CN103172709A - IBDV (Infectious Bursal Disease Virus) VP2 protein and IBD subunit vaccine - Google Patents
IBDV (Infectious Bursal Disease Virus) VP2 protein and IBD subunit vaccine Download PDFInfo
- Publication number
- CN103172709A CN103172709A CN2011104427816A CN201110442781A CN103172709A CN 103172709 A CN103172709 A CN 103172709A CN 2011104427816 A CN2011104427816 A CN 2011104427816A CN 201110442781 A CN201110442781 A CN 201110442781A CN 103172709 A CN103172709 A CN 103172709A
- Authority
- CN
- China
- Prior art keywords
- ibdv
- albumen
- bursal disease
- virus
- silkworm
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to an IBDV (Infectious Bursal Disease Virus) VP2 protein. The mass production of the expressive IBDV VP2 protein in a silkworm larva or a pupae is succeeded for the first time by performing the subcutaneous injection of a restructured autographa californica nuclear polyhedrosis virus into the silkworm larva or the pupae. However, in the prior art, the expression of the restructured autographa californica nuclear polyhedrosis virus containing the IBDV VP2 in the silkworm larva or the pupae always cannot be performed successfully. The invention further provides a method for producing the IBDV VP2 protein and an IBD subunit vaccine by utilizing a silkworm bioreactor. The IBDV VP2 protein can be applied to the preparation of the vaccine for treating and preventing the IBD as well as the preparation of an IBD diagnostic reagent.
Description
Technical field
The present invention relates to a kind of infectivity bursa of Fabricius virus VP 2 albumen and preparation method thereof, refer to that especially a kind of this infectivity bursa of Fabricius virus VP 2 albumen that utilizes prepares infectious bursal disease subunit vaccine and method thereof, belongs to biological technical field.
Background technology
Infectious bursal disease (IBD) is one of three large Infectious Diseases that endanger at present world's aviculture.China is the big country of birds, beasts and eggs production of poultry meat in the world, and industrial scale occupies the first in the world.This disease, in China's popularity complexity, is classified as two class deadly infectious diseases of preferential defense by the Ministry of Agriculture.Infectious bursal disease virus (IBDV) is mainly attacked the chick in 3～10 week age, especially the chicken in 4 week age.Sick chicken group's mortality ratio reaches 5%～30%.The organ of this virus infringement is mainly the fabricius bursa, causes that the fabricius bursa is congested, hemorrhage and downright bad, and causes the pathology such as the hemorrhage and kidney urate deposition of leg muscle.After chicken infects bursal disease virus, other transmissible diseases of Chang Jifa and death.The classical vaccine of preventing and treating this disease is weak malicious seedling and deactivation vaccine, and attenuated vaccine has its potential infectivity and pathogenic, and the immune effect of deactivation vaccine is uncertain.
Researcher utilizes multiple expression system to express IBDV VP2 albumen with preparation IBDV VP2 protein subunit vaccine both at home and abroad, as utilizing escherichia expression system, Macreadie etc., Rong Jun etc. utilize yeast expression system expression, Bayliss etc. to utilize recombinant fowlpox virus expression system, Darteil etc. to utilize the expression of IBDV VP2 albumen that utilized the recombinant fowl adenovirus expression system to carry out such as recombinant herpesvirus of turkeys expression system, Sheppard, but these method major parts all need a large amount of culturing cells, the cost costliness, be unfavorable for that plant accepts.Although Dybbing etc. successfully utilize Recombinant AcMNPV AcNPV at expressed in insect cells IBDV VP2 albumen,, the insect cell substratum is still expensive.
Although Lu looks for the expression of IBDV VP2 albumen that utilized recombinant Bombyx mori baculovirus/silkworm larva expression system to carry out of good grade, but build, the recombinant Bombyx mori baculovirus program is complicated, virus is recombinated complexity and success ratio is low, and need in the bombyx mori cell foster in difficulty, cultivate the breeding recombinant virus, also limit the application of recombinant virus aspect production IBDV VP2 albumen and IBD subunit vaccine.
Summary of the invention
Think that traditionally autographa california nuclear polyhedrosis virus AcNPV can not infected silkworm larva or silkworm chrysalis, but the present inventor finds the AcNPV of restructuring IBDV VP2 gene and can infect by the subcutaneous injection mode silkworm larva and the silkworm chrysalis thereof that kind is ' making greatly ' in research process, this just provides a kind of simple efficient mode for utilizing silkworm biological reactor to produce IBDV VP2 albumen.The contriver also finds with the natural structure of silkworm larva or pupa expression restructuring IBDV VP2 albumen and natural IBDV VP2 albumen very approaching, the natural radioactivity and the antigenicity that have kept VP2 albumen have been avoided the residual shortcoming of recombinant protein intracellular toxin of escherichia coli expression simultaneously.Silkworm larva or silkworm chrysalis also can stimulate body to produce the material that some can stimulate body immune system after infecting recombinant virus, strengthen the immune efficacy that recombinant VP 2 albumen is prepared into the IBD subunit vaccine.
Therefore, main purpose of the present invention is to provide a kind of infectivity bursa of Fabricius virus VP 2 albumen mass-produced method in silkworm larva or silkworm chrysalis, realizing comparatively at an easy rate, scale operation infectious bursal disease subunit vaccine.
Technical scheme
At first the present invention provides a kind of infectivity bursa of Fabricius virus VP 2 albumen, for using Recombinant AcMNPV, and through subcutaneous injection silkworm larva or pupa, the IBDV VP2 albumen that success is expressed in silkworm larva or pupal cell first.And in the prior art, people will be contained the Recombinant AcMNPV of IBDV VP2 protein coding gene with failing all the time, the middle expression in silkworm larva or pupal cell.
Preferably, the kind of the silkworm larva of production IBDV VP2 of the present invention albumen or pupa is for making greatly;
The clinical diagnosis that IBDV strain of the present invention is the Foundation of Luoyang censorship is to extract the strain that screening obtains in the sick fabricius bursa pathological material of disease of infections chicken cloacal bursa (IBD), the Infectious bursal disease virus VP2 immunogenicity that this strain obtains is better, IBDV virus to domestic variation has higher immunogenicity, and the encoding gene of this IBDV VP2 albumen is SEQ ID NO.1.Therefore, the coding gene sequence of the preferred IBDV VP2 of the present invention albumen is SEQ ID NO.1; And other Infectious bursal disease virus VP2 also can be used method of the present invention to prepare.
Preferably, IBDV VP2 albumen of the present invention is soluble proteins, has antigenic activity.Be that the contriver finds that expressing restructuring IBDV VP2 albumen with silkworm larva or pupa can form and the similar active structure of natural structure, shows as non-fusion rotein and is applicable to expressing, and having kept activity and the antigenicity of natural VP2 albumen.Therefore, IBDV VP2 albumen of the present invention is particularly suitable for preparing the infectious bursal disease subunit vaccine; And also be particularly suitable for preparing the diagnostic reagent of infectious bursal disease or test kit etc., the infection conditions about infectious bursal disease virus can be provided easily and quickly.
Another object of the present invention is to provide a kind of method of utilizing silkworm biological reactor to produce infectivity bursa of Fabricius virus VP 2 albumen, comprised the following steps:
1) coding gene sequence of clone IBDV VP2 albumen, upper to the expression transfer vector pFastBacI of insect nuclear polyhedrosis virus (AcNPV), obtains recombinant expressed transfer vector;
2) recombinant expressed transfer vector is transformed in the competent escherichia coli cell DH10Bac that contains baculovirus vector, obtains the recombinant baculovirus genome shuttle vectors that contains IBDV VP2 gene;
3) above-mentioned recombinant baculovirus genome shuttle vectors is transfected in insect cell, obtains the restructuring insect nuclear polyhedrosis virus with this IBDV VP2 gene;
4) by above-mentioned restructuring insect nuclear polyhedrosis virus through subcutaneous injection silkworm larva or pupa, express in vivo VP2 albumen.
Preferably, the coding gene sequence of the IBDV of clone described in aforesaid method of the present invention VP2 albumen is SEQ ID NO.1.
Preferably, the kind of silkworm larva or pupa described in aforesaid method of the present invention is for making greatly.
Preferably, insect cell described in aforesaid method of the present invention is the insect cells such as sf9, sf21, High Five.
The present invention also provides a kind of method of utilizing silkworm biological reactor to produce the infectious bursal disease subunit vaccine, after having obtained IBDV VP2 albumen, also comprise the IBDV VP2 albumen will obtained, add vaccine adjuvant, be prepared as the step of infectious bursal disease subunit vaccine.
The present invention has also obtained a kind of infectious bursal disease subunit vaccine that uses the IBDV VP2 albumen of silkworm biological reactor production and prepare.
Therefore, as subsequent embodiment of the present invention is proved, the application that IBDV VP2 albumen of the present invention can be in preparation treatment or infection prevention bursal disease vaccine.
Therefore, IBDV VP2 albumen of the present invention also can be provided in the application prepared in the infectious bursal disease diagnostic reagent.
As seen from the above, with the production of existing IBDV VP2 albumen, compare, the present invention has following advantage at least:
(1) China is also having the history of several thousand aspect the silkworm cultivation, silkworm artificial breeding technology maturation, and the artificial breeding of throughout the year carrying out extensive silkworm under aseptic condition can realize; (2) the expression cost of silkworm larva and silkworm chrysalis is well below insect cell expression system, and the expressing quantity of every silkworm is equivalent to the expression amount of albumen in 100ml-1000ml insect cell substratum; (3) production unit is simple, easy handling; (4) the insect nuclear polyhedrosis virus without infectivity, is very safe expression system to people and livestock and poultry.(5) the restructuring AcNPV virus formulation provided in the present invention is easier, screen more intuitive and convenient, the restructuring IBDV VP2 albumen that injection silkworm or pupa can directly be expressed solubility, simplified the loaded down with trivial details step and many wheel plaque screening purification of Recombinant virus and complicated technology and the technology of purifying and separating VP2 albumen that build recombinant Bombyx mori baculovirus.(6) encoding gene of IBDV VP2 albumen of the present invention, from the clinical diagnosis of Foundation of Luoyang censorship, be that the sick fabricius bursa pathological material of disease of infections chicken cloacal bursa (IBD) carries out the IBDV LYZS strain virus that separation screening obtains after homogenate, the IBDV VP2 protein immunization power of this encoding viral is better, is more suitable for prevention and treatment or the diagnosis of the IBDV infection of domestic big area appearance.
The accompanying drawing explanation
Fig. 1 is infectious bursal disease virus provided by the invention (IBDV) VP2 gene PCR amplification figure;
Fig. 2 is IBDV VP2 albumen agp antigen assay figure as a result in silkworm hemolymph.
Embodiment
Further set forth the present invention below in conjunction with embodiment.Should be understood that these embodiment are only for the present invention is described, but not limit the scope of the invention.In the following example, the reagent of the experimental technique of unreceipted actual conditions and undeclared formula is according to method and the condition of normal condition or manufacturer's suggestion and carries out or configure.
The present embodiment has been described the preparation method of infectious bursal disease virus provided by the invention (IBDV) VP2 gene, and cloning process is nido RT-PCR, comprises the following steps:
(1) extraction of infectious bursal disease virus (IBDV) LYZS pnca gene group RNA:
IBDV LYZS strain virus is for being the sick fabricius bursa pathological material of disease of infections chicken cloacal bursa (IBD) carries out homogenate from the clinical diagnosis of Foundation of Luoyang censorship, extracts the test kit specification sheets according to the TIANamp viral RNA of TIANGEN Biotech (Beijing) Co., Ltd. and carries out the extraction of IBDV geneome RNA.
(2) clone of IBDV LYZS strain VP2 gene:
1. a pair of primer that is positioned at the external zones of VP2 gene order of sequence selection conserved regions design of the IBDV strain of registering according to GenBank, the large fragment that comprises the VP2 gene for amplification, purpose is by the sequence information of the entirely accurate of definite VP2 gene that checks order.Then design suitable primer amplification according to sequencing result and go out the VP2 gene.The upstream primer called after WVP2F of peripheral primer pair, its sequence information is 5`-GGTTAGTAGAGATCAGACAAAC-3`, downstream primer called after WVP2R, its sequence information is 5`-GCATGGGCTAGGGGAGCGGCAGG-3`.The RNA extracted in step () is for reverse transcription, utilize Reverse Transcriptase XL (the AMV) (article No.: D2620) carry out the synthetic of the first chain of precious biotechnology (Dalian) company limited, in the PCR of 0.2ml pipe, add containing the above-mentioned RNA of 1 microgram, add 1 microlitre (10pm/ microlitre) downstream primer WVP2R as the reverse transcription primer, add 4 microlitre 5 * Reverse Transcriptase XL Buffer and 1 microlitre Reverse Transcriptase XL (5U/ μ l), then add distilled water to 20 microlitre, 42 ℃ of water-bath effects 60 minutes, obtain the cDNA chain of IBDV VP2 gene.
2. take WVP2F and WVP2R as the upstream and downstream primer, take above-mentioned cDNA as template, utilize the TaKaRa Ex Taq of precious biotechnology (Dalian) company limited
(article No.: DRR001A) carry out pcr amplification VP2 gene and peripheral sequence thereof, response procedures is as follows: 95 ℃ of denaturation 3min, and 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 1.5min, carry out 35 circulations, and 10min is extended in 72 ℃ of insulations.The PCR product checks order and measures the sequence information of VP2 gene.
According to above-mentioned VP2 gene sequencing consequence devised pair of primers for increasing the expressed sequence of VP2 gene, upstream primer called after VP2F, its sequence information is 5`-CGC
gGATCCaTGGCATTCCTCATCTGCTTGATAA-3`, downstream primer called after VP2R, its sequence information is 5`-CCC
aAGCTTtCAAAATTCTGCGAAGTAATCTG-3`, and in primer primer suitable restriction enzyme site recognition sequence (underscore), the 5` end of upstream primer is introduced BamHI restriction enzyme enzyme recognition site sequence " G ↓ GATCC ", the 5` end of downstream primer is introduced HindIII restriction enzyme enzyme recognition site sequence " A ↓ AGCTT ", the PCR product of take in step 2 is template, carry out PCR by primer VP2F and VP2R, response procedures is as follows: 95 ℃ of denaturation 2min, 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 1.5min, carry out altogether 30 circulations, 10min is extended in 72 ℃ of insulations.
4. utilize the PCR product described in nucleic acid restriction endonuclease BamHI and HindIII double digestion step 3, and be cloned in the site of the BamHI of donor plasmid pFastBacI and HindIII, obtain recombinant vectors pFastBac-VP2, and the VP2 gene in recombinant vectors is carried out to sequential analysis.In sequence table, SEQ ID NO.1 is infectious bursal disease virus provided by the invention (IBDV) VP2 gene order, in sequence table, SEQ ID NO.2 is infectious bursal disease virus provided by the invention (IBDV) VP2 protein sequence, with reference to Fig. 1, is infectious bursal disease virus provided by the invention (IBDV) VP2 gene PCR amplification.
The present embodiment has been described the method for production infectious bursal disease virus (IBDV) VP2 albumen, and it expresses said gene albumen with silkworm or pupa, and comprises the steps:
(1) the recombinant vectors pFastBac-VP2 of the above acquisition is transformed into to the competent escherichia coli cell DH10Bac (Invitrogen that contains baculovirus full-length gene group shuttle vectors, Catalog:10361012) in, in Bacillus coli cells, recombinate, screened recombinant shuttle vector by blue hickie, recombinant shuttle vector called after Bacmid-VP2.
(2) by the described recombinant shuttle vector Bacmid-VP2 of step () transfection insect sf9 cell, (Invtrogen company Catalog:11496015), produces recombinant baculovirus, called after vBac-VP2: prepare 5 * 10 in cell
5the sf9 cell suspension of cells/ml, in 6 porocyte culture plates, access sf9 cell 2ml/ hole, make cell attachment more than 27 ℃ of standing 1h.Get the centrifuge tube of an aseptic 1.5ml, add 1 μ g recombinant shuttle plasmid Bacmid-VP2, dilute and mix with the SF900II SFM substratum of 100 μ L, the standing 30min of room temperature, get another aseptic 1.5ml centrifuge tube and add 8 μ L transfection reagent Cellfectin
iI Reagent (Invitrogen, article No.: 10362100), dilute and mix with the SF900II SFM substratum of 100 μ L, the standing 30min of room temperature, mix transfection reagent diluent and recombinant shuttle plasmid diluent in above-mentioned two centrifuge tubes, mix gently the standing 10min of room temperature with pipettor.Discard the culture supernatant of the sf9 cell in ready 6 orifice plates, add above-mentioned mixing transfection liquid, and add SF900II SFM to 1ml, 27 ℃ of incubators are cultivated 6h, then discard transfection liquid, add fresh SF900II SFM substratum, continue to cultivate collecting cell culture supernatant after 5 days, and then by the infected insect sf9 cell recombinant virus that increases.Recombinant virus is by virus plaque measuring recombinant virus titre.
(3) with step (two) described restructuring insect nuclear polyhedrosis virus infected silkworm larva (or pupa), scale operation chicken infectivity bursa of Fabricius virus (IBDV) VP2 albumen.
With reference to Fig. 2, preparing virus titer is 5 * 10
7the restructuring insect nuclear polyhedrosis virus of pfu/ml, with 25 μ L microsyringes in silkworm (purchased from Inst. of Silkworm, Chinese Academy of Agricultural Sciences, kind: make greatly) the subcutaneous intersegmental membrane of 5 day old larva is inoculated above-mentioned recombinant virus, every silkworm inoculates 5 μ L, within the 3rd, 4,5,6,7,8 days after inoculation, collect silkworm body hemolymph, the recycling high pressure homogenizer is processed, and 4 ℃, 800bar pressure treatment 2 times.Measure the expression of VP2 albumen in silkworm larva by agar diffusion test, result shows, AGP (agar diffusion test) the antigen amount that the 6th giant silkworm body hemolymph is expressed VP2 albumen after inoculation reaches maximum value 1: 3200, and (standard serum is purchased from China medicine supervision for animals institute, concentration is 1: 8, every bottle of 2 milliliters of dresses).
The present embodiment has been described IBDV genetic engineering subunit vaccine provided by the invention.It is vaccinate: diluent, immunological adjuvant, sanitas and the stablizer etc. that comprise the hemolymph of the silkworm larva that the above infects recombinant virus, above-mentioned silkworm hemolymph lymph utilizes PBS (0.01M, pH=7.4) the agp antigen concentration that is diluted to VP2 albumen reaches 1: 16, (V: V) mix, immunological adjuvant comprised mineral oil, Si Ben-80, tween-80, aluminum stearate and ISCOM etc. with 1: 1～1: 3 for the diluent obtained and immunological adjuvant.The ratio of hemolymph diluent and immunological adjuvant can be 1: 2.Its production method is, collects above-mentioned silkworm hemolymph, and high pressure homogenizer is processed, 4 ℃, and 800bar pressure treatment 2 times, the centrifugal 30min of 12000rpm, collect supernatant, utilizes PBS (0.01M, pH=7.4) to carry out 200 times of above-mentioned supernatant liquors of dilution.The oil phase preparation: get 94 parts, injection mineral oil, 1.5 parts of aluminum stearates, be placed in the oil phase preparation tank and be heated to 80 ℃, then add Si Ben-806 part, reaches 121 ℃ to temperature and maintain 30 minutes, cooling rear standby; Water preparation: get 4 parts of tween-80s, 121 ℃ of sterilizings 30 minutes, cooling, then add above-mentioned hemolymph diluent, fully stir, until tween-80 dissolves fully; Use the laboratory room small-sized emulsifying device to carry out emulsification, press water: oil phase=1: 2, with 17500r/min, stir 5 minutes, add 1% Thiomersalate solution before stopping stirring, making its ultimate density is 0.01%.The vaccine outward appearance of preparation is evenly emulsion of white, and in vaccine, antigen part AGP antigen concentration is 1: 16.
With above-mentioned IBDV genetic engineering subunit vaccine inoculated with subcutaneous injections nonimmune chick in 4 week age, every 0.25ml, 10 of immune group, set up 10 as a control group simultaneously, wherein poison contrast (No. 1-5) is attacked in 5 conducts, do not attack poison as normal control (No. 6-10) for another 5, control group is immunization not, with the condition breeding observing.Separately, in order studying with the immune effect of similar vaccine, to compare, to select the commercial IBD subunit vaccine A of certain company as the parallel test group, the IBDV VP2 antigen that this vaccine contains escherichia coli expression, its AGP antigen concentration >=1: 16.Blood sampling respectively in the 14th, 21 days after vaccine inoculation, separation of serum, detect the IBD antibody titers with agar diffusion test (AGP).After vaccine inoculation 21 days, immune group, parallel test group and attack malicious control group chicken, attacked respectively IBDV BC6/85 virulent strain virus liquid through the eye droppings approach, only attacks the toxic agent amount and be 100BID/.After attacking poison; day by day observe the chicken clinical manifestation; record morbidity and death condition; cut open inspection and observe dead chicken bursa pathology, slaughtered the total Test chicken to 96 hours, by only cuing open inspection; observed and recorded test chicken injection site, subcutaneous, chest muscle, each organ of internal organ and fabricius bursa pathology; calculate protection ratio, each group test chicken weighed before cuing open inspection simultaneously, respectively organize test chicken body weight difference.The Immunization result is judged: chest muscle or leg flesh strip are hemorrhage; The fabricius bursa is hemorrhage, oedema, jaundice; In the fabricius bursa, there are more than one pathologies such as gel-shaped or cheesy secretory product to be judged to and infect the positive.Tire and be more than or equal to 3Log2 when Serum Antibody, it is qualified to be judged to be.When cuing open, the inspection result is negative, and in immune latter 21 days simultaneously 8/10 above immune chicken serums, antibody titer is more than or equal to 3Log2, judges that the vaccine immunity protection is qualified.The results are shown in Table 1,2.
Test chicken serum antibody (AGP) result of tiring after table 1. immunity
After table 2. is attacked poison, test chicken cuts open the inspection result of determination
"+" means to cut open the inspection result and is judged to be the positive; "-" means to cut open the inspection result and is judged to be feminine gender.
As table 2, attack each immune group chicken of the rear observation of poison, the mental status, diet etc. are all normal, and control group is attacked 5 chickens of poison, fluffy and disorderly, depressed by hair, and control group is not attacked 5 malicious chickens, and the mental status, drinking-water etc. are all normal.Cut open inspection and respectively organize chicken to 96h after attacking poison, immune group, contrast do not attack malicious group and parallel test group chicken is showed no fabricius bursa pathology as chest muscle or leg flesh strip is hemorrhage, fabricius bursa enlargement or atrophy, jaundice, in gel-shaped secretory product etc. is arranged, attack poison contrast chicken see fabricius bursa enlargement, jaundice, in gel-shaped is arranged, even cheesy secretory product.
Interpretation of result: according to test-results, control group antibody is all negative, attack malicious control group and attack rear 5/5 positive of poison, attacking the poison contrast sets up, not attacking malicious control group, to cut open inspection all normal, and after 10 chicken immunes of immune group, 21 days antibody horizontals 10/10 are more than or equal to 3Log2, judges that 10/10 is negative after attacking poison, after 10 chicken immunes of parallel test group, 21 days antibody horizontals 9/10 are more than or equal to 3Log2, attack poison and judge that afterwards 10/10 is negative.Result shows; control group is set up; immune group all can provide the malicious protection of attacking of test with parallel experimental group vaccine; by antibody horizontal evaluation after immunity; IBDV genetic engineering subunit vaccine prepared by the present invention not only can provide good protection for the attack of IBDV BC6/85 virulent strain; and immune efficacy and antibody horizontal all be better than the parallel laboratory test group, to compare difference not obvious for immune group and blank group and parallel laboratory test group test chicken body weight simultaneously.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (12)
1. an infectivity bursa of Fabricius virus VP 2 albumen, is characterized in that, described Infectious bursal disease virus VP2 is to use Recombinant AcMNPV, through subcutaneous injection silkworm larva or pupa, and the VP2 albumen of expressing in vivo.
2. IBDV VP2 albumen according to claim 1, is characterized in that, the kind of described silkworm larva or pupa is for making greatly.
3. IBDV VP2 albumen according to claim 1, is characterized in that, the coding gene sequence of described IBDV VP2 albumen is SEQ ID NO.1.
4. IBDV VP2 albumen according to claim 1, is characterized in that, described IBDV VP2 albumen is soluble proteins, has antigenic activity.
5. a method of utilizing silkworm biological reactor to produce infectivity bursa of Fabricius virus VP 2 albumen, is characterized in that, comprises the following steps:
1) coding gene sequence of clone IBDV VP2 albumen, upper to the expression transfer vector pFastBacI of insect nuclear polyhedrosis virus (AcNPV), obtains recombinant expressed transfer vector;
2) recombinant expressed transfer vector is transformed in the competent escherichia coli cell DH10Bac that contains baculovirus vector, obtains the recombinant baculovirus genome shuttle vectors that contains IBDV VP2 gene;
3) above-mentioned recombinant baculovirus genome shuttle vectors is transfected in insect cell, obtains the restructuring insect nuclear polyhedrosis virus with this IBDV VP2 gene;
4) by above-mentioned restructuring insect nuclear polyhedrosis virus through subcutaneous injection silkworm larva or pupa, express in vivo VP2 albumen.
6. method according to claim 4, is characterized in that, the coding gene sequence of described clone IBDV VP2 albumen is SEQ ID NO.1.
7. method according to claim 4, is characterized in that, the kind of described silkworm larva or pupa is for making greatly.
8. method according to claim 4, is characterized in that, described insect cell is the insect cells such as sf9, sf21, High Five.
9. a method of utilizing silkworm biological reactor to produce the infectious bursal disease subunit vaccine, it is characterized in that, also comprise the albumen by the described IBDV VP2 of claim 1-4 any one, add vaccine adjuvant, be prepared as the step of infectious bursal disease subunit vaccine.
10. the infectious bursal disease subunit vaccine prepared by the described IBDV VP2 of claim 1-4 any one albumen.
11. the application of the described IBDV VP2 of claim 1-4 any one albumen in treatment or infection prevention bursal disease vaccine.
12. the application of a claim 1-4 any one described IBDV VP2 albumen in preparing the infectious bursal disease diagnostic reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104427816A CN103172709A (en) | 2011-12-26 | 2011-12-26 | IBDV (Infectious Bursal Disease Virus) VP2 protein and IBD subunit vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011104427816A CN103172709A (en) | 2011-12-26 | 2011-12-26 | IBDV (Infectious Bursal Disease Virus) VP2 protein and IBD subunit vaccine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103172709A true CN103172709A (en) | 2013-06-26 |
Family
ID=48632957
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011104427816A Pending CN103172709A (en) | 2011-12-26 | 2011-12-26 | IBDV (Infectious Bursal Disease Virus) VP2 protein and IBD subunit vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103172709A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017814A (en) * | 2014-06-20 | 2014-09-03 | 西南大学 | Chicken infectious bursal disease virus structural protein VP2 applicable to silkworm middle silk gland expression as well as expression vector and application thereof |
| CN106046123A (en) * | 2016-05-30 | 2016-10-26 | 广东温氏大华农生物科技有限公司 | Chicken infectious bursal disease virus VP2 protein and preparation method and application thereof and kit |
| CN109134668A (en) * | 2018-09-19 | 2019-01-04 | 天康生物股份有限公司 | Fusion protein of chicken infectivity bursa of Fabricius virus and preparation method thereof, application, expression system and vaccine |
| CN111548394A (en) * | 2019-12-23 | 2020-08-18 | 乾元浩生物股份有限公司 | Fowl bursa virus gene engineering vaccine and its preparation method and use |
| CN112048484A (en) * | 2020-07-27 | 2020-12-08 | 华南农业大学 | Gene VII-type Newcastle disease recombinant virus for expressing infectious bursal disease virulent strain VP2 protein and vaccine |
| CN113088539A (en) * | 2021-02-26 | 2021-07-09 | 华南农业大学 | Recombinant adenovirus expressing IBDV VP2 protein, preparation and application thereof |
-
2011
- 2011-12-26 CN CN2011104427816A patent/CN103172709A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| GENBANK: "ACCESSION：AAR14320", 《GENBANK》 * |
| GUO T.等: "Productive infection of Autographa californica nucleopolyhedrovirus in silkworm Bombyx mori strain Haoyue due to the absence of a host antiviral factor", 《VIROLOGY》 * |
| 卢觅佳: "传染性法氏囊病病毒多聚蛋白在家蚕中的表达及其免疫原性研究", 《中国博士学位论文全文数据库》 * |
| 季平 等: "异源多角体蛋白对家蚕核型多角体病毒粒子的包装", 《微生物学报》 * |
| 曹翠平: "家蚕新型高效杆状病毒表达***的开发和应用研究", 《中国博士学位论文全文数据库》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017814A (en) * | 2014-06-20 | 2014-09-03 | 西南大学 | Chicken infectious bursal disease virus structural protein VP2 applicable to silkworm middle silk gland expression as well as expression vector and application thereof |
| CN104017814B (en) * | 2014-06-20 | 2016-08-24 | 西南大学 | Be suitable to chicken infectivity bursa of Fabricius virus Viral structural protein VP2 and expression vector and application that silkworm middle division of silkgland is expressed |
| CN106046123A (en) * | 2016-05-30 | 2016-10-26 | 广东温氏大华农生物科技有限公司 | Chicken infectious bursal disease virus VP2 protein and preparation method and application thereof and kit |
| CN109134668A (en) * | 2018-09-19 | 2019-01-04 | 天康生物股份有限公司 | Fusion protein of chicken infectivity bursa of Fabricius virus and preparation method thereof, application, expression system and vaccine |
| CN111548394A (en) * | 2019-12-23 | 2020-08-18 | 乾元浩生物股份有限公司 | Fowl bursa virus gene engineering vaccine and its preparation method and use |
| CN112048484A (en) * | 2020-07-27 | 2020-12-08 | 华南农业大学 | Gene VII-type Newcastle disease recombinant virus for expressing infectious bursal disease virulent strain VP2 protein and vaccine |
| CN113088539A (en) * | 2021-02-26 | 2021-07-09 | 华南农业大学 | Recombinant adenovirus expressing IBDV VP2 protein, preparation and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106946995A (en) | The I group of type aviadenovirus genetic engineering subunit vaccine of serum 4, its preparation method and application | |
| CN103122352B (en) | Porcine circovirus II-type recombinant baculovirus as well as preparation method and application thereof | |
| CN103172709A (en) | IBDV (Infectious Bursal Disease Virus) VP2 protein and IBD subunit vaccine | |
| CN106636012B (en) | One boar lid his virus stain, vaccine combination and its preparation method and application | |
| CN106591242B (en) | One plant of canine parvovirus poison strain CPV-YH and its application | |
| CN102066407A (en) | Novel avian astrovirus | |
| CN107841507A (en) | A kind of porcine circovirus 2 type Cap cell-penetrating peptides antigen-4 fusion protein gene of high efficient expression and its application | |
| CN108384762B (en) | Porcine alpha-enterocoronavirus and culture method and application thereof | |
| CN109439634A (en) | Pseudorabies virus genetic engineering attenuated vaccine strain and its application | |
| CN102352347A (en) | Porcin circovirus type 2 (PCV2) recombinant baculovirus construction method and subunit vaccine preparation method thereof | |
| CN107551267A (en) | A kind of Goose Parvovirus subunit vaccine and its preparation method and application | |
| CN106282130A (en) | A kind of I group 4 type aviadenovirus, inactivated vaccine and preparation method thereof | |
| CN113491767A (en) | Triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis and preparation method thereof | |
| CN116217678A (en) | Virus-like particle vaccine for resisting H5N1 subtype avian influenza and preparation method and application thereof | |
| CN102304529B (en) | Preparation method of rabbit hemorrhagic fever virus empty capsid antigen | |
| CN106421771A (en) | H7N9 subtype avian influenza genetic engineering vaccine taking baculovirus as carrier as well as preparation method and application of vaccine | |
| CN103505724A (en) | Swine fever and porcine pseudorabies bivalent vaccine as well as preparation method and application thereof | |
| CN112940084A (en) | Serum type4 avian adenovirus subunit vaccine and application thereof | |
| CN113384692A (en) | Duck reovirus and duck circovirus bivalent inactivated vaccine and preparation method thereof | |
| CN102363770A (en) | Recombinant baculovirus capable of expressing porcine circovirus type 2 Cap protein and somatostatin in fusion manner, and subunit vaccine thereof | |
| CN104274829B (en) | A kind of vaccine combination and its preparation method and application | |
| CN109550045A (en) | 3 type of pig circular ring virus, pig parvoviral and swine flu triple inactivated vaccine and preparation method thereof | |
| CN109943576A (en) | A kind of recombinant rabies virus of chimeric canine distemper virus principal immune gene and its application | |
| CN103820398B (en) | A kind of mink enteritis virus recombinant subunit vaccine and preparation method thereof | |
| CN106916832A (en) | O-shaped foot and mouth disease virus recombinant nucleic acid, recombinant vaccine strain and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130626 |