CN106148358A - A kind of method utilizing escherichia expression system to prepare pig parvoviral virus sample particle subunits vaccine and application - Google Patents

A kind of method utilizing escherichia expression system to prepare pig parvoviral virus sample particle subunits vaccine and application Download PDF

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CN106148358A
CN106148358A CN201610557798.9A CN201610557798A CN106148358A CN 106148358 A CN106148358 A CN 106148358A CN 201610557798 A CN201610557798 A CN 201610557798A CN 106148358 A CN106148358 A CN 106148358A
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ppv
expression
albumen
protein
pig parvoviral
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张改平
刘运超
王爱萍
陈玉梅
邢广旭
姬鹏超
刘文英
王娟
冯景
刘东民
邓瑞广
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Henan Zhongze Biological Engineering Co ltd
Henan Academy of Agricultural Sciences
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Henan Zhongze Biological Engineering Co ltd
Henan Academy of Agricultural Sciences
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Abstract

The invention discloses the encoding gene of a kind of PPV VP 2 protein, the method for prokaryotic expression VP2 prion sample granule and the application in prepared by vaccine thereof.Sequence is optimized, synthetic VP2 gene, the gene of synthesis is inserted pET28a carrier, then enters in BL21 (DE3) Host Strains with chaperone plasmid corotation, make VP2 albumen and chaperone coexpression, to promote the correct folding of VP2 albumen.It is demonstrated experimentally that the VP2 albumen expressed by this recombinant bacterium can realize self assembly in vitro, and there is good immunogenicity.Use virus-like particle subunit vaccine immune mouse prepared by the VP2 albumen expressed by the present invention and Cavia porcellus the hemagglutination inhibition antibody of induced high levels and neutralizing antibody to produce, and this vaccine can preventing Guinea from the infection of pig parvoviral poison by force.The recombinant bacterium using the present invention can efficiently prepare pig parvoviral virus sample granule, and production cost is low, simple to operate, have more preferable biological safety.

Description

It is single that one utilizes escherichia expression system to prepare pig parvoviral virus sample granule Asia The method of position vaccine and application
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of PPV VP 2 protein solubility expression method, Encoding gene, further relates to the preparation method of a kind of pig parvoviral virus sample granule, and this virus-like particle is as subunit The application of vaccine.
Background technology
Pig parvoviral (Porcine parvovirus, PPV) is one of Etiological causing pig breeding dysfunction, its Be mainly characterized by causing first farrowing sow to miscarry, stillborn fetus, mummy tire and cause the death of newborn piglet.From 1966 Since Mayr and Mahnel finds and confirm that PPV exists and be pathogenic, Chinese scholars has carried out grinding extensively and profoundly to it Study carefully.In recent years, PPV infects in expanding ascendant trend, causes huge economic loss to whole world pig industry.In China, Pan Xue Pearl is isolated to PPV first in nineteen eighty-three.Hereafter, several strain PPV it is isolated to successively in various places, although different separation strains belong to Same serotype, but various clinical symptom can be caused, and all to cause breeding difficulty.It addition, pig parvoviral is frequent With some other virus, the such as mixed infections such as the breeding of porcine circovirus 2 type and pig is viral with respiratory system syndrome, cause breaking Milk piglet multisystem syndrome, dermatitis, respiratory syndrome etc..At present, China's Suprapubic arch sling and breeding boar PPV positive rate are up to More than 80%.
PPV belongs to Parvoviridae, and parvovirus belongs to, and the genome of PPV is sub-thread minus-strand dna, altogether 3 kinds of structure eggs of coding White: VP1, VP2 and VP3,3 kinds of non-structural proteins: NS1, NS2 and NS3.VP2 albumen therein is the capsid egg that this virus is main In vain, there is the ability that self assembly is virus-like particle (virus-like particle, VLP), be good antigen delivery Carrier, also has good immunogenicity simultaneously, effectively can produce immunoreation by induced animal body.
Many researchs and clinical practice show that vaccine immunity is to control a popular very effective method of PPV, pig body Mainly resist PPV by humoral immune reaction after immunity to infect.China is used for preventing the vaccine of PPV to be mainly inactivated vaccine, Although PPV inactivated vaccine can be effectively protected the infection from PPV of the pig body, but have that production cost is high, inactivation of virus is the completeest Entirely and the shortcoming such as immune effect is unstable;It addition, inactivator and residual viral DNA may property dangerous to immune swine body.Mesh Before, novel PPV virus-like particle subunit vaccine obtains to be studied widely, VP2 albumen the virus-like particle formed has The structure similar to PPV virion, and without virulent hereditary material, it is impossible to independently replicate, there is no infectivity, can be with sky So conformation and pattern present antigen, effective stimulus humoral and cellular immune response, induction pig body produces good immunoreation.
At present, the mainly insect cell expression system for expressing PPV virus-like particle of research report, but it produces Relatively costly.By contrast, prokaryotic expression system expressing quantity is high, production cost is low, and production operation is simple, but grasps in reality In work, most albumen form inclusion body owing to can not correctly fold, and bring obstacle for follow-up research work and application.
Summary of the invention
An object of the present invention is to provide the DNA sequence of the pig parvoviral VP2 gene of a kind of manually modified synthesis, as Shown in SEQ ID NO.1.
The two of the purpose of the present invention are to provide a kind of recombinant vector expressing PPV VP 2 protein, this restructuring are carried Body imports after escherichia coli, it is possible to solubility expression VP2 albumen, this VP2 albumen in vitro can self assembly, and have and be similar to Natural viral coagulation.
The three of the purpose of the present invention are to provide a kind of recombinant bacterium expressing PPV VP 2 protein, and this recombinant bacterium contains VP2 recombiant plasmid and chaperone plasmid, it is possible to solubility expression VP2 albumen, this VP2 albumen has coagulation and excellent resisting Originality, may be used for preparing swine parvovirus vaccine.
The four of the purpose of the present invention are to provide a kind of side utilizing bacterium coli solubility to express PPV VP 2 protein Method, the method production cost is low, simple, is suitable for large-scale production.
The five of the purpose of the present invention are to provide the condition that a kind of PPV VP 2 protein assembles in vitro, under the conditions of being somebody's turn to do The virus-like particle formed has and is similar to the size of natural fine small virus, shape and coagulation.
The six of the purpose of the present invention are to provide the application in preparation subunit vaccine of a kind of PPV VP 2 protein, Body will can be induced after this vaccine immunity animal to produce specific immune response, possess the characteristic of anti-PPV poison by force.
In order to achieve the above object, the present invention takes techniques below measure:
Design a kind of recombinant bacterium expressing PPV VP 2 protein, prepared by following key step:
(1) the manually modified synthesis of VP2 gene
With reference to the VP2 gene order (GenBank:AY583318) of pig parvoviral Chinese strain, according to e. coli codon Preferences, the pig parvoviral VP2 gene that synthetic is complete, its nucleotides sequence is classified as shown in SEQ ID NO.1.
(2) structure of recombinant vector
After synthesis pig parvoviral VP2 gene, it is carried out PCR amplification, insert pET28a carrier, product will be connected and convert In JM109 cell, the monoclonal of screening kalamycin resistance, and it is carried out PCR and enzyme action qualification, it is thus achieved that restructuring pET28a- VP2 carrier.
(3) structure of recombinant bacterium
PET28a-VP2 vector is entered in BL21 (DE3) cell, the monoclonal of screening kalamycin resistance, and it is carried out PCR identifies, it is thus achieved that containing the recombinant bacterium of VP2 expression vector;Then (CaCl is passed through2-MgCl2Method) this recombinant bacterium is made sense By state, and chaperone plasmid is proceeded to, it is thus achieved that the final recombinant bacterium expressing solubility VP2 albumen.
The determination of the outer assembling condition of VP2 albuminous body: the VP2 albumen of abduction delivering, after affinitive layer purification, is carried out at 4 DEG C Dialyse to remove imidazoles and then assembling, by changing the salt ionic concentration of dialysis solution, grope VP2 albumen and assemble in vitro Good condition, the albumen after dialysis passes through Electronic Speculum, and dynamic light scattering and blood coagulation tests detect.
The application in preparation subunit vaccine of the above-mentioned pig parvoviral virus sample granule, research shows this vaccine immunity Body can be induced after animal to produce specific immunoreation, there is the characteristic of anti-PPV strong virus attack.Concrete scheme refers to specifically Embodiment.
The method have the benefit that:
Present invention firstly provides employing chaperone and PPV VP2 albumen coexpression in escherichia coli, expressed VP2 albumen Solubility is significantly improved, and this VP2 albumen can be self-assembled into VLP under optimum conditions, and the vaccine made with this VLP is exempted from Epidemic disease mice and Cavia porcellus can induce good specific immune response, can be used for producing the vaccine of prevention pig parvoviral.
(1) present invention is according to the preferences of e. coli codon, and combines substantial amounts of practical studies, manually modified synthesis The DNA sequence of pig parvoviral VP2 gene, the VP2 albumen applying this sequence to express has good immunogenicity.
(2) present invention by pig parvoviral VP2 gene insert pET28a carrier, and with chaperone coexpression, improve The expression of solubility VP2 albumen;It is poly-that this selected molecular chaperones is possible not only to prevent protein from being formed in folding process Collection thing or inactive structure, improve correct folding ratio, and can also affect on folding path.
(3) the pig parvoviral VP2 gene expressed by the present invention can form high-quality pig parvoviral virus sample granule.
(4) the pig parvoviral virus sample granule expressed by the present invention prepares subunit vaccine, can induce after immune animal Body produces the specific immune response for pig parvoviral.
Accompanying drawing explanation
Fig. 1: for pET28a Vector map;
PET28a carrier is started by T7 promoter, with kalamycin resistance gene.
Fig. 2: the enzyme action for prokaryotic expression carrier pET28a-VP2 identifies collection of illustrative plates;
M:DL2000 DNA Marker;1&2:pET28a-VP2 bacterium solution PCR;3&4:pET28a-VP2 double digestion.
Fig. 3: analyze the schematic diagram of VP2 protein expression for SDS-PAGE;
M: protein marker;After 1:BL21 (DE3) empty bacterium induction;After the induction of 2:pET28a empty carrier;3:pET28a-VP2 weight Supernatant after group carrier induction;4:pET28a-VP2 recombinant vector induction postprecipitation;5:pET28a-VP2 recombinant vector and companion's egg Supernatant after white coexpression;6:pET28a-VP2 recombinant vector and chaperone coexpression postprecipitation.
Fig. 4: analyze the schematic diagram of VP2 protein purification for SDS-PAGE;
M: protein marker;After 1:BL21 (DE3) empty bacterium induction;After the induction of 2:pET28a empty carrier;3:pET28a-VP2 weight Supernatant after group carrier and chaperone coexpression;Purified product after 4:pET28a-VP2 recombinant vector and chaperone coexpression.
Fig. 5: analyze the schematic diagram of VP2 protein purification for Western-blot;
After the induction of 1:pET28a empty carrier;Supernatant after 2:pET28a-VP2 recombinant vector and chaperone coexpression;3:pET28a- Purified product after VP2 recombinant vector and chaperone coexpression.
Fig. 6: for the Electronic Speculum testing result schematic diagram of VP2 albumen assembled in vitro;
After VP2 albumen after purification is to different buffer dialysis, observe with transmission electron microscope after phosphotungstic acid negative staining.
Fig. 7: for the dynamic light scattering testing result schematic diagram of VP2 albumen assembled in vitro;
After VP2 albumen after purification is to different buffer dialysis, detect with Malvern Nano-ZS nano particle size instrument.
Fig. 8: for the hemagglutination activity testing result schematic diagram of VP2 albumen assembled in vitro;
After VP2 albumen after purification is to different buffer dialysis, 0.8% mouse red blood cell is applied to detect its hemagglutination activity.
Detailed description of the invention
According to following embodiment, the present invention can be better understood from.Should be understood that following example are merely to illustrate the present invention Not for limiting the scope of the present invention.Technical scheme of the present invention, if not otherwise specified, is the routine side of this area Case, described reagent, if not otherwise specified, it is commercialization or published reagent material.
Embodiment 1
A kind of method of prokaryotic expression PPV VP 2 protein, the method by PPV VP2 albumen and chaperone coexpression, Solubility high recombinant expression protein good, active can be obtained.It specifically comprises the following steps that
The structure of 1.1 recombinant vector pET28a-VP2
1.1.1 the manually modified synthesis of PPV main immunogenic gene VP2
With reference to PPV China strain VP2 gene order (GenBank:AY583318), according to colibacillary codon-bias Being optimized sequence, synthetic VP2 gene, its nucleotides sequence is classified as shown in SEQ ID NO.1.
1.1.2 the structure of pET28a-VP2 carrier
As follows for expanding the primer sequence of VP2 protein coding gene:
F:5 '-GGATCCATGTCGGAAAATGTGGAACA-3’ (BamHI)
R:5 '-AAGCTTATACAGTTTCCGTGGAATGA-3’ (HindIII)
Wherein, forward primer carriesBamHI restriction enzyme site;Reverse primer carriesHindIII digestion site.Primer is by giving birth to Work biological engineering (Shanghai) limited company synthesizes.
Use above-mentioned primer, with synthesis VP2 gene as template, carry out PCR amplification, reaction condition is: 95 DEG C of denaturations 3min;95 DEG C of degeneration 30 s, 56 DEG C of annealing 30 s, 72 DEG C extend 1 min;Totally 30 circulations;72 DEG C extend 10 min.Use 1% The genetic fragment of agarose gel electrophoresis detection amplification, result is as in figure 2 it is shown, genes of interest size is about 1700bp, with expection Result is consistent;With sky root gel, amplified fragments being reclaimed test kit reclaim, description is shown in concrete operations, reclaims product and pET28a Plasmid is respectively through restricted enzymeBamHI andHindIII is digested, and reaction condition is 37 DEG C, 4h.
It is separately recovered the double digestion product of pET28a carrier and VP2 gene subsequently, then connects overnight with T4 ligase 16 DEG C. To connect product and convert e. coli jm109 (TaKaRa) competent cell, detailed process refers to test kit description.Due to Having kalamycin resistance gene on pET28a carrier, the cell after converting coats the LB containing 50 g/mL kalamycin resistances Agar plate.Single bacterium colony on random picking flat board, is inoculated in the LB culture medium with kalamycin resistance, 37 DEG C of cultivations 12h.Identify through bacterium solution PCR and enzyme action and obtain positive colony (result is as shown in Figure 2), the named pET28a-VP2 of positive plasmid, Sending company to check order recombiant plasmid pET28a-VP2, result shows that Insert Fragment sequence is correct, not sudden change and insertion side To correctly.
The structure of 1.2 PPV VP2 E. coli expression strains and expression
1.2.1 the structure of PPV VP2 E. coli expression strains
By thin for positive pET28a-VP2 plasmid transformation escherichia coli BL21 (DE3) (TaKaRa) competence of acquisition in above-mentioned 1.1 Born of the same parents, converted product coats the LB agar plate containing 50 g/mL kalamycin resistances, picking list bacterium colony, identifies through bacterium solution PCR, Screening positive clone is the bacterial strain containing recombinant expression plasmid.
In order to realize the coexpression of VP2 albumen and chaperone, to increase the expression of solubility VP2 albumen, by above-mentioned Recombinant bacterium is inoculated in the LB culture medium that 50mL contains kanamycin, and 37 DEG C of cultivation to OD values are 0.6, then 4 DEG C, 5000rpm Centrifugal 5min collects bacterial sediment, passes through CaCl2-MgCl2Experienced of method, is prepared as competent cell, then by companion's egg White matter grain proceeds to wherein, makes this BL21 (DE3) cell i.e. contain pET28a-VP2 expression plasmid, contains again chaperone plasmid, Owing to two kinds of plasmids are compatible plasmid, transcription and translation each other does not interferes with, so VP2 albumen and companion's egg can be realized White coexpression, coats converted product containing 50 g/mL kanamycin and the LB agar plate of 25 g/mL chloromycetin, picking Single bacterium colony, this bacterium colony is coexpression VP2 albumen and the recombinant bacterial strain of chaperone.
1.2.2 the expression of prokaryotic expression bacterial strain and qualification
The PPV VP2 expression strain 1:100 of above-mentioned acquisition is seeded to containing 50 g/mL kanamycin and 25 g/mL chloromycetin In LB liquid medium, it is simultaneously introduced the expression of 50ng/mL tetracycline induction chaperone, when 37 DEG C of cultivations are to OD value about 0.6 Add the IPTG(isopropylthiogalactoside induction of final concentration of 1.0mmol/L), the expression of induction VP2 albumen, 25 DEG C lure Centrifugal collection thalline after leading 12h.
According to the weight in wet base of above-mentioned thalline, add buffer A (50mmol/L PB, 250mmol/L with the ratio of 1:10 (W/V) NaCl, pH7.0) thalline is resuspended, then put ultrasonic degradation on ice, 12000rpm is centrifuged 20min, cleer and peaceful precipitation in separation, so Rear sampling, identifies expression and the solubility situation of albumen with SDS-PAGE and Western-blot.Result as it is shown on figure 3, according to This figure result, has chaperone and the solubility expression of VP2 albumen in this expression strain.
The preparation of embodiment 2 pig parvoviral virus sample granule
The purification of 2.1 VP2 albumen and the mensuration of VLP
2.1.1 the purification of recombinant VP 2 albumen
Being purified by Ni-NTA chromatographic column (Merck) by the supernatant of ultrasonic degradation, detailed process is: Ni-NTA buffer After A balance, sample is slowly flowed across chromatographic column, then with the buffer B of at least 10 times of column volumes (50mmol/L PB, 250mmol/L NaCl, 30mmol/L imidazole, pH7.0) cross post, clean foreign protein, finally with buffer C (50mmol/ L PB, 250mmol/L NaCl, 300mmol/L imidazole, pH7.0) eluting destination protein, collect elution fraction, and use SDS-PAGE and Western-blot detect, result such as Fig. 4, VP2 albumen after affinitive layer purification purity up to 95% with On, and His monoclonal antibody has good reaction.
2.1.2 albumen assembling in vitro and detection
Albumen after purification is loaded in bag filter, dialyses at 4 DEG C, explore by changing the salt ionic concentration of dialysis solution The optimum condition that VP2 albumen assembles in vitro, set salt ionic concentration is respectively as follows: 50mmol/L, 100mmol/L, 150mmol/ L、200mmol/L.The form of the VLP then formed by transmission electron microscope observing, by the particle diameter of nano particle size instrument detection VLP Distribution, and the hemagglutination activity of VLP is detected by blood coagulation tests.Result is as it is shown in figure 5, can be seen that prokaryotic expression according to result It is similar with natural PPV virus with in terms of hemagglutination activity in form, size that VP2 can be assembled into VLP, this VLP in vitro.Enter one Step, this VLP is at 50mmol/L NaCl, and the quality formed during pH7.0 is preferable, and formation rate is higher.
Concrete detection method is as described below:
Transmission electron microscope (TEM) detects: the PPV VLP obtained by above-mentioned purification, through 2% phosphotungstic acid negative staining, utilizes transmission electron microscope observing The form of VLP, its diameter is about 24-25nm, and size is uniform, is rendered as hollow form, and result is as shown in Figure 6.
Dynamic light scattering (DLS) detects: the PPV VLP obtained by above-mentioned purification is detected by nano particle size instrument, knot Fruit such as Fig. 7, shows according to DLS result, and the VLP purity obtained in the present invention is high, and size is homogeneous, structural integrity.
Hemagglutination activity (HA) detects: take 96 hole V-type micro-reaction plates, and every hole adds 50 μ L PBS, and each hole of first row adds 50 μ L antigens, then carry out 2 times of serial dilutions, until the 11st hole discards 50 μ L by antigen.Then to add 0.8% mice red in every hole Cell suspension 50 μ L, with trace oscillator mix homogeneously, puts effect 1h under room temperature.During result of determination, with 100% red cell agglutination Antigen highly diluted multiple as judge terminal.
Embodiment 3 PPV virus-like particle immunogenicity detects
The mensuration of 3.1 VP2 protein concentrations and the mensuration of endotoxin content
The concentration of the VP2 albumen of purification is measured, specifically with BCA protein assay kit (Pierce Biotechnology) Step is with reference to its description.The concentration of measured VP2 albumen is 0.57mg/mL, and expression is higher, it is possible to meet industry metaplasia The requirement produced.
With endotoxin assay test kit (ToxinSensorTMEndotoxin Detection System, GenScript) measuring the endotoxin content of purifying protein, concrete steps are with reference to its description, the endogenous toxin of measured VP2 albumen Cellulose content is less than 0.3mU/mg.
3.2 animal immune
With reference to the method in the Pharmacopoeia of the People's Republic of China (version in 2005), the VLP obtained in embodiment 2 is prepared as epidemic disease Seedling.
3.2.1 the immunity of mice
Virus-like particle, in the presence of Freund adjuvant, divides 5 μ g/, the Kunming mouse of 10 μ g/ only immunity 3-4 week old, sets simultaneously Pig parvoviral inactivated vaccine immune group and phosphate buffer inoculation group, as control comparisons, often organize 5 mices of immunity.Immunity altogether 2 times, with Freund's complete adjuvant, carry out weekly tail vein blood after exempting from incomplete Freund's adjuvant, head, separate blood for the 2nd time for the 1st time Clear and preserve, in order to the hemagglutination inhibition antibody in subsequent detection mice serum and NAT, result shows, expressing protein The virus-like particle formed, can stimulate mice to produce the specific antibody of pig parvoviral, and the antibody horizontal produced is bright Aobvious higher than inactivated vaccine immune group.
3.2.2 the immunity of Cavia porcellus
24 body weight are the female healthy guinea pig of 400-500g, are randomly divided into 4 groups, often group 6, A group and B group oil adjuvant, point The most immune 50 μ g/, 25 μ g/, C group immunity PPV inactivated vaccine is as positive control, and D group immunity PBS is as negative control.Exempt from Epidemic disease is divided 2 times and is carried out, and interval time is two weeks, within after first immunisation every 14 days, carries out Culling heart blood, separates Virus monitory serum antibody, And (often group 2) aseptic spleen of winning after lethal in 64 days, separating spleen lymphocyte detection PBMC propagation and cytokine Secretion situation, then subcutaneous injection PPV standard poison, carries out cuing open killing for after counteracting toxic substances 7 days, gathers the tissues such as conscience spleen lung kidney, passes through RT- PCR detection tissue toxic amount.Result show formed virus-like particle can induce Cavia porcellus produce good humoral immunization and Cell immune response, can be effectively protected the body attack from pig parvoviral poison by force.
3.3 antibody horizontal detections
3.3.1 hemagglutination inhibition antibody detection
The preparation of 0.8% mouse red blood cell:
With sterilizing syringe draw the fresh Mouse Blood of 1mL in the sterile centrifugation tube containing the heparin of 1000U in;To above-mentioned centrifugal Pipe adds in 9mL PBS and is centrifuged 5min with 1500rpm, abandon supernatant;With the resuspended blood cell of 10mL PBS, 2000rpm is centrifuged 10min, abandons supernatant, so by red blood cell washing three times;Finally according to institute's expense, it is made into 0.8% (v/v) mice with PBS red carefully Born of the same parents' suspension, used within 5 days.
HA determines 4 unit poison valencys:
Carrying out on 96 hole Microhemagglutination plates, the most each hole adds 50 L PBS;50 L virus liquids are added in hole, left side the 1st, mixed After closing uniformly, inhaling 50 L to the 2nd hole, doubling dilution is to the 10th hole the most successively, inhales and abandons 50 L;11st, 12 holes are erythrocyte pair According to;Add 0.8% mouse red blood cell suspension 50 L successively to each hole from right-to-left after dilution, mixing of vibrating on the oscillator, Room temperature places observed result after 1h, and with the viral highest dilution of erythrocyte 100% coagulation as terminal, these dilution 4 times are 4 unit poison.
Antibody in HI detection serum:
Adding 25 L PBS in each hole of V-type Sptting plate, standard 96 hole, the sample that often detection of row hole is different, often in row 1st hole adds the serum to be checked of 25 L, takes out 25 L and add to the 2nd hole after mixing, and doubling dilution is to the 10th hole successively, abandons 25 L, 11st hole adds positive serum and does positive control, and the 12nd hole adds negative serum and does negative control;Then every hole adds 4 units of 25 L Poison, vibration mixing, incubated at room 30min;Rear every hole adds the mouse red blood cell suspension of 50 L 0.8%, and incubated at room 2h is observed As a result, to completely inhibit the serum highest dilution blood clotting suppression titer as this serum of red cell agglutination.As shown in Figure 8, Blood clotting suppression titer is 1:5120.
3.3.2 neutralizing antibody detection
Pig parvoviral standard strain poison valency measures:
Seed culture of viruses for this test method is pig parvoviral standard strain, and purchased from China Veterinary Drugs Supervisory Inst., this strain can be at PK- Breed on 15 cells, good to the immunogenicity of pig.Viral level measures: PK-15 cell is with 2 × 105/ mL is inoculated in 96 porocytes Culture plate, until at the bottom of cell covers with 80% hole time, i.e. can be used for virus inoculation;With DMEM culture fluid, seed culture of viruses made continuous 10 times of series Dilution, each dilution factor takes 100 L and adds in above-mentioned 96 porocyte culture plates, and each dilution factor makees 8 repetitions, and sets normal thin Born of the same parents cultivate comparison, put 37 DEG C, containing 5% CO2After cultivating 2h in incubator, discarding virus liquid, every hole adds 100 L and contains 3% FBS DMEM maintain liquid, put 37 DEG C, containing in 5% CO2 incubator continue cultivate;Day by day observation of cell pathological changes, cell pyknosis, assemble, Granule increases, and some cells merge, come off, occur that more space is then judged to CPE.Observe 3, record cytopathic hole count, The TCID of virus is calculated according to Reed-Muench method50.Result: every milliliter of virus liquid is not less than 106.0TCID50
The detection of serum neutralizing antibody:
The detection of pig parvoviral neutralizing antibody is carried out with reference to the standard in the Pharmacopoeia of the People's Republic of China (version in 2005), tool Body embodiment is: Sample serum inactivates 30min in 56 DEG C, then with 1:10 for initial concentration times in 96 porocyte culture plates Ratio is diluted to the 10th hole, and every hole adds 7909 strain pig parvoviral 100 TCID subsequently50, mixing is placed on 37 DEG C, containing 5% CO2 Incubator is hatched 2h, then takes 100 L and be inoculated in cut-and-dried length and have 96 porocyte culture plates of 80% PK-15 cell In, it is placed in 37 DEG C, cultivates after 2 days containing in 5% CO2 incubator, observation of cell pathological changes, with the serum of 50% cell generation pathological changes High dilution is as this serum NAT.Result display serum NAT is about 1:420.
SEQUENCE LISTING
<110>Henan Academy of Agricultural Sciences
<120>a kind of method utilizing escherichia expression system to prepare pig parvoviral virus sample particle subunits vaccine and
Application
<130> /
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1443
<212> DNA
<213>artificial sequence
<400> 1
atgcacgctt ctcgtctgat ccacctgaac atgccggaac acgaaaccta caaacgtatc 60
cacgttctga actctgaatc tggtgttgct ggtcagatgg ttcaggacga cgctcacacc 120
cagatggtta ccccgtggtc tctgatcgac gctaacgctt ggggtgtttg gttcaacccg 180
gctgactggc agctgatctc taacaacatg accgaaatca acctggtttc tttcgaacag 240
gaaatcttca acgttgttct gaaaaccatc accgaatctg ctacctctcc gccgaccaaa 300
atctacaaca acgacctgac cgcttctctg atggttgctc tggacaccaa caacaccctg 360
ccgtacaccc cggctgctcc gcgttctgaa accctgggtt tctacccgtg gctgccgacc 420
aaaccgaccc agtaccgtta ctacctgtct tgcatccgta acctgaaccc gccgacctac 480
accggtcagt ctcagcagat caccgactct atccagaccg gtctgcactc tgacatcatg 540
ttctacacca tcgaaaacgc tgttccgatc cacctgctgc gtaccggtga cgaattctct 600
accggtatct accacttcga caccaaaccg ctgaaactga cccactcttg gcagaccaac 660
cgttctctgg gtctgccgcc gaaactgctg accgaaccga ccaccgaagg tgaccagcac 720
ccgggtaccc tgccggctgc taacacccgt aaaggttacc accagaccat caacaactct 780
tacaccgaag ctaccgctat ccgtccggct caggttggtt acaacacccc gtacatgaac 840
ttcgaatact ctaacggtgg tccgttcctg accccgatcg ttccgaccgc tgacacccag 900
tactacgacg acgaaccgaa cggtgctatc cgtttcacca tgggttacca gcacggtcac 960
ctgaccacct cttctcagga actggaacgt tacaccttca acccgcagtc taaatgcggt 1020
cgtgctccga tgcagcagtt caaccagcag gctccgctga acctggaaaa caccaacaac 1080
ggtaccctgc tgccgtctga cccgatcggt ggtaaatcta acatgcactt catgaacacc 1140
ctgaacacct acggtccgct gaccgctctg aacaacaccg ctccggtttt cccgaacggt 1200
cagatctggg acaaagaact ggacaccgac ctgaaaccgc gtctgcacgt taccgctccg 1260
ttcgtttgca aaaacaaccc gccgggtcag ctgttcgtta aaatcgctcc gaacctgacc 1320
gacgacttca acgctgactc tccgcagcag ccgcgtatca tcacctactc taacttctgg 1380
tggaaaggta ccctgacctt caccgctaaa atgcgttctt ctaacatgtg gaacccgatc 1440
taa 1443
<210> 2
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<212> DNA
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ggatccatgt cggaaaatgt ggaaca 26
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<213>artificial sequence
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aagcttatac agtttccgtg gaatga 26

Claims (9)

1. encoding a gene for PPV VP 2 protein, its sequence is as shown in SEQ ID NO.1.
2. an expression vector, containing the gene order described in claim 1.
3. a host cell, containing the expression vector described in claim 2, and can be with the companion of this expression vector coexpression Protein expression vector.
4. host cell as claimed in claim 3, it is characterised in that: described PPV VP 2 protein is solubility expression.
5. the solubility expression method of a PPV VP 2 protein, it is characterised in that: by PPV VP 2 protein with Chaperone co expression.
6. the solubility expression method of as claimed in claim 5 PPV VP 2 protein, it is characterised in that specifically include with Lower step:
A. design and synthesize codon optimized pig parvoviral VP2 gene, be attached with pET28a carrier, build protokaryon table Reach carrier;
B. VP2, the E. coli recombinant stain of chaperone coexpression are built;
C. recombinant bacterial strain inoculation while add tetracycline, 37 DEG C cultivate 2~3h, to OD value reach 0.6 time, addition The expression of 1.0mmol/L isopropylthiogalactoside induction VP2 albumen.
7. the expression of a pig parvoviral virus sample granule, it is characterised in that comprise the following steps:
By claim 6 gained inducible strain ultrasonic degradation, the supernatant after cracking passes through affinitive layer purification, by the product of purification Thing buffer is dialysed, and to obtain final product.
8. the expression of pig parvoviral virus sample granule as claimed in claim 7, it is characterised in that: the ring of described dialysis Border condition is: 4 DEG C, 50mmol/L NaCl, pH7.0.
9. the application in preparation subunit vaccine of the pig parvoviral virus sample granule expressed by claim 7.
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CN108318684A (en) * 2018-04-04 2018-07-24 南京农业大学 A kind of the visible protein chip preparation method and detection method of detection pig parvoviral antibody
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CN110845580A (en) * 2019-11-05 2020-02-28 中国农业科学院兰州兽医研究所 Method for assembling porcine parvovirus-like particles and identifying immunogenicity thereof
CN111606978A (en) * 2020-06-09 2020-09-01 河南省农业科学院 Method for removing endotoxin in escherichia coli recombinant expression porcine parvovirus VP2 protein
CN111606978B (en) * 2020-06-09 2023-03-17 河南省农业科学院 Method for removing endotoxin in escherichia coli recombinant expression porcine parvovirus VP2 protein
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Application publication date: 20161123