CN109456391B - A kind of VLP vaccine and its preparation method and application preventing goose astrovirus infection - Google Patents

Abstract

The invention discloses a kind of VLP vaccines and its preparation method and application for preventing goose astrovirus infection, belong to veterinary biologics field.The present invention passes through clone, amplification Capsid albumen full genome；GoAsV/Bac plants of recombinant baculovirus of the antigen protein are expressed using insect cell-baculovirus expression system building, recombinant virus high efficient expression antigen protein in insect cell HF；Adjuvant emulsion is added after extraction purification, BEI inactivation, vaccine is made.The preparation method is simple, can largely prepare goose astrovirus Capsid albumen, and time-consuming short, expression quantity is high, substantially reduces production cost, is conducive to be mass produced.It include the goose astrovirus VLP vaccine of Capsid albumen prepared by preparation method of the present invention, immune effect is good, and immunizing dose is small, can effectively prevent the infection of goose astrovirus.

Description

A kind of VLP vaccine and its preparation method and application preventing goose astrovirus infection

Technical field

The present invention relates to a kind of VLP vaccines and its preparation method and application for preventing goose astrovirus infection, belong to for animals Field of biological product.

Background technique

Goose astrovirus mainly causes gaggle that serious severe visceral pain wind and arthragra, and goose throughout the country occurs There is generation in factory.According to the literature and raiser's oral account, this disease betide 2011 earliest, and Hebei province introduces France Ao Erweiya Corporate investment creates Anser anser field, occurs as soon as gout symptom from the Anser anser seedling of Ao Er NIVEA Corp December current year, later by Year spreads at home, and by 2016~2017 years, case all occurred in all goose kinds, all feeding goose places at home.The disease mainly exists It falls ill on young goose, infection age in days is 2~25 ages in days, and young goose is susceptible within predominantly 15 ages in days.This disease occurs throughout the year, annual November It is peak season to May next year, it may be with related, other time morbidity reduction of divulging information, keep the temperature.The infected age in days of case fatality rate, after The factors such as hair infection influence, and for gaggle infection rate 80% or more, disease incidence reaches as high as 50%, which has become harm and support goose Industry develops in a healthy way a kind of Important Infectious Diseases.

Currently, vaccine inoculation is prevention, controls and even eliminate one of mainly arranging for goose astrovirus, subunit vaccine, especially It is viroid sample particle vaccines (Virus like particles, VLPs), and this kind of vaccine does not contain nucleic acid substances, safety Preferably, persistent infection or latent infection will not be generated after inoculation, the immune response of generation can mutually be distinguished with wild virus infection, favorably In the control and elimination of epidemic disease.

Therefore the good goose astrovirus subunit of a kind of low production cost, high production efficiency and immune effect of vaccine is developed The production method of vaccine has important practical significance.

Summary of the invention

The first purpose of the invention is to provide a kind of for preventing the antigen of goose astrovirus infection, and the antigen contains Amino acid sequence shown in SEQ ID NO.1.

In one embodiment of the invention, the amino acid sequence of the antigen is as shown in SEQ ID NO.1.

A second object of the present invention is to provide the DNA for encoding the antigen.

Third object of the present invention is to provide the compositions for containing the antigen.

In one embodiment of the invention, the antigen is inactivated by BEI.

Fourth object of the present invention is to provide goose astrovirus virus vlps vaccine, contains the antigen and vaccine adjuvant.

In one embodiment of the invention, the content of the antigen is 25~100 μ g/mL.

In one embodiment of the invention, the adjuvant is oil adjuvant.

Fifth object of the present invention is to provide the antibody that application antigen immune response obtains.

Sixth object of the present invention is to provide the antibody in terms of the drug of preparation prevention and treatment goose astrovirus infection Using.

The pharmaceutical composition containing the antibody in conjunction with the antigentic specificity is also claimed in the present invention.

The utility model has the advantages that the present invention is by the recombinant baculovirus rBac-GoAsV- of the goose astrovirus Capsid albumen of expression Capsid, access insect cell efficiently express Capsid albumen, remove cell fragment by centrifugation, after BEI inactivation is added, add Enter oil adjuvant mixing and emulsifying and vaccine is made.Vaccine prepared by the present invention has the advantages that efficient, safety is good, can effective stimulus kind Goose generates active immunity, young goose generates passive immunity, and the antibody level generated can help young goose to resist lethal dose virus Attack.Using the young goose of vaccine immunity prepared by the present invention, after 4~5 weeks yolk GoAsV fine jade expand antibody titer average value >= 3.0log2；After the susceptible laying breeding geese 28d of immune health each group goose goose astrovirus fine jade expand antibody titer average value >= 3.6log2, passive immunity group young bird goose serum GoAsV fine jade expand antibody titer average value >=2.7log2, attack protective rate on the 14th after poison Up to 100%.

Detailed description of the invention

Fig. 1 is the PCR amplification result figure of Capsid entire open reading frame；Wherein, M:DL5000 DNA Marker；1: Control；2:PCR product；

Fig. 2 is transfer vector building PCR qualification result figure；Wherein, M:DL5000 DNA Marker；1~7:7 different Bacterium colony PCR；

Fig. 3 is restructuring rod granule PCR qualification result figure；Wherein, M:DL5000 DNA Marker；1~7:7 different bacterium colony PCR；

Fig. 4 is SDS-PAGE detection recombinant baculovirus expression product；Wherein, M: pre- dsred protein Marker；1:Sf9 is thin Born of the same parents' culture；2: wild baculovirus infection sf9 cell；3:F3 is for recombinant baculovirus；

Fig. 5 is Western Blot identification recombinant baculovirus expression product；Wherein, M: pre- dsred protein Marker；1: Infect the Sf9 cell of empty baculoviral；2:F3 is for recombinant baculovirus；

Fig. 6 is recombinant virus sample particle electromicroscopic photograph.

Specific embodiment

Embodiment 1: the building of recombinant baculovirus

1, goose astrovirus RNA is extracted: extracting LH plants of total serum IgEs of goose astrovirus with TRIzol Reagent kit；

2, goose astrovirus cDNA is synthesized: according to HiScript II One Step RT-PCR Kit kit (Nanjing promise Wei Zan Biotechnology Co., Ltd, product number: P611-01) operating instruction progress cDNA synthesis.

3, design of primers and synthesis: announced according to GenBank goose astrovirus Capsid gene order (accession number: NC_034567 a pair of of specific primer) is designed, and adds EcoR I and Xho I restriction enzyme site respectively at primer both ends, it is amplifiable The target fragment of 2124bp out.Primer sequence is respectively as follows:

P1:CCGGAATTCATGGCCGACAAGGTCACTGTCTCAG

P2:CCGCTCGAGTTAATCAAACTCTTGTCCGCCCTTC

4, Capsid gene fragment amplification and recycling

(1) PCR amplification: using the DNA of extraction as template, the primer in step 2 is PCR.PCR reaction system is (total volume 25 μ L): 0.5 μ L of DNA profiling, P1 and P2 11 μ L of 0.5 μ L, 12.5 μ L of archaeal dna polymerase and sterile water.PCR reaction condition are as follows: 95 DEG C, 5min；95 DEG C of 30s, 65 DEG C of 30s, 72 DEG C of 120s, 30 circulations；72℃10min.1% agarose gel electrophoresis shows, at Function amplifies the specific band of about 2124bp, is consistent (Fig. 1) with expected size.

(2) target fragment glue recycles: by PCR product through 1% agarose gel electrophoresis, gel extraction purpose in the UV lamp Segment.Concrete operations are carried out referring to plastic recovery kit specification.

4, target gene and transfer plasmid connect: by pFastBac I and capsid gene amplification fragment with EcoR I and It is separately recovered, purifies after Xho I digestion, overnight with 4 DEG C of T4DNA ligase connections.

5, connection product transformed competence colibacillus cell: connection product is aseptically converted in T1 competent cell, is mixed Even, 800uL LB culture is added based on 37 DEG C of concussions under aseptic condition in ice bath 30min, 42 DEG C of heat shock 90s, immediately ice bath 2min Cultivate 60min.Culture 14000rpm is centrifuged 1min, 800uL supernatant is drawn, remaining culture is coated on LB (benzyl containing ammonia Penicillin resistance) in solid medium, 37 DEG C are incubated overnight.Picking single colonie makees bacterium colony PCR identification, positive plasmid inspection sequencing (Fig. 2).Correct recombinant plasmid is sequenced and is named as pFastBac I-GoAsV-capsid.

6, recombinant baculovirus constructs: will identify that correct transfer plasmid pFastBac I-GoAsV-capsid is transferred to greatly In enterobacteria DH10Bac competent cell, selects positive colony M13 primer and make PCR identification.

M13-F:TGTAAAACGACGGCCAGT

M13-R:CAGGAAACAGCTATGAC

PCR reaction system is (25 μ L of total volume): 0.5 μ L of DNA profiling, M13-F and M13-R 0.5 μ L, archaeal dna polymerase 11 μ L of 12.5 μ L and sterile water.PCR reaction condition are as follows: 93 DEG C, 5min；94 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 5min, 30 circulations； 72℃10min.1% agarose gel electrophoresis shows that Successful amplification goes out the specific band of about 4500bp, is consistent with expected size (Fig. 3).Positive restructuring rod granule is named as rBacmid-GoAsV-capsid.

7, restructuring rod granule transfects sf9 cell: the method for the restructuring rod granule liposome transfection of purification is turned restructuring rod granule Sf9 cell is contaminated, concrete operation method is said referring to the cellfectin transfection reagent of silent your scientific and technological (China) Co., Ltd of winged generation of match Bright book carries out, and obtains f1 for recombinant baculovirus rBac-GoAsV-capsid.

Embodiment 2: the preparation of recombinant C apsid albumen

1, recombinant baculovirus expand: by recombinant baculovirus rBac-GoAsV-capsid be inoculated with sf9 insect cell, 27 DEG C culture 4 days, collect culture, centrifuging and taking supernatant i.e. obtain f2 for recombinant baculovirus；

2, Identification of Fusion Protein is expressed:

(1) above-mentioned f2 is accessed into sf9 insect cell, 27 DEG C of cultures for recombinant baculovirus with the inoculum concentration of MOI=5~10 4 days, culture is collected, centrifuging and taking supernatant obtains recombinant C apsid albumen；

(2) SDS-PAGE is identified: above-mentioned supernatant is carried out SDS-PAGE electrophoresis；After electrophoresis, is dyed and decolourized After find, about in the position 80kDa, molecular weight is consistent with theoretical size, illustrates to express successfully (Fig. 4), through being sequenced, the albumen Amino acid sequence as shown in SEQ ID NO.1.

(2) Western Blot is identified: being taken gel after SDS-PAGE electrophoresis, is directly used BIO-LAB transfer device by its turn It prints on NC film, after transfer, carries out Western blot identification according to a conventional method.It is referred to goose astrovirus positive serum Product (1:200) are used as primary antibody；Use the rabbit-anti goose IgG (1:2000) of horseradish peroxidase-labeled as ELIAS secondary antibody；Finally use TMB develops the color (green skies biotechnology research institute).The result shows that occur 1 apparent specific band at 80kDa, and it is negative Property control without this specific reaction, illustrate that the recombinant protein can identify by the antibody in goose astrovirus positive serum, with good Good specificity and reactionogenicity (Fig. 5).

10, the great expression of recombinant C apsid albumen: it will identify that correct recombinant virus connects malicious amount with MOI=1~10 It is inoculated with HF cell mass propgation, culture solution supernatant is collected by centrifugation, that is, obtains and contains a large amount of recombinant C apsid albumen, by expression Recombinant C apsid albumen carries out Electronic Speculum detection (Fig. 6).

Embodiment 3: vaccine preparation

1, it inactivates: the recombinant C apsid albumen largely prepared in embodiment 2 being added in inactivation tank, is added final concentration of 0.2%~0.5% inactivator BEI, for 24 hours in 37 DEG C of inactivations.

2, the inspection of semifinished product

(1) steriling test: steriling test is carried out by existing " Chinese veterinary pharmacopoeia " annex.

(2) determining the protein quantity: protein content is detected by BCA method.

(3) inactivation is examined: the protein liquid after inactivation being taken sf9 insect cell, 27 DEG C is placed in and continues culture 72 hours.Observation No lesion occurs, and it is qualified to determine that inactivation is examined.

3, VLP vaccine preparation:

Carrying out vaccine preparation by semi-finished product proteantigen after the assay was approved, (each liquid component presses volume in following preparation Than meter).

(1) oil is mutually prepared: 95 parts of white oil for animals are taken, 1 part of aluminum stearate, is placed in oily phase preparation tank after being heated to 80 DEG C, Again plus 5 parts of Si Ben -80, it until when temperature reaches 115 DEG C, maintains, it is spare after cooling.

(2) recombinant protein is used normal saline dilution at 50 μ g/mL by water phase preparation.5 parts of Tween-80s after taking sterilizing, It is added in Agitation Tank, while being added 95 parts of seedling protein liquid, stir 20~30min, be completely dissolved Tween-80.

(3) emulsification takes 2 parts of oily phase to be put in high-speed shearing machine, starts the stirring of motor slow rotation, while water being added slowly 1 part of phase, with 10000rpm emulsification 5 minutes.After emulsification, 10mL is taken, 15min is centrifuged with 3000rpm, the water phase that tube bottom is precipitated should not More than 0.5mL.

, embodiment 4: vaccine product inspection

1, character

Appearance: vaccine should be milky Virgin's milk agent, free from admixture and qualification is answered in outer packing；

Dosage form: water-in-oil type.A cleaning suction pipe is taken, a small amount of vaccine is drawn and instills in cold water, in addition to the first drop, should all not be expanded It dissipates.

Stability: drawing vaccine 10mL and be added in centrifuge tube, is centrifuged 15min with 3000rpm, water phase Ying Buchao is precipitated in tube bottom Cross 0.5mL.

Viscosity: it is carried out by existing " Chinese veterinary pharmacopoeia " annex, regulation should be met.

2, it loading quantity inspection: is carried out by existing " Chinese veterinary pharmacopoeia " annex, regulation should be met.

3, it steriling test: is carried out by existing " Chinese veterinary pharmacopoeia " annex, regulation should be met.

4, safety verification:

Goose astrovirus VLP vaccine is prepared as described in Example 3, and lot number is respectively rCapsid-001P, rCapsid- 002P, rCapsid-003P totally 3 batches.3~5 age in days young bird geese, 1 monthly age goose, 7 monthly ages that goose astrovirus fine jade expands negative antibody open production Kind goose, September age laying breeding geese.Every batch of vaccine chooses 3~5 age in days young bird geese 10 respectively, 1 monthly age goose 10,7 monthly ages open a production kind goose 10, September age laying breeding geese 10, every chest muscle of young goose inject 0.2mL, and every chest muscle of remaining goose vaccinates 1.0mL, every group of goose for separately taking 10 identical sources, identical age in days, as nonimmune control group.21 are observed continuously after injection Day, daily situations such as observing each group goose mental status, drinking-water, food-intake；7 days after immune, 14 days, touchs on the 21st check each be immunized Whether group goose injection site has the reaction of the locally injectings such as redness；It weighs before immune with after being immunized 21；Laying breeding geese statistics is laid eggs Situation appoints 20 pieces of the produced goose embryo of kind goose on the 21st after taking every batch of vaccine immunity respectively, marks, whether compare goose embryo hatching rate It is variant；It cuts open and kills all geese, check injection site vaccine absorbing state, whether have pathological change；10 young birds of every group of random screening Raising is to 1 monthly age after goose weighing, counts survival rate and opposite daily gain, relative immunity group and control group young bird goose survival rate and average The difference of opposite daily gain.

5, efficacy test:

(1) it is grouped and immune

It chooses August age health and opens production kind goose 80 (goose astrovirus fine jade expands negative antibody), be randomly divided into 4 groups, every group 20 Only.Optional three groups are immunized rCapsid-001P, rCapsid-002P, rCapsid-003P batch vaccine, chest muscle note respectively Penetrate 1mL/ only, remaining one group is nonimmune control group, and each experimental group goose marks respectively.

(2) goose serum, Yolk antibody detection are planted

Weekly by only taking a blood sample to after being immunized 28 after each experimental group goose is immune, serum is separated, detection GoAsV fine jade expands antibody； Appoint respectively for every group and take immune latter 4~5 weeks interior 10 pieces of produced goose eggs of kind of goose (together with control group), it is anti-to detect GoAsV fine jade expansion in yolk Body.

Respectively by three batches of vaccines with 1mL/ dose immunization, the susceptible laying breeding geese of immune health, the goose star of each group goose after 28d Shape virus fine jade expand antibody titer average value >=3.6log2, the antibody of nonimmune control group is feminine gender, and concrete outcome is shown in Table 1.

Serum GoAsV fine jade expands antibody after a kind of goose of table is 28 days immune

The detection of goose egg Yolk antibody:

Appoint in 4~5 weeks after every batch of vaccine immunity group is immune and take 10 pieces of goose eggs, it is flat that yolk GoAsV fine jade expands antibody titer Mean value >=3.0log2, it is feminine gender that 10 pieces of goose egg yolk GoAsV fine jades of nonimmune control group, which expand antibody, the results are shown in Table 2.

Goose egg yolk GoAsV fine jade expands antibody titer in 4~5 weeks after table 2 is immune

(3) 3 age in days young bird goose passive immunity challenge viral dosages

Every experimental group is appointed respectively takes immune latter 4~5 weeks interior 30 pieces of produced hatching eggs (together with control group), marks, sets 38 DEG C incubator is hatched 29~30, the young goose that immune group is hatched optional 20, control group 20, is put on footnote, is placed in and attacks poison It is raised in area's isolator 3, every group of young bird goose blood sampling separation serum, detection GoAsV fine jade expands antibody.Carry out challenge viral dosage simultaneously: GoAsV attacks malicious group, attacks malicious (every milliliter of allantoic fluid viral level 10 with GoAsV-LH plants of F3 generations^4.03ELD₅₀/ 0.2mL), every young bird Goose takes orally 0.5mL, and 0.5mL is subcutaneously injected in neck, attacks poison 10；Immune group and control group are individually insulated breeding observing 14, remember Record morbidity, dead and protection situation.

The results show that the 3 batches of vaccine passive immunity groups every group 10 young goose serum GoAsV fine jades expand antibody titer average values >= 2.7log2 the results are shown in Table 3.

33 age in days young bird goose passive immunizing agent GoAsV fine jade of table expands antibody titer

(4) 3 age in days young bird goose passive immunity challenge viral dosages:

3 batches of vaccine passive immunity group every group 10 young geese search for food after attacking poison, drink water, the state of mind is normal, and no goose is starlike Viral clinical symptoms occur, and attack every group of equal 10/10 strong work on the 14th after poison.Non-passive immunized controls group young bird goose attacks essence on the 3rd after poison Mind starts to occur that spirit is depressed, anorexia, astasia, peels off, and arranges dilute canescence or yellow green excrement, attacks the 5th day after poison and occur Death, until attacking total dead 5 young geese on the 10th after poison.Every goose dissect of dying of illness, plantar, instep, toe joint enlargement subcutaneously have a large amount of uric acid Salt；There is lime sample uric acid mineralization in entire heart and partial liver；Kidney enlargement, hyperemia, surface are covered with lime sample lithate Deposition；Lungs surface distribution table hydrochlorate deposition；Glandular stomach spreads uric acid mineralization；Spleen bleeding is serious, there is canescence necrosis region etc.. Passive immunity, which attacks poison protection, the results are shown in Table 4.

43 age in days young bird goose passive immunity of table attacks malicious (LH plants of goose astrovirus)

The above results show that 3 batch vaccines have good immune effect to kind of goose, make to contain in serum when young goose hatching The GoAsV antibody of higher level can effectively resist the attack of goose astrovirus.

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

SEQUENCE LISTING

<110>Yangzhou You Bang biologics Co., Ltd

<120>a kind of VLP vaccine and its preparation method and application for preventing goose astrovirus infection

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 504

<212> PRT

<213>GoAsV LH plants of Capsid protein amino acid sequences

<400> 1

Met Ala Asp Lys Val Thr Val Ser Val Lys Lys Ala Lys Ala Arg Gly

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Arg Thr Arg Ser Arg Ser Arg Ser Arg Ser Arg Ser Arg Ser Arg Gly

20 25 30

Lys Arg Val Ile Ile Lys Lys Lys Lys Ala Lys Thr Ile Lys Lys Lys

35 40 45

Arg Lys Lys Arg Val Arg Lys Val Arg Arg Ile Lys Gly Arg Val Ser

50 55 60

Asp Thr Lys Thr Thr Val Thr Gly Arg Val Gly Asn Asp Asp Ser Ser

65 70 75 80

Arg Lys Val Asn Thr Met Lys Asn Asp Ala Gly Ser Ala Ser Ser Thr

85 90 95

Ile Arg Ala Ser Tyr Gly Trp Arg Ile Val Cys Val Arg Thr Ala Gly

100 105 110

Ala Ala Asn Val Gly Thr Val Val Ala Asp Ser Gly Val Ala Thr Ala

115 120 125

Ser Asp Thr Ile Lys Ala Arg His Val Ser Val Gly Met Arg Tyr Val

130 135 140

Trp Lys Ile Lys Arg Gly Arg Gly Trp Trp Asn Met Asp Thr Gly Asp

145 150 155 160

Asp Thr Asn Ser Gly Ala Ile Ser Trp Thr Tyr Arg Thr Val Ala Ser

165 170 175

Ser Thr Asn Ser Val Gly Gly Ile Val Ala Asn Val Arg Tyr Ser Asn

180 185 190

Tyr Thr Lys Asn Ala Met Asn Arg Ile Thr Val Lys Ser Gly Ile Thr

195 200 205

Lys Asn Asp Thr Asp Gly Ser Val Val Met Val Asp Ala Arg Ala Ile

210 215 220

Asp Asn Arg Arg Thr Asn Ala Ser Gly Gly Lys Trp Ala Val Ser Thr

225 230 235 240

Thr Val Val Asp Val Val Ala Asp Ala Ile Gly Trp Gly Trp Lys Gly

245 250 255

Gly Trp Trp Val Ile Arg Lys Ile Gly Ala Ala Gly Asn Ala Ser Lys

260 265 270

Tyr Ala Ile Tyr Ala Ser Val Asp Ala Lys Asn Asn Ser Lys Ile Val

275 280 285

Lys Ser Ile Ser Asn Arg Val Ile Thr Ile Asn Val Asn Ser Gly Ile

290 295 300

Ala Thr Thr Gly Gly Ala Ala Val Ala Ile Tyr Arg Ile Ala Asn Asn

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Ile Tyr Asp Val Asp Arg Val Asp Gly Asp Trp Tyr Cys Asn Asn Gly

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Ile Ala Thr Thr Val Thr Ala Ser Ser Ala Met Gly Thr Ala Asn Val

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405 410 415

Asn Met Gly Trp Val Val Ser Lys His Lys Val Arg Val Tyr Trp Thr

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ccgctcgagt taatcaaact cttgtccgcc cttc 34

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tgtaaaacga cggccagt 18

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Claims (7)

1. a kind of antigen, which is characterized in that amino acid sequence is as shown in SEQ ID NO.1.

2. encoding the DNA molecular of antigen described in claim 1.

3. the composition containing antigen described in claim 1.

4. composition according to claim 3, it is characterised in that antigen is inactivated by binary ethylenimine.

5. a kind of vaccine for preventing and treating goose astrovirus VLP, which is characterized in that helped containing antigen described in claim 1 and vaccine Agent.

6. vaccine according to claim 5, which is characterized in that the content of the antigen is 25~100 μ g/mL.

7. vaccine according to claim 5 or 6, which is characterized in that the adjuvant is oil adjuvant.