CN103740743A

CN103740743A - Recombined rhabdovirus for expressing porcine bocavirus VP2 protein and application thereof

Info

Publication number: CN103740743A
Application number: CN201310750497.4A
Authority: CN
Inventors: 肖少波; 方六荣; 刘丽枝; 马俊; 曾松林; 王荡; 罗锐; 陈焕春
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2013-12-30
Filing date: 2013-12-30
Publication date: 2014-04-23
Anticipated expiration: 2033-12-30
Also published as: CN103740743B

Abstract

The invention discloses a recombined rhabdovirus for expressing a porcine bocavirus VP2 protein and application thereof. A porcine bocavirus VP2 gene is amplified by PCR (polymerase chain reaction), and the nucleotide sequence of the porcine bocavirus VP2 gene is represented by SEQ ID NO.1. The amplified porcine bocavirus VP2 gene is inserted into BamHI and XhoI locuses of a rhabdovirus transmissible plasmid pFastBacTMHTB to construct a recombined transmissible plasmid pFastBac HTB-VP2; the recombined transmissible plasmid is converted into a DH10Bac competent cell comprising a rhabdovirus framework plasmid; the VP2 gene is integrated into the rhabdovirus framework plasmid Bacmid through homologous recombination; an sf9 cell is transfected by extracting the DNA of the recombined Bacmid to obtain the recombined rhabdovirus rBV-VP2 of which the preservation number is CCTCC V201401. A large quantity of VP2 protein and virus sample particles are expressed and produced in insect cells, and an effective PBoV VLPs subunit vaccine is developed.

Description

Express recombinant baculovirus and the application thereof of pig bocavirus VP2 albumen

Technical field

The present invention relates to animal virology and genetic engineering technical field, refer to particularly a kind of recombinant baculovirus and application thereof of expressing pig bocavirus VP2 albumen.

Background technology

Pig bocavirus (Porcine Bocavirus) suffered from pmws (Post-weaning Multisystemic Wasting Syndrome in Sweden first in 2009, PMWS) and in the sick pig body of porcine circovirus 2 type coinfection find (the .Detection of a novel porcine boca-like virus in the background of porcine circovirus type2induced postweaning multisystemic wasting syndrome.Virus Res2009 such as Blomstrom AL, 146:125-129).China's reported first in 2011 the nearly full-length gene group sequence of pig rich card sample virus (PBo-likeV), and confirmed that PBo-likeV belongs to bocavirus and belongs to, called after pig bocavirus WH1 strain from phylogenetic analysis.By epidemiology, detect to analyze and find, confirm that pig bocavirus also exists popular .Complete coding sequences and phylogenetic analysis of porcine bocavirus.J Gen Virol, 92:784-788 such as () Zeng S widely in China.

Pig bocavirus is the single-stranded DNA viruses without cyst membrane; volume is little; simple in structure; diameter is about 25～30nm; belong to Parvoviridae parvovirus subfamily bocavirus and belong to (the .In vivo such as Moron VG; dendritic cells can cross-present virus-like particles using an endosome-to-cytosol pathway.J Immunol2003,171:2242-2250;

.Identification and nearly full-length genome characterization of novel porcine bocaviruses.PLoS One, the 5:e13583 such as Cheng WX).This belongs to other member and comprises bovine parvovirus (Bovine Parvovirus), dog microvirus (Canine Minute virus), human bocavirus (Human Bocavirus).Pig bocavirus genome total length is about 5kb, three open reading frame (Open Reading Frames encode, ORFs), wherein ORF1 and the ORF3 viral Nonstructural Protein (NS1, NP1) of being responsible for encoding, ORF2 coding structure albumen (overlapping VP1/VP2).ORF3 is the distinctive open reading frame of bocavirus, and its function is still not clear.As the main structural protein of bocavirus, the capsid protein of ORF2 genes encoding---VP2 albumen in vitro self assembly becomes virus-like particle (VLPs) and may contain special epitope .Evidence of prior exposure to human bocavirus as determined by a retrospective serological study of404serum samples from adults in the United States.Clin Vaccine Immunol2009,16:597-604 such as () Cecchini S.Teng Ling side etc. after expressed in insect cells human bocavirus VP2 albumen for the examination of clinical samples, confirm VP2 albumen have certain antigenicity (Teng Lingfang etc. bocavirus VP2 gene is in sf9 expressed in insect cells and clinical samples examination. China's experiment and clinical virology magazine 2009,23 (6)).Martinez C etc. can excite special immune response (the .Production of porcine parvovirus empty capsids with high immunogenic activity.Vaccine10:684-690 such as Martinez C) after utilizing PPV VP 2 protein (VLPs) the immune swine body of insect cell expression, shows that VP2 albumen has good immunogenicity.

Now there are some researches show that bocavirus is relevant with breeding difficulty, digestive tube and respiratory tract disease, especially in You Ling colony, sickness rate is higher.Sweden scholar passes through the EPDML investigation to pig bocavirus and finds, there is the pig of pmws (PMWS), the infection rate of its pig bocavirus is twice (the .Studies of porcine circovirus type2 such as Blomstrom AL that does not suffer from the pig of PMWS, porcine boca-like virus and torque teno virus indicate the presence of multiple viral infections in postweaning multisystemic wasting syndrome pigs.Virus Res, 152:59-64).In addition, according to people such as Zhai Shaolun, from there being sample detection collected in the ill pig body of respiratory symptom, find, pig bocavirus ubiquity, there is higher prevalence rate (69.7%) (.High prevalence of a novel porcine bocavirus in weanling piglets with respiratory tract symptoms in China.Arch Virol, the 155:1313-1317 such as Zhai S).

At present, occurred a series of pig bocavirus varient in swinery, also at the early-stage to its research, it is independent pathogenic unknown.In the animal of generation pmws, there is multiple virus (porcine circovirus 2 type, pig bocavirus, pig parvoviral, PRRS virus and Pestivirus suis etc.), this shows that pig bocavirus may work as a kind of cofactor in advancing of disease process.But most (PBoV1, PBoV2 and PBoV4) is located away from the sick pig that suffers from PMWS, and in the recall rate of sick pig higher than health pig, this shows that pig bocavirus is to the certain dependency of having of multisystemic exhaustion syndrome after weaning, also may there is more serious disease with the cause of disease polyinfection such as porcine circovirus 2 type, PRRS virus and Pestivirus suis in it, cause respiratory tract infection and diarrhoea, and cause miscarriage, stillborn foetus, even dead.

As a newfound virus, etiology effect and it potential pathogenic of pig bocavirus in the diseases such as digestive tube, respiratory tract and diarrhoea is unknown to a great extent.Therefore, the close supervision to pig bocavirus and effective prevention and control are by the development trend that is future studies.

Summary of the invention

Technical problem to be solved by this invention is just to provide the recombinant plasmid that contains pig bocavirus VP2 gene.

As preferred version, described recombinant plasmid is the pFastBac HTB-VP2 recombinant transfer plasmid that contains pig bocavirus VP2 gene.

Described recombinant plasmid also comprises with recombinant transfer plasmid pFastBac HTB-VP2 and carries out the baculovirus shuttle plasmid after homologous recombination, this recombinant shuttle plasmid called after rBacmid-VP2.

The present invention also provides a kind of recombinant baculovirus that contains pig bocavirus VP2 gene, and the pig bocavirus VP2 albumen of its expression can form virus-like particle (VLPs) after expressed in insect cells.

As preferred version, by pcr amplification one boar bocavirus VP2 gene, its nucleotide sequence is as shown in sequence table SEQ ID NO.1.By amplification to pig bocavirus VP2 gene be inserted into baculovirus transferring plasmid pFastBac ^tMthe BamH I of HTB and Xho I site are configured to recombinant transfer plasmid pFastBac HTB-VP2, then this recombinant transfer plasmid is converted into the DH10Bac competent cell that contains baculovirus skeleton plasmid, by homologous recombination by VP2 gene integration to baculovirus skeleton plasmid Bacmid, obtain recombinant baculovirus rBV-VP2 after extracting restructuring Bacmid DNA transfection sf9 cell.This recombinant baculovirus, called after: the recombinant baculovirus E2/rBV-VP2Autographa californica multiple nucleopolyhedrovirus E2/rBV-VP2 that contains pig bocavirus VP2 gene, it is preserved in Chinese Typical Representative culture collection center (CCTCC), preservation date is: on December 20th, 2013, preserving number is: CCTCC NO:V201401.

The present invention also provides the application of above-mentioned recombinant baculovirus rBV-VP2 in preparation pig bocavirus vaccine.

As preferred version, described pig bocavirus vaccine is subunit vaccine.Its preparation method is: after recombinant baculovirus rBV-VP2 is infected to sf9 cell, express the subunit vaccine that VP2 albumen (VLPs) obtains.

Select the theoretical basis of baculovirus:

Baculovirus (Baculovirus) is the virus that a class infects invertebrates, is shaft-like, has higher host specificity.Many embedding nuclear polyhedrosis virus of autographa california (Autographa califonica multiple nuclear polyhedrosis virus, AcMNPV) genome is the virus covalently closed circular double-stranded DNA of about 130kb, be widely used most and have distinctive baculovirus most, many castes be can infect, A. noctuid, the greedy noctuid (noctuid) in meadow, cabbage looper (wild cabbage looper) etc. comprised.

Bac-to-Bac baculovirus vector expression system (Bac-to-Bac Baculovirus Expression Vector System, BEVS) use one or more promotors, external source goal gene is inserted into promotor downstream, obtain recombinant virus, this recombinant virus is expressed foreign gene when copying in insect cell or insect body.On the whole, Bac-to-Bac baculovirus vector expression system has many good qualities:

(1) recombinant baculovirus is easy to build, easily operation, and biological safety is higher, has very single host range.

(2) baculovirus is a kind of powerful expression vector, and genome own is very large, can hold larger external source fragment, has very high protein expression level, now has been widely used for expressing the foreign gene that molecular weight is larger.Therefore baculovirus can expressed fusion protein also can be for expressing multiple genes such as goal gene and reporter gene .Protective immunity against influenza H5N1virus challenge in mice by intranasal co-administration of baculovirus surface-displayed HA and recombinant CTB as an adjuvant.Virology2008,380:412-420 such as () Prabakaran M.

(3) recombinate shape virus infection efficiency is higher, infectious titer particle easily obtains, can infect non-division cells, and can effectively mediate the delivery of vivo gene, have very high target cell and tissue specificity (Hu Shaomin etc. baculovirus is as mammalian cell expression vector and the application in medical science thereof. foreign medical science parasitosis fascicle 2004,31 (5): 222-226).

(4) host insect cell can suitably be modified the albumen of baculovirus expression, its mode similar with animal mammalian cell (.Insect cells as hosts for the expression of recombinant glycoproteins.Glycoconj J1999, the 16:109-123 such as Altmann F).It is reported, the product of expressing through baculovirus expression system, tends to posttranslational modification, has and the similar biological activity of native protein.

Nowadays; take baculovirus as vector expression recombinant protein; prepare subunit vaccine and obtained good effect (the .Expression of foot-and-mouth disease virus capsid proteins in silkworm-baculovirus expression system and its utilization as a subunit vaccine.PLoS One2008 such as Li Z, 3:e2273; The .Evaluation of efficacy of mammalian and baculovirus expressed E2subunit vaccine candidates to bovine viral diarrhoea virus.Vaccine2009 such as Thomas C, 27:2387-2393; The .Delivery of vaccine peptides by rapid conjugation to baculovirus particles.Vaccine2008 such as Wilson S; 26:2451-2456); in field of gene research, also obtain some progress (the .Virus-like particles as a vaccine delivery system:myths and facts.Hum Vaccin2008 such as Roy P, 4:5-12; .Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutininneuraminidase proteins.J Vet Sci2008, the 9:301-308 such as Lee YJ).Therefore, baculovirus becomes a kind of good expression vector, is widely used in biomedical each field.

More detailed scheme is as described below:

One, the structure of recombinant baculovirus transfer vector pFastBac HTB-VP2

1, the design of primer

According to the special primer VP2-F/VP2-R of PBoV genome sequence (GenBank accession number is HQ223038.1) the design pair for amplification VP2 gene of this laboratory report, and in upstream primer, introduce BamH I restriction enzyme site, in downstream primer, introduce Xho I restriction enzyme site.Primer is synthetic by Dalian precious biotechnology company limited.Primer sequence is as follows:

VP2-F:5'-TTT GGATCCATGTCCGCACACAGGGGGGC-3'

VP2-R:5'-GGG CTCGAGTTACAACACTTGGTTGAT-3'

2, pcr amplification

The pig bocavirus genome being separated to take this laboratory is template amplification VP2 gene fragment, and amplification condition is: after 95 ℃ of sex change 5min, enter circulation; Loop parameter is: 95 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 2min; 30 rear 72 ℃ of extension 10min of circulation.Reaction finishes rear use 0.8% agarose gel electrophoresis and detects amplification.

3, the structure of recombinant transfer plasmid pFastBac HT B-VP2

Reclaim PCR positive products, by BamH I and Xho I restriction enzyme site, the VP2 gene clone that amplification is arrived is to donor plasmid pFastBac ^tMin HT B carrier (Invitrogen, the U.S.), through enzyme, cut and check order and identify that confirmation builds correctly, obtains positive recombinant plasmid pFastBac HTB-VP2.Recombinant plasmid pFastBac HTB-VP2 enzyme is cut qualification result as shown in Figure 2.

Two, the screening purifying of recombinant shuttle plasmid rBacmid-VP2

With reference to Bac-to-Bac expression system specification sheets, by building 10 correct μ L recombinant transfer vector pFastBac HTB-VP2, be converted in the DH10Bac competent cell that contains baculovirus skeleton plasmid Bacmid.Mix and be placed on 30min on ice.42 ℃ of water-bath 45s, then ice bath 2min.Add 900 μ L LB to cultivate based on 37 ℃, 225rpm jolting 4-6 hour.By 10 ^-1, 10 ^-2, 10 ^-3after dilution competent cell, getting respectively 100 μ L dilution recovery bacterium liquid coats and contains kantlex, gentamicin, tsiklomitsin, on the high salt LB flat board of IPTG and X-gal, cultivate 48 hours for 37 ℃, under the booster action of Helper plasmid, there is homologous recombination in recombinant transfer vector pFastBac HTB-VP2 and baculovirus skeleton plasmid Bacmid, external source goal gene is incorporated into formation restructuring Bacmid plasmid in baculovirus skeleton plasmid.Through the blue hickie of two-wheeled, the positive bacterium colony of screening purifying white.

Three, the extraction of recombinant shuttle plasmid (rBacmid-VP2)

Aseptic picking falls within and contains 50 μ g/mL kantlex containing the positive bacteria of restructuring Bacmid, 7 μ g/mL gentamicins, in the high salt LB liquid nutrient medium of 10 μ g/mL tsiklomitsins, 37 ℃ of 300r/min overnight incubation, centrifugal 1 minute collecting cell precipitation of 12000rpm, with 0.3mL solution I (15mM Tris-Hcl, PH8.0; 10mM EDTA, 100 μ g/mL RNase; Filtration sterilization, 4 degree store) resuspended precipitation, add 0.3mL solution II (0.2M NaOH, 1%SDS, now with the current) slightly mix, the standing 5min of room temperature, slowly adds 0.3mL solution III (3M Potassium ethanoate, PH5.5) mix, ice bath 5-10min, the centrifugal 10min of 12000r/min, supernatant is added in 0.8mL Virahol, mix ice bath 5-10min, the centrifugal 15min of room temperature 12000rpm, 70% washing with alcohol precipitation, is dissolved in 40 μ L1 × TE after being dried, can use immediately or 4 ℃ of preservations, avoid multigelation.The Bacmid plasmid extracting utilizes VP2 upstream primer VP2-F and M13 downstream primer to carry out PCR evaluation.PCR qualification result as shown in Figure 2.

Four, the acquisition of recombinant baculovirus

Utilize liposome-mediated infection protocol, the recombinant shuttle vector (rBacmid-VP2) after screening, purifying is transfected in sf9 insect cell, in 27 ℃ of cultivations, after there is cytopathy, collecting cell culture supernatant can obtain recombinant baculovirus.

(1) prepare Bacmid DNA plasmid, and measure its concentration.Good form, eugonic Sf9 cell is accessed to 6 porocyte culture plates, in 27 ℃ of incubators, cultivate, treat that cell grows to 70～80% individual layers and prepares transfection.

(2) preparation cell transfecting liquid: get two aseptic centrifuge tubes of 1.5mL, A pipe adds 5～8 μ L liposomes and 100 μ L serum-free Grace ' s nutrient solutions, mixes the standing 5min of room temperature.In B pipe, add 100 μ L serum-free Grace ' s nutrient solutions and 3～5 μ g to treat transfection Bacmid DNA therebetween, mix the standing 5min of room temperature.

(3) mixed solution in A pipe is dropped to pipe B in, after mixing in the standing 20min of room temperature.Inhale and abandon the substratum for the treatment of transfectional cell hole therebetween, use serum-free Grace ' s nutrient solution washing 3 times.

(4) inhale and abandon serum-free Grace ' the s nutrient solution for the treatment of transfectional cell hole, 200 μ L mixed solutions in B pipe are added drop-wise on cell monolayer, add 800 μ L serum-free Grace ' s nutrient solutions, shake up gently, then cell plate are placed in to 27 ℃ of incubators and cultivate.

(5) cultivate after 5h, inhale and abandon transfection mixed solution, add 3mL Grace ' s growth media and continue to cultivate 72h.

In 27 ℃ of cultivations, after there is cytopathy, collecting cell culture supernatant can obtain recombinant baculovirus.And by the recombinant baculovirus of the first-generation.First-generation recombinant baculovirus is continued to inoculation Sf9 passage, can obtain the recombinant baculovirus of high titre.Recombinant baculovirus is continued to inoculation insect cell and expand, collect viral supernatant, the centrifugal 5min of 500 × g, shifts supernatant to sterile tube, is sub-packed in-80 ℃ of preservations, and with BacPAK Baculovirus Rapid Titer Kit(Clontech) measure its titre.

Five, the detection of expression of recombinant protein and the electron microscopic observation of virus-like particle

(1) expression of western blot test testing goal albumen

Recombinant baculovirus was inoculated to sf9 cell after 72 hours, collecting cell sample, with PBS(pH7.4) piping and druming cell makes it to disperse to come off, then low-speed centrifugal (the centrifugal 10min of 2000rpm), collecting cell precipitation, wash after 1 time with PBS, add equivalent 2 × SDS-PAGE sample-loading buffer (containing 50mM DTT) resuspended, mix 95 ℃ of water-bath 5min, then put and wait for loading on ice, carry out SDS-PAGE and Western blot and analyze.The primary antibodie that in the present invention, Western blot is used is anti-PBoV VP2 monoclonal antibody and anti-His label monoclonal antibody (the green skies, Beijing) prepared by this laboratory, and it is the sheep anti-mouse igg of HRP mark that two of use resists.

(2) expression of indirect immunofluorescence assay (IFA) testing goal albumen

The baculovirus transduction of amplification is inoculated in to the sf9 insect cell of six orifice plates, establishes the contrast of wild-type baculovirus cell.After 48 hours, every hole PBS(pH7.4) wash 3 times, with the fixing 10min of 80% the acetone soln that is placed in advance-20 ℃, PBS washing 3 times, the monoclonal antibody that adds the anti-His albumen of suitable dilution is primary antibodie, 37 ℃ act on 1 hour, PBS washes each 5min 3 times, then add the sheep anti-mouse igg of fluorescein isothiocyanate (FITC) mark anti-as two, 37 ℃ of effect 45min, PBS washes 3 times.After this with tinfoil paper lucifuge parcel, use PBS room temperature rinsing three times, each 10 minutes.Under Laser Scanning Confocal Microscope, observe the fluorescence of transfectional cell and take a picture.

(3) electron microscopic observation of virus-like particle (VLPs)

On the sf9 cell that grows up to individual layer, infect inoculation recombinant baculovirus rBV-VP ₂, after 72 hours, collect sick cell, with PBS, wash twice, 1000rpm15min and be resuspended in 25mmol/L NaHCO after centrifugal ₃(pH8.3), in, after ultrasonic treatment, the centrifugal 15min of 10000rpm, contains the VP of expression in supernatant ₂albumen.Supernatant, by sucrose concentration gradient centrifugation, is dyeed by phospho-wolframic acid negativity after desugar, utilize transmission electron microscope observe and take a picture.

Six, the production technique of subunit vaccine

Sf9 cell is seeded to 175cm ²in Tissue Culture Flask, until Growth of Cells when being paved with 70-80%, the dosage that P4 is pressed to MOI=5 for recombinant baculovirus rBV-VP2 infects sf9 cell, cultivate 72 hours for 27 ℃, collecting cell sample, PBS washing 1 time, ultrasonic degradation cell 20min on ice, lysate is through 12000rpm, 4 ℃, centrifugal 15min removes cell debris, and collection supernatant utilizes the VP2 albumen of affinity chromatography method purifying His label, utilize the protein concentration after spectrophotometric determination purifying, get the supernatant and the adjuvant that contain purifying protein and be mixed with subunit vaccine.

Seven, the immune efficacy of subunit vaccine check

By the subunit vaccine VP2(200 μ g/mL of two groups of various dose and 20 μ g/mL) and PBS respectively through the back leg intramuscular injection BALB/c mouse in 6 week age, 6 every group, every mouse 100 μ L, altogether immunity 2 times, 2 weeks, interval; Use the negative control of phosphate buffered saline buffer PBS as protein immunization simultaneously.After

first immunisation

3,6 weeks through tail vein negative pressure hemostix, separation of serum, detection specificity VP2 antibody; In head, exempt from latter 6 weeks, by cervical vertebra, put to death all immune mouses, aseptic taking-up spleen, utilize lymphocyte separation medium (Beijing Da Kewei Bioisystech Co., Ltd) to separate splenic lymphocyte, carry out cellular immunization (comprise stimulate proliferation splenic lymphocyte the mRNA of exponential sum IFN-γ transcribes, protein excretion level) and detect.Result VP2 subunit vaccine immune group induction ELISA antibody horizontal and cell immune response, all apparently higher than PBS control group, illustrate that PBoV VP2 albumen has good immunogenicity.

Beneficial effect of the present invention is:

The present invention utilizes a strain pig bocavirus WH1 strain that this laboratory the is separated to VP2 gene that increases, its homologous recombination to rhabdovirus expression vector is configured to recombinant baculovirus rBV-VP2, and great expression produces VP2 albumen and virus-like particle in insect cell, be developed to a kind of effectively PBoV VLPs subunit vaccine.

Accompanying drawing explanation

Fig. 1 has shown the design of graphics that includes PBoV (GenBank HQ223038.1) WH1 strain VP2 gene transfer vector pFastBac HTB-VP2 in the present invention;

Fig. 2 has shown that the enzyme of the recombinant transfer vector pFastBac HTB-VP2 that includes VP2 gene in the present invention is cut evaluation and the PCR qualification result of the rBacmid-VP2 that recombinates;

Fig. 3 has shown that the recombinant baculovirus that includes VP2 gene in the present invention detects at IFA and the western blot of sf9 cells VP2 albumen;

Fig. 4 has shown that the recombinant baculovirus that includes VP2 gene in the present invention is at the electron microscopic observation of sf9 cells VP2 albumen formation virus-like particle;

Fig. 5 shown after PBoV (GenBank HQ223038.1) WH1 strain VP2 gene subunit vaccine is with various dose immunity BALB/c mouse for the specific ELISA antibody horizontal of VP2;

Fig. 6 has shown the splenic lymphocyte stimulation index of inducing after PBoV (GenBank HQ223038.1) WH1 strain VP2 gene subunit vaccine is with various dose immunity BALB/c mouse;

After Fig. 7 has shown that PBoV (GenBank HQ223038.1) WH1 strain VP2 gene subunit vaccine is with various dose immunity BALB/c mouse, with fluorescent quantitative RT-PCR method, detecting mouse spleen lymphocyte is stimulated the mRNA relative level of rear IFN-γ by specific antigen;

After Fig. 8 has shown that PBoV (GenBank HQ223038.1) WH1 strain VP2 gene subunit vaccine is with various dose immunity BALB/c mouse, with solid phase sandwich ELISA method, detecting mouse spleen lymphocyte is stimulated the level of rear secretion of gamma-IFN by specific antigen.

Embodiment

In order to explain better the present invention, below in conjunction with specific embodiment, further illustrate main contents of the present invention, but content of the present invention is not only confined to following examples.

Embodiment 1: the recombinant plasmid that contains pig bocavirus VP2 gene and the preparation of recombinant baculovirus

The structure of the 1.1 baculovirus transferring plasmid pFastBac HTB-VP2 that comprise PBoV VP2 gene

The pig bocavirus genome being separated to take this laboratory is template, according to the special primer of PBoV genome sequence (GenBank accession number is HQ223038.1) the design pair for amplification VP2 gene of this laboratory report, and in upstream primer, introduce BamH I restriction enzyme site, in downstream primer, introduce Xho I restriction enzyme site, pcr amplification is with the PBoV VP2 gene of specific restriction enzyme site.Primer is synthetic by Dalian precious biotechnology company limited.Sequence is as follows:

VP2-F:5'-TTT GGATCCATGTCCGCACACAGGGGGGC-3'

VP2-R:5'-GGG CTCGAGTTACAACACTTGGTTGAT-3'

The PCR product of VP2 gene is reclaimed, use respectively restriction enzyme BamH I and Xho I double digestion PCR product, Direct Cloning is to the carrier pFast that cuts processing by same enzyme ^tMthe corresponding site of HT B, obtains recombinant plasmid pFastBac HTB-VP2, and its structure is shown in Fig. 1, and enzyme cuts evaluation and sequencing result confirms that recombinant transfer plasmid pFastBac HTB-VP2 builds correctly.Enzyme is cut evaluation figure and be the results are shown in Figure 2.

The structure of 1.2 recombinant shuttle vector rBacmid-VP2 and PCR identify

The recombinant transfer plasmid pFastHTB-VP2 of purifying is transformed and enters DH10Bac ^tMecoli, coats and includes 50 μ g/mL kantlex, 7 μ g/mL gentamicins, and 10 μ g/mL tsiklomitsins, 100 μ g/mL X-gal, on the LB agarose plate of 40 μ g/mL IPTG.DH ₁₀baculovirus shuttle vectors (Bacmid DNA) in Bac intestinal bacteria contains and carries LacZ gene, when foreign gene inserts, and the inactivation of LacZ gene, DH under X-gal and IPTG exist ₁₀bac intestinal bacteria form white colony.If do not insert foreign gene, can form blue colonies.Picking white colony is streak culture, after twice purifying, chooses white clone and extracts recombinant baculovirus shuttle plasmid rBacmid-VP2DNA.As follows with PBoV VP2 upstream primer (VP2-F) and M13 downstream primer (M13-R):

VP2-F5'-TTT GGATCCATGTCCGCACACAGGGGGGC-3'

M13-R5'-CAGGAAACAGCTATGAC-3'

Carry out pcr amplification, 0.8% agarose gel electrophoresis detected magnitude is 2400bp left and right (size of the PCR product of restructuring Bacmid should be that 1665bp adds that the 745bp of external source segment is 2400bp left and right altogether), and PCR result conforms to expection size.Confirm that VP2 gene has been incorporated in the Bacmid of recombinant baculovirus.PCR qualification result is shown in Fig. 2.

The preparation of 1.3 recombinant baculovirus rBV-VP2 and propagation

By freshly extd restructuring rBacmid-VP2DNA, utilize liposome-mediated infection protocol, transfection sf9 insect cell individual layer.27 ℃ of cultivations, observe by sky, and after there is cytopathy, (cell becomes greatly, becomes circle, comes off gradually), collects culture supernatant.The centrifugal 10min of 2000rpm, removes cell debris, has obtained first-generation recombinant baculovirus rBV-VP2.The supernatant of collection is infected to normal sf9 insect cell, collect supernatant, be extended to continuously P4 generation.By each generation rBV-VP2 packing ,-80 ℃ of refrigerators save backup for a long time.

1.4 recombinant baculovirus titer determinations

With reference to BacPAKTM Baculovirus Rapid Titer Kit(Clontech, the U.S.) mensuration recombinant baculovirus titre.

(1) by sf9 cell early stage logarithm according to 6.5 × 10 ⁴cells/well is seeded to 96 orifice plates.Encase flat board with the plastics bag with wet gauze, cultivate 1 hour for 27 ℃.

(2) in the Grace ' s of 900 μ L perfect medium (Grace ' s substratum+10%FBS), add the viral sample of 100 μ L, carry out successively doubling dilution, finally obtain 10 ^-7, 10 ^-8, 10 ^-9three dilution viral suspension samples.In dilution, each extent of dilution will fully mix.

Cultivate 1 hour, treat that cell density reaches 3～4 × 10 for (3) 27 ℃ ⁵during cell/mL, inhale and abandon substratum in 96 orifice plates.

(4) respectively three dilution viral suspensions of difference are joined in 96 orifice plates according to 25 μ L/ holes, each extent of dilution is established 3 repetitions, adds 25 μ L diluents in negative control hole simultaneously.Jog flat board is even to spread.

(5), with encasing flat board with the plastics bag of wet gauze, cultivate 1 hour for 37 ℃.

(6) inhale and abandon viral suspension gently, note not scraping bottom cellular layer.

(7) every hole adds 50 μ L methylcellulose gum to cover cellular layer, with encasing flat board with the plastics bag of wet gauze, cultivates 43-47 hour for 27 ℃.

(8) every hole adds the acetone of the precooling of the fresh configuration of 150 μ L, incubated at room 10min.

(9) get rid of liquid in plate, wash 3 times 5min at every turn with 200 μ L PBS+0.05% polysorbas20s.

(10) every hole adds the sheep blood serum after 50 μ L dilutions, room temperature incubation 5min.

(11) get rid of liquid in plate, on thieving paper, control moisture in dry hole.

(12) every hole adds the mouse source GP64 monoclonal antibody after 25 μ L dilutions, hatches 25min for 37 ℃.

(13) get rid of liquid in plate, on thieving paper, control moisture in dry hole.Wash 3 times each 5min with 200 μ LPBS+0.05% polysorbas20s.

(14) it is anti-that every hole adds the sheep anti mouse two of the HRP mark after 50 μ L dilutions, hatches 25min for 37 ℃.

(15) get rid of liquid in plate, on thieving paper, control moisture in dry hole.Wash 3 times each 5min with 200 μ LPBS+0.05% polysorbas20s.

(16) every hole adds the blue peroxidase substrate of 50 μ L, incubated at room 3 hours.

(17) utilize opticmicroscope to calculate the plaque number after dyeing, application of formula is calculated the titre that can obtain recombinant baculovirus.

Average plaque number × extension rate × 40 in virus titer (IFU/ml)=every hole

Embodiment 2: the evaluation of recombinant baculovirus expression foreign protein

The IFA of 2.1 recombinant baculovirus identifies

Recombinant baculovirus rBV-VP2 inoculation is paved with to the sf9 cell of individual layer, establishes wild-type baculovirus WTBV contrast simultaneously.Observe cell and occur, after pathology, discarding substratum, with the fixing 5min of 80% acetone of precooling, PBS washing 3 times; With confining liquid and the diluent of making IFA containing the PBS(of 10% lowlenthal serum) sealing 30min; The mouse monoclonal antibody that adds the anti-His albumen of suitable dilution is primary antibodie, 37 ℃ of effect 1h, and PBS washes 3 times, each 5min; Then add two anti-(sheep anti-mouse iggs of FITC mark), after this, with 37 ℃ of effect 45min of tinfoil paper parcel, PBS washes 3 times, each 10 minutes.Under Laser Scanning Confocal Microscope, observe the fluorescence of transfectional cell and take a picture.

Result shows that the metainfective sf9 cell of recombinant baculovirus rBV-VP2 can detect obvious specificity fluorescent reaction, and negative control group there is not specificity fluorescent reaction, and recombinant baculovirus rBV-VP2 successful expression VP2 albumen in sf9 cell is described.IFA result as shown in Figure 3.

The western blot of 2.2 recombinant baculovirus identifies

2.2.1 preparation of samples

Recombinant baculovirus was inoculated to sf9 cell after 72 hours, collecting cell sample, with PBS(pH7.4) piping and druming cell make it disperse come off, then low-speed centrifugal (the centrifugal 10min of 2000rpm), collecting cell precipitation, wash after 1 time with PBS, according to document (Pehanorm Brooker etc., 1996) described method is carried out the processing of sample, add equivalent 2 × SDS-PAGE sample-loading buffer (containing 50mM DTT or not containing 50mM DTT) resuspended, mix 95 ℃ of water-bath 5min, then put and wait for loading on ice, carry out SDS-PAGE and Western blot and analyze.

2.2.2SDS-PAGE electrophoresis

(1) join glue: by (1996) methods such as Pehanorm Brookers, prepare 12% separation gel and 5% collection layer glue.

(2) fall glue: clip the sheet glass of recording polyacrylamide gel, 12% the about 5mL of SDS acrylamide separation gel making is poured into rapidly in the gap of two glass plates, reserve perfusion spacer gel requisite space, on separation gel, add one deck isopropylcarbinol, put 37 ℃ of greenhouse polymerization 45 min, after separation gel polymerization, incline and tectum liquid, wash gel top for several times with deionized water, with the paper handkerchief residual liquid that exhausts, again 5% spacer gel just having made is poured on separation gel, insert immediately comb, put 37 ℃ of greenhouse polymerization 45min, after the whole polymerizations of glue, shift out comb, water washes away the residual liquid of loading slot, the glue that pulls out loading slot with syringe needle straightens tooth, be fixed on electrophoresis apparatus.

(3) electrophoresis: add appropriate electrophoretic buffer (25mM Tris-base, 250mM glycine, 0.1%SDS) toward electrophoresis apparatus, get the sample loading of handling well, 20 μ L samples on every hole.Switch on power, voltage is transferred to 80V, when bromophenol blue indicator is gone to separation gel, voltage is transferred to 120V, and power-off while waiting bromophenol blue indicator to go to separation gel bottom, finishes electrophoresis.

2.2.3Western blot detects analysis

(1) electricity turns: after SDS-PAGE electrophoresis finishes, get the gel after SDS-PAGE electrophoresis, and not dyed, directly use Bio-RAD company

tetra Cell transfer device is transferred to PVDF membrane (PVDF) above by charged albumen one, and 12V is wet turns 12 hours.Concrete operations are as follows: put on one's gloves, prepare the nitrocellulose filter of two 5.8mm × 8.8mm filter paper and 1 formed objects.PVDF is submerged into completely in methyl alcohol and soaks 5min to remove the upper residual bubble of PVDF.In a pallet, add a small amount of electricity to turn damping fluid, two filter paper and sponge are soaked in wherein.The installation transfer device that puts on one's gloves, the filter paper of placing a sponge and soaked on operating panel, Accurate align, drives bubble out of, from electrophoresis chamber, takes off gel, transfers to rinsing in transferring film liquid and once, then accurately lies against on filter paper, and assurance is alignd, and there is no bubble.Pvdf membrane is put to gel top, finally placed filter paper and sponge, guarantee that equally Accurate align does not stay bubble.Clamping plate are put into wet turn trough according to positive and negative electrode circuit and connect, in wet turn trough, pour transferring film liquid (48mM Tris-base, 39mM glycine, 20% methyl alcohol) into and switch on power and adjust voltage 12V, wetly turn 12 hours.

(2) sealing: pvdf membrane rinsing in TBST, is put into plastics casing once, add appropriate confining liquid (containing the TBST of 5% skim-milk), lie against room temperature on shaking table and shake gently incubation 1～2h.

(3) primary antibodie is combined with target protein: after sealing, with TBST, pvdf membrane is washed 3 times to each 5min.Add the primary antibodie (anti-PBoV VP2 monoclonal antibody or anti-His label monoclonal antibody) through the dilution of TBST dilution, lie against room temperature incubation 4h on shaking table.Wash 3 times each 5min with TBST.

(4) two anti-and anti-bindings: add the sheep anti-mouse igg of the HRP mark diluting with TBST anti-as two, lie against room temperature incubation 1h on shaking table.Again pvdf membrane is washed 3 times to each 5min with TBST.

(5) add substrate colour developing: the equivalent mixed liquor of pvdf membrane being put into Enhanced chemiluminescence Western blot detection substrate A and B liquid carries out, after lucifuge colour developing 1～5min, blotting the unnecessary substrate of pvdf membrane and directly putting into Molecular Image Station2000MM(Kodak) expose and take a picture.

Western blot result show cell sample that rBV-VP2 infects both can with His monoclonal antibody reaction also can with VP2 monoclonal antibody generation specific reaction, the cell sample of wild-type contrast is without any specific protein band, recombinant baculovirus rBV-VP2 successful expression VP2 albumen in sf9 cell is described, and this albumen and VP2 monoclonal antibody has good specific reaction.Western blot result as shown in Figure 3.

The formation of 2.3 virus-like particles

By recombinant baculovirus rBV-VP2(preserving number: CCTCC V201401) inoculation sf9 cell great expression recombinant protein VP2, the 4 ℃ of centrifugal 30min results of 12000rpm supernatants after harvested cell cracking.Supernatant 25000rpm4 ℃ of centrifugal 5h, precipitation is resuspended with PBS, be laid on 20-60% gradient sucrose 35000rpm, 4 ℃ of centrifugal 5h, draw the interfacial sample of different gradients, carry out western blot evaluation, method as previously mentioned, is chosen protein content the purest the highest sample and is again carried out ultracentrifugation and remove sucrose, TEN solution (10mMTris-HCl, PH8.0 for precipitation; 1mM EDTA, PH8.0; 0.1M NaCl) resuspended, carry out electron microscopic observation, detect the formation of virus-like particle.Electronic Speculum result shows that PBoV virus-like particle (VLPs) size is at 22～27nm, quite similar (the McKillen J.Isolation in cell cultures and initial characterisation of two novel bocavirus species from swine in Northern Ireland.Vet Microbiol of virus particle belonging to natural bocavirus on size and geometric, 152:39-45.), show that recombinant baculovirus rBV-VP2 can form PBoV virus-like particle after sf9 cells VP2 albumen.Electronic Speculum result is as shown in 4.

Embodiment 3: the preparation of recombinant subunit vaccine

Sf9 cell is seeded to 175cm ²in Tissue Culture Flask, until Growth of Cells when being paved with 70-80%, the dosage that P4 is pressed to MOI=5 for recombinant baculovirus rBV-VP2 infects sf9 cell, cultivates 72 hours collecting cell sample for 27 ℃, PBS washing 1 time, ultrasonic degradation cell 20min on ice, lysate is through 12000rpm, 4 ℃, centrifugal 15min removes cell debris, collects supernatant by the VP2 albumen of affinity chromatography method purifying His label.Utilize the protein concentration after spectrophotometric determination purifying, get the supernatant and the adjuvant that contain purifying protein and be mixed with subunit vaccine.

Embodiment 4: subunit vaccine of the present invention and the control vaccine biological experiment to mouse immune effect

1) immune programme for children of BALB/c mouse

4-6 BALB/c mouse in age in week is divided into 3 groups, is respectively VP2 recombinant protein vaccine immune group (200 μ g/mL and 20 μ g/mL) and the PBS control group of various dose, 6 every group, adopt back leg intramuscular injection, every mouse 100 μ L, immunity 2 times altogether, 3 weeks, interval.Within 3,6 weeks after head exempts from, through tail vein negative pressure hemostix, separation of serum, detects and surveys the ELISA antibody for VP2 albumen in the rear serum of immunity.In head, exempt from latter 6 weeks, by cervical vertebra, put to death all immune mouses, aseptic taking-up spleen, utilize lymphocyte separation medium (Beijing Da Kewei Bioisystech Co., Ltd) to separate splenic lymphocyte, carry out cellular immunization detection (comprise stimulate proliferation splenic lymphocyte the mRNA of exponential sum IFN γ transcribes, protein excretion level).

2) in serum, the ELISA antibody horizontal of the anti-PBoV VP2 of specificity detects

By prokaryotic expression plasmid pET-30a, (+) – VP2 transforms the VP2 albumen of induction escherichia coli BL21(DE3) expression and purifying and makes envelope antigen.

(1) after antigen is suitably diluted with coating buffer work, coated 96 hole enzyme plates, 100 μ L/ holes, put wet box interior after 37 ℃ of incubation 1h, are positioned over 4 ℃ spend the night (more than 8h).

(2) take out enzyme plate, dry, with washings washing 3 times, 3min at every turn, after last drying, pats plate hole totally towards being placed down on filter paper repeatedly.

(3) every hole adds 150 μ L confining liquids (insulation liquid), 37 ℃ of sealing 30min.Repeat (2).

(4) with confining liquid, serum to be checked is carried out to 2 times of doubling dilutions from 1:100 on enzyme plate, every hole adds 100uL, and adds negative serum as negative control, after 37 ℃ of effect 2h, repeats (2).

(5) with confining liquid, ELIAS secondary antibody is suitably diluted, 100 μ L/ holes, 37 ℃ of 30min, repeat (2).

(6) add freshly prepared substrate solution (tmb substrate) 100 μ L, the standing 15～20min of room temperature lucifuge.

(7) finally add the HF stop buffer of 50 μ L1%SDS/0.25% with termination reaction, the standing 5min of room temperature measures OD value under 630nm wavelength by microplate reader.The antibody titers that to detect inverse that hole and negative control hole OD ratio is more than or equal to 2.1 high dilution be this serum.

Result shows that head exempts from latter the 3rd week, in 20 μ g/mL, two immunizing dose group Mice Bodies of 200 μ g/mL, produced VP2 antibody, after booster immunization, ELISA antibody horizontal continues to raise for the second time, and between twice immunization experiment group and control group, all there were significant differences (P<0.05, t-test), test-results as shown in Figure 5.

3) splenic lymphocytes test

Adopt MTS method measure the stimulation index of mouse spleen lymphocyte (.MTS such as Ye Ping and mtt assay detect the comparison of mouse NK and specific cytotoxic t lymphocytes activity. China's experiment and clinical virology magazine, 1998,12(1): 85-86).

Add baculovirus expression in 96 porocyte culture plates after, the VP2 albumen of purifying is done to stimulate former.To be 4.0 × 10 with perfect medium dilution ⁶the lymphocyte suspension of cell/mL adds in each hole, every hole 100 μ L.96 porocyte culture plates are placed to 37 ℃, 5%CO ₂in incubator, cultivate 72h.Then every hole adds 20 μ L MTS, mixes rear continuation and cultivates 4～6h.By automatic enzyme mark determinator survey OD492nm value.With stimulation index (Stimulation Index, SI), judge lymphopoiesis titre.The calculating of SI value: test holes OD ₄₉₂nm average/control wells OD ₄₉₂nm average.The cellular immune level of inducing after the dosage immune mouse of result with 200 μ g/mL is the highest, be significantly higher than PBS immune group (P<0.05, t-test), the VP2 albumen that shows expression can be induced stronger specificity lymphopoiesis.Confirmed that recombinant VP 2 albumen can induce significant cellullar immunologic response in Mice Body.Test-results as shown in Figure 6.

4) fluorescence relative quantification RT-PCR method detects lymphocyte is stimulated the mRNA level of rear IFN γ by specific antigen

Employing relative fluorescence quantitative RT-PCR method (Zhang Jingbo etc. Cell Biology Experiment technology. Chemical Industry Press, 2006) detect mouse spleen lymphocyte by the mRNA level of the rear IFN-γ of specific antigen stimulation.Mouse spleen lymphocyte after purified rear VP2 albumen stimulates in vitro, extracts total RNA, utilizes oligo dT that mRNA in-vitro transcription is become to cDNA.Utilize primer as follows:

β-actin-F:5'-GGTCATCACTATTGGCAACG-3'

β-actin-R:5'-TCCATACCCAAGAAGGAAGG-3'

Amplification mouseβ-actin gene β-actin, and set it as the internal reference of relative quantification RT-PCR; Utilize primer as follows:

IFNγ-F:5'-AGCTCTTCCTCATGGCTGTT-3'

IFNγ-R:5'-TTTGCCAGTTCCTCCAGATA-3'

Amplification mouse IFN-γ gene.Quantitative fluorescent PCR reaction system (25 μ L) comprising: the each 0.5 μ L(10 μ M of IFN γ and β-actin upstream and downstream primer),

green Realtime PCR Master Mix12.5 μ L(comprises reaction buffer, dNTP, MgCl ₂, SYBR Green I, Taq enzyme), distilled water 11 μ L, cDNA sample 0.5 μ L.Each sample does three repetitions.Reaction amplification condition is: 50 ℃ of 2min, 94 ℃ of denaturation 10min and then 94 ℃ of sex change 15s of 40 circulations and 60 ℃ of annealing with extend 1min.The variation of the fluorescent signal in whole reaction process is detected by ABI Prism7500 real-time fluorescence quantitative PCR instrument (Applied Biosystems, the U.S.).Result demonstration, the IFN γ level that 200 μ g/mL immune group activate is the highest, is secondly 20 μ g/mL immune group, is all significantly higher than PBS control group, illustrates that VP2 albumen has good immunogenicity.Test-results as shown in Figure 7.

5) ELISA method detects the IFN γ level of splenic lymphocyte secretion

Adopt mouse IFN γ solid phase sandwich ELISA test kit (R & D company, the U.S.) to detect the ability of secreting IFN γ after mouse spleen lymphocyte is stimulated by specific antigen.

(1) press the method that real-time fluorescence quantitative PCR detects the IFN γ relative expression level in immune mouse spleen cell and stimulate the mouse spleen lymphocyte separating, 37 ℃, 5%CO ₂in incubator, cultivate 3d.

(2) from the each 50 μ L culture supernatant of drawing of each culture hole, add the sample diluting liquid of equivalent, and then add and use in the coated enzyme plate of anti-mouse IFN γ monoclonal antibody; Meanwhile, the standard substance of different concns are added to other hole (100 μ L/ hole) of same enzyme plate, so that drawing standard curve.

(3) enzyme plate is hatched after 1h in 37 ℃, washes plate 4 times.

(4) add biotinylated antibody working fluid 100 μ L/ holes, hatch after 1h for 37 ℃, wash plate 4 times.

(5) add enzyme conjugates working fluid 100 μ L/ holes, hatch after 1h for 37 ℃, wash plate 4 times.

(6) add developer 100 μ L/ holes, 37 ℃ of lucifuges are hatched 15-20min.

(7) add stop buffer 100 μ L/ holes, after mixing, at once survey OD450 value.

(8) according to the OD value of standard substance and the concentration drawing standard curve of correspondence and the linear relationship of OD value and concentration, the IFN γ content of calculation sample.

Result shows that the IFN γ level of 200 μ g/mL immune group mouse boosting cell secretions is the highest, reaches 657pg/mL, and secondly 20 μ g/mL immune group, reach 515pg/mL, and two groups are all significantly higher than PBS immune group.Two groups of experimental group contrast control groups of statistical analysis all reach difference utmost point conspicuous level (P<0.01, t-test).Test-results as shown in Figure 8.

Term definition

English name	Chinese
		pFastBac?HTB-VP2	Recombinant transfer plasmid
rBacmid-VP2	Recombinant shuttle plasmid
		rBV-VP2	Recombinant baculovirus
WTBV	Wild-type baculovirus

Other unspecified part is prior art.Although above-described embodiment has been made detailed description to the present invention; but it is only the present invention's part embodiment; rather than whole embodiment, people can also obtain other embodiment according to the present embodiment under without creative prerequisite, and these embodiment belong to protection domain of the present invention.

Claims

1. a recombinant plasmid vector, is characterized in that: this recombinant plasmid vector is the plasmid vector that contains pig bocavirus VP2 gene.

2. recombinant plasmid vector according to claim 1, is characterized in that: described plasmid vector is pFastBac ^tMhTB, this recombinant plasmid called after pFastBac HTB-VP2.

3. recombinant plasmid vector according to claim 1 and 2, is characterized in that: described plasmid vector is baculovirus shuttle plasmid Bacmid, this recombinant shuttle plasmid called after rBacmid-VP2.

4. contain the recombinant baculovirus of pig bocavirus VP2 gene claimed in claim 3.

5. recombinant baculovirus according to claim 4, it is characterized in that: described recombinant virus is recombinant baculovirus rBV-VP2, this recombinant baculovirus is preserved in Chinese Typical Representative culture collection center (CCTCC), preservation date is: on December 20th, 2013, preserving number is: CCTCC V201401.

6. the application of any one in preparation pig bocavirus vaccine in a claim 1～4.

7. the application described in 6 as requested, is characterized in that: described pig bocavirus vaccine is subunit vaccine.

8. a preparation method for subunit vaccine described in claim 6, is characterized in that: after recombinant baculovirus rBV-VP2 is infected to sf9 cell, express the subunit vaccine that VP2 albumen (VLPs) obtains.