CN101792742A - Recombinant baculovirus expressing IBDV (Infectious Bursal Disease Virus) immunogen gene and preparation method and application thereof - Google Patents

Abstract

The invention discloses a recombinant baculovirus expressing IBDV (Infectious Bursal Disease Virus) immunogen genes and a preparation method and application thereof, particularly relates to a recombinant baculovirus comprising a sequence shown as SEQ ID NO.1 and displaying structural proteins VP2 of the IBDV on the surface. The preparation method comprises steps of utilizing the recombinant baculovirus to inoculate insect cells, culturing for 48-72h, collecting infection cells, carrying out freeze thawing at 40DEG C below zero/37DEG C, centrifuging, taking supernate to test virus PFU and adjusting the titer of the virus to 109PFU/ml to obtain a safe and effective gene engineered subunit vaccine for preventing IBD.

Description

Express recombinant baculovirus and the preparation and the application of IBDV immunogen gene

Technical field

The present invention relates to a kind of recombinant baculovirus and preparation and application of the IBDV of expression immunogen gene.

Background technology

IBD (infectious bursal disease) is a kind of acute, the height contagious disease of the harm chick that caused by IBDV (infectious bursal disease virus), is one of main transmissible disease of harm world aviculture.Central immune organ-fabricius bursa of main infringement chick causes immunosuppression, thereby enhancing body is to the susceptibility and the reactivity that reduces other vaccine of other pathogenic agent.

The IBD that takes place in the world has two serotypes at present: serum I type and serum II type.Serum II type strain can be divided into classical strains (also claiming standard serum I type), variant (also claiming the hypotype strain) and highly virulent strain again to the chicken no pathogenicity in the serum I type.Classical strains is a feature with the oedema that causes the fabricius bursa, is widely current all over the world; Variant causes the rapid atrophy of the fabricius bursa, does not see the process that oedema is arranged, and has very strong immunosuppressive action; Highly virulent strain causes the severe haemorrhage of the fabricius bursa, and outward appearance is like " purple grape " sample, and mortality ratio is up to more than 70%.There is three types IBDV strain simultaneously in China, thereby has caused enormous economic loss for China's aviculture.

IBDV belongs to birnavirus section, Avibirnavirus, and genome is by A, and two double-stranded RNA sections of B are formed.A sheet segment length 3.3kp comprises two complete open-reading frames (ORF), the three kinds of virus structural proteins of VP2-VP4-VP3 of encoding respectively; B sheet segment length 2.9kp has a successive open reading frame (ORF), coding VP1 albumen.Wherein VP2 is the primary structure albumen of IBDV, contains type specificity, can induce the antigen decision family of neutralizing antibody, is main host protective antigen.

IBDV is a kind of important virus of present serious threat aviculture development.Because this virus central immune organ-fabricius bursa of mainly encroaching on chick finally causes immunosuppression, thus enhancing body to the susceptibility of other pathogenic agent with reduce reactivity to other vaccine, the result increases the weight of epidemic situation.IBDV variant and highly virulent strain is popular in recent years, and traditional weak malicious seedling and deactivation vaccine immuning failure often have generation.

At present the control of IBD disease is not still had effective method, the vaccine of anti-system IBD mainly contains deactivation vaccine and weak malicious seedling, and IBDV variant and highly virulent strain is popular in recent years, and traditional weak malicious seedling and deactivation vaccine immuning failure often have generation.Therefore developing new generation vaccine, to control IBDV be the task of top priority.

Summary of the invention

In order to address the above problem, the invention provides a kind ofly in order to address the above problem, the invention provides a kind of recombinant baculovirus and can be used as the subunit vaccine that prevention IBD infects.

A kind of recombinant baculovirus of expressing the IBDV immunogen gene provided by the invention has the sequence shown in the SEQ IDNO.1.Described recombinant baculovirus surface display IBDV structural protein VP2.

The invention provides a kind of preparation method of above-mentioned recombinant baculovirus, step comprises:

A, preparation VP2 gene.The preparation method is: with the PCR method IBDV Shaanxi strain VP2 gene that increased, and it is cloned into the pMD18-T carrier, has made up the pMD18T-VP2 plasmid;

B, with the VP2 gene of IBDV, be inserted into the p10 promotor downstream of baculovirus surface display transposon vector pBacSC, make up the recombinant baculovirus swivel base plasmid pBacSC-VP2 of the VP2 gene contain IBDV;

C, above-mentioned recombinant baculovirus swivel base plasmid pBacSC-VP2 changed over to carry out homologous recombination in the intestinal bacteria, obtain the recombinant baculovirus genomic dna.As preferably, described intestinal bacteria are competence intestinal bacteria DH10Bac.

D, with the recombinant baculovirus DNA transfection insect cell that step c obtains, obtain recombinant baculovirus.Preferably, recombinant baculovirus DNA is changed among the insect cell Sf9 by liposome-mediated method, pack out recombinant baculovirus.

The invention provides a kind of above-mentioned application of recombinant baculovirus in preparation chicken infectivity bursa of Fabricius virus vaccine.

The invention provides a kind of method for preparing the chicken infectivity bursa of Fabricius virus vaccine: recombinant virus inoculation insect cell, cultivate 48～72h, collect cells infected ,-40 ℃/37 ℃ freeze thawing, centrifugal, to get supernatant and measure viral PFU, the titre of adjusting virus makes it reach 10 ⁹PFU/ml.The preferred Sf9 insect cell of above-mentioned insect cell.

Advantage of the present invention and positively effect are:

But 1, the recombinant baculovirus BacSC-VP2 surface display IBDV structural protein VP2 that obtains of the present invention, also just obtained target protein when directly obtaining baculovirus, can avoid expression system in the prior art (comprising traditional baculovirus expression system) to express the complicated processes of target protein separation and purification through ultracentrifugation.And; the target protein of expressing in the expression system of prior art is to be secreted in the substratum of Sf9 cell; need come the purifying target protein by protein purification system; waste time and energy like this; cost dearly; can not large-scale application in the large-scale production of target protein, this also is the most troubling place of present protein expression.

2, add the eGFP gene in the transposon vector, can simplify the mensuration of virus titer greatly by the production that detects recombinant virus that has or not of fluorescent microscope direct viewing fluorescence.

3, the albumen of this recombinant baculovirus surface display can with IBDV antibody generation specific reaction.

4, the strain recombinant baculovirus BacSC-VP2 that obtains of the present invention can stimulate mouse to produce effective humoral immunization and cellular immunization.

5, the strain recombinant baculovirus BacSC-VP2 that obtains of the present invention is safe to laboratory animal, does not have any pathological phenomena.

6, the strain recombinant baculovirus that obtains of the present invention has good genetic stability, and foreign gene is not lost after repeatedly going down to posterity.

7, goal gene used in the present invention is the domestic strain isolated of IBDV (highly virulent strain separates from Shaanxi), thereby the recombinant baculovirus that makes up can be used as the subunit vaccine of China prevention IBD.

Description of drawings

Fig. 1 is that reorganization swivel base plasmid pBacSC-VP2 makes up schema.

Fig. 2 is the expression that recombinant baculovirus BacSC-VP2 infects Sf9 cell green fluorescent protein.

Fig. 3 is the displaying of VP2 albumen on the Sf9 cytolemma that the conjugation focusing microscope is analyzed recombinant baculovirus BacSC-VP2.

Fig. 4 is that the proteic Western blot of the VP2 of recombinant baculovirus BacSC-VP2 analyzes.

Fig. 5 is that immuno-electron microscope is analyzed the displaying of VP2 albumen on recombinant baculovirus BacSC-VP2 cyst membrane.

Label declaration among Fig. 5: A. recombinant baculovirus BacSC-VP2; B. negative control group, recombinant baculovirus BacSC.

Embodiment

The invention will be further described below in conjunction with the drawings and specific embodiments, can be implemented so that those skilled in the art can better understand the present invention also, but illustrated embodiment is not as a limitation of the invention.

The present invention is with the PCR method IBDV Shaanxi strain VP2 gene (concrete sequence see SEQ IDNO.1) that increased, and it is cloned into the pMD18-T carrier, made up the pMD18T-VP2 plasmid.Enzyme is cut the pMD18T-VP2 plasmid, reclaims the VP2 gene.The IBDV structural protein gene VP2 that reclaims is inserted into the p10 promotor downstream of baculovirus surface display transposon vector pBacSC, makes up the recombinant baculovirus swivel base plasmid pBacSC-VP2 of the VP2 that contains IBDV; The recombinant plasmid that makes up is changed in baculovirus/insect expression system competence intestinal bacteria DH10Bac, make it carry out homologous recombination, and carry out resistance screening with kantlex, gentamicin and tsiklomitsin.(PCR), the immune protein marking (western blot), laser confocal microscope and immunoelectron microscope test detect recombinant virus through the polymerase chain reaction respectively, and screening obtains positive recombinant baculovirus; With the screening immune respectively BALB/c mouse of positive recombinant baculovirus and chicken and carried out the detection of amynologic index.

The building process of pBacSC carrier:

1.1 primer design

Gp64 genome sequence and green fluorescent protein (eGFP) gene (GenBank:EU048697) genome sequence according to the baculovirus AcMNPV (GenBank:AY542374) that delivers design 3 pairs of Auele Specific Primers (P1/P2, P3/P4, P5/P6).

Upstream primer P1 5 '-TCA CCCGGGATGCTACTAGTAAATCAGTCACACCAAGGCTTCAATAAGGAACACACAAGCAAGAT GGTAAGCGCTATTGTTTTATATGTGCTTTTG-3 ', 5 ' end adds the SmaI restriction enzyme site; Downstream primer P2 5 '-GTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGCGCACCACCA CCACCACCACGCGCGCCCATGGGCTAGCGCATGCGCATGC-3 '.Upstream primer P3 5 '-GCGCGCCCATCGCATGCTTCAGGGCTAGTGTTTGGTCATGTA-3 '; Downstream primer P4 5 '-CCC GGTACCTTAATATTGTCTATTAC-3 ', 5 ' end adds Kpn I restriction enzyme site.Expection amplification segment is 267bp, contains GP64 signal peptide (SP) sequence, 6 Histidines (His6) label, and 4 multiple clone site (BsshI, NcoI, NheI, SphI), GP64 strides film district (TM) and GP64 cytoplasmic region (CTD) gene.

Upstream primer P5 5 '-GCT GGATCCATGGTGAGCAAGGGCGAGGAGCTGT-3 ', 5 ' end adds the BamHI restriction enzyme site.Downstream primer P6 5 '- AAGCTTCTTGTACAGCTCGTCCATGCCGAGA-3 ', 5 ' end adds the HindIII restriction enzyme site.Expection amplification segment is 717bp, contains complete green fluorescence protein gene (eGFP) gene.

1.2SP-His6-the amplification of four restriction enzyme site-TM-CTD sequences (concrete sequence is seen SEQ IDNO.2)

The PCR reaction system is as follows: 10 * PCR Buffer, 5 μ L, dNTPs 4 μ L, P1/P2, P3/P4 each 1 μ L, Taq DNA polymerase 1 μ L add water to 50 μ L.Response procedures: 95 ℃ of sex change 5min; 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 30sec, totally 30 circulations; Last 72 ℃ are extended 10min.Get 10 μ L PCR products after reaction finishes and add 1 μ L, 6 * Loading Buffer in 0.8% agarose gel electrophoresis, analysing amplified product

1.3eGFP the amplification of gene

The PCR reaction system is as follows: 10 * PCR Buffer, 5 μ L, dNTPs 4 μ L, P5/P6 each 1 μ L, TaqDNA polymerase 1 μ L, pEGFP-C1 plasmid (BD Biosciences Clontech) 1 μ L, add water to 50 μ L.Response procedures: 95 ℃ of sex change 5min; 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 2min, totally 30 circulations; Last 72 ℃ are extended 10min.Get 10 μ L PCR products after reaction finishes and add 1 μ L, 6 * Loading Buffer in 0.8% agarose gel electrophoresis, analysing amplified product.

1.4PCR product is connected with pFastBacTM Dual carrier

Give birth to worker UNIQ-10 glue with reference to Shanghai and reclaim test kit specification sheets recovery goal gene.BamHI and HindIII restriction enzyme site place with the multiple clone site (MCS) in the Polyhedfin promotor downstream of eGFP gene insertion vector pFastBacTM Dual (Life Technologies).With quadrat method four restriction enzyme sites of SP-His6--TM-CTD gene is inserted into the SmaI and the KpnI restriction enzyme site place of the another one p10 promotor downstream multiple clone site (MCS) of the pFastBacTM Du al carrier that has inserted the eGFP gene, thereby successfully constructs the pBacSC carrier.Concrete working method, parameter all are to carry out according to the molecular biology ordinary method.

Narrate specific implementation method of the present invention below:

1.IBDV the clone of Shaanxi strain VP2 gene

1.1 primer design

According to the genome sequence of the I BDV OKYM strain of delivering (GenBank:D49706.1), design 1 pair of Auele Specific Primer (P1/P2).Upstream primer P1 5 '-GCTCTCGAGATGACAAACCTGCAAGATC-3 ', 5 ' end adds Xh oI restriction enzyme site.Downstream primer P2 5 '-GGGGAATTCCCTTAGGGCCCGGATTATG-3 ', 5 ' end adds EcoR I restriction enzyme site.Expection amplification segment is 1356bp, contains complete VP2 gene.

1.2 virus genomic amplification

Take out the IBDV Shaanxi of-80 ℃ of preservations and separate the wild poison of IBDV, extract test kit according to Trizol and extract viral RNA.Get RNA and make template 3 μ L, add VP2 upstream and downstream primer P1/P2 each 1 μ L, dNTP 4 μ L, RNase-Inhibitor 0.5 μ L, AMV 0.5 μ L, 5 * AMV Buffer, 4 μ L, add DEPC H ₂O to 20 μ L, 42 ℃ of 1h promptly get the cDNA template;

The PCR reaction system is as follows: 10 * PCR Buffer, 5 μ L, dNTPs 4 μ L, P1/P2 each 1 μ L, rTaq1 μ L, cDNA 6 μ L, add water to 50 μ L.Response procedures: 95 ℃ of sex change 5min; 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 2min, totally 30 circulations; Last 72 ℃ are extended 10min.Get 10 μ L PCR products after reaction finishes and add 1 μ L, 6 * Loading Buffer in 0.8% agarose gel electrophoresis, analysing amplified product.

1.3PCR the purifying of product and recovery

Give birth to worker UNIQ-10 glue with reference to Shanghai and reclaim test kit specification sheets recovery goal gene.

2. the preparation of competent escherichia coli cell (Calcium Chloride Method)

Scrape with the aseptic inoculation ring and to get frozen intestinal bacteria bacterial classification in-70 ℃ of refrigerators, streak inoculation is cultivated about 16h for 37 ℃ in the LB agar plate that does not contain penbritin (Amp).The single bacterium colony of picking, be inoculated in the 100mlLB substratum, 37 ℃, 250r/min jolting are cultivated OD600=0.4～0.6, under aseptic condition, inoculum transferred in two aseptic and polypropylene centrifuge tubes (following operation all needs aseptic) with the ice precooling, ice bath 10min makes culture be cooled to 0 ℃; 4 ℃, the centrifugal 10min of 2000r/min abandon supernatant, with the ice-cold 75mmol/L CaCl of 10ml ₂, the resuspended precipitation of 10mmol/L (pH6.5) solution, ice bath 10min, 4 ℃, the centrifugal 10min of 2000r/min abandons supernatant, and is ice-cold with 2ml, contain 15%v/v) the 75mmol/L CaCl of glycerine ₂, the resuspended every pipe precipitation of 10mmol/L TrisCl (pH6.5) solution, competent cell is sub-packed in the aseptic Eppendorf tube every pipe 200 μ l with aseptic suction nozzle; Indicate bacterial strain, volume and date, it is frozen standby to put-70 ℃ of refrigerators.

3.VP2 the ligation of gene fragment and pMD18-T carrier

Get 0.5 μ g pMD18-T carrier, the VP2DNA fragment that adds 3～5 times of molar weights, 2 μ l 5 * connection damping fluid adds water and is settled to 10 μ l, add the T4DNA of 1Weiss unit ligase enzyme at last, mixing is also centrifugal, 16 ℃ connect 1～4h, get 7 μ l ligation liquid and transform above-mentioned E co.li competent cell.

4. transform

(volume: plasmid＜1 μ l connects product＜10 μ l, DNA＜50ng) add in the above-mentioned competent cell of 200 μ l, mixing gently, ice bath 30min will to connect product D NA in right amount; 42 ℃ of water-bath thermal shocking 90s, ice bath cooling 2min; The LB substratum that adds 37 ℃ of preheatings of 200 μ l, 50min is cultivated in 37 ℃ of 150r/min joltings; Get nutrient solution and coat the LB agar plate that contains Amp (50 μ g/ml), behind 37 ℃ of cultivation 14h～16h, obtain transforming bacterium colony.

5. the extraction of plasmid (alkaline lysis)

The single above-mentioned conversion bacterium colony of picking is inoculated into 2ml and contains in the LB nutrient solution (down together) of 50 μ g/ml Amp, and 12～16h are cultivated in 37 ℃ of 250r/min joltings; 1.5ml is changed in the Eppendorf tube, and the centrifugal 30s of 12000r/min abandons supernatant.Solution I (50rnmol/L glucose with the precooling of 200 μ l ice; 25mmol/L TrisCl, pH8.0; 10mmol/L EDTA, pH8.0) resuspended precipitation, add the new preparation of 200 μ l solution II (0.2mol/L NaOH, 1%SDS), put upside down mixing for several times, add the ice-cold solution III of 200 μ l (3mol KAc, 5mol/L glacial acetic acid), put upside down mixing, 4 ℃ of centrifugal 10min of 12000r/min, get supernatant, use the saturated phenol/chloroform of equivalent/primary isoamyl alcohol (25: 24: 1) respectively, each extracting of chloroform/primary isoamyl alcohol (24: 1) once; Add 2 times of cold dehydrated alcohols of volume, mix, put-20 ℃ of 30min, 4 ℃ of centrifugal 10min of 12000r/min abandon supernatant, and precipitation is drained with 70% cold washing with alcohol.With 20 μ l contain final concentration 20 μ g/ml RNA enzymes (no DNA enzyme) TE (10mmol/L Tri sHCl, pH8.0,1mmol/LEDTA, pH8.0, down with) dissolution precipitation, and in 37 ℃ of water-baths, act on 30min, agarose gel electrophoresis inspection or-20 ℃ of preservations.

6. the enzyme of recombinant plasmid pMD18T-VP2 is cut evaluation

With 1.0 μ g plasmid DNA and suitable quantity of water mixing, making its cumulative volume is 18 μ l, add 2～3 EcoR I of unit and Xho I restriction enzyme and the corresponding 10 * restriction enzyme reaction damping fluid of 1 μ l respectively, flick tube wall mixing and centrifugal, put optimal reactive temperature water-bath 2～3h, the agarose gel electrophoresis inspection.Enzyme cut the result all with estimate identical person, be the purpose recombinant plasmid.Behind the complete degestion ,-20 ℃ of preservations are in order to further identifying or reclaim the usefulness of fragment.

7. carry the structure of VP2 gene recombination baculovirus transposon vector plasmid

With XhoI and EcoRI double digestion pMD18T-VP2 plasmid, reclaim the VP2 gene, be inserted into same baculovirus transposon vector pBacSC, carry out ligation, make up the baculovirus transposon vector pBacSC-VP2 that contains the VP2 gene through XhoI and EcoRI double digestion.Concrete test operation process is identical with the pMD18T-VP2 construction of recombinant plasmid.

8. homologous recombination

8.1 preparation " three is anti-" LB flat board

LB liquid nutrient medium behind the high pressure steam sterilization (containing 1.5% agar powder) is put in the super clean bench, treat that its temperature adds following three kinds of microbiotic to final concentration and is when being cooled to 50 ℃ of left and right sides: kantlex (Kan) 50 μ g/mL, gentamicin (Gm) 7 μ g/ml and tsiklomitsin (Tet) 10 μ g/ml, and (final concentration is that the shop system is dull and stereotyped immediately behind 150 μ g/mL and IPTG (final concentration the is 40 μ g/mL) mixing to add X-gal.

8.2 conversion process

Getting recombinant plasmid pBacSC-VP2 10 μ L (4 μ g) adds in the competence intestinal bacteria DH10Bac pipe that 100mL prepares, mixing gently, centrifuge tube is put into mixture of ice and water ice bath 30min, centrifuge tube is put into 42 ℃ of heat-shocked 2min, move to rapidly again and place 5min in the frozen water.The LB liquid nutrient medium 900mL that adds sterilization, 37 ℃ of shaking table 200rpm/min shaking culture 1h add kantlex (final concentration is 50 μ g/mL), gentamicin (final concentration is 7 μ g/ml) back continuation shaking table 200rpm/min shaking culture 4h.The centrifugal 10min of 6000rpm/min, retain bacteria precipitation and the about 300 μ L of LB liquid, with fresh LB liquid nutrient medium with it with 10 ^-1, 10 ^-2With 10 ^-3Different dilutions.Respectively get 100 μ L then and evenly coat " three is anti-" LB flat board respectively, plate is inverted in 37 ℃ is cultivated 24～48h, observe the growing state of blue white bacterium colony.

8.3 hickie is identified

5～10 of toothpick pickings with sterilization are separated good white colony, are inoculated in once more on the flat board that contains " three is anti-", are inverted for 37 ℃ and cultivate 24h, observe the variation of bacterium colony phenotype, further turn out to be white colony.Again with sterilization 6 white colonies of toothpick picking and 2 blue colonies, put into the test tube that 6ml LB substratum is housed respectively, add kantlex (Kan) 50 μ g/mL, gentamicin (Gm) 7 μ g/ml and tsiklomitsin (Tet) 10 μ g/ml again, three kinds of microbiotic, 37 ℃ of shaking table 200rpm/min shaking culture are spent the night.

8.4 the extraction of recombinant baculovirus DNA and preparation

Above-mentioned cultivation bacterium liquid branch is filled in the Eppendorf pipe of 1.5mL the centrifugal 1min of 12000rpm/min.Abandon clean supernatant, add 300 μ L solution I (it is standby to sterilize for 50mmol/L Tris-HCL pH8.0,10mmol/L EDTA), resuspended precipitation.(0.2mol/L NaOH, 1%SDS), gentleness is put upside down mixing, puts 5min in the ice-water bath, makes the bacterium cracking to add 300 μ L solution II.(water 28.5ml pH5.5), slightly shakes in the adition process, places 10min on ice for 5mol/L potassium acetate 60ml, glacial acetic acid 11.5ml slowly dropwise to add 300 μ L solution III.The centrifugal 10min of 12000r/min with good another the clean Eppendorf pipe of markers, adds 800 μ L Virahols.Gently supernatant is transferred in the new pipe that has added Virahol, notes not sucking albumen precipitation, centrifuge tube is put upside down mixing for several times, place room temperature 15min, the centrifugal 10min of 12000r/min abandons supernatant.With each washing of 75% and 100% ethanol once, the centrifugal 5min of 14000r/min abandons supernatant respectively, and air-dry (under the aseptic condition) 1h is dissolved in 40 μ L TE damping fluids.

9. recombinant baculovirus DNA transfection Sf9 cell

In 6 orifice plates, add Sf9 insect cell 9 * 10 in each hole ⁵Cells, substratum 2mL Sf900IISFM, containing two anti-concentration is 0.5 * final concentration (50units/ml penicillin, 50 μ g/ml streptomycin).Cell is from the logarithmic phase that is in of cultivating 3～4 days, and vigor is greater than 97% cell, and places 27 ℃ of incubator 1h; In this 1h, prepare following experiment.Get 2 1.5ml Eppendorf pipes and be designated as A, B, add the Sf-900II SFM nutrient solution of 100 μ L antibiotic-frees respectively, add the above-mentioned recombinant baculovirus DNA of 5 μ L in the A pipe, mixing.Add 6 μ L lipofectamine Cellfectin in the B pipe, mixing (noticing that liposome is a greasy suspended substance, may precipitate that put upside down pipe before the use 5～10 times, it is even that it is mixed).Mix A pipe and B pipe, mixing is put room temperature 45min gently, makes it form the Cellfectin-DNA mixture.Get the good Sf9 monolayer cell of adherent growth, abandon old nutrient solution, use the Sf900II SFM nutrient solution of antibiotic-free to wash 1 time.Get above-mentioned Cellfectin-DNA mixture, adding 0.8ml does not contain antibiotic Sf900II SFM and mixes.From cell, inhale and abandon the substratum that cleans usefulness, liposome and the recombinant DNA mixture that mixes is taped against on the cell, cover cell, places 28 ℃ of cultivation 6h after.Mixture is abandoned in suction, adds to contain antibiotic Sf900II SFM nutrient solution continuation cultivation, observation of cell form performance every day.Collecting cell is used for the analysis of expressing protein, collects culture supernatant simultaneously as former seed culture of viruses, divides tubule to be stored in-20 ℃, is used for infecting again the Sf9 cell.

10. the PCR of recombinant baculovirus BacSC-VP2 identifies

Get the cell culture 2mL of virus, multigelation 3 times, the centrifugal 15min of 12000r/min gets supernatant 437.5 μ L, adds 12.5 μ L Proteinase Ks (20mg/mL) and 50 μ L SDS liquid (10%), 37 ℃ of water-bath 30min; Equal-volume phenol/chloroform extracting is once got supernatant; The equal-volume chloroform is carried again, gets supernatant; Add 1/10 volume NaAc (2mol/L) and 2 times of volume dehydrated alcohols, place 2h for-20 ℃; 4 ℃ of centrifugal 15min of 15000r/min get precipitation; 70% washing with alcohol once precipitates with the dissolving of 30 μ L TE solution, is the DNA of extraction, and-70 ℃ of preservations are standby.Getting 5 μ L during PCR gets final product.After the PCR reaction conditions is 95 ℃ of pre-sex change 5min, by 95 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 2min totally 30 circulations, and last 72 ℃ are extended 10min.

11. the amplification of recombinant baculovirus amount is with concentrated

The suspension culture insect cell treats that cell concn reaches 1 * 10 ⁶Cell/ml, (Multiplicity ofinfection, recombinant baculovirus amount infected insect cell MOI) utilize virus to duplicate in the host insect cell and reach the purpose that virus quantity amplifies with MOI=1.Infect centrifugal cell and the cell debris removed after 3～4 days, collect the amplification that supernatant liquor is promptly finished virus quantity.

12. the detection of expression of recombinant virus product

12.1 fluorescence microscope is identified recombinant virus

Have the eGFP gene on the reorganization pBacSC-VP2 transposon vector, can expressing green fluorescent protein, can confirm the transfection positive cell by the cell quantity that fluorescence microscope sends fluorescence.After the transfection behind the 48h cell present tangible CPE and change, fluorescence appears in transfectional cell.(seeing accompanying drawing 2) produced the recombinant baculovirus BacSC-VP2 with infection activity.

12.2SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

With the virus of the purification of Recombinant after amplification BacSC-VP2, be inoculated in 10ml culturing bottle 1 * 10 respectively with 10MOI ⁶In individual/ml Sf9 cell, establish the contrast of baculovirus negative control group and cell simultaneously, when obvious pathology appears in 75% cell, abandon nutrient solution, with TEN (40mmol/L TrisCl pH7.5, the 1mmol/L EDTA of 1ml, 150mmol/L NaCl) wash-out cell, be collected in the Eppendorf pipe, the centrifugal 5min of 3000r/min abandons supernatant, cell precipitation is washed 3 times with PBS, add 60 μ l lysis buffer (10mmol/LTrisCl, pH7.4,1mmol/L MgCl ₂, 0.5%NP40,20 μ g/ml DNase I) and cracking, ice bath 30min boils 3min, the centrifugal 5min of 5000r/min ,-20 ℃ are frozen.

Get 30 μ l cell pyrolysis liquids and equivalent 2 * sample buffer (100mmol/L Tris Cl, pH6.8,4%SDS, 0.2% tetrabromophenol sulfonphthalein, 20% glycerine, adding 2.5% beta-mercaptoethanol before using) mixing, carry out the SDS-PAGE electrophoresis in 10% gel.

12.3 immunoblotting (Western blot)

Behind the SDS-PAGE protein isolate, with its electrotransfer to nitrocellulose filter.5% skimming milk or 3% bovine serum albumin damping fluid (10mmol/L Tri s Cl, pH7.5,150mmol/L NaCl, 0.05%Tween20) 37 ℃ of sealing 2h, lavation buffer solution (10mmol/L TrisCl pH7.5,150mmol/LNaCl, 0.05%Tween20) washing is 3 times, and each 5～10min dilutes the anti-IBDV positive serum of chicken on (1: 300) mulch film with the sealing damping fluid, 2h is made in the room temperature sense, wash 3～5 times, 2h is made in the anti-chicken IgG of the rabbit of horseradish peroxidase-labeled (1: 4000) room temperature sense, wash 3～5 times after, each 5～10min, clean once with distilled water at last, press from both sides out nitrocellulose filter and dry slightly, preparation ECL (enhancedchemiluminescence) working fluid, incubated at room film number minute under visible light is stuck blotting membrane and is fixed in X-ray sheet exposure magazine with the preservative film bag.Change the darkroom then over to X-ray film was pressed in the several seconds of exposing on the film by several minutes, protein band can clearly be presented on the X-ray film behind the developing fixing.

12.4 laser conjugation focusing microscope is analyzed

Put into aseptic slide glass in 6 well culture plates, the Sf9 cell is inoculated in earlier on the aseptic slide glass, and cell access amount is that every hole adds 1 * 10 ⁶Behind the cell absorption 1h, inhale and abandon substratum, add recombinant baculovirus and under MOI=10, infect.Inhale after 2 days and abandon substratum, add 1ml methyl alcohol again: acetone (1: 1) mixed solution is fixed 5 minutes under-20 ℃ of refrigerators, adds 1ml PBS (phosphate-buffered saline) shaking speed 50r/min afterwards again and cleans 2 times, each 5min.Then the 2%BSA (bovine serum 26 albumin) with 1ml reacts 30min down at 37 ℃.Add 1ml PBS (phosphate-buffered saline) shaking speed 50r/min afterwards again and clean 2 times, each 5min.Next add the anti-IBDV positive serum of chicken (1: 100) respectively and react 1h down at 37 ℃.After cleaning 3 times with the PBS of 1ml, 37 ℃ of anti-chicken IgG of rabbit (1: 100) that add the FITC mark again are reaction 1h down.Last clean 3 times with the PBS of 1ml again.(Leica Germany) observes down for confocal microscope, TCS SP2, has VP2 recombinant protein cytolemma and can be dyeed by FITC and present green fluorescence at laser confocal microscope.

12.5 immunoelectron microscope detects

The insect baculovirus drop of getting 15 μ l purifying has the copper mesh of carbon membrane to have one of carbon membrane to face down envelope and is suspended on the insect baculovirus liquid level on stencil plate, absorption 30min.Splash into contain 1%BSA 1 of PBS liquid on stencil plate, with copper mesh have the one side of carbon membrane frivolous on drop the room temperature 20min that blockades, then with PBS liquid washing 3 times, each 5min.The method of washing is identical with the method for blockading, and water is blotted at relations with network with filter paper.Copper mesh is floated on (about 15 μ l) on the anti-IBDV positive serum of chicken (1: the 50) drop respectively, and room temperature 30～60min. sets up negative control group simultaneously, does not add first antibody, replaces with PBS.The PBS rinsing is washed 3 times, each 5min, and filter paper blots.Copper mesh is suspended on the anti-chicken IgG of the rabbit second antibody drop of colloid gold label (1: 50 times of dilution) incubated at room 30～60min.The PBS rinsing is washed 3 times, each 5min, and filter paper blots.Copper mesh is suspended in negative staining 2min on 2% the phospho-wolframic acid drop, in drying at room temperature, electron microscopic observation.

13. the detected result of recombinant baculovirus BacSC-VP2 expression product

Behind the recombinant baculovirus BacSC-VP2 inoculation Sf9 insect cell that screening obtains, cytolemma place at cells infected, can be observed a special circle yellow-green fluorescence, illustrate that expression product is special and expression product is illustrated on the cytolemma of Sf9 insect cell (seeing accompanying drawing 3).Analyze through Western blot, the cell culture of reorganization poison inoculation has occurred being the special band of 40kD (seeing accompanying drawing 4) with the anti-IBDV positive serum of pig bonded size, has proved that VP2 albumen has obtained expression in the Sf9 insect cell.The malicious BacSC-VP2 immunoelectron microscope of recombinating detects and the anti-IBDV positive serum of chicken bonded gold particle, and the gold particle position is at the envelope protein place that recombinant virus expands, and shows that VP2 albumen is illustrated on the cyst membrane of baculovirus (seeing accompanying drawing 5).

14. the immunogenicity research of recombinant virus BacSC-VP2

14.1 recombinant virus BacSC-VP2 immune mouse

Recombinant virus BacSC-VP2 inoculation Sf9 insect cell is cultivated 48～72h, collects cells infected.-40 ℃/37 ℃ multigelations 3 times, the centrifugal 10min of 3000r/min gets supernatant and measures viral PFU, and the titre of adjusting virus makes it reach 10 ⁹PFU/ml.

12 of BALB/c female mices, body weight 18～20g divides 2 groups at random, takes the abdominal injection mode, injects recombinant baculovirus BacSC-VP2 respectively and does not contain the baculovirus BacSC0.5ml of VP2 gene.Above-mentioned 2 groups of equal immune secondaries, 14 days at interval, plucked eyeball in the 14th day after the last immunity and get blood, the cervical vertebra dislocation is put to death, and the blood coagulation separation of serum detects the antigenic IgG antibody of anti-IBDV for ELISA and carries out the serum NAT that neutralization test detects immune mouse.Aseptic its spleen of getting carries out the quantity that the t lymphocyte subset class was tested and detected with flow cytometry analysis in the increment of T lymphocyte respectively.

14.2 the detection of spleen amynologic index

14.2.1 spleen list lymphocyte suspension preparation

Mouse is put to death in the cervical vertebra dislocation, and aseptic condition takes out spleen down, places the plate that fills RPMI 1640 substratum, grinds with slide, and 200 order nylon net filters are made single cell suspension, 1500r/min, and centrifugal 5min abandons supernatant.With the centrifugal cell twice of washing of Hanks liquid, be resuspended in the RPMI RPMI-1640 that contains 10%NBS, counting, it is standby to transfer to 2 * 107/ml.

14.2.2 the detection of spleen t lymphocyte subset class quantity

Extracting spleen cell suspension 0.1ml, add 5ml PBS, 1500r/min, centrifugal 10min washes cell twice, adds fluorescent mark rat anti-mouse CD4+ and CD8+ monoclonal antibody (this antibody dilutes by 1: 10 with PBS) room temperature lucifuge respectively and place 30min in 0.5ml PBS cell suspending liquid, adding 5ml PBS again washes once, the centrifugal 10min of 1500r/min will manage floor cells and suspend with 200 μ l PBS, treat that the upflowing cell instrument detects.FACS detects 10000 cells, and the gained data are carried out statistical procedures.

14.2.3T lymphocyte increment test

(1) preparation of splenocyte: spleen, grinding, filtration are catched and killed, take a blood sample, got to mouse, use the washing of Hank ' s liquid for the last time, abandon supernatant, with the resuspended splenocyte of RPMI-1640 1ml that contains 10% calf serum, adjust cell concn to 2 * 10 again ⁶Individual/ml.

(2) every hole adds lymphocyte suspension 100 μ l in 96 orifice plates, adding the former ConA of differential stimulus (10 μ g/ml) 100 μ l in the corresponding aperture respectively is that test group and 1640 substratum, 100 μ l are non-stimulator antigen hole as positive controls, the former 100 μ l of 20 μ g/ml recombinant baculovirus differential stimuluses, 3 holes of the former repetition of different stimulated; There is not lymphocytic hole as background only to add 200 μ l, 1640 substratum.

(3) behind the 68h, every hole adds MTT (5mg/ml) 20 μ l, and lucifuge is cultivated 4h; Every hole is inhaled and is abandoned 100 μ l nutrient solutions, adds DMSO 100 μ l again, measures a hole OD value with enzyme-linked immunosorbent assay instrument 630nm in the 10min.

(4) result calculates: SI=(average OD value-background values that special stimulation is former)/(average OD value-background values that non-stimulation is former)

14.3ELISA anti-IBDV antibody in the mensuration immune serum

Envelope antigen in the ELISA test kit is the totivirus of deactivation, and two anti-are the sheep anti-mouse igg antibody of horseradish peroxidase-labeled.Measure each hole OD value at enzyme linked immunological instrument 450nm.

14.4 recombinant virus is in the immunology research of body animal chicken

Immunology and detection method with mouse.

Detected result:

The recombinant baculovirus BacSC-VP2 that obtains extracts the laggard performing PCR amplification of genome, must expect the purpose fragment of size.Laser confocal microscope viewing test, immune golden electron microscope observation and Westernblot detect and are positive findings, illustrate that the recombinant baculovirus of structure can be expressed IBDV VP2 capsid protein respectively.Recombinant baculovirus can be in expressed in insect cells, and recombinant virus has good genetic stability.With carrying out the mouse immune experiment after a large amount of amplifications of recombinant baculovirus, can in immune serum, detect the antibody of anti-IBDV, show that recombinant virus stimulates the mouse body to produce specific humoral immunity.The detection and the lymphocyte proliferation assay result of immune mouse splenic t-cell subclass quantity show that all mouse has produced specific cellular immunity.

Result of study shows that the constructed recombinant baculovirus of the present invention is a kind of safe and effective genetic engineering subunit vaccine, has the good prospect that exploitation becomes the vaccine that is used to prevent IBD.

The above embodiment is the preferred embodiment that proves absolutely that the present invention lifts, and protection scope of the present invention is not limited thereto.Being equal to that those skilled in the art are done on basis of the present invention substitutes or conversion, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.

Sequence table

＜110〉Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology

＜120〉recombinant baculovirus and the preparation and the application of expression IBDV immunogen gene

<130>1

<160>2

<170>PatentIn?vetsion?3.5

<210>1

<211>1356

<212>DNA

＜213〉infections chicken cloacal bursa virus VP2 gene (Infectious bursal disease virus vp2 gene)

 

<400>1

atgacaaacc?tgcaagatca?aacccaacag?attgttccgt?ccatacggag?ccttctgatg 60

ccaacaaccg?gaccggcgtc?cattccggac?gacaccctag?agaagcacac?tctcaggtca 120

gagacctcga?cctacaattt?gactgtgggg?gacacagggt?cagggctaat?tgtctttttc 180

cctggtttcc?ctggctcaat?tgtgggtgct?cactacacac?tgcagagcaa?tgggaactac 240

aagttcgatc?agatgctcct?gactgcccag?aacctaccgg?ccagctacaa?ctactgcagg 300

ctagtgagtc?ggagtctcac?agtgaggtca?agcacactcc?ctggtggcgt?ttatgcacta 360

aacggaacca?taaacgccgt?gaccttccaa?ggaagcctga?gtgaactgac?agatgttagc 420

tacattgggt?tgatgtcagc?aacagccaac?atcaacgaca?aaatcgggaa?cgtcctagta 480

ggggaagggg?taaccgtcct?cagcttaccc?acatcatatg?atcttgggta?tgtgagactc 540

ggtgacccca?ttcccgctat?agggctcgac?ccaaaaatgg?tagcaacatg?tgatagcagt 600

gacaggccca?gagtctacac?cataactgca?gccgatgatt?accaattctc?atcacagtac 660

caagcaggtg?gagtaacaat?aacactgttc?tcagctaata?tcgatgccat?cacaagcctc 720

agcatcgggg?gagaactcgt?gtttcaaaca?agcgtccaag?gccttatact?gggtgctacc 780

atctacctta?taggctttga?tgggactgcg?gtaataacca?gagctgtggc?cgcagacaat 840

gggctaacgg?ccggcactga?caaccttatg?ccattcaata?ttgtgattcc?aaccggcgag 900

ataacccagc?caatcacatc?catcaaactg?gagatagtaa?cctccaaaag?tggtggtcag 960

gcgggggatc?agatgtcatg?gtcagcaagt?gggagcctag?cagtgacgat?ccacggtggc 1020

aactatccag?gggccctccg?tcccgtcaca?ctagtagcct?acgaaagagt?ggcaacagga 1080

tctgtcgtta?cggtcgccgg?ggtgagcaac?ttcgagctga?tcccaaatcc?tgaactagca 1140

aagaacctgg?tcacagaata?cggccgattt?gacccaggag?ccatgaacta?cacaaaattg 1200

atactgagtg?agagggaccg?tcttggcatc?aagaccgtat?ggccaacaag?ggagtacaca 1260

gactttcgcg?agtacttcat?ggaggtggcc?gacctcaact?ctcccctgaa?gattgcagga 1320

gcatttggct?tcaaagacat?aatccgggcc?ctaagg 1356

 

<210>2

<211>252

<212>DNA

＜213〉four restriction enzyme sites of SP-His6--TM-CTD gene (signal peptide sequence, sex histidine tag, four restriction site, transmembrane domain, cytomere domain gene)

 

<400>2

atgctactag?taaatccacc?agtcaaaggc?ttcaataagg?agcaagatgg?taagtgtttt 60

caacacacag?ctatatatgt?gcttttggcg?gcggcggcgc?attctgcctt?tgcggcgcac 120

caccaccacc?accacgcgcg?cccatgggct?agcgcatgct?gtagttaact?tcatgtttgg 180

tcatattaat?tgttttgtaa?ttagatttta?tttttgtact?gtatgattag?aaaccgtaat 240

agacaatatt?aa 252

Claims (9)

1. a recombinant baculovirus of expressing the IBDV immunogen gene is characterized in that, described recombinant baculovirus has the sequence shown in the SEQ ID NO.1.

2. recombinant baculovirus according to claim 1 is characterized in that, described recombinant baculovirus surface display IBDV structural protein VP2.

3. the preparation method of claim 1 or 2 described recombinant baculovirus is characterized in that step comprises:

A, preparation VP2 gene;

B, with above-mentioned VP2 gene, be inserted into the p10 promotor downstream of baculovirus surface display transposon vector pBacSC, make up the recombinant baculovirus swivel base plasmid pBacSC-VP2 of the VP2 gene contain IBDV;

C, above-mentioned recombinant baculovirus swivel base plasmid pBacSC-VP2 changed over to carries out homologous recombination in the intestinal bacteria, recombinant baculovirus DNA;

D, with the recombinant baculovirus DNA transfection insect cell that step c obtains, obtain recombinant baculovirus.

4. the preparation method of recombinant baculovirus according to claim 3, it is characterized in that, the preparation method of described VP2 gene is: with the PCR method IBDV Shaanxi strain VP2 gene that increased, and it is cloned into the pMDl8-T carrier, made up the pMDl8T-VP2 plasmid, enzyme is cut the pMDl8T-VP2 plasmid, reclaims the VP2 gene.

5. the preparation method of recombinant baculovirus according to claim 3 is characterized in that, the method for the described recombinant baculovirus DNA of steps d transfection insect cell is: recombinant baculovirus DNA is changed in the insect cell by liposome-mediated method.

6. the preparation method of recombinant baculovirus according to claim 3 is characterized in that, the described intestinal bacteria of step c are competence intestinal bacteria DH10Bac.

7. the application of recombinant baculovirus as claimed in claim 1 or 2 in preparation chicken infectivity bursa of Fabricius virus vaccine.

8. a method for preparing the chicken infectivity bursa of Fabricius virus subunit vaccine is characterized in that, described recombinant virus inoculation insect cell, cultivate 48～72h, collect cells infected ,-40 ℃/37 ℃ freeze thawing, centrifugal, to get supernatant and measure viral PFU, the titre of adjusting virus makes it reach 10 ⁹PFU/ml.

9. method according to claim 8 is characterized in that, described insect cell is the Sf9 insect cell.

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