CN102703390A - Grass carp hemorrhage antigenic protein, preparation method and application thereof - Google Patents

Abstract

The invention discloses a grass carp hemorrhage antigenic protein, a preparation method and an application thereof, wherein the preparation method comprises the steps: A. according to nucleotide complete sequence of a grass carp reovlrus VP6 gene, designing a primer to perform reverse transcription-polymerase chain reaction; cloning to obtain a VP6 protein gene; B. using EcoRI and SacII to perform enzyme digestion on a product obtained from reverse transcription-polymerase chain reaction in step A and a pPICZB carrier; constructing a pPICZB-VP6 expression carrier; C. electrically transferring recombinant obtained in step B to pichia yeast; coating the pichia yeast on the YPDS flat plate containing ZEOCIN antibiotic, and screening to obtain a high-copy recombinant yeast containing a target gene; and D. obtaining the used antigenic protein by separation and purification. The method is simple and is easy to operate, can be used for carrying out large-scale industrial production by a fermentation tank; the protein is safe and reliable, has immunological competence and can be fully used as the grass carp hemorrhage antigenic protein.

Description

A kind of hemorrhagic disease of grass carp antigen protein and preparation method and application

Technical field

The present invention relates to the bioprotein preparing technical field; More specifically relate to a kind of hemorrhagic disease of grass carp antigen protein; The preparation method who also relates to a kind of hemorrhagic disease of grass carp antigen protein simultaneously also relates to the application of a kind of hemorrhagic disease of grass carp antigen protein in preparation grass carp hemorrhage disease vaccine.

Background technology

Grass carp (Grass carp, Ctenopharyngodon idellus) is one of important cultured freshwater fish of China, in China's freshwater aquiculture, accounts for critical role.Wide, the fast growth in feed of grass carp source, delicious meat is liked by numerous culturists and human consumer deeply.But the grass carp disease is many, the breed surviving rate is low, has hindered the development of grass carp aquaculture.Hemorrhagic disease of grass carp is a kind of virus disease of serious harm grass carp fingerling, and its popular scope is wide, velocity of propagation is fast, and mortality ratio is high, and all cause enormous economic loss to the grass carp aquaculture every year.For a long time, the Study of Prevention Technology of hemorrhagic disease of grass carp is the important content of China's aquiculture disease Study of Prevention Technology always, and the research of hemorrhagic disease of grass carp Prevention Technique not only has important significance for theories, and has broad application prospects.

The main pathogen of hemorrhagic disease of grass carp is a GCRV.At present; There is not grass carp to use the grass carp hemorrhage disease vaccine on the market; But some related patent U.S. Patent No. have been applied for; For example, Chinese invention patent application 200410060881.2 disclosed a kind of GCRV antigen subunit vaccine preparations are that employing grass carp kidney cell line is a CIK cell proliferation GCRV; Through centrifuging, the cell virus suspension that has infected GCRV is carried out purifying and concentrates; The virus of surfactant treatment purifying is disintegrated the intact virus structure, forms viral nucleic acid and capsomere component; Adopt nuclease digestion, degrade free virus genome dsRNA; Through dialysis treatment, do not contained the GCRV proteantigen subunit preparation of nucleic acid.And for example Chinese invention patent application 201110189906.9 disclosed a kind of reovirus genes of grass carps engineered vaccine preparing methods are the recombinant baculovirus (vAcGCRV-VP5/VP7) that adopt sf9 insect cell propagation reorganization GCRV outer capsid albumen VP5 and VP7; Through the sf9 of freezing-thawing and cracking recombinant virus infection cell, adopt sucrose layer centrifuging separating recombinant proteins component, obtain the vivoexpression VP5 and the VP7 albumen composition of purifying thus; VP5 and VP7 albumen through with purifying carry out fish body protection experiment, confirm that it has good immunoprotective effect.But subunit vaccine since complicated process of preparation, cost high, be difficult to wide popularization and application.

Seek that a kind of technology is simple, cost is low, the grass carp hemorrhage disease vaccine that is easy to extensively promote guarantees food safety, environmental safety to safeguarding national security, and promotes that the Sustainable development of grass carp aquaculture is significant.

Summary of the invention:

One object of the present invention is to provide a kind of GCRV, GCRV GCRV-104, and CCTCC NO:V201217 submits preservation on April 18th, 2012.

The GCRV particle is to be the symmetric icosahedron of 5:3:2; Diameter range has double capsid at 55-80nm, no cyst membrane; This virus particle has certain resistance to soda acid (pH3-10) and pyroprocessing; Insensitive to chloroform and ether, handle for 56 ℃ and still have virulence half a hour, virus infectivity obviously descends but handle after one hour then.The buoyant density of GCRV particle in CsCl and SDGC is for being respectively 1.37g/cm ³And 1.305g/cm ³, settling ratio is 550S.Because this nucleoid can synthesize RNA polymerase, and this enzyme optimum temperuture is 28 ℃, so GCRV popular has seasonality.GCRV 104 strains pathology in the CIK cell is obvious, begins to occur pathology after inoculating 24h approximately, and pathology reaches 90% when inoculating 5-6 days.Exhale lonely other strains of intestines different with grass carp, netted pathology such as roll does not appear in GCRV GCRV-104 strain in CIK, but cell rounding, brightening comes off afterwards.

Another object of the present invention is to provide a kind of hemorrhagic disease of grass carp antigen protein; Hemorrhagic disease of grass carp antigen protein of the present invention is a GCRV VP6 recombinant protein; GCRV VP6 albumen can produce antigen-reactive; But do not have pathogenicly, its use is safer than prior art.

A further object of the invention is the preparation method who has been to provide a kind of hemorrhagic disease of grass carp antigen protein, and technology is simple, cost is low, is easy to the grass carp hemorrhage disease vaccine, particularly through engineering approaches productions in a large number extensively promoted.

A further object of the invention has provided the application of a kind of hemorrhagic disease of grass carp antigen protein in preparation grass carp hemorrhage disease vaccine.

In order to realize above-mentioned purpose, the present invention adopts following technical measures:

A kind of GCRV GCRV-104, its preparation process is:

Doubtful trouble hemorrhagic disease of grass carp grass carp appearance is gathered in the literary composition village in Jianglin County, Jingzhou City, Hubei Province; The viscera tissues such as liver,spleen,kidney of getting the disease fish are in aseptic petridish; With aseptic eye scissors it is shredded; The PBS that adds 10 times of volumes (V/W) grinds to form tissue homogenate together with organizing fragment to change in the lump in the glass homogenizer under the condition of ice bath.Tissue homogenate is freezing in 80 ℃ of ﹣, melt under the room temperature, so multigelation is 3 times.With the centrifugal 30min of 5000 * g, supernatant is with 0.22 μ m filter filtration sterilization under 4 ℃ of conditions.Get the tissue homogenate of 1mL, in the CIK cell that 10 μ g help infectious agent Polybrene to be inoculated into to grow to confluent monolayer, place 28 ℃ of incubators to adsorb 1h, during every 20min gently the reciprocal inclination culturing bottle be beneficial to evenly absorption of virus for several times.After absorption finishes, discard viral liquid, every bottle adds people 5mL cell maintenance medium (the MEM substratum that contains 2% serum), places 25 ℃ of incubators to continue to cultivate, day by day observation of cell pathology situation.When 70%-80%CPE appears in cell monolayer, harvested cell culture, (20 ℃-25 ℃ of 80 ℃-room temperature conditions of ﹣; As follows) down freeze thawing 2 times; Under 4 ℃ of conditions, with the centrifugal 20min of 4000 * g, discard deposition, it is subsequent use that viral liquid is sub-packed in the frozen pipe 80 ℃ of preservations of ﹣.The applicant delivers this GCRV GCRV-104 to the Chinese typical culture collection center (CCTCC) in the Wuhan University of Wuhan City, Hubei Province on April 18th, 2012, and deposit number is CCTCC NO:V201217.

A kind of hemorrhagic disease of grass carp antigen protein preparation method the steps include:

Hemorrhagic disease of grass carp antigen protein of the present invention is a GCRV VP6 recombinant protein.

A. the virus total RNA of extracting from GCRV is a template;

Using GCRV GCRV-104, therefrom extract virus total RNA, is template rt grass carp GCRV VP6 gene with total RNA of this virus;

The a pair of primer of complete nucleotide sequence (GeneBank NO.HM234682) design according to GCRV VP6 gene carries out RT-polymerase chain reaction, and the clone obtains the VP6 protein gene; Used primer sequence is:

P1:5 '-CGG AATTCATGGCACGTGTGGTTTAT-3 ' (line place is an EcoR I restriction enzyme site) P2:5 '-TCC CCGCGGGTGTGGTTACGCGGGTCAG-3 ' (line place is a Sac II restriction enzyme site).

Reaction conditions is: 48 ℃ of rts 45 minutes; 94 ℃ of deactivations and preparatory sex change 5 minutes; Again through 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute, react 40 circulations; 72 ℃ were extended 10 minutes.

B. change Yeast expression carrier then over to, obtain recombinant expression vector;

Aforementioned RT-polymerase chain reaction product and pPICZ B carrier are used EcoR I and Sac II double digestion respectively, two products are connected, thereby be built into pPICZ B-VP6 expression vector.Cutting evaluation through polymerase chain reaction evaluation and enzyme adds their confirmation.Identify that sure positive plasmid pPICZ B-VP6 is the recombinant plasmid that has complete GCRV VP6 protein gene.Sequencing result shows that positive plasmid pPICZ B-VP6 contains the reading frame of GCRV VP6 protein gene.

C. again it is imported in the yeast expression bacterium, obtain the restructured Pichia pastoris in expression bacterium, breed and carry out abduction delivering restructured Pichia pastoris in expression bacterium;

The recon electricity that obtains among the step B is changed in the pichia spp; Get 200 μ l conversion products be coated on the ZEOCIN concentration gradient be 0.5,1.0,2.0,3.0,4.0 and the YPDS flat board of 5.0mg/mL on; Put 30 ℃ of constant temperature culture 2～4 days; The high resistant strain of step-sizing ZEOCIN obtains the high copy of goal gene recombinant bacterial strain.

The high copy of picking screening colony inoculation is in 100ml BMGY substratum from the YPD-ZEOCIN flat board, and 28 ℃～30 ℃ shaking culture 16～18h are when OD600=2～6; The centrifugal 10min collecting cell of room temperature 1500g; The cell of collecting is suspended in the triangular flask of 250ml with 20ml BMMY substratum, and three layers of sterile gauze tying guarantee excellent air permeability; 28 ℃ of shaking culture abduction deliverings; Every 24h adds methanol concentration to 0.5% in substratum, and the 24h sampling once, and sampling amount 1ml expresses to guarantee successive induction.

By the hemorrhagic disease of grass carp antigen protein that the restructured Pichia pastoris in expression bacterium is expressed, its sequence is the aminoacid sequence shown in the SEQ ID NO.1.

D. obtain used antigen protein through separation and purification: collect the bacterium liquid that obtains among the step C and it is powdered through lyophilize; PBS suspension with 100 μ l pH7.4 makes powder dissolving as far as possible; Ultrasonic treatment is translucent to bacterium liquid; Behind the SDS-PAGE electrophoresis, obtain the target protein of purifying, the 46kDa place promptly is.

Research shows that GCRV VP6 molecular weight of albumen is 46kDa, is in one and antigen; Research shows that this albumen has immunization; Can bring out immunoreation,, but not have pathogenic because GCRV VP6 albumen can produce antigen-reactive.And yeast expression system is a kind of simple eukaryotic expression system; Can express foreign protein by stability and high efficiency; And can translate post-treatment and modification to expression product, simultaneously, its sophisticated high density fermentation technology provides guarantee for the suitability for industrialized production of target protein; Therefore adopting GCRV VP6 albumen is the deficiency that subunit vaccine can overcome prior art fully, and its use is safer than prior art.

Albumen of the present invention adopts RT-polymerase chain reaction to clone, and through the expression of yeast reorganization bacterium, makes it possess the condition of large-scale commercial prodn again.

The application of a kind of hemorrhagic disease of grass carp antigen protein in preparation grass carp hemorrhage disease vaccine the steps include:

The Westen-blot of recombinant expression protein analyzes:

(1). transfer printing: cut 8 filter paper and 1 nitrocellulose filter (NC); Size equates with gel; In transfering buffering liquid, soak 3min; Order by four metafiltration paper, NC film, SDS-PAGE running gel, four metafiltration paper on positive plate stacks neatly, confirms negative plate on the no bubble bonnet, and 110mA shifted 1 hour.

(2). sealing: after shifting completion, take off the NC film, downcut standard molecular weight Marker, put in the amino black staining fluid and contaminate 5min, take out the back, take off most until the background blueness with the rinsing decolouring.All the other NC films wash with PBS, add the 10mL confining liquid, and room temperature is jolting 1-2hr gently.

(3). with combining of antibody: after sealing finishes; Towards Xian NC film 4-5 time, add the proteic polyclonal antibody 10mL of mouse-anti GCRV 104 strain VP6 that PBST 1:200 doubly dilutes with PBS, 37 ℃ combine 1.5 hours; With PBST flushing 4-5 time, slowly shake at shaking table at every turn.The sheep anti-mouse antibody of the horseradish peroxidase-labeled that adding 1:2000 doubly dilutes (two is anti-), jog was hatched 1 hour under the room temperature, took out with PBST flushing 3-4 time each 10min.

(4). the substrate colour developing: the NC film is transferred in the 10mL substrate solution, and room temperature lucifuge jog 3min observes the colour developing situation, waits when band occurring, changes in the PBST damping fluid room temperature preservation immediately over to.

(5). recombinant expression protein immunity qualification result

Reorganization pPICZ B-VP6 expression product carries out western blot test through being transferred to behind the SDS-PAGE on the NC film; Good antigen one antibody response can take place with the proteic polyclonal antibody of mouse-anti GCRV 104 strain VP6 in the VP6 albumen of expressing; VP6 albumen has immunocompetence, can be used as the hemorrhagic disease of grass carp vaccine antigen protein fully.

Compared with prior art, the present invention also has following advantage:

1. the vaccine antigen of the present invention's production is the neutrality antigen protein, rather than the GCRV of deactivation or attenuation, so this albumen is reliable as vaccine safety.

2. the vaccine that the present invention relates to is a protein with antigen, compare with other new generation vaccines (like live vector vaccine, nucleic acid vaccine, gene-deleted vaccine) the recombination problem that possibly occur can not take place, so there is not the biological safety problem in the present invention.

3. the present invention cultivates simply with the expression vector of yeast as target protein, can use the fermentor tank large-scale production, can realize quality control, guarantees that the vaccine safety of being produced is effective.

4. the present invention compares with escherichia coli prokaryotic expression with the expression vector of yeast as target protein, and it is high that expression amount is wanted, and its activity will be far above inclusion body protein.

5. the expressed target protein of the present invention can directly be used to prepare the antigen of GCRV VP6 monoclonal antibody.

6. the relative currently available vaccines, existing vaccines technology of method of the present invention wants simple.

Description of drawings

Fig. 1 is a kind of electrophoresis and Western-blot synoptic diagram of pPICZ B-VP6 yeast expression product.

Figure 1A is a kind of electrophoresis synoptic diagram of pPICZ B-VP6 yeast expression product.

Wherein: M is the molecular weight of albumen standard; The 1st, pPICZB-VP6/KM71 cracking deposition; The 2nd, pPICZB-VP6/K M71 cracking supernatant; The 3rd, pPICZB/KM71 cracking supernatant.

Figure 1B is that a kind of pPICZ B-VP6 yeast expression product Western-blot analyzes synoptic diagram.

Wherein: M is the molecular weight of albumen standard; The 1st, pPICZB-VP6/KM71 cracking deposition; The 2nd, pPICZB-VP6/K M71 cracking supernatant; The 3rd, pPICZB/KM71 cracking supernatant.

Embodiment

The main raw that the present invention uses

Virus: GCRV GCRV-104 deposit number CCTCC NO:V201217.

Expressing bacterium: KM71 is the yeast of destination gene expression, must handle making it have susceptibility when being used for the plasmid conversion through sorbyl alcohol, becomes competent cell, and this bacterial strain can be bought from Invitrogen company and obtain.

PPICZ B carrier: be Yeast expression carrier, available from Invitrogen company.

Primer: all gene amplifications are synthetic by the precious biological ltd in Dalian with the primer of transforming usefulness.

Enzyme and reagent: various restriction enzymes and dna ligase are available from Promega company; Taq archaeal dna polymerase, 10 * PCR Buffer, dNTPs, DNA marker are the TaKaRa Company products; Sepharose DNA reclaims test kit, DNA extracts test kit in a small amount and cerevisiae dna extraction test kit is an OMEGA Bio-Tek Company products; TRIzol LS Reagent, foetal calf serum, rt test kit and Zeocin ^TMAvailable from Invitrogen company; Protein molecular weight standard is available from Fermentas company; The proteic polyclonal antibody of mouse-anti GCRV 104 strain VP6 is an AB CLONAL Company products available from the sheep anti-mouse antibody of the green spring bio tech ltd of Beijing health, horseradish peroxidase-labeled; Biochemical reagents such as sorbyl alcohol, methyl alcohol are homemade analytical pure.

Embodiment 1: the separation of GCRV GCRV-104

Doubtful trouble hemorrhagic disease of grass carp grass carp appearance is gathered in the literary composition village in Jianglin County, Jingzhou City, Hubei Province; The viscera tissues such as liver,spleen,kidney of getting the disease fish are in aseptic petridish; With aseptic eye scissors it is shredded; The PBS that adds 10 times of volumes (V/W) grinds to form tissue homogenate together with organizing fragment to change in the lump in the glass homogenizer under the condition of ice bath.Tissue homogenate is freezing in 80 ℃ of ﹣, melt under the room temperature, so multigelation is 3 times.With the centrifugal 30min of 5000 * g, supernatant is with 0.22 μ m filter filtration sterilization under 4 ℃ of conditions.Get the tissue homogenate of 1mL, in the CIK cell that 10 μ g help infectious agent Polybrene to be inoculated into to grow to confluent monolayer, place 28 ℃ of incubators to adsorb 1h, during every 20min gently the reciprocal inclination culturing bottle be beneficial to evenly absorption of virus for several times.After absorption finishes, discard viral liquid, every bottle adds people 5mL cell maintenance medium (the MEM substratum that contains 2% serum), places 25 ℃ of incubators to continue to cultivate, day by day observation of cell pathology situation.When 70%-80%CPE appears in cell monolayer, the harvested cell culture, freeze thawing is 2 times under the 80 ℃-room temperature condition of ﹣, under 4 ℃ of conditions, with the centrifugal 20min of 4000 * g, discards deposition, and it is subsequent use that viral liquid is sub-packed in the frozen pipe 80 ℃ of preservations of ﹣.This virus is submitted preservation, GCRV GCRV-104, CCTCC NO:V201217 on April 18th, 2012.

Embodiment 2: extract the total RNA of GCRV

GCRV 104 strains are type strains that China preserves: GCRV GCRV-104 deposit number CCTCC NO:V201217.

Cultivate the grass carp kidney cell to confluent monolayer, the sucking-off nutrient solution is 0.1 dosage with infection multiplicity; Inoculation 1mL removes the GCRV cell toxicant material of cell debris through the centrifugal 5min of 4000r/min, adds the Polybrene (final concentration 10 μ g/ml) of 10 μ L, places 28 ℃ of incubators to adsorb 1h; Make virus absorption onto cell, every 20min rocks culturing bottle once gently in the adsorption process, makes that viral liquid and cell monolayer are full and uniform to be contacted; Behind the absorption 1h, inhale and abandon viral liquid, add the nutrient solution of 5mL 2% foetal calf serum (V/V); Put 28 ℃ of cultivations; Tangible cytopathic effect occurs until cell, the collecting cell lesion material is put-80 ℃ of refrigerators and is preserved subsequent use.

Cell culture and virus-80 is ℃ to room temperature freeze thawing 3 times, and the centrifugal 30min of 4000r/min gets the centrifugal 2h of supernatant 20000r/min, abandons supernatant, with the seedless aqueous suspension of 250 μ L, extracts the RNA of GCRV with Trizol LS.

(1) viral suspension and TRIzol LS Reagent press the 1:3 mixed, place 5min at ambient temperature.

(2) every 0.75mL TRIzol LS Reagent adds the 0.2mL chloroform, covers tight lid, acutely rocks 15s with hand, places 2 ~ 15min then at ambient temperature.

(3) at 2 ~ 8 ℃, 12000g, centrifugal 15min gets upper phase and moves in the new pipe.

(4) every 0.75mL TRIzol LS Reagent adds the 0.5mL Virahol, at room temperature places 10min.

(5) at 2 ~ 8 ℃, 12000g, centrifugal 15min.

(6) remove supernatant, 75% ethanol (V/V) that adds seedless water preparation cleans RNA (every 0.75mL TRIzol LS Reagent adds 1mL ethanol at least), vortex mixing.

(7) at 2 ~ 8 ℃, 10000g, centrifugal 10min in super clean bench or vacuum-drying 5 ~ 10min, obtains the total RNA of GCRV.

(8) with an amount of seedless water (20 μ L) the total RNA of suspension GCRV, available rifle head is pressure-vaccum gently, and 55 ~ 60 ℃ of water-bath 10min hydrotropies are put into-80 ℃ of refrigerators and preserved subsequent use.

Selecting GCRV 104 strains in the present embodiment for use, therefrom extract virus total RNA, is template with this total RNA, carries out RT-polymerase chain reaction (RT-PCR), and the primer is to being:

P1:5 '-CG GAATTCATGGCACGTGTGGTTTAT-3 ' (containing EcoR I restriction enzyme site) P2:5 '-TCC CCGCGGGTGTGGTTACGCGGGTCAG-3 ' (containing Sac II restriction enzyme site).

Reaction conditions is: 48 ℃ of rts 45 minutes; 94 ℃ of deactivations and preparatory sex change 5 minutes; Again through 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute, react 40 circulations; 72 ℃ were extended 10 minutes.

Amplified fragments comprises the reading frame of GCRV VP6 gene.

Embodiment 3: construction recombination plasmid pPICZ B-VP6 and transformed yeast bacterium

1. the clone of goal gene:

At first aforementioned RT-polymerase chain reaction product and pPICZ B carrier are used EcoR I and Sac II double digestion respectively, two products are connected, thereby be built into pPICZ B-VP6 expression vector.Cutting evaluation through polymerase chain reaction evaluation and enzyme adds their confirmation.Identify that sure positive plasmid pPICZ B-VP6 is the recombinant plasmid that has complete GCRV VP6 protein gene.Sequencing result shows that positive plasmid pPICZ B-VP6 contains the reading frame of GCRV VP6 protein gene.

2.1 the preparation of reagent

10 * YNB: take by weighing 13.4g YNB (YNB is no amino acid yeast nitrogen, yeast culture base moity) and be dissolved in the 100ml deionized water, being heated to YNB dissolves fully, filtration sterilization, and 4 ℃ of preservations can be put 1 year.

500 * B:20mg vitamin H is dissolved in the 100ml deionized water, filtration sterilization, and 4 ℃ of preservations can be put 1 year.

10 * D:200g glucose is dissolved in the 1000ml deionized water, and high pressure (121 ℃, 0.105PKa) 15min or filtration sterilization, 4 ℃ of preservations can be put 1 year.

10 * M:5ml methyl alcohol is dissolved in the 100ml deionized water, filtration sterilization, and 4 ℃ of preservations can be preserved February.

10 * GY:10ml glycerine is dissolved in the 90ml deionized water, and high pressure (121 ℃, 0.105PKa) 20min sterilization, 4 ℃ of preservations can be put more than 1 year.

1M phosphate buffered saline buffer (pH6.0): 1M K ₂HPO ₄132ml, 1M KH ₂PO ₄868ml, H ₃PO ₄Transfer to pH6.0, and autoclaving (121 ℃, 0.105PKa), 4 ℃ of preservations.

YPD and YPD are dull and stereotyped: the 1g yeast extract, and the 2g peptone is dissolved in the 90ml deionized water, autoclaving (121 ℃, 0.105PKa), the cooling back adds 10ml 10 * D, and making sheet adds the 1.5g agar powder.

The BMGY:1g yeast extract, the 2g peptone is dissolved in the 70ml deionized water, and (121 ℃, 0.105PKa), the cooling back adds 10ml 1M phosphate buffered saline buffer, 10ml 10 * YNB, 0.2ml 500 * B, l0ml 10 * GY to autoclaving.

The BMMY:1g yeast extract, the 2g peptone is dissolved in the 70ml deionized water, and (121 ℃, 0.105PKa), the cooling back adds 10ml 1M phosphate buffered saline buffer, 10ml 10 * YNB, 0.2ml 500 * B, 10ml 10 * M to autoclaving.

2.2 pPICZ B-VP6 expression vector alkaline lysis is extracted plasmid in a small amount.

2.3 evaluation to recombinant plasmid pPICZ B-VP6

Evaluation comprises following aspect:

(1) recombinant plasmid pPICZ B-VP6 agarose gel electrophoresis is identified;

(2) pcr amplification of recombinant plasmid pPICZ B-VP6; Promptly with the P1 that contains following two restriction enzyme sites, P2 primer recombinant plasmid being carried out pcr amplification identifies;

P1:5 '-CGG AATTCATGGCACGTGTGGTTTAT-3 ', line place is an EcoR I restriction enzyme site; P2:5 '-TCC CCGCGGGTGTGGTTACGCGGGTCAG-3 ', line place is a Sac II restriction enzyme site;

(3) enzyme of recombinant plasmid pPICZ B-VP6 is cut evaluation; Promptly identify with EcoRI and Sac II double digestion;

(4) order-checking of recombinant plasmid pPICZ B-VP6 conclusive evidence;

(5) with DNAstar software sequence is analyzed, and carried out homology relatively with reported sequence.

The correct goal gene that obtains through above assay certificate is connected on the expression vector accurately, and successful structure p PICZ B-VP6 recombinant expression vector.

2.4 the preparation of pichia spp competent cell, conversion and screening

(1) with the single colony inoculation of streak culture pichia spp KM71 in 3ml YPD substratum, 28 ℃, 250r/min sways and cultivates 24 ~ 28h.

(2) get substratum 100 μ l and transfer in the 20mlYPD substratum, 28 ℃, the 250r/min shaking culture is spent the night, OD600=1.3～1.5 o'clock, 4 ℃ of centrifugal 5min harvested cells of 1500g.

(3) with the 1M sorbyl alcohol washing of 100ml, 50ml ice precooling water and the precooling of 20ml ice, 4 ℃ of centrifugal 5min of 1500g with the 1M sorbyl alcohol suspension thalline of 0.8ml ice precooling, put ice bath to cell at last successively.

(4) recombinant vectors 10 μ l (100 μ g) that get linearization of Sal I and 80 μ l pichia spp competent cell mixings are transferred in the electrotransfer cup of 0.2cm of ice precooling then, place 5min on ice.

(5) the electrotransfer cup is placed on the electroporation apparatus with the pulsed current electric shock once electric commentaries on classics condition: voltage 1500V, electric capacity 25F, resistance 200 Ω, time 5ms, 0 ℃ of temperature.

(6) electric shock finishes; The 1M sorbyl alcohol that adds the precooling of 1ml ice immediately; Mixing, get 200 μ l conversion products be coated on ZE OCIN concentration gradient be 0.5,1.0,2.0,3.0,4.0 and the YPDS flat board of 5.0mg/mL on, put 30 ℃ of constant temperature culture 2～4 days; The high resistant strain of step-sizing ZEOCIN obtains the high copy of goal gene recombinant bacterial strain pPICZB-VP6/KM71.

Embodiment 4: the expression of recombinant protein pPICZB-VP6 albumen in the yeast expression bacterium

The high copy of picking screening colony inoculation is in 100ml BMGY substratum from the YPD-ZEOCIN flat board, and 28 ℃～30 ℃ shaking culture 16～18h are when OD600=2～6; The centrifugal 10min collecting cell of room temperature 1500g; The cell of collecting is suspended in the triangular flask of 250ml with 20ml BMMY substratum, and three layers of sterile gauze tying guarantee excellent air permeability; 28 ℃ of shaking culture abduction deliverings; Every 24h adds methanol concentration to 0.5% (V/V) in substratum, and the 24h sampling once, and sampling amount 1ml expresses to guarantee successive induction.

The bacterium liquid of collecting powders it through lyophilize, and suspend with the PBS of 100 μ l pH7.4 dissolves powder as far as possible.

1. recombinant protein pPICZ B-VP6 electrophoresis detection

The preparation of (I) SDS-PAGE solution:

Tris-glycine buffer: 25mmol/L Tris, 250mmol/L glycocoll (pH 8.0), 0.1%SDS.

The last appearance of 2 * SDS buffer:100mmol/L Tris.Cl (pH8.0), 200mmol/L mercaptoethanol, 4%SDS, 0.2% tetrabromophenol sulfonphthalein (W/V), 20% glycerine (V/V).

Amino black staining fluid (100ml): 0.5g amino black 10B is dissolved in 60ml water, 30ml ethanol, the 10ml glacial acetic acid.

Confining liquid: 10ml PBST+0.3g BSA.

Substrate solution (30ml): the DAB (diaminobenzidine) of dissolving 15mg among the 30ml PBST, 9mgCoCl6H ₂O adds 10 μ l 30%H ₂O ₂Use immediately behind the mixing.

5 * nucleic acid sample-loading buffer: 50% glycerine (V/V), 50mmol/L EDTA pH8.0,0.125% tetrabromophenol sulfonphthalein (W/V), 0.125% YLENE cyanogen (W/V).

Coomassie brilliant blue staining liquid: dissolving 0.25g coomassie brilliant blue R250 in 45ml methyl alcohol, 45ml water, the l0ml Glacial acetic acid min. 99.5.

Destainer: 30% methyl alcohol (V/V), 10% glacial acetic acid (V/V), the zero(ppm) water complement is long-pending to 100ml.

PBST solution: 5.8g Na ₂HPO ₄12H ₂O, 0.4g KCl, 0.4g KH ₂PO ₄, 16g NaCl, 1ml tween 20, the zero(ppm) water complement is long-pending to 2000ml.

(II)SDS-PAGE

(1) cleans sheet glass, be fixed on the electrophoresis chamber, with 2.0% agarose edge sealing.Press the separation gel 20ml of the formulated 15% of " molecular cloning ", inject rapidly between two sheet glass, Jiao Ding covers the 1ml distilled water.Treat that separation gel solidifies hypsokinesis and goes out tectum liquid, with deionized water rinsing gel top for several times, be inverted empty dry liquids, add the spacer gel solution of 2ml 5%, plug comb, vertically place electrophoresis chamber it is solidified.

(2) carefully remove comb, between the upper and lower groove of electrophoresis apparatus, add the Tris-glycine buffer, with the irrigation with syringe well for several times, power cathode is linked to each other with last groove.

(3) the bacterium liquid of collecting powders it through lyophilize, and with the PBS suspension of 100 μ l pH7.4, ultrasonic treatment is translucent to bacterium liquid; Add the last appearance of isopyknic 2 * SDS buffer, mixing, 5min is boiled in water-bath; With appearance 20 μ l on the microsyringe, opening power gets into separation gel with 80V voltage electrophoresis to tetrabromophenol sulfonphthalein; Bring up to 120V to voltage, continue electrophoresis to tetrabromophenol sulfonphthalein and arrive the gel bottom.

(4) dyeing: take off gel and use distilled water flushing,, be placed on room temperature dyeing 3h on the decolorization swinging table with the coomassie brilliant blue staining liquid immersion gel of 5 times of gel volumes.

(5) decolouring: the mixed solution with 30% (V/V) methyl alcohol, 10% (V/V) glacial acetic acid, on decolorization swinging table, decolour and spend the night, change staining fluid therebetween 3～4 times.After treating that blue background purifies fully, gel is immersed termination decolouring in the zero(ppm) water.

Embodiment 5: the application of recombinant protein pPICZB-VP6 in preparation grass carp hemorrhage disease vaccine

(I) preparation of Western-blot analytical solution:

Washings solution: 150mmol/L NaCl

50mmol/L Tris-HCl pH7.5

Transfering buffering liquid: 39mmol/L glycocoll

48mmol/L Tris alkali

0.037% SDS（W/V）

20% methyl alcohol (V/V)

Amino black staining fluid (100mL): 0.5g amino black 10B is dissolved in 40mL distilled water, 50mL methyl alcohol, the 10mL glacial acetic acid.

Confining liquid: 10mL PBST+0.3g BSA

Substrate solution (30mL): the DAB (diaminobenzidine) and the 9mg CoC126H that in 30mL PBST, add 15mg ₂0 makes its dissolving, adds 10 μ l 30%H ₂0 ₂Use immediately behind the mixing.

(II) Westen-blot of recombinant expression protein analyzes

(1). transfer printing: cut 8 filter paper and 1 nitrocellulose filter (NC); Size equates with gel; In transfering buffering liquid, soak 3min; Order by four metafiltration paper, NC film, SDS-PAGE running gel, four metafiltration paper on positive plate stacks neatly, confirms negative plate on the no bubble bonnet, and 110mA shifted l hour.

(2). sealing: after shifting completion, take off the NC film, downcut standard molecular weight Marker, put in the amino black staining fluid and contaminate 5min, take out the back, take off most until the background blueness with the rinsing decolouring.All the other NC films wash with PBS, add the 10mL confining liquid, and room temperature is jolting 1-2hr gently.

(3). with combining of antibody: after sealing finishes; Towards Xian NC film 4-5 time, add the proteic polyclonal antibody 10mL of mouse-anti GCRV 104 strain VP6 that PBST 1:200 doubly dilutes with PBS, 37 ℃ combine 1.5 hours; With PBST flushing 4-5 time, slowly shake at shaking table at every turn.The sheep anti-mouse antibody of the horseradish peroxidase-labeled that adding 1:2000 doubly dilutes (two is anti-), jog was hatched 1 hour under the room temperature, took out with PBST flushing 3-4 time each 10min.

(4). the substrate colour developing: the NC film is transferred in the 10mL substrate solution, and room temperature lucifuge jog 3min observes the colour developing situation, waits when band occurring, changes in the PBST damping fluid room temperature preservation immediately over to.

(5). recombinant expression protein immunity qualification result

Reorganization pPICZB-VP6 expression product carries out western blot test through being transferred to behind the SDS-PAGE on the NC film; Tangible positive reaction band appears in the result about 46kDa; Explain that expression product can be discerned by the proteic polyclonal antibody of mouse-anti GCRV 104 strain VP6, have immunity.

More than good antigen one antibody response can take place with the proteic polyclonal antibody of mouse-anti GCRV 104 strain VP6 in the VP6 albumen of test explanation expression, and VP6 albumen has immunocompetence, can be used as the hemorrhagic disease of grass carp vaccine antigen protein fully.

SEQUENCE LISTING

< 110>Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences

< 120>a kind of hemorrhagic disease of grass carp antigen protein and preparation method and application

< 130>a kind of hemorrhagic disease of grass carp antigen protein and preparation method and application

<160> 1

<170> PatentIn version 3.1

<210> 1

<211> 447

<212> PRT

< 213>GCRV GCRV-104

<400> 1

Met Ala Arg Val Val Tyr Val Phe Tyr Gly Ser Gln Trp Phe Thr Leu

1 5 10 15

Gln Asn Pro Ala Phe Asn Val Asp Asp Val Ser Val Ile Ser Thr Ser

20 25 30

Gly Ala Gly Thr Trp Gly Lys Leu Ala Glu Val Asn Ala Ile Asp Gly

35 40 45

Val Arg Gln Thr Thr Gly Asn Asp Arg Ser Pro Leu Phe Ile Ser Arg

50 55 60

Leu Leu Lys Leu Gly Gln Leu Ala Thr Val Trp Leu Pro Ser Leu Ser

65 70 75 80

Arg Ala Leu Gln Phe Arg Phe Glu Gln Tyr His Gly Ala Ile Leu Arg

85 90 95

Ser Pro Ala Ile Asp Ala Leu Val Leu Val Pro Arg Ala Arg Val Pro

100 105 110

Pro Ala Val Val Pro Ala Glu Ala Gly Leu Tyr Asp Leu Met Asn Phe

115 120 125

Pro Arg Phe Thr Glu Leu Pro Gly Gln Met Trp Glu Met Val Leu Asp

130 135 140

Ser Cys Gly Ile Thr Met Asp Ala Met Ala Val Gln Ser Met Pro Leu

145 150 155 160

Tyr Ile Gly Phe Glu Asn Gln Ala Pro Val Ala Asp Leu Met Ser Gln

165 170 175

Met Pro Thr Phe Val Gly Arg Ser Ile Thr Ser Ile Ala Ala Ile Leu

180 185 190

Tyr Ser Gln Ser Arg Asn Ser Ser Gly Val Pro Asp Pro Leu Ile Gly

195 200 205

Arg Thr Ala Arg Leu Leu Ser Thr Ile Thr Leu Leu Ser Phe Thr Gly

210 215 220

His Met His Asn Asp Ala Thr Tyr Tyr Gly Phe Tyr Val Ser Arg Pro

225 230 235 240

Arg Glu Thr Lys Ser Val Glu Gly Ala Ile Leu Met Tyr Gln Gln Gly

245 250 255

Ala Asn Ile Ile Pro Leu Thr Asn Ala Asn Pro Asn Gly Ala Tyr Thr

260 265 270

Ala Val Gly Ile Gly Asp Pro Leu Trp Gln Leu Ser Pro Asp Leu Tyr

275 280 285

Ala Met Phe Leu Leu Asn Ile Val Tyr Ala Ala Asn Cys Val Thr Pro

290 295 300

Val Leu Asp Thr Thr His Leu Ala Pro Met Arg Thr Trp Ala Val Ala

305 310 315 320

Gly Gly Met Asn Met Tyr Arg Leu Asn Trp Arg Asp Ala Leu Arg Arg

325 330 335

Lys Ile Arg Val Asp Arg Gln Arg Gly Ile Ile Thr Gln Pro Glu Glu

340 345 350

Asn Asp Cys Leu Gln Ala Val Asp Asp Tyr Ala Arg Asp Cys Ala Ala

355 360 365

Leu Leu Asp Asp Val Leu Ala Arg Gln Thr Ala Phe Asn Arg Ala Asn

370 375 380

Pro Gly Gly Glu Thr Met Arg Val Lys Pro Phe Ala Gln Ala Asp Tyr

385 390 395 400

Arg Asn Pro Phe Glu Phe Val Pro Ala Met Ile Ser Leu Thr Arg Val

405 410 415

Thr Thr Pro Ala Ala Ala Ala Ser Phe Leu Glu Gln Lys Leu Ile Ser

420 425 430

Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His His His

435 440 445

Claims (4)

1. a GCRV is characterized in that: GCRV GCRV-104, CCTCC NO:V201217.

2. a kind of hemorrhagic disease of grass carp antigen protein that GCRV according to claim 1 makes is characterized in that: its sequence is the aminoacid sequence shown in the SEQ ID NO.1.

3. the preparation method of the described a kind of hemorrhagic disease of grass carp antigen protein of claim 2 the steps include:

A. rt grass carp GCRV VP6 gene:

Using GCRV GCRV-104, therefrom extract virus total RNA, is template rt grass carp GCRV VP6 gene with total RNA;

Design a pair of primer according to the complete nucleotide sequence of GCRV VP6 gene and carry out RT-polymerase chain reaction, the clone obtains the VP6 protein gene; Used primer sequence is:

P1:5 '-CGG AATTCATGGCACGTGTGGTTTAT-3 ', line place does EcoRThe I restriction enzyme site;

P2:5 '-TCC CCGCGGGTGTGGTTACGCGGGTCAG-3 ', line place does SacThe II restriction enzyme site;

Reaction conditions is: 48 ℃ of rts 45 minutes; 94 ℃ of deactivations and preparatory sex change 5 minutes; Again through 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute, react 40 circulations; 72 ℃ were extended 10 minutes;

B. make up the yeast expression recon:

Steps A RT-polymerase chain reaction product and pPICZ B carrier are used respectively EcoRI with SacThe II double digestion makes two products connect then under the effect of ligase enzyme, is built into pPICZ B-VP6 expression vector, cuts evaluation through polymerase chain reaction evaluation and enzyme and adds their confirmation;

C. make up the restructured Pichia pastoris in expression bacterium, breed and carry out abduction delivering restructured Pichia pastoris in expression bacterium;

The recon electricity that obtains among the step B is changed in the pichia spp; Getting 200 μ l conversion products is coated on the YPDS flat board that the ZEOCIN concentration gradient is 0.5,1.0,2.0,3.0,4.0 and 5.0 mg/mL; Put 30 ℃ of constant temperature culture 2～4 days; The high resistant strain of step-sizing ZEOCIN obtains the high copy of goal gene recombinant bacterial strain;

The high copy of picking screening colony inoculation is in 100 ml BMGY substratum from the YPD-ZEOCIN flat board, and 28 ℃～30 ℃ shaking culture 16～18 h are when OD600 is 2～6; The centrifugal 10 min collecting cells of room temperature 1500 g; The cell of collecting is suspended in the triangular flask of 100 ml with 20 ml BMMY substratum, and three layers of sterile gauze tying guarantee excellent air permeability; 28 ℃ of shaking culture abduction deliverings; Every 24h adds methanol concentration to 0.5%V/V in substratum, and 24 h sampling once, and sampling amount 1 ml expresses to guarantee successive induction;

D. obtain used antigen protein through separation and purification: collect the bacterium liquid that obtains among the step C and it is powdered through lyophilize; PBS suspension with 100 μ l pH7.4 makes powder dissolving as far as possible; Ultrasonic treatment is translucent to bacterium liquid, and the 46KDa place is target protein behind the SDS-PAGE electrophoresis.

4. the application of the described a kind of hemorrhagic disease of grass carp antigen protein of claim 2 in preparation grass carp hemorrhage disease vaccine.

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