a) is obtained is inserted among the pichia vector pPICZ α A, obtains recombinant expressed pichia vector, called after pPICZ α A-chIL-2-HN;
(c) efficiently express the structure of the engineering strain of chIL-2-HN fusion rotein
Above-mentioned chIL-2-HN fusion gene PCR product and yeast expression vector pPICZ α A are all used
KpnI,
NotThe I double digestion, T4DNALigase connects, and connects product Transformed E .coliDH5 α, and recombinant expression plasmid carries out
KpnI and
NotThe I double digestion is identified; Enzyme is cut with PCR and is accredited as the order-checking of male plasmid, called after pPICZ α A-chIL-2-HN; Adopt electrotransformation, recombinant plasmid pPICZ alpha A-chIL-2-HN conversion is entered among the Pichia yeast X-33, identify, obtain the genetically engineered pichia spp X-33 bacterial strain of express recombinant fusion rotein by high resistance screening and PCR;
(d) the chIL-2-HN fusion rotein of acquisition solubility
Screen the recombinant yeast pichia pastoris X-33 bacterium that carries the chIL-2-HN antigen-4 fusion protein gene and be inoculated among the 5mLBMGY 30 ℃ of about 22h to OD of 230r/min shaking culture above-mentioned
600 Reach 3 ~ 6; The centrifugal 2min of room temperature 3000r/min collects thalline and is resuspended in the 25mLBMMY substratum, carries out 28 ℃ of abduction deliverings, and 250r/min cultivates 60h, during every 24h to add final concentration be 1% methyl alcohol; Behind the 72h, the centrifugal 10min of 5000r/min collects the nutrient solution supernatant, is the chIL-2-HN fusion rotein of acquisition.
Reorganization chIL-2-HN fusion rotein of the present invention is the body fluid and the cellullar immunologic response of enhancing body effectively, the present invention uses the chIL-2-HN fusion protein immunization chicken of reorganization, the antibody subtype measurement result shows that chIL-2-HN fusion protein immunization group chicken physical efficiency produces an equilibrated IgG1 and IgG2a antibody response.Cytokine ELISA test-results shows that the fusion protein immunization group mainly stimulates Th1 differentiation (FNI-g), but the differentiation that also can stimulate Th2 (IL-4).
The strong malicious challenge test of Avian pneumo-encephalitis virus shows: the chIL-2-HN fusion rotein can obviously improve the malicious protection ratio of attacking of body, and chIL-2-HN the fusion rotein effectively body fluid and the cellullar immunologic response of enhancing body of reorganization is described.
The present invention is easy to realize mass production: the chIL-2-HN fusion rotein of the present invention's reorganization is selected pichia yeast expression system when producing, its advantage is that technology is simple, and cost is low, the output height, and the energy large scale fermentation is easy to realize mass production.
The chIL-2-HN fusion rotein that the present invention designs and develops not only has the specific antibody that can produce higher level behind the immune chicken, and strong virus attack is had good provide protection; The cytokine immunoadjuvant function that also has interleukin II in addition.The present invention provides feasible technical route for preparation reorganization chIL-2-HN fusion rotein, chIL-2-HN fusion rotein by present method preparation can be used as newcastle disease novel gene engineered vaccine, also can be used as the immunologic adjuvant of newcastle disease conventional vaccine, in newcastle disease prevention and treatment, have broad application prospects.
Description of drawings
Fig. 1 recombinant plasmid pPICZ alpha A-chiIL2-HN enzyme is cut and PCR identifies;
Lane1-2. negative control; The Lane3.PCR amplified production; 4.
KpnI/
NotI double digestion product; LaneM. λ DNA/
EcoRI+
HindIIImarker
The PCR of Fig. 2 transformant genomic dna identifies;
Lane1. negative control; The Lane2.X-33PCR amplified production; Lane3.X-33/pPICZ α APCR amplified production; Lane4.X-33/pPICZ α A-chiIL2-HNPCR amplified production
The SDS-PAGE of expression product analyzes in Fig. 3 recombination yeast fermentation supernatant;
Lane1.X-33/pPICZ α A; Lane2-6.X-33/pPICZ α A-chiIL2-HN 0,24,48,72, the 96h fermented liquid supernatant; LaneM. low molecular weight protein (LMWP) Marke
Expression product Westernblot analyzes in Fig. 4 recombination yeast fermented supernatant fluid;
Lane1.X-33/pPICZ α A; Lane2-6X-33pPICZ α A-IL2-IL2-HN 0,24,48,72,96h induces supernatant; LaneM. dye low molecular weight protein (LMWP) Marker in advance
Fig. 5 a, Fig. 5 b, Fig. 5 c are NDV-HN antibody and antibody subtype analysis;
Lymphocyte increment situation behind Fig. 6 immunity recombinant protein;
Fig. 7 cytokines measurement.
Embodiment:
Obtain coding
ChIL-2-HNThe gene order of fusion rotein:
The nucleotide sequence (number of including: AY029588), utilize DNAStar, 2 primer fragments of primerpremier software design F according to fowl IL-2 among the GenBank
1, F
2Amplification fowl IL-2 gene.Primer is synthetic by Dalian Bao Bio-Engineering Company.Primers F 15 ' end is introduced
KpnThe I restriction enzyme site, F25 ' end is introduced flexible joint.Primer sequence is as follows:
F1:SEQIDNO.1；
5’-CGGGTACCATGTGCAAAGTACTGATCTTTGGC-3’
F2:SEQIDNO.2；
5’-GCTGCCGCCGCCGCCTTTTTGCAGATATCTCAC-3’
With NDV-I is that vaccine is done 50 times of dilutions, is inoculated in instar chicken embryo on the 10th by allantoic cavity, and 0.2mL/ piece, 37 ℃ are continued hatching.48-72h takes out not dead chicken embryo in the inoculation back, gets the aseptic grinding of spleen, and carries out extraction and the amplification of total mRNA by the requirement of test kit immediately.Total mRNA is a template with chicken embryo spleen, RT-PCR amplification chicken IL-2 gene.By the requirement of RT-PCR test kit, in 50 μ L reaction systems, add 10 * buffer5 μ L respectively, MgCl210 μ L, dNTP mixture 5 μ L, RNA enzyme inhibitors 1 μ L, ThermoScript II 1 μ L, Taq enzyme 1 μ L, each 2 μ L of upstream and downstream primers F 1 and F2, template 5 μ L, no RNA enzyme water 18 μ L.50 ℃ of 30min reverse transcriptions, 94 ℃ of 2min deactivation ThermoScript II.Adopt touchdown PCR to increase, parameter is: 94 ℃ of 50s, and 58 ℃ of 45s, 72 ℃ of 100s circulate 6 times, 94 ℃ of 50s, 56 ℃ of 50s, 72 ℃ of 100s circulate 27 times, and 72 ℃ are extended 10min.(EthidiumBromide after EB) 1% agarose gel electrophoresis is identified, downcuts the purpose band to amplification PCR products, reclaims by glue recovery test kit (Dalian TaKaRa company) operation instruction subsequently and is fowl IL-2 gene fragment through containing ethidium bromide.
The nucleotide sequence (number of including: GU573799.1), utilize DNAStar, 2 primer fragments of primerpremier software design F according to newcastle disease virus HN gene among the GenBank
3, F
4Amplification newcastle disease virus HN gene, primers F 35 ' end is introduced flexible joint, and F45 ' holds introducing
NotThe I restriction enzyme site.Primer sequence is as follows:
F3:SEQ.ID.NO.3；
5’-GGCGGCGGCGGCAGCATGGACCGTGTAGTTAGC-3’
F4:SEQ.ID.NO.4；
5’-TAGCGGCCGCAACTCTATCATCCTTGAGGATCTCAAC-3’
Frozen NDV strain after melting, 37 ℃ of water-baths is inoculated the non-immune chicken embryos of 9～10 ages in days (each embryo 0.2mL), 37 ℃ of hatchings by the allantoic cavity approach.Chicken embryo dead in the 24h discards, and the allantoic fluid of the dead chicken embryo of aseptic collection 24～48h (having red corpuscle or yolk to make allantoic fluid become muddy chicken blastochyle discards) behind multigelation 3 times, 8000r/min4 ℃ centrifugal 15min, is got supernatant liquor, and 20 ℃ of preservations are stand-by.The extraction of viral RNA is extracted the test kit explanation according to the precious biotechnology RNA of company limited in Dalian and is operated.Total RNA4 μ L with extraction is a template, adds MgCl
23 μ L10 * reverse transcription buffer2.5 μ L, dNTPMixture (10mmol/L) 2.5 μ L, RnaseInhititor0.5 μ L (20U), RT enzyme 0.5 μ L, Taq enzyme 0.5 μ L, primers F 31 μ L, primers F 41 μ L, sterilization dH
2O9.5 μ L, total system 25 μ L.Carry out the RT-PCR reaction by following reaction conditions: 50 ℃ of 40s, 94 ℃ of 2min, 94 ℃ of 60s, 55 ℃ of 60s, 72 ℃ 2min11 circulation; 94 ℃ of 60s, 53 ℃ of 60s, 72 ℃ 2min12 circulation; 94 ℃ of 60s, 51 ℃ of 60s, 72 ℃ 2min10 circulation; 72 ℃ of 10min.The PCR product downcuts the purpose band after containing the evaluation of ethidium bromide 1% agarose gel electrophoresis, reclaim by glue recovery test kit operation instruction subsequently and be the newcastle disease virus HN gene.
Fowl IL-2 gene and newcastle disease virus HN gene with above-mentioned acquisition are template, utilize F1 and F4 primer, by overlapping PCR method amplification chIL-2-HN fusion gene.
The PCR reaction conditions: 94 ℃ of pre-sex change 2min enter PCR circulation: 94 ℃ of 30s, and annealing temperature is from 55 ℃, every circulation 1min, totally 30 circulations; 72 ℃ are extended 10min, and the PCR product downcuts the purpose band after containing the evaluation of ethidium bromide 1% agarose gel electrophoresis, reclaim by glue recovery test kit operation instruction subsequently and are the chIL-2-HN fusion gene.（SEQ.ID.NO.5）。
ChIL-2-HNThe structure of fusion gene recombinant yeast expression vector
With
KpnI,
NotThe I restriction endonuclease carries out double digestion to above-mentioned chIL-2-HN fusion gene PCR product and yeast expression vector pPICZ α A, and places 37 ℃ of water-bath effect 2h, enzyme to cut product equally after 1% agarose gel electrophoresis is identified, glue reclaims test kit and reclaims evaluation.The chIL-2-HN fusion gene that the process enzyme is cut spends the night for 4 ℃ by the mol ratio of 1:3 with yeast expression vector pPICZ α A and is connected.Get the adding of connection product and contain in the polypropylene centrifuge tube of 100 μ L competence DH5 α, gently ice bath 30min behind the mixing.Polypropylene centrifuge tube is taken out back 42 ℃ of heat-shocked 90sec from ice, then ice bath 2min immediately.The LB substratum that adds 800 μ L37 ℃ preheatings is in 37 ℃ of jolting (100～150rpm) 45min.Get 100 μ L bacterium liquid evenly coating contain the agar LB flat board of penbritin (Amp) 50 μ g/mL, 37 ℃ just putting 20min after, be inverted and cultivate 16 ~ 20h.By the alkaline lysis method of extracting plasmid on " molecular cloning experiment guide ".Carry out extracting plasmid
KpnI and
NotThe I double digestion is identified.Cut out the positive plasmid of plasmid (Fig. 1) of big or small dna fragmentation about existing 2000bp with enzyme.And with positive plasmid called after pPICZ α A-chIL-2-HN.Serve the order-checking of extra large Invitrongen company.
Express the structure of the engineering strain of chIL-2-HN fusion rotein:
Get the male recombinant yeast expression vector pPICZ α A-chIL-2-HN that embodiment 2 obtains and carry out the SacI linearizing, linearizing recombinant expression vector pPICZ α A-chIL-2-HN5 μ g mixes mutually with 80 μ L competence Pichia yeast X-33, be transferred to the 0.2cm electricity revolving cup of precooling, put 5min on ice, 1.5kV, 25 μ F, 200 Ω the electric shock, the 1mol/L sorbyl alcohol that adds the 1mL precooling is immediately got 200 μ L and is coated on the YPDS flat board, and 30 ℃ are cultured to single bacterium colony and occur; Detailed step is with reference to PichiaExpressionKit; Adopt PCR method to analyze the pichia spp transformant,--freeze--with boiling that cooking method prepares pcr template, usefulness primers F 1 and F4: reaction system is the same, the PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 45s, 48 ℃ of 45s, 72 ℃ of 45s, 25 circulations; 72 ℃ of 6min; Evaluation amplifies the clone that size is about 2200bp and is decided to be pichia spp positive transformant (Fig. 2), is the genetically engineered pichia spp X-33 bacterial strain of expressing the chIL-2-HN fusion rotein.
The abduction delivering of reorganization chIL-2-HN fusion rotein:
Be inoculated among the 5mLBMGY 30 ℃ of about 22h to OD of 230r/min shaking culture with screening the recombinant yeast pichia pastoris bacterium X-33 bacterium that carries the chIL-2-HN antigen-4 fusion protein gene
600Reach 3 ~ 6; The centrifugal 2min of room temperature 3000r/min collects thalline and is resuspended in the 25mLBMMY substratum, carries out 28 ℃ of abduction deliverings, and 250r/min cultivates 60h, during every 24h to add final concentration be 1% methyl alcohol; Behind the 72h, the centrifugal 10min of 5000r/min collects the nutrient solution supernatant, is the chIL-2-HN fusion rotein of acquisition.The molecular weight of chIL-2-HN fusion rotein can identify that concrete steps are as follows by the SDS-PAGE running gel:
Above-mentioned nutrient solution supernatant 100 μ L with collecting add isopyknic 2 * sds gel sample loading buffer (100mmol/LTrisCl (pH6.8); 200mmol/L dithiothreitol (DTT) (DTT); 4%SDS (electrophoresis level); 0.2% tetrabromophenol sulfonphthalein; 20% glycerine), 100 ℃ are boiled 5min so that the protein distortion is got 10 μ L application of samples and carried out the SDS-PAGE gel electrophoresis, and the preparation of SDS-PAGE running gel and deposition condition are with reference to the molecular cloning handbook.After running gel prepared, in electrophoresis chamber, pour Tris-glycine electrophoretic buffer (25mmol/LTris into; 250mmol/L glycine (electrophoresis level) (pH8.3); 0.1%SDS), application of sample finishes back connection power supply.Groove on the power cathode termination, groove under the anodal termination.Voltage is 80V in concentrating glue, enters that voltage is adjusted into 120V in the separation gel.Arrive back, separation gel bottom powered-down up to sample, take out gel, use coomassie brilliant blue staining 1h, the 1-2h that on decolorization swinging table, decolours subsequently, observations.About 100KDa(Fig. 3 of chIL-2-HN fusion protein molecule amount of reorganization).Test as an anti-Western-blot that carries out with the NDV positive serum simultaneously, the swimming lane of sample has a specificity purpose band on the nutrient solution supernatant of recombinant yeast pichia pastoris bacterium X-33 bacterium, and band (Fig. 4) does not appear in the last sample swimming lane of the nutrient solution supernatant of negative control Pichia yeast X-33 bacterium, the result shows, reorganization chIL-2-HN fusion rotein has obtained successful expression, and has good immunogenicity.
The application of reorganization chIL-2-HN fusion rotein:
1) animal immune
With above-mentioned reorganization chIL-2-HN fusion protein immunization chicken, estimate its immunological characteristic.200 of the healthy chicks of 20 ages in days are divided into 5 groups at random, 40/group.First group of negative contrast, immune 200 μ LPBS; Second group, the 3rd group, the rIL-2 of the 4th group of immune 200ppm of difference, rHN, rIL-2-HN recombinant protein (with 200 μ LPBS dilution).Adopt the chest muscle injection system to carry out immunity.The 5th group for the newcastle disease I is vaccine contrast, adopts the approach immunity 200 μ L of collunarium, eye droppings; All groups two all immunity at interval once are total to immunity three times.And respectively at respectively randomly drawing 5 hearts blood samplings, separation of serum after the immunization the 7th day, 14 days, 21 days, 28 days, 35 days the time.Simultaneously, the aseptic thymus gland of each test chicken of taking is used for carrying out lymphocyte proliferation assay.In last immunity one week of back, get and respectively organize 15 application of test chicken F simultaneously
48E
9The strong poison of standard is attacked (1000ELD
50/ only), to attack the poison back and observed 7 days continuously, morbidity, the death condition of test chicken respectively organized in record.
2) newcastle disease virus HN antibody and antibody subtype detect
Detect NDV-HNIgG production of antibodies after every group of immunity by ELISA, find immune group head exempt from 1 week the back just can detect the IgG antibody response.Antibody produces constantly behind second time booster immunization increases.With respect to the IgG level, the rHN immune group is exempted from and for the first time will be a little less than the NDVVaccine immune group during booster immunization at head, but behind second time booster immunization, both differences not obvious (p〉0.05).With respect to IgG1 level and IgG2a antibody response, IgG1 antibody is the main antibody subtype of rHN immune group chicken, and IgG2a antibody is the main antibody subtype of NDVVaccine immune group chicken, yet, use chIL-2-HN fusion protein immunization group chicken can produce an equilibrated IgG1 and IgG2a antibody response.Particularly behind second time booster immunization, observe the level of dominant IgG1 and IgG2a antibody, show that IL-2 can help to regulate Th1 type immune response (Fig. 5 a, Fig. 5 b, Fig. 5 c).
3) tetramethyl-azo azoles blue laws (MTT) is analyzed
From the aseptic weekly thymus gland of taking each test chicken of the 1st, 3,5 weeks of back of immunity for the first time, detect the lymphocytic propagation situation of thymus gland T.ChIL-2-HN fusion protein immunization group induced thymus gland T lymphopoiesis ability the strongest when the result was presented at for the 5th week, and other group is followed successively by rIL-2 immune group, NDVVaccine immune group, rHN immune group and PBS control group.The rIL-2 immune group the 1st the week and induced thymus gland T lymphopoiesis ability to be lower than the NDVVaccine immune group in the 3rd week, but the 5th week apparently higher than the NDVVaccine immune group, significant difference (
p＜0.05) (Fig. 6).
4) mensuration of IL-4 and IFN-γ in the serum
Use quantitative ELISA and the IL-4 in the serum and IFN-γ are carried out quantitative analysis find that chIL-2-HN fusion protein immunization group induces the secretion of IL-4 and IFN-γ the strongest in each immune group, difference extremely significantly (
p＜0.01).Next is the rIL-2 immune group, the secretion that NDVVaccine immune group and rHN immune group are mainly induced IL-4.It induces the secretion level of two kinds of cytokines to be starkly lower than the rIL-2 immune group, significant difference (
p＜0.05) (Fig. 7).
5) challenge test result
After immunization, the 35th day the time, each immune group residue chicken is got 15 use the F48E9 strong virus attacks.The result is as shown in table 1, and 15 test chickens of rHN immune group had 5 morbidities in 7 day observation period, and protection ratio only is 66.7%, wherein 2 death; And the NDVvaccine immune group is fallen ill 1, and protection ratio is 93.3%.15 test chickens of chIL-2-HN fusion protein immunization group only had 2 morbidities in 7 day observation period, protection ratio reaches 86.7%(table 1).
It should be noted last that: above embodiment is only in order to explanation, and unrestricted technical scheme of the present invention, although the present invention is had been described in detail with reference to the foregoing description.Those of ordinary skill in the art is to be understood that: still can make amendment or be equal to replacement the present invention, and not break away from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Sequence table
＜110〉University Of Science and Technology Of He'nan
＜120〉fowl IL-2 and newcastle disease virus HN gene recombinant fusion protein and application thereof
<170>?patentinversion3.3
<210>?1
<211>?32
<212>?DNA
＜213〉synthetic
＜223〉primers F 1
<400>?1
cgggtaccatgtgcaaagtactgatctttggc 32
<210>?2
<211>?33
<212>?DNA
＜213〉synthetic
＜223〉primers F 2
<400>?2
gctgccgccgccgcctttttgcagatatctcac 33
<210>?3
<211>?33
<212>?DNA
＜213〉synthetic
＜223〉primers F 3
<400>?3
ggcggcggcggcagcatggaccgtgtagttagc 33
<210>?4
<211>?37
<212>?DNA
＜213〉synthetic
＜223〉primers F 4
<400>?4
tagcggccgcaactctatcatccttgaggatctcaac 37
<210>?5
<211>?2154
<212>DNA
＜221〉gene of chIL-2-HN fusion rotein
<400>?5
ggagcatctctatcatcagcaaaaaggaaacctcttcaaacattaataaaggatttagaa 120
atattggaaaatatcaagaacaagattcatctcgagctctacacaccaactgagacccag 180
gagtgcacccagcaaactctgcagtgttacctgggagaagtggttactctgaagaaagaa 240
actgaagatgacactgaaattaaagaagaatttgtaactgctattcaaaatatcgaaaag 300
aacctcaagagtcttacgggtctaaatcacaccggaagtgaatgcaagatctgtgaagct 360
aacaacaagaaaaaatttcctgattttctccatgaactgaccaactttgtgagatatctg 420
caaaaaggcggcggcggcagcatggaccgtgtagttagcagagtcgtgctggagaatgag 480
gaaagagaagcaaagaacacatggcgcctggttttccggatcgcagtcttacttttaatg 540
gtaatgactctagctatctccgcagctgccctggcatatagcacgggggccagtacgccg 600
catgacctcgcaggcatatcgactgtgatctccaagacagaagataaggttacgtcttta 660
ctcagttcaagtcaagatgtgatagataggatatacaagcaggtggctcttgaatccccg 720
ctggcgctactaaacactgaatctataattatgaatgcaataacctctctttcttatcaa 780
attaacggggctgagaacaatagcggatgtggtgcgcctgttcatgacccagattatatc 840
ggggggataggcaaagaactcatagtggacgacaccagtgatgtcacatcattttatcct 900
tctgcatatcaagaacacttgaatttcatcccggcgcccaccacaggatccggttgcact 960
cggataccctcatttgacatgagcgccacccattattgttatactcacaatgtgatacta 1020
tccggttgcagagatcactcacactcacatcaatacttagcacttggtgtgcttcggaca 1080
tctgcaacagggagggtattcttttctactctgcgctccatcaatttagatgacacccaa 1140
aatcggaagtcctgcagtgtgagtgcaacccctttaggttgtgatatgctgtgctctaag 1200
gtcacagggactgaagaggaggattacaagtcaattgcccccacatcaatggtgcacgga 1260
aggctagggtttgacggtcaataccatgagaaggacttagacaccacggtcttatttaaa 1320
gattgggtggcaaattacccgggagtgggaggagggtcttttattgacgaccgtgtatgg 1380
ttcccagtttacggagggctcaaacccaattcacccagtgacactgcacaagaagggaaa 1440
tatgtaatatacaagcgccataacaacacctgccccgatgaacaagattaccaaattcgg 1500
atggctaagtcttcatataaacccgggcgatttggtggaaagcgcgtacagcaagccatc 1560
ctatccatcaaagtgtcaacatccctgggtaaggacccggtgctgactattccacctaat 1620
acaatcacactcatgggagccgaaggcagaatcctcacagtagggacatctcacttcttg 1680
taccaacgagggtcttcctatttctcccctgccttattatatcccatgacagtaaataac 1740
aaaacggctacactccatagtccttacacgtttaatgctttcactcggccaggtagtgtc 1800
ccttgccaggcatcagcaagatgccccaactcatgcatcactggggtctataccgatcca 1860
tatcccttaatcttccataggaatcatactctacgaggggtcttcgggacgatgcttgat 1920
gatgagcaagcgaggcttaaccccgtatctgcggtattcgacaacatatctcgcagtcgt 1980
gtcactcgggtgagttcaagcagcaccaaggcagcatacacgacatcgacatgttttaaa 2040
gttgtcaagaccaataaagcttattgtcttagtatcgcagaaatatccaataccctattc 2100
ggggaattcaggatcgttcccttattagttgagatcctcaaggatgatagagtt 2154
<210>?6
<211>?5
<212>PRT
＜221〉flexible Linker peptide
<400>?6
<210>?7
<211>?718
<212>?PRT
＜221〉chIL-2-HN fusion rotein
<400>?7
ectqqtlqcylgevvtlkketeddteikeefvtaiqnieknlksltglnhtgseckicea 120
nnkkkfpdflheltnfvrylqkggggsmdrvvsrvvleneereakntwrlvfriavlllm 180
vmtlaisaaalaystgastphdlagistvisktedkvtsllsssqdvidriykqvalesp 240
lallntesiimnaitslsyqingaennsgcgapvhdpdyiggigkelivddtsdvtsfyp 300
sayqehlnfipapttgsgctripsfdmsathycythnvilsgcrdhshshqylalgvlrt 360
satgrvffstlrsinlddtqnrkscsvsatplgcdmlcskvtgteeedyksiaptsmvhg 420
rlgfdgqyhekdldttvlfkdwvanypgvgggsfiddrvwfpvygglkpnspsdtaqegk 480
yviykrhnntcpdeqdyqirmakssykpgrfggkrvqqailsikvstslgkdpvltippn 540
titlmgaegriltvgtshflyqrgssyfspallypmtvnnktatlhspytfnaftrpgsv 600
pcqasarcpnscitgvytdpyplifhrnhtlrgvfgtmlddeqarlnpvsavfdnisrsr 660
vtrvsssstkaayttstcfkvvktnkayclsiaeisntlfgefrivpllveilkddrv 718
Claims (10)
1.chIL-2-HN fusion rotein is characterized in that: comprise the fowl leukocyte plain 2(chIL-2 that is situated between) aminoacid sequence, Avian pneumo-encephalitis virus hemagglutinin-neuraminidase (HN albumen) aminoacid sequence and in be situated between flexible Linker peptide ammino acid sequence between plain 2 aminoacid sequences and the proteic aminoacid sequence of newcastle disease virus HN of fowl leukocyte.
2. chIL-2-HN fusion rotein according to claim 1 is characterized in that: by the fowl leukocyte plain 2(chIL-2 that is situated between) gene and newcastle disease virus HN gene be connected in series by flexible Linker peptide and form.
3. chIL-2-HN fusion rotein according to claim 1 and 2 is characterized in that: this albumen has the aminoacid sequence shown in the SEQIDNO.7.
4. chIL-2-HN fusion rotein according to claim 1 and 2 is characterized in that: described flexible Linker peptide has the aminoacid sequence shown in the SEQIDNO.6.
5. separated DNA sequence is characterized in that: coding has the fusion rotein that sequence is the aminoacid sequence shown in the SEQIDNO.7.
6. dna sequence dna according to claim 5 is characterized in that: have the nucleotide sequence shown in the SEQIDNO.5.
7. recombinant yeast pichia pastoris carrier, it is characterized in that: it contains the described dna sequence dna of claim 6.
8. host cell, it is characterized in that: it contains the described dna sequence dna of claim 6.
9. recombination fusion protein is characterized in that: obtained by following method:
(a) gene order of acquisition coding chIL-2-HN fusion rotein;
(b) gene order of the chIL-2-HN fusion rotein that step (a) is obtained is inserted among the pichia vector pPICZ α A, obtains recombinant expressed pichia vector, called after pPICZ α A-chIL-2-HN;
(c) the recombinant expressed pichia vector pPICZ α A-chIL-2-HN that step (b) is obtained, electric shock is transformed in the Pichia yeast X-33 competent cell, high resistance screening and PCR identify, obtain the gene pichia spp X-33 bacterial strain of express recombinant fusion rotein;
(d) the gene engineering yeast X-33 bacterial strain of the express recombinant fusion rotein that step (c) is obtained is cultivated under the suitable culture condition, and separation and Culture liquid supernatant obtains recombination fusion protein.
10. as the application of recombination fusion protein as described in the claim 9 in newcastle disease prevention and treatment.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102180959A (en) * | 2011-03-17 | 2011-09-14 | 天津康莱森生物科技集团有限公司 | Improved chook Interleukin-2 protein and preparation method thereof |
| CN102603898A (en) * | 2012-02-23 | 2012-07-25 | 广西壮族自治区兽医研究所 | Fowl adenovirus group I immunity related fusion protein as well as encoding gene and application thereof |
| CN104357408A (en) * | 2014-03-13 | 2015-02-18 | 哈尔滨博翱生物医药技术开发有限公司 | Recombined newcastle disease virus and application thereof |
| CN104357409A (en) * | 2014-11-07 | 2015-02-18 | 东北农业大学 | Recombinant Newcastle disease virus for expressing chicken IL2 and application of virus in vaccines |
| CN110488011A (en) * | 2018-05-14 | 2019-11-22 | 洛阳中科生物芯片技术有限公司 | Newcastle disease virus antibody assay kit |
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2010
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| 《兽医研究》 20081231 王强等 新城疫病毒（NDV）的分子生物学特征及基因工程疫苗的研究进展 第6-11页 1-10 , 第12期 2 * |
| 《河南科技大学学报：自然科学版》 20090831 刘一尘等 新城疫病毒HN蛋白表达条件的优化、纯化及其活性鉴定 第72-77页 1-10 第30卷, 第4期 2 * |
| 《病毒学报》 20080930 梁俊文等 新城疫病毒分离株HN和P基因分子进化及其相关研究 第390-395页 1-10 第24卷, 第5期 2 * |
| 《福建农林大学学报（自然科学版）》 20090930 程太平等 一株新城疫病毒HN基因的克隆及序列分析 第512-516页 1-10 第38卷, 第5期 2 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102180959A (en) * | 2011-03-17 | 2011-09-14 | 天津康莱森生物科技集团有限公司 | Improved chook Interleukin-2 protein and preparation method thereof |
| CN102603898A (en) * | 2012-02-23 | 2012-07-25 | 广西壮族自治区兽医研究所 | Fowl adenovirus group I immunity related fusion protein as well as encoding gene and application thereof |
| CN104357408A (en) * | 2014-03-13 | 2015-02-18 | 哈尔滨博翱生物医药技术开发有限公司 | Recombined newcastle disease virus and application thereof |
| CN104357409A (en) * | 2014-11-07 | 2015-02-18 | 东北农业大学 | Recombinant Newcastle disease virus for expressing chicken IL2 and application of virus in vaccines |
| CN104357409B (en) * | 2014-11-07 | 2018-01-09 | 东北农业大学 | Express chicken IL2 recombinant Newcastle disease virus and its application in vaccine |
| CN110488011A (en) * | 2018-05-14 | 2019-11-22 | 洛阳中科生物芯片技术有限公司 | Newcastle disease virus antibody assay kit |
| CN110488011B (en) * | 2018-05-14 | 2022-11-01 | 洛阳中科生物芯片技术有限公司 | Newcastle disease virus antibody detection kit |
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