CN103936839A - Recombinant porcine parvovirus VP2 protein, recombinant polyhedrosis virus for expressing protein as well as application of protein in vaccine preparation and virus diagnosis - Google Patents

Recombinant porcine parvovirus VP2 protein, recombinant polyhedrosis virus for expressing protein as well as application of protein in vaccine preparation and virus diagnosis Download PDF

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CN103936839A
CN103936839A CN201410142613.9A CN201410142613A CN103936839A CN 103936839 A CN103936839 A CN 103936839A CN 201410142613 A CN201410142613 A CN 201410142613A CN 103936839 A CN103936839 A CN 103936839A
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porcine parvovirus
virus
albumen
cell
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崔尚金
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Harbin National Biological Polytron Technologies Inc
Harbin Veterinary Research Institute of CAAS
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HARBIN ANIMAL BIOLOGICAL PRODUCTS NATIONAL ENGINEERING RESEARCH CENTER CO LTD
Harbin Veterinary Research Institute of CAAS
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Abstract

The invention discloses a recombinant porcine parvovirus VP2 protein, a recombinant polyhedrosis virus for expressing the protein as well as an application of the protein in vaccine preparation and virus diagnosis. An original VP2 protein is modified, so that the HA titer of the modified VP2 protein is improved from 213 to 218. Furthermore, the invention also discloses the recombinant polyhedrosis virus for expressing the recombinant porcine parvovirus VP2 protein. The experiment proves that the recombinant VP2 protein expressed by the virus has the advantages of high immunogenicity, high titer and the like. The first farrowing healthy negative sows are immunized by using porcine parvovirus VP2 subunit vaccines prepared from the VP2 protein, so that diseases caused by the porcine parvovirus can be prevented, and the porcine parvovirus VP2 protein has the advantages of safety, fast speed of antibody generation, lasting immunity period and the like. In addition, the modified VP2 protein serves as an antigen-coated ELISA plate, an indirect ELISA method for porcine parvovirus antibodies is established, the sensitivity is high, the specificity is high, and a good technical means is provided for diagnosis and detection of the porcine parvovirus.

Description

Recombination porcine parvovirus VP2 albumen, the restructuring polyhedrosis virus of expressing this albumen and the application in vaccine preparation and viral diagnosis thereof
Technical field
The present invention relates to a kind of recombination porcine parvovirus VP2 albumen, express restructuring polyhedrosis virus and the application in vaccine preparation and viral diagnosis thereof of this albumen, be particularly related to the restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 of the expression VP2 albumen that a strain prepares voluntarily), this virus can realize the high efficient expression to VP2 albumen in cell, the invention still further relates to recombination porcine parvovirus VP2 albumen and is preparing pig parvoviral VP2 subunit vaccine and the application in the reagent of preparation diagnosis or detection porcine parvovirus infection.The invention belongs to detection and the diagnostic field of biovaccine and pig parvoviral.
Background technology
Pig parvoviral (Porcine parvovirus, PPV) be one of main pathogen of causing pig breeding dysfunction, the main object that PPV infects is pregnant pig, progestational first farrowing sow particularly, farrowing sow miscarriage be can cause, stillborn foetus, fetus mummification etc. produced, and parent lacks clinical symptom conventionally, also can cause other symptoms in addition.From Mayr in 1966 and Mahnel find and confirm that PPV exists and pathogenic since, Chinese scholars has been carried out research extensively and profoundly to it.In recent years, PPV infects and is expansion ascendant trend, has caused huge financial loss to global pig industry.In China, Pan Xuezhu has been separated to PPV first in nineteen eighty-three.After this, several strain PPV in various places, have been separated to successively, at present, it is found that pig parvoviral often with some other virus, polyinfections such as porcine circovirus 2 type and pig breeding and respiratory system syndrome virus, cause weanling pig multisystem syndrome, dermatitis, respiratory syndrome etc., cause very large loss to the pig industry in the world.Although different isolates all belongs to same serotype, can cause various clinical symptom, but all take, cause breeding difficulty as main.At present, China's multiparity sow and herd boar PPV positive rate are up to more than 80%.
Because PPV breeds difficulty in cell, tire not high, and its main immunogens is VP2 albumen, so the present inventor is separated to a strain pig parvoviral on 2005 pig farms, Nian Cong Heilongjiang Province, through laboratory qualification, be virulent strain, after domestication clone by this strain called after PPV BQ-C strain.This virus strain has been documented in application number for " 201210166453.2 ", in the patent application of denomination of invention for " pig parvoviral BQ-C strain and the application in preparing porcine parvovirus inactivated vaccines thereof ".By the VP2 gene of PPV BQ-C strain, be cloned in restructuring polyhedrosis virus expression vector, prepared the restructuring polyhedrosis virus of the expression VP2 of high-titer, and carried out the development work of porcine parvovirus VP2 subunit inactivated vaccine, the restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 that immunogenicity is good, tire expression VP2 stable is cultivated in screening), and selected the most applicable inactivator, adjuvant and other conditions of preparing vaccine called after restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 strain).The production technique of goods, security, protection ratio, immune programme for children, minimum dose, antibody extended period and preservation period etc. have all been carried out to test determination, and obtained a large amount of testing datas, result proves that this vaccine safety is effective.On this basis, carried out middle trial production, through sampling inspection, all meet the quality standard of drafting, and pilot product has been carried out to proof test and potency test, its result also proved vaccine can be prevented the sow breeding difficulty being caused by pig parvoviral effectively, on the basis of laboratory test, interim test, the present invention has developed pig parvoviral (BQ-C strain) VP2 subunit inactivated vaccine, its result also proved vaccine can be prevented the sow breeding difficulty being caused by pig parvoviral effectively, has further verified every quality standard of this vaccine.Meanwhile, utilize the VP2 albumen of expressing, prepared ELISA detection method corresponding to vaccine.The method susceptibility is good, specificity is high, and yin and yang attribute threshold value is: 0.181+3 * 0.030=0.271, finally determines and judges that scope 0.25-0.3 is as suspicious.When OD450nm > 0.3 is judged to the positive; OD450nm < 0.25 is negative.With the comparison coincidence rate of French LSI test kit be 96.7%.
Summary of the invention
The advantages such as one of object of the present invention is to provide a kind of improved recombination porcine parvovirus VP2 albumen, and this albumen has the height of tiring, and immunogenicity is good;
Two of object of the present invention is to provide the restructuring polyhedrosis virus AcMNPV of the above-mentioned recombination porcine parvovirus VP2 of a kind of stably express albumen;
Three of object of the present invention is to provide described improved recombination porcine parvovirus VP2 albumen and detects or diagnose the application in pig parvoviral antibody reagent, particularly ELISA detection reagent at the preparation prevention tiny medicine of pig and in preparation.
The restructuring polyhedrosis virus AcMNPV that four of object of the present invention is to provide described stably express recombination porcine parvovirus VP2 albumen prevents the application in the tiny medicine of pig in preparation.
In order to achieve the above object, the present invention has adopted following technique means:
On the basis that the present inventor is template in the patent strain PPV BQ-C pnca gene group of utilizing existing invention, vp2 gene order is analyzed to rear discovery with software, vp2 gene order is lower in sf9 cells efficiency, analyzing reason may be that this sequence derives from virus, cannot be in insect cell due to species variation fine table
Reach.Therefore, for obtain can be in cell the native protein sequence of high expression level, improve antigen effective content simultaneously, inventor has carried out analyzing transformation to the invalid antigen of VP2 region, by software analysis and in conjunction with specific experiment, verify, gene order (shown in SEQ ID NO.2) and the coding protein sequence (shown in SEQ ID NO.1) thereof of improved vp2 have finally been determined, improved sequence has been carried out again synthetic, then synthetic vp2 gene after transformation is proceeded in restructuring polyhedrosis virus transfer vector pFastBacI, construction recombination plasmid pFastPVP2, again this recombinant plasmid is proceeded in DH10Bac competent cell, by blue hickie screening picking white bacterial plaque, also by the method for PCR, identify positive bacterium colony, extract genome, be restructuring Polyhedrosis gene-rod granule.Use transfection reagent Cellfectin (Invitrogen) that restructuring rod granule is proceeded in sf9 cell, thereby obtain the polyhedrosis virus of recombinating.By the restructuring polyhedrosis virus inoculation sf9 cell obtaining, harvested cell after 96 hours, after-20 ℃ of multigelations, by carrying out western blot, IPMA, identify, find the expression that VP2 albumen is succeeded, and, by above transformation, the improved VP2 albumen HA expressing under similarity condition is tired by 2 before transformation 13bring up to 2 18.Express the restructuring polyhedrosis virus AcMNPV of this VP2 albumen, called after pFastBac-VP2.
Therefore, the present invention proposes a kind of improved recombination porcine parvovirus VP2 albumen, the aminoacid sequence of described recombination porcine parvovirus VP2 albumen is as shown in SEQ ID NO.1.And
The nucleotide sequence of the recombination porcine parvovirus VP2 albumen that coding is described, preferred, described nucleotide sequence is as shown in SEQ ID NO.2.
The expression vector that contains described nucleotide sequence and the host cell that contains described expression vector are also within protection scope of the present invention.
Further, the invention allows for described recombination porcine parvovirus VP2 albumen and preparing pig parvoviral VP2 subunit vaccine and the application in the reagent of preparation diagnosis or detection porcine parvovirus infection.
A kind of pig parvoviral VP2 of the present invention subunit inactivated vaccine, is characterized in that containing described recombination porcine parvovirus VP2 albumen.
In pig parvoviral VP2 subunit inactivated vaccine of the present invention, preferred, the sub-albumen of described pig parvoviral (BQ-C strain) VP2 adopts following methods to carry out deactivation:
Described pig parvoviral VP2 subunit inactivated vaccine is by 0.2%(w/w) divinyl imines (BEI) deactivation after 96 hours under 32 ℃ of conditions, add 0.02%(w/w) Sulfothiorine stops, after deactivation, adopting 15%(v/v) the commercialization MontanideTM ISA11R adjuvant of France match BIC Corp after emulsification, obtain.
In the present invention, emulsifying process is the commercialization MontanideTM ISA11R adjuvant that adopts France match BIC Corp, can be directly used in vaccine emulsification preparation after degerming; The mixing of same batch of virus liquid of deactivation being thawed before emulsification, stirs with 200r/min at aseptic indoor refiner, then according to water antigen, mixes according to volume ratio 8.5:1.5 proportioning with oil phase adjuvant; Emulsification program stirred for first water being put into emulsor, then slowly adds oil phase adjuvant, with 800r/min continuously stirring 30 minutes; The vaccine that production obtains belongs to oil-in-water formulation, for stable interface and elimination bubble, obtains optimum emulsification effect, needs packing after static 30 minutes.
The present invention is a kind of for diagnosing or detect the ELISA diagnostic kit of porcine parvovirus infection, it is characterized in that containing described recombination porcine parvovirus VP2 albumen.
The present invention utilizes the VP2 albumen of expression, has prepared corresponding ELISA detection method.The method is responsive, special, and yin and yang attribute threshold value is: 0.181+3 * 0.030=0.271, finally determines and judges that scope 0.25-0.3 is as suspicious.When OD450nm > 0.3 is judged to the positive; OD450nm < 0.25 is negative.With the comparison coincidence rate of French LSI test kit be 96.7%.
Further, the present invention proposes a kind of restructuring polyhedrosis virus AcMNPV that expresses PPV VP 2 protein, restructuring polyhedrosis virus AcMNPV of the present invention, for after going down to posterity on sf9 cell the 10th generation virus strain, this virus strain called after pFastBac-VP2, Classification And Nomenclature is for expressing the restructuring polyhedrosis virus of PPV VP 2 protein, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC No.9004, preservation date is on March 26th, 2014.The aminoacid sequence of the PPV VP 2 protein of wherein, being expressed by this virus strain is as shown in SEQ ID NO.1.
The key parameter of virus high efficient expression VP2 in Sf9 cell described in the present invention also provides.By changing efficient infection multiplicity (MOI), the serum-concentration of virus, and then changed the growth kinetics curve of virus replication, made viral offspring's quality, extracellular environment, recombinant protein output all changes to some extent.The HA of VP2 tires by 2 13bring up to 2 18.
A kind of described restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 that utilizes of the present invention) express the method for PPV VP 2 protein, it is characterized in that comprising the following steps:
(1) suspension culture Sf9 cell, treats that cell density reaches 5-10 * 10 5during individual cells/ml, stop cultivating, prepare to connect poison;
(2) keep shaking speed under the condition of 120rpm, by described restructuring polyhedrosis virus AcMNPV(pFastBac-VP2) take infection multiplicity MOI and through step (1), cultivate as 10PFU/ cell access the Sf9 cell obtaining, 120rpm cultivates results virus after 24-48 hour;
(3) purifying VP2 albumen, obtains.
Further, the present invention also provides the application in prepare pig parvoviral VP2 subunit inactivated vaccine of described virus.
Further, the present invention also provides the purposes in the biological products of the disease that described VP2 subunit inactivated vaccine causes by pig parvoviral in preparation prevention.Especially, described disease is the sow breeding difficulty that the healthy negative sow of primiparity occurs.
Accompanying drawing explanation
Fig. 1 is effective and invalid antigenic region comparison diagram before and after the transformation of antigenic region; Wherein Figure 1A is for before transforming; Figure 1B is for after transforming;
Fig. 2 is for expressing the restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 strain of VP2) IPMA detected result after cultivating on sf cell;
IPMA detected result after the wild-type restructuring polyhedrosis virus that Fig. 2 A does not express VP2 for inoculation is cultivated on sf cell; Fig. 2 B expresses the restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 strain of VP2 for inoculation) IPMA detected result after cultivating on sf cell;
Fig. 3 is VP2 Protein Detection and purification result.
Fig. 3 A is albumen preliminary purification result; Fig. 3 B is that first protein sample is through Ni 2+electrophoresis result purity of protein after filler affinity chromatography is 20%, and total protein concentration is very high; Fig. 3 C is that second batch protein sample is through Ni 2+electrophoresis result after filler affinity chromatography; Fig. 3 D is through Ni 2+albumen after filler affinity chromatography is the protein electrophoresis result after 30kD ultrafiltration is centrifugal again, and purity of protein is 30%, and total protein concentration is very high.
Embodiment
Below by experiment, also the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiment, only for the object of illustration, never limit the scope of the invention.
Embodiment 1:VP2[Porcine parvovirus] transformation of GenBank:ACF39501.1 expressing gene
In order to improve antigen effective content, the present invention, according to gene software analysis, has carried out analyzing transformation to the invalid antigen of VP2 region, has improved antigen effective area.Concrete analysis transformation process is as follows:
1) protein sequence before transformation:
GenBank accession number: ACF39501.1
2) before transformation, Effective Antigens district (bobbin upper zone) and invalid antigenic region (region below bobbin) analytical results, as shown in accompanying drawing 1A, transforms front 1 and transform front 2 and be respectively and transform the position 1 of presequence and the analytical results at position 2.
3) the present invention is directed to inactive area transformation, transform rear Effective Antigens district (bobbin upper zone) and invalid antigenic region (region below bobbin) result as shown in accompanying drawing 1B, position 1 and the position 2 of sequence after transformation rear 1 and transformation rear 2 are respectively and transform.From Fig. 1 result, can find out that improved Effective Antigens region has expanded more than 2 times.
4) need the sequence of optimization and transformation: according to above analytical results, we determine that the regional sequence that needs transformation and optimize is: GVSTGSFNNQTEFQXEINLVSFEQXSATSPPTKIYNNDLTXETLGFYPWLPTKPTQ YRYYLSCTRXETLGFYPWLPTKPTQYRYYLSCTRXVGYNTPYMNFXYQHGQLTT SSQELERYTFXMNTLNTYGPLTALNNTAPVFPNGQIWDKELDTDLKPRLHVTAPF VCKNNPPGQLFVKIAXFNADSPQQPRIITYSNFWWKXAENIGNYIPTNIGGIKMFP EYSQLIPRKLY, the sequence that wherein X representative can further be optimized in different carriers.
5) optimization of sequence and synthetic
By software analysis and in conjunction with specific experiment, verify, finally determined improved vp2 gene order (shown in SEQ ID NO.2) and coding protein sequence (shown in SEQ ID NO.1) thereof.
By above transformation, the VP2 albumen HA expressing under similarity condition tires by 2 13bring up to 2 18, improved VP2 albumen HA tires and is improved significantly as can be seen here.
Embodiment 2: preparation and evaluation the restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 strain of expression VP2)
1 seed culture of viruses source and standard
According to new biological product requirements of customs declaration, the a large amount of testing data obtaining in conjunction with the present invention, with reference to < < Chinese veterinary pharmacopoeia > > (version in 2005), seedling is identified with seed culture of viruses, now seed culture of viruses standard in Trial Regulation (draft) is carried out to following explanation.
1.1 seed culture of viruses source and cultural methods
Seed culture of viruses of the present invention is for expressing the restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 strain of VP2), that to utilize PPV BQ-C pnca gene group be template to the inventor, after VP2 gene is transformed, VP2 gene (shown in SEQ ID NO.2) is proceeded to the plasmid pFastPVP2 that builds restructuring in restructuring polyhedrosis virus transfer vector pFastBacI, again this recombinant plasmid is proceeded in DH10Bac competent cell, by blue hickie screening picking white bacterial plaque, also by the method for PCR, identify positive bacterium colony, extract genome, be restructuring Polyhedrosis gene-rod granule.Use transfection reagent Cellfectin (Invitrogen) that restructuring rod granule is proceeded in sf9 cell, thereby obtain the polyhedrosis virus of recombinating.By the restructuring polyhedrosis virus inoculation sf9 cell obtaining, harvested cell after 96h, after-20 ℃ of multigelations, by carrying out western blot, IPMA, identify, find the expression that VP2 albumen is succeeded, express the restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 of VP2) IPMA detected result after cultivating on sf cell is as shown in Figure 2.This expresses the restructuring polyhedrosis virus of VP2, for after going down to posterity on sf cell the 10th generation virus strain, called after pFastBac-VP2, Classification And Nomenclature is for expressing the restructuring polyhedrosis virus of PPV VP 2 protein, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, and its culture presevation is numbered: CGMCC No.9004, preservation date is on March 26th, 2014.The aminoacid sequence of the PPV VP 2 protein of wherein, being expressed by this virus strain is as shown in SEQ IDNO.1.
Cultivate: sf cell density is 5-10 * 10 5individual cells/ml, the MOI of infection is 10PFU/ cell, and guarantees synchronously to infect in 250mL shaking flask (rotating speed 120RPM).Within 42 hours, results are viral later, and now cell infection amount is more than 90%, and it is 2 that the HA of VP2 tires 18, than 2 of general bibliographical information 13high 32 times.
1.2 seeds culture of viruses (pFastBac-VP2 strain) standard
1.2.1 red cell agglutination valency
Get the 96 U-shaped micro-reaction plates in hole, every hole adds 25 μ l PBS(0.01mol/L, pH value 7.0～7.4), in each hole of first row, add 25 μ l antigens, then antigen is carried out to 2 times of serial dilutions, until the 11st hole discards 25 μ l.Every hole adds 25 μ l PBS, then adds 0.6% guinea-pig red blood cell suspension 25 μ l, with micro oscillator, mixes, and puts under room temperature and acts on 1 hour.During result of determination, using the highly diluted multiple of antigen of 50% red cell agglutination as judging terminal.
Result: seed culture of viruses should be not less than 1:512 to guinea-pig red blood cell agglutination titer.
1.2.2 viral level
With nutrient solution, seed culture of viruses is made to continuous 10 times of serial dilutions, each extent of dilution is got 100 μ l and is added in 96 porocyte culture plates, and each extent of dilution is done 8 repetitions, the sf9 cell suspension that adds subsequently digestion to disperse, and every hole 100 μ l(cell contents are with 3 * 10 5/ mL), and establish normal cell and cultivate contrast, put 22 ℃, the CO containing 5% 2in incubator, cultivate, observation of cell pathology (CPE) day by day, cell pyknosis, assembles, and particle increases, and some cytogamy, comes off, occurs that more space is judged to CPE.Observe 7, record cytopathic hole count, according to Reed-Muench method, calculate viral TCID 50.
Result: every milliliter of virus liquid is not less than 10 6.0tCID 50.
1.2.3 immunogenicity
Seed culture of viruses is inoculated to sf9 cell and breed, results virus liquid, purifying obtains VP2 albumen, with after BEI deactivation, adds Montanide tMiSA15AVG adjuvant mixing and emulsifying is made oil-in-water-type inactivated vaccine, with 5 of healthy susceptible first farrowing sows (PPV HI antibody titer is not higher than 1:8), the above-mentioned inactivated vaccine 2ml preparing of intramuscular injection before breeding, after gestation 35～40 days, together with 5 of the identical contrast pigs of condition, collunarium through each 2ml(10 of intramuscular injection pig parvoviral BQ-C strain 6.0tCID 50), attack malicious cuing open for latter 40 days and kill all pigs.Should at least 4 there is the breeding difficulty symptoms such as stillborn foetus, mummy tire or fetus heavily absorb in control group, immune group should at least 4 head protections.
1.2.4 pure check
Restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 strain to the expression VP2 setting up) primordial seed is criticized 10th～11 generations, basic bacteria and is criticized 12nd～21 generations and seeding and criticized for 22nd～25 generations and carried out bacterium, mould, mycoplasma check, and the 11st, 12 and 24 generation seeds culture of viruses have been carried out to the check of exogenous virus, result shows, the primordial seed that the present invention sets up is criticized, basic bacteria is criticized, seeding is criticized all and pollutes without bacterium, mould, mycoplasma and exogenous virus, all pure.
1.2.5 seed culture of viruses is used generation
By the restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 strain of the original expression VP2 obtaining), on sf9 cell, pass continuously 40 generations (being numbered on the bacterial strain basis of XX 30 generations of continuous passage in culture presevation), measured the TCID of every generation virus 50, the VP2 of selection the 15th, 20,25,30,35,40 generation expressing viral, with after BEI deactivation, adds respectively Montanide tMiSA15AVG adjuvant mixing and emulsifying is made oil-in-water-type inactivated vaccine, 5 monthly age of inoculation replacement gilt, gestation is attacked poison 35 days time.
Result shows, virus goes down to posterity on sf9 cell, after the 10th generation (comprise the 10th generation virus, i.e. culture presevation is numbered the virus strain of CGMCC No.9004) each generation viral level is stable, is all not less than 10 6.0tCID 50, and expressing protein HA tires all 2 9above, reached the standard of making vaccine.
Challenge test shows, the vaccine immunity pig of the 10th, 15,20,25,30 generations virus preparation is in the protectiveness that BQ-C strain is attacked without significant difference, and fetus is healthy survival all.According to above result, for guarantee producing good immunogenicity and the security of seed culture of viruses, the present invention has determined that the high reps of going down to posterity of virus is in 30 generations, and original seed culture of viruses was 10～13 generations, basis seed culture of viruses was 14th～25 generations, produced and was limited in 3 generations (25th～27 generation) with the highest passage number of seed culture of viruses.
1.2.6 seed culture of viruses preservation period determines
To express the restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 strain of VP2) the wet poison of strain is kept at-20 ℃ and-70 ℃, and after preserving, different time sampling carries out that HA tires and TCID 50measure, result show wet poison in-20 ℃ frozen 18 months, when preserving 30 months for-70 ℃, poison valency starts obvious decline; Freeze-drying poison is kept at-20 ℃ and-70 ℃, and after preserving, annual sampling carries out that HA tires and TCID 50measure.Viral the tiring that result is preserved 36 months at-20 ℃ slightly declines, and the HA that preserves 48 months virus tires and TCID 50value obviously declines, and preserve 48 months viral HA at-70 ℃, tires and TCID 50significantly do not change, preserve and decline obviously for 60 months.Consider other unfavorable factors that exist when virus is preserved, guarantee viral preservation period reliability, so will express the restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 strain of VP2) wet poison is decided to be 12 months the preservation period of-20 ℃, the preservation perives of-70 ℃, is decided to be 24 months.
2 express recombination porcine parvovirus VP2 albumen
Utilize described restructuring polyhedrosis virus AcMNPV(pFastBac-VP2 strain) express the method for PPV VP 2 protein, comprise the following steps:
(1) suspension culture Sf9 cell, treats that cell density reaches 5-10 * 10 5during individual cells/ml, stop cultivating, prepare to connect poison;
(2) keep shaking speed under the condition of 120rpm, described restructuring polyhedrosis virus AcMNPV be take to infection multiplicity MOI and through step (1), cultivate the Sf9 cell obtaining as 10PFU/ cell access, 120rpm cultivates results virus after 42 hours, now cell infection amount is more than 90%, and it is 2 that the HA of VP2 tires 18, than 2 of general bibliographical information 13high 32 times;
(3) purifying VP2 albumen, adopts His label protein purification kit (the one-stop His labelled protein trace of sky, Beijing bounties (http://www.tiandz.com) purifying suit) to prepare albumen, and concrete grammar is with reference to specification sheets.After purifying, carry out determination of protein concentration.Obtain.
The preparation of embodiment 3VP2 subunit inactivated vaccine
Preparation technology's flow process of pig parvoviral VP2 subunit inactivated vaccine is according to the commercialized vaccine preparation technology of maturation preparation at present.The 3 batches of vaccines of preparing from laboratory and 5 batches of vaccine quality inspection results of pilot-scale production, the vaccine product steady quality of preparing with this technical process, effect inspection result meets formulated quality standard completely.
The preparation of 1VP2 subunit liquid
(culture presevation is numbered: CGMCC No.9004) to get restructuring polyhedrosis virus AcMNPV the 10th generation virus pFastBac-VP2 strain preparing, according to infection multiplicity, it is 10PFU/ cell inoculation Sf9 protein-free medium, add 3% calf serum, cell density is 10 * 10 5individual cells/ml, cultivates 42h and receives poison, and now cell infection amount is more than 90%.Adopt His label protein purification kit (the one-stop His labelled protein trace of sky, Beijing bounties (http://www.tiandz.com) purifying suit) to prepare albumen, concrete grammar is with reference to specification sheets.After purifying, carry out determination of protein concentration.
2 inactivation technologies
Selecting final concentration is that 0.1%, 0.2%, 0.3%, 0.4% formaldehyde solution or final concentration are that 0.1%, 0.2%, 0.3%, 0.4% divinyl imines (BEI) DuiVP2 subunit liquid carries out respectively deactivation comparison test, test-results shows, BEI 0.2%(w/w) can be by VP2 and other residual microbial complete inactivations after 96 hours under 32 ℃ of conditions, and test-results shows that BEI is less to the damage of cell; Antigen adds 0.3% formaldehyde solution, within 48 hours, also can reach the object of complete inactivation virus through 37 ℃ of deactivations; But formaldehyde has strong and stimulating, if residual free formaldehyde enters animal body in vaccine, can produce untoward reaction.Thereby final definite 0.2%(w/w that uses) BEI deactivation under 32 ℃ of conditions, after 96 hours, adds 0.02%(w/w) Sulfothiorine termination deactivation.
3 emulsifying process
Adopt the commercialization Montanide of France match BIC Corp tMiSA11R adjuvant, can be directly used in vaccine emulsification preparation after degerming; Before emulsification, the Tong Pi VP2 subunit liquid lyolysis of deactivation is frozen to mixing, at aseptic indoor refiner, with 200r/min, stir, then according to water antigen, mix according to the proportioning of volume ratio 8.5:1.5 with oil phase adjuvant; Emulsification program stirred for first water being put into emulsor, then slowly adds oil phase adjuvant, with 800r/min continuously stirring 30 minutes; The vaccine that production obtains belongs to oil-in-water formulation, for stable interface and elimination bubble, obtains optimum emulsification effect, needs packing after static 30 minutes.
4 inspection of semifinished product quality standards
4.1 steriling test
By existing < < Chinese veterinary pharmacopoeia > > appendix, test, answer asepsis growth.
4.2VP2 assay
Adopt HA assay method.Every milliliter of VP2 liquid is not less than 2 9hA.
4.3 deactivation checks
Get the virus liquid after deactivation, (cell content is with 2 * 10 in virus liquid and the growth media ratio of 1: 10, to be inoculated in 3 bottles, sf9 cell 6/ ml), cultivate to observe 5 for 22 ℃, to without pathology person 3 generations of blind passage again, establish virus control simultaneously.Pathology that result is acellular, is deactivation complete.3 batches of work in-process of Laboratory Production are all qualified, and pig parvoviral VP2 subunit inactivated vaccine 1201,1202 and 1203.
5 about inspection after construction quality standard
5.1 safety verification standards
In order to guarantee the security of vaccine, 5 batches of vaccines that the present invention is prepared laboratory have successively carried out " safety testing to small white mouse ", " safety testing to the inoculation of piglet single dose, single dose repeated inoculation, disposable overdose (2 multiple dose) inoculation ", " safety testing to the inoculation of replacement gilt single dose, single dose repeated inoculation, disposable overdose (2 multiple dose) inoculation ".Test-results shows: after each immune animal single dose inoculation, single dose repeated inoculation, disposable heavy dose of inoculation, pig state is all good, and the drinking-water of searching for food is normal, without abnormal response; Breeding function to pregnant sow after vaccine inoculation also has no significant effect.More than test all proves that vaccine is safe.By experimental observation the present invention, sum up as long as there is not local redness, ulcer, erosion and neuratorphy, the phenomenons such as minimizing of searching for food after vaccination in 21 days, just there will not be the untoward reaction causing because of vaccine inoculation, just can prove the security of vaccine later.Therefore, regulation in Trial Regulation (draft): with healthy susceptible piglet (the not high 1:8 of HI antibody titer) 2 of 10～20kg, each deep intramuscular injection vaccine 4ml, 2 pigs that the condition of separately getting is identical do not inoculate in contrast, Continuous Observation 21 days.Observation period planted agent, there is not any part or the systemic adverse reactions that by vaccination, are caused.
The formulation of 5.2 efficacy test standards
5.2.1 antibody titer and Immunization are protected correlation test
By 1 batch of qualified pig parvoviral VP2 subunit inactivated vaccine 1201 of safety testing, according to various dose 0.5ml/ head part, 1ml/ head part, 2ml/ head part, the healthy replacement gilt (PPVHI antibody is not higher than 8) at respectively immune 3～5 monthly ages of 4ml/ head part, and with 10 6.0tCID 50/ ml PPV BQ-C strain strong malicious 4ml of the 10th generation attacks, to determine the dependency of HI antibody titers and Immunization protection.Test-results shows, when serum HI antibody titer is when being not less than 1:64, sows farrowing surviving rate reaches 97.5%, wherein has a porkling miscarriage in one group, but PCR detects piglet virus isolated rate, is 0, also without other, causes the pathogen infection of sow miscarriage.Above result shows under test conditions, when serum HI antibody titer is when being not less than 1:64, can effectively stop virus vertically to pass to fetus through parent.Thereby during the check of definite vaccine potency, after inspecting standard be immune 28 days, its serum HI antibody titer is not less than 1:64.
5.2.2 minimum immune dosage test
By 3 batches of qualified pig parvoviral VP2 subunit inactivated vaccines 1201,1202 and 1203 of safety testing, according to various dose 0.5ml/ head part, 1ml/ head part, 1.5ml/ head part, the healthy replacement gilt (PPV HI antibody is not higher than 8) at respectively immune 3～5 monthly ages of 2ml/ head part, in immunity blood sampling in latter 28 days, carry out HI antibody test.Result shows, with 1ml/ doses immune swine after 28 days HI antibody titer mean value be not less than 1:64, and the above dosage immune swine of 1.5ml/ head part after 28 days HI antibody titer be all not less than 1:64.According to HI antibody titer with attack poison protection correlation test result, determine at latter 28 days pig parvoviral HI antibody titers of immunity and reach 1:64 when above, can protect the attack of the strong poison of farrowing sow opposing pig parvoviral completely.The present invention is defined as 1.5ml/ head part by the minimum immune dosage of pig parvoviral VP2 subunit liquid inactivated vaccine.
6 antibody Fluctuations and immune duration test
By the qualified 3 batches of pig parvoviral VP2 subunit inactivated vaccines 1201,1202 and 1203 of safety testing immune health replacement gilts, multiparity pig and boar respectively.For further grasping immune efficacy and immune duration, formulate rational immune programme for children, guarantee that immune swinery keeps high and lasting antibody horizontal, carries out antibody test to immune swine different time, to grasp its antibody Fluctuation.
Test-results shows; 3～5 monthly ages first farrowing sow immunity 1.5ml/ head part; after 2～3 weeks, booster immunization once; multiparity sow is in previous month immune 1.5ml/ head part of breeding, and after 28 days, its serum HI antibody is not less than 1:64, and after immunity, antibody peak is in 2～6 months; thereafter antibody horizontal declines gradually; to 9 months, still reach 1:64, for guaranteeing the immune protective effect of vaccine, determine that its immune duration is 7 months.
Determining of 7 immune programme for children
According to the time length of piglet maternal antibody, initial immunity should be at 5～6 monthly ages, because at this moment maternal antibody has dropped to lower level (HI antibody titer is lower than 1:64).And replacement gilt breeding time was generally for 6 monthly ages, when 5～6 monthly age, immunity also just in time can avoid sow when gestation, to suffer strong virus attack like this.Sustainable existence for multiparity sow due to its internal antibody; pig parvoviral does not often constitute a threat to it; so former, generally do not carry out immunity; but in recent years due to the complicacy of clinical onset; as pig parvoviral, tend to infection increasing the weight of pig circular ring virus etc.; so the present invention advocates multiparity sow to be carried out before gestation to immunity, can protect like this pregnant sow in the whole Gestation period, to avoid the attack of the strong poison of pig parvoviral.
According to antibody Fluctuation, the duration of immunity after the breeding time of replacement gilt, replacement gilt immunity, combination is simultaneously the practical situation of porcine parvovirus infection clinically, the present invention has determined that the immune programme for children of pig parvoviral VP2 subunit inactivated vaccine is: first farrowing sow 5～6 monthly age immunity 1 time, and after 2～4 weeks, booster immunization is 1 time; Multiparity sow is in breeding immunity in first 3～4 weeks 1 time; Twice of the annual immunity of boar.Each immunity is 1.5ml/ head.
The preservation period of 8 vaccines
3 batches of VP2 subunit inactivated vaccines 1201,1202 and 1203 preservation perives that the present invention is prepared laboratory have been carried out experimental study, 3 batches of vaccines are preserved 9,12,15,18 months under 2～8 ℃ of conditions, in each time period, sampled respectively its proterties, security and immune efficacy are detected.Result demonstration, 3 batches of vaccines are preserved 18 months proterties considerable change are not occurred under 2～8 ℃ of conditions, and steriling test and security all meet the requirement of quality standard.In efficacy test, the 1201 batches of vaccines preserve that within 18 months, after sample immune guinea pig, to have a cavy HI antibody titer be 1:32; 1201 and 1202 batches of vaccine preservations all have a pig HI antibody titer for 18 months after sample immune swine be 1:32, but the average HI antibody horizontal of immune swine is 1:64, consider the loss of tiring that vaccine causes in transportation and use procedure, the present invention is decided to be the preservation period of vaccine under 2～8 ℃ of conditions and preserves 15 months.
9 with the immune efficacy comparison of totivirus deactivation commercial seedling
By the VP2 subunit inactivated vaccine 1201,1202 of laboratory trial-production and 1203 and totivirus deactivation commercial seedling; distinguish healthy replacement gilt of immune 350g～400g healthy guinea pig and 5～6 monthly ages; after 28 days, its serum HI antibody titer is all not less than 1:64; reach and attack poison protection requirement, vaccine HI antibody mean value prepared by laboratory is all a little more than commercially available totivirus deactivation commercial seedling.
The preparation of embodiment 4rVP2-ELISA test kit
1, utilize recombinant VP 2 albumen (aminoacid sequence is as shown in SEQ ID NO.1) after purifying as envelope antigen, tentatively set up the indirect ELISA method that detects PPV antibody, VP2 Protein Detection and purification result are as shown in Figure 3, reaction conditions is optimized: coating buffer and confining liquid are respectively 2mM pH8.0TrisHcl and 5% skimming milk, the coated concentration of the best of antigen is 2ug/mL simultaneously; The extension rate of serum the best is 1:200, and the working concentration of ELIAS secondary antibody is 1:6000, and primary antibodie, two anti-working hours are 60min, and developing time is 15min.
Test-results shows: the yin and yang attribute threshold value of the ELISA method of setting up is: 0.181+3 * 0.030=0.271, finally determines that working as OD450nm > 0.3 is judged to the positive; OD450nm < 0.25 is negative, judge scope when 0.25-0.3 as suspicious.
Applying this detection method detects 596 parts of clinical serum in the areas such as Heilungkiang, Shandong, Tianjin, positive rate is 83.5%～91.1%, average positive rate is 87.03%, chooses 92 parts of clinical samples and French LSI test kit is compared, and both coincidence rates are 96.7%.
2, conclusion
Above experimental result shows that indirect ELISA method specificity, susceptibility and the repeatability of the detection PPV antibody that the present invention sets up is all more satisfactory.
Therefore, a kind of indirect ELISA method that detects PPV antibody that the present invention sets up, for PPV antibody test provides effective means, for the development of porcine parvovirus assay kit provides basic substance.
The foregoing is only preferred embodiment of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, revise, and even equivalence change, but all will fall within the scope of protection of the present invention.

Claims (10)

1. a recombination porcine parvovirus VP2 albumen, is characterized in that the aminoacid sequence of described recombination porcine parvovirus VP2 albumen is as shown in SEQ ID NO.1.
2. the nucleotide sequence of coding recombination porcine parvovirus VP2 albumen claimed in claim 1, preferred, described nucleotide sequence is as shown in SEQ ID NO.2.
3. an expression vector, is characterized in that containing nucleotide sequence claimed in claim 2.
4. a host cell.It is characterized in that containing expression vector claimed in claim 3.
5. recombination porcine parvovirus VP2 albumen claimed in claim 1 is being prepared pig parvoviral VP2 subunit vaccine and the application in the reagent of preparation diagnosis or detection porcine parvovirus infection.
6. a pig parvoviral VP2 subunit inactivated vaccine, is characterized in that containing recombination porcine parvovirus VP2 albumen claimed in claim 1.
7. for diagnosing or detect an ELISA diagnostic kit for porcine parvovirus infection, it is characterized in that containing recombination porcine parvovirus VP2 albumen claimed in claim 1.
8. a restructuring polyhedrosis virus AcMNPV who expresses PPV VP 2 protein, called after pFastBac-VP2, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCC No.9004, the aminoacid sequence of the PPV VP 2 protein of being expressed by this virus strain is as shown in SEQ ID NO.1.
9. the application of restructuring polyhedrosis virus AcMNPV claimed in claim 8 in preparing pig parvoviral VP2 subunit inactivated vaccine.
10. express a method for PPV VP 2 protein, it is characterized in that comprising the following steps:
(1) suspension culture Sf9 cell, treats that cell density reaches 5-10 * 10 5during individual cells/ml, stop cultivating, prepare to connect poison;
(2) keep shaking speed under the condition of 120rpm, restructuring polyhedrosis virus AcMNPV claimed in claim 8 be take to infection multiplicity MOI and as 10PFU/ cell access, cultivate the Sf9 cell obtaining through step (1), 120rpm cultivates results virus after 24-48 hour;
(3) purifying VP2 albumen, obtains.
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CN106148358A (en) * 2016-07-15 2016-11-23 河南省农业科学院 A kind of method utilizing escherichia expression system to prepare pig parvoviral virus sample particle subunits vaccine and application
CN107991499A (en) * 2017-12-08 2018-05-04 中牧实业股份有限公司 Pig parvoviral structural proteins antibody ELISA immunity detection reagent
CN108918870A (en) * 2018-07-07 2018-11-30 广东省实验动物监测所 The liquid-phase chip detection method of pig parvoviral
WO2020116815A1 (en) * 2018-12-05 2020-06-11 대한민국(농림축산식품부 농림축산검역본부장) Recombinant porcine parvovirus antigenic protein and use thereof
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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
CN113150126A (en) * 2021-04-20 2021-07-23 开江县动物疫病预防控制中心 Rabbit-derived porcine parvovirus 6-type VP2 protein antibody and preparation method thereof
CN114106113A (en) * 2021-11-13 2022-03-01 江苏南农高科技股份有限公司 Recombinant baculovirus expressing porcine parvovirus VP2 protein, preparation method and application
CN114106113B (en) * 2021-11-13 2024-04-19 江苏南农高科技股份有限公司 Recombinant baculovirus for expressing porcine parvovirus VP2 protein, preparation method and application
CN117866053A (en) * 2024-01-10 2024-04-12 武汉珈创生物技术股份有限公司 Polyclonal antibody for simultaneously detecting multiple porcine parvoviruses and preparation and application thereof

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