CN109880839A - Porcine mycoplasmal pneumonia and pig circular ring virus bigeminy recombinant vaccine preparation method - Google Patents
Porcine mycoplasmal pneumonia and pig circular ring virus bigeminy recombinant vaccine preparation method Download PDFInfo
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- CN109880839A CN109880839A CN201910200702.7A CN201910200702A CN109880839A CN 109880839 A CN109880839 A CN 109880839A CN 201910200702 A CN201910200702 A CN 201910200702A CN 109880839 A CN109880839 A CN 109880839A
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Abstract
The present invention relates to technical field of vaccines, the preparation method and application of porcine mycoplasmal pneumonia and porcine circovirus 2 type bigeminy recombinant vaccine more particularly, to a kind of baculovirus expression.A kind of porcine mycoplasmal pneumonia and porcine circovirus 2 type bigeminy recombinant vaccine, which is characterized in that the recombinant baculovirus vaccine includes the recombinant protein of the coding of the gene as shown in SEQ ID NO.1 and the recombinant protein that the gene as shown in SEQ ID NO.2 encodes.Above-mentioned bigeminy recombinant vaccine can generate effective immanoprotection action after animal is immunized, and have a good application prospect for prevention and control porcine mycoplasmal pneumonia and porcine circovirus 2 type disease.
Description
Technical field
The present invention relates to technical field of vaccines, more particularly, to a kind of porcine mycoplasmal pneumonia and porcine circovirus 2 type bigeminy
Recombinant vaccine preparation method and applications.
Background technique
Mycoplasma hyopneumoniae (Mycoplasma hyopneumonia, Mhp) is a kind of disease of chronic respiratory infectious disease
Substance can cause porcine mycoplasmal pneumonia (Mycoplasmal pneumoniae of swine, MPS), also known as " swine enzootic pneumonia "
Or " porcine enzootic pneumonia (porcine enzootic pneumonia, PEP) ", and the disease has high infection rate and high biography
Metachromia brings huge economic loss to world's pig breeding industry.The pathogen such as mycoplasma hyopneumoniae Chang Huiyu pig circular ring virus (PCV)
Coinfection increases the seriousness and potential persistence of disease, aggravates respiratory disease, cause in the sick pig death rate
It rises.Studies have shown that the specific adhesion of Mhp and porcine respiratory epithelial cell cilium is the key that cause porcine mycoplasmal pneumonia.?
Adhesion factor antigen on Mhp cell membrane can cause the immunological effect for mycoplasma, it has now been found that P97, P46 and P42 are
The memebrane protein of three kinds important adhesive functions.
Porcine circovirus 2 type (PCV2) is a kind of pathogen that can cause porcine circovirus type 2 infection series of disease.Infection
Swinery immunity power declines afterwards, and can concurrent or secondary other bacteriums or virus infection.PCV2 can cause weanling pig to decline
It exhausts, is dead.Studies have shown that the nucleocapsid protein (Cap) of porcine circovirus 2 type ORF2 coding virus is its main immunogene,
The generation that can induce neutralizing antibody, diagnosis and vaccine research to pig circular ring virus have important meaning, and develop gene work
The ideal target antigen of journey subunit vaccine.
Baculoviral (Baculovirus) belongs to insect viruses, is widely used in as a kind of high efficiency gene expression vector
The expression study of foreign gene.The expression system is able to produce the protein product with posttranslational modification, close to natural egg
White structure and biological activity.It is simple and easily to be also equipped with safety, preparation in addition to the advantage for having viral vectors for baculovirus vector
In obtain the virion of high titre the advantages that.GP64 albumen is as widely used external source in current baculoviral display systems
The fusion object of gene is the main membrane glycoprotein of baculoviral, and energy mediate retroviral is merged with infected cell.The albumen
Signal peptide sequence (SP), intermediate mature sequence and the c-terminus GP64 cross-film knot being mainly made of 20 amino acid residues of aminoterminal
3 part of structure domain (TMD) constitute, wherein SPGP64 is responsible for guiding the GP64 of intracellular expression to cell membrane, TMDGP64 be responsible for by with
Its virus nucleocapsid contacted superscribes the cell membrane containing GP64 albumen.With the development of baculoviral surface display technologies,
Foreign protein is often blended between SPGP64 and certain transmembrane domain, to realize foreign protein on recombinant baculovirus surface
Efficient displaying.
In order to study the bigeminy recombinant vaccine product that can prevent porcine mycoplasmal pneumonia and porcine circovirus 2 type simultaneously,
The present invention by baculovirus expression system co-expressed Mhp P97R1, P46 and P42 chimeric protein and PCV2 Cap egg
It is white, identify that BALB/c mouse and piglet is immunized in correct recombinant baculovirus, through antibody level and/or lymphocyte proliferation assay
Equal detections prove that the recombinant baculovirus immune effect of vaccine of building is good, therefore this method can be used as and a kind of prepare Mhp and PCV2
Bigeminy recombinant vaccine method use.
Summary of the invention
Above-mentioned purpose of the invention is achieved through the following technical solutions:
A kind of nucleic acid, gene order is as shown in SEQ ID NO.1 or its gene order is as shown in SEQ ID NO.2；Or
Contain the nucleic acid as shown in SEQ ID NO.1 and the nucleic acid as shown in SEQ ID NO.2 simultaneously.
A kind of recombinant expression carrier, which is characterized in that contain the gene as shown in SEQ ID NO.1；Or containing such as SEQ
Gene shown in ID NO.2；Or contain the gene as shown in SEQ ID NO.1 and the gene as shown in SEQ ID NO.2 simultaneously；
Preferentially, the carrier of the channel genes is pFastBac Dual.
A kind of recombinant baculovirus, which is characterized in that expression has amino acid sequence as shown in SEQ ID NO.3 or ammonia
Base acid sequence is as shown in SEQ ID NO.4；Or simultaneously containing amino acid sequence recombinant protein as shown in SEQ ID NO.3 and
The recombinant protein as shown in SEQ ID NO.4.
A kind of porcine mycoplasmal pneumonia and porcine circovirus 2 type bigeminy recombinant vaccine, which is characterized in that the recombination bar
Shape viral vaccine includes simultaneously above-mentioned recombinant protein.Above-mentioned vaccine further includes vaccine adjuvant, suspending agent, surface-active
One or more of agent, antigens inactive agent or preservative.
Some of embodiments, the vaccine also include vaccine adjuvant, such as the acceptable aqueous adjuvants of veterinary science or oiliness
Adjuvant.Preferably, aqueous adjuvants include but is not limited to aluminium salt series of adjuvants, Montanide IMS series of adjuvants or Montanide
GEL series of adjuvants, propolis, immunostimulating complex, cytokine class adjuvant, nucleic acid and its derivative species adjuvant, lecithin lipid
Adjuvant.The Montanide IMS series of adjuvants includes 1313VG, 251C VG, 2215VG；The Montanide GEL series
Adjuvant is GEL 01PR or Montanide PET GEL A；The oil-in-water series of adjuvants includes MF59, Montanide
ISA15A VG etc.；The cytokine class adjuvant includes interleukin (IL-1, IL-2, IL-4, IL-12), interferon (IFN-γ,
IFN-α, IFN-β) etc.；The nucleic acid and its derivative species adjuvant include immunostimulatory sequence DNA (CpG DNA) or CpG oligomerization
Deoxynucleotide etc..It is further preferred that aqueous adjuvants preferred embodiment be related to IMS1313N VG, IMS2215VG, Gel01,
One or more of compositions of carbomer, aluminium hydroxide gel.Some of embodiments, the vaccine include not formula Freund's complete adjuvant
And/or not formula Freund's incomplete adjuvant.
Some of embodiments, the concentration range of the adjuvant are from 5 ~ 50%V/V, preferably 20 ~ 30%V/V, further
It is preferred that 25% V/V.
Some of embodiments, the oil adjuvant include but is not limited to white oil, saualane or squalene, Drake oil
(Drakeoil) and other animal oil, vegetable oil or mineral oil.Above-mentioned oil adjuvant, can also be with either natural origin
It is by artificial synthesized acquisition.
Some of embodiments, the vaccine further include suspending agent, surfactant, antigens inactive agent or preservative.Institute
Stating suspending agent may include, for example, aluminum stearate and other available suspending agents of technical field.The surfactant
May include, for example, Sorbitan Monooleate (TWEE series), sapn (SPAN) and technical field it is available its
His surfactant.The antigens inactive agent includes but is not limited to, such as formalin, beta-propiolactone etc..The preservative
Including, such as thimerosal.The application method and dosage of above-mentioned substance are well known to the skilled person.
The present invention also provides the preparation methods of recombinant baculovirus, which comprises the steps of:
Building contains the expression vector of above-mentioned nucleic acid；
Amplification, purifying and the detection of recombinant baculovirus.
The present invention also provides above-mentioned nucleic acid, recombinant vector, recombinant baculovirus, vaccines to prevent, treat, diagnose pig branch original
Application in body pneumonia and/or porcine circovirus 2 type disease.
The present invention has following beneficial effect:
(1) combination of more immunogenic protein fragments can induce the immune effect for generating collaboration in the bigeminy vaccine；
(2) after mouse and piglet is immunized in the bigeminy vaccine, it can generate and be equal to or similar to using individually commercial vaccine
The body fluid and/or cell immunoreceptor shown shows to cause in mouse and piglet body after this recombinant viral vaccine is immune
Generate the immunological effect of specificity.
Detailed description of the invention
Fig. 1 is pFastBac dual-P97R1P46P42-Cap construction of recombinant vector schematic diagram.
Fig. 2 is the Western Blot identification of recombinant protein.
Fig. 3 is the electromicroscopic photograph figure of recombinant baculovirus.
Fig. 4 is that serum ELISA testing result after mouse is immunized in recombinant baculovirus.
Fig. 5 is that spleen lymphocyte proliferation index testing result after mouse is immunized in recombinant baculovirus.
Fig. 6 is that serum ELISA testing result after piglet is immunized in recombinant baculovirus.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should apply example
It is only to aid in the understanding present invention, should not be regarded as a specific limitation of the invention.
It is the ordinary skill in the art if not otherwise specified in technical solution of the present invention；The reagent or material
Material, if not otherwise specified, is purchased from commercial channel.
Some agent prescriptions that technical solution of the present invention refers to:
PBS:8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4It is dissolved in ddH2O is settled to 1 L；
Antigen coat buffer: 1.59 g Na2CO3, 2.93 g NaHCO3, ddH2O is settled to 1 L；
ELISA cleaning solution: the PBS solution of 0.05% Tween-20；
ELISA confining liquid: 5% skimmed milk power, the PBS solution of 5%BSA；
ELISA antibody diluent: 2% skimmed milk power, the PBS solution of 1%BSA；
ELISA terminate liquid: 2 M H2SO4。
Embodiment 1
The acquisition of target gene and the building of expression vector
Shandong strain PCV2 Cap gene order (accession number: KY656098.1) is searched in ncbi database, deletes nuclear location letter
Number peptide carries out baculovirus expression system codon optimization to the nucleic acid sequence of 579 bp of residue.Same method is searched
The P97R1(MHP168_110 of Mhp168 plants (accession number: CP002274.1)), P46(MHP168_522) and P42(MHP168_
069) gene order carries out baculovirus expression system codon optimization to it.Sequence selection according to carrier and gene is appropriate
Restriction enzyme site and connection peptide sequence, artificial synthesized following fusion: 1. I-SP-Cap-TMD-6 of Xho × His-TAA-Kpn
I, wherein TAA is terminator codon；2. I-SP-P97R1-P46-P42-TMD-6 of BamH × His-TAA-Hind III, wherein
It is connected with flexible joint GGSG between P97R1, P46 and P42, TAA is terminator codon.1. 2. two sequences are respectively such as
Shown in SEQ ID NO.1 and SEQ ID NO.2.
The successively 1. and 2. enzyme by sequence respectively with III two groups of restriction endonuclease of Xho I and Kpn I, BamH I and Hind
It cuts, is connected into pFastBac Dual carrier using molecular biology method.Obtain pFastBac Dual-P97R1P46P42- Cap
Recombinant vector, Vector map are as shown in Figure 1.
Embodiment 2
The preparation of restructuring rod granule, the detection of eukaryotic expression and the collection of virus
By recombinant plasmid pFastBac Dual-P97R1P46P42-Cap and pFastBac the Dual empty plasmid of above-mentioned acquisition
It is transformed into respectively and passes through CaCl2In the DH10Bac competent cell of method preparation, positive colony is filtered out by blue hickie.It extracts
Positive baculovirus plasmid passes through FuGENE6(Promega company) to be each separately transfected into density be about 2 × 10 to reagent6Sf9 it is thin
In born of the same parents, cell is blown and beaten with sterile PBS solution after 96 h, cell precipitation is collected by centrifugation, total protein is extracted, respectively with anti-
P97R1, anti-P46, anti-P42, anti-Cap and anti-6 × His are primary antibody, are detected by Western Blot method
The expression of recombinant protein, as a result as shown in Figure 2；Cells and supernatant is collected, as P1 is respectively designated as recombinant virus
RVAc-P97R1P46P42-Cap and rVAc-dual.
Embodiment 3
Amplification, purifying and the detection of recombinant baculovirus
P1 is obtained into virus after two generations of continuous amplification for virus amplification is carried out in recombinant virus inoculation Sf9 cell according to MOI=0.1
The metastable P3 of virulence is for recombinant virus.By P3 generation virus with the suspension Sf9 cell of the dose inoculation shaking flask culture of MOI=0.1,
96 h collect viral supernatants by low-speed centrifugal after inoculation.35000 rpm, 4 DEG C of 2 h of ultracentrifugation collect precipitating；Precipitating is used
PBS is added slowly to complete the sucrose solution (60-40-20%(w/v) of density gradient in advance after being resuspended) upper layer, 30000 rpm, 4 DEG C
2 h of ultracentrifugation collects the milky turbidity band between 40-60% sucrose liquid level；After the liquid of collection is resuspended with PBS, 35000
Rpm, 4 DEG C of 2 h of ultracentrifugation remove sucrose solution, are resuspended with PBS collect recombinant baculovirus again.Virus liquid is loaded to
On 200 mesh copper mesh, in electric microscopic observation form after phosphotungstic acid dyeing, as shown in Figure 3.
Embodiment 4
Animal immune experiment
1, mouse immune is tested
The female BAl BIc of 30 6-8 week old/c mouse is randomly divided into following five groups: 1. PBS group；2. rVAc-dual group；③Mhp
Positive vaccine group (auspicious times suitable-prosperous) 4. PCV2 positive vaccine group (the net promise of circle) 5. rVAc-P97R1P46P42-Cap viral vaccine
Group, every group 6.Using subcutaneous routes, in the 0th, 14 and 28, d is immunized respectively.Wherein every mouse injection of PBS group
100 μ L corresponding commodity vaccines are immunized in 200 μ L of PBS, every mouse of positive vaccine group respectively, and virus organizes every mouse difference
Immune 1 × 108PFU recombinant virus rVAc-dual and rVAc-P97R1P46P42-Cap.Tail is carried out in the 14th and 28 d respectively
Portion takes blood, carries out endocanthion to every group of 3 mouse respectively in the 35th and 42 d and blood and spleen lymphocyte proliferation is taken to test.
Specific detecting step is as follows:
(1) antibody level detects: in order to assess mouse for humoral immunity effect caused by various antigenic stimulus, using indirect
ELISA method, respectively with concentration is 1 μ g/mL antigen rP97, rP46, rP42 after buffer dilutes and Cap protein is coated with,
The closing of ELISA confining liquid, be incubated for respectively serum that above tetra- periods of 14,28,35 and 42 d acquire (antibody diluent 1:
100 dilutions), then it is incubated for secondary antibody, developing solution is added to develop the color, wherein use ELISA cleaning solution board-washing 4 times between every two step, every time 1
min.It is eventually adding the reaction of ELISA terminate liquid color development stopping, detects the OD value under 450 nm wavelength.As a result as shown in figure 4, its
Middle * * indicates that P < 0.01, * * * indicate P < 0.001.The mice serum of recombinant virus immune group can be shown for four kinds of antigens
Significant specific IgG reaction, and extremely significant for the reaction of rP42 albumen, show after mouse is immunized in recombinant viral vaccine
It has good immune performance in vivo.
(2) spleen lymphocyte proliferation is tested: in order to assess mouse for cell immunoreceptor caused by various antigens, being made
Mice spleen lymph with mouse lymphocyte separating liquid (it is Bioisystech Co., Ltd that Beijing, which reaches section) separation 35 d and 42 d is thin
Born of the same parents, concrete operations process are shown in product description.Each group splenic lymphocytes after counting separation, it is dilute that 100 μ L are added in the every hole of 96 orifice plates
It releases to 4 and releases to 1 × 106cell/mL.After cell is adherent, the hybrid antigen after 100 μ L dilute is added in every hole respectively
(mixture of rP97, rP46, rP42 and Cap, wherein four kinds of antigen final concentrations are 10 μ g/mL), 1640 culture mediums and the positive
It compares (canavaline, 10 μ g/mL of final concentration).Cell is put into CO220 μ g MTT are added in every hole after 42 h of incubator culture
(5 mg/mL) continues to cultivate 4 h, abandons cell culture supernatant to the greatest extent, and every hole is added 100 μ L DMSO solutions, shakes on oscillator
5 min are mixed, the OD value under 490 nm wavelength is finally detected, calculating stimulus index, (SI=stimulation hole OD value/does not stimulate hole OD
Value).As a result as shown in figure 5, wherein NS indicates that there was no significant difference, * * * indicates P < 0.01.The mouse of viral vaccine group compares
In other control groups, higher stimulus index can be shown for corresponding antigens stimulation, can be drawn after showing this virus vaccine
Send out the cell immunoreceptor that specificity is generated in Mice Body.
2, piglet immunological is tested
9 4-5 week old piglets are randomly divided into following three groups: 1. rvAc-dual zero load virus immunity group, 2. positive vaccine immunity
Group (auspicious times of suitable-prosperous+circle Ke Xin)RvAc-P97R1P46P42-Cap virus vaccine group, every group 3.Using leg muscle
Injection, is immunized in the 0th d and the 14th d respectively.Wherein positive auspicious times of the piggy injection commercialized vaccine of vaccine group every it is suitable-
Prosperous and circle each 1 mL of Ke Xin, unloaded every piglet of virus immunity group are immunized 1 × 10 respectively9PFU recombinant virus rvAc-dual, disease
Malicious every piglet of vaccine immunity group is immunized 1 × 10 respectively9PFU recombinant virus rvAc-P97R1P46P42-Cap.Respectively the 0th,
Venous blood sampling is carried out with 28 d, separates serum.It is same to carry out indirect method with tetra- kinds of albumen coatings of rP97, rP46, rP42 and Cap
The antibody level of ELISA detection serum.As a result as shown in fig. 6, wherein * * * indicates P < 0.001.Recombinant virus rvAc-
The piglet serum of P97R1P46P42-Cap vaccine immunity group is directed to P97R1, P46, P42 and pig circular ring virus 2 of mycoplasma hyopneumoniae
Poison 2 type Cap, tetra- kinds of antigens can show significantly to be similar to the specific IgG reaction (P < 0.001) after positive vaccine immunity,
Show that recombinant virus rvAc-P97R1P46P42-Cap vaccine also has good immune performance on ontology animal piglet.
In conclusion through baculovirus expression system prepare recombinant virus rVAc-P97R1P46P42-Cap be immunized it is small
Preferable immune effect can be played after mouse and piglet, it was demonstrated that development can be used as by recombinant baculovirus prepared by the method
A kind of method of porcine mycoplasmal pneumonia and porcine circovirus 2 type bigeminy recombinant vaccine.
The above is only the preferred embodiment of the present invention, not does limitation in any form to the present invention, though
So the present invention is disclosed above with preferred embodiment, and however, it is not intended to limit the invention, any technology people for being familiar with this profession
Member, in the range of not departing from technical solution of the present invention, when the technology contents using the disclosure above make a little change or repair
Decorations are the equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, technology according to the present invention are real
Matter any simple modification, equivalent change and modification to the above embodiments, still fall within the range of technical solution of the present invention
It is interior.
Sequence table
<110>Hangzhou Hong Sheng Biotechnology Ltd.
Institutes Of Technology Of Zhejiang
Hangzhou Hong Qiao Zhong Ke gene technology Co., Ltd
<120>porcine mycoplasmal pneumonia and pig circular ring virus bigeminy recombinant vaccine preparation method
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 882
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ctcgagatgg tgtcagccat cgtgttgtac gttctgttgg ccgccgctgc ccattcagct 60
tttgctaacg gcatcttcaa cactaggctg tccagaactt tcggatacac cgtgaagaag 120
accactgtcc gcacccctag ctggaaggtg gacatgatgc gtttcaacat caacgacttc 180
ctgcctcccg gtggcggatc taacccactg tcagtccctt tcgagtacta ccgcatccgt 240
aaggtgaagg tcgaattctg gccttgctcc cccatcacac aaggtgaccg cggtgtggga 300
tccagcgctg tcatcctgga cgacaacttc gtgaccaagg ctactgccct gacctacgac 360
ccctacgtca actactcttc aaggcacacc atcactcagc cattctccta ccacagccgc 420
tacttcactc ccaagccagt gctggactct accatcgact acttccagcc taacaacaag 480
cgcaaccagc tgtggctgcg tctgcagacc actggcaacg tggaccacgt cggactgggt 540
actgccttcg agaactcaat ctacgaccag gaatacaaca tcagggtgac catgtacgtc 600
cagttcagag agttcaacct gaaggaccca cctctgaacc ccaagggtga cactggactg 660
tctaagaacc caatcgagtt ggtggaggga tggttttctt cttggaaatc atctattgct 720
tcattttttt tcattatcgg tctgattatt ggactgttcc tggtgctgcg cgtgggtatt 780
catctgtgca ttaaactgaa gcacactaag aagcgccaga tctatactga tattgaaatg 840
aacagactgg gaaagcatca tcaccaccac cactaaggta cc 882
<210> 2
<211> 1512
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggatccatgg tgtcagccat cgtgttgtac gttctgttgg ccgccgctgc ccattcagct 60
tttgctgaag gcaagcgcga ggaagtcgac aagaaggtga aggagctgga caacaagatc 120
aagggaatcc tgcctcagcc tcccgctgct aagcctgagg ctgccaagcc agtggctgcc 180
aagcctgaag ctgccaagcc cgtcgctgcc aagccagagg ctgccaagcc tgtggctgcc 240
aagcccgaag ctgccaagcc agtcgctgcc aaaccagaag ccgctaaacc tgaggccgcc 300
aagccagtcg ctaccaacac taacaccaac actggtttct ctctgaccaa caagcctaag 360
gaggactact tccctatggc tttctcctac aagctggagt acaccgacga aaacaagctg 420
agcctgaaga ctcccgagat caacgtgttc ctggaactgg gtggctcagg ccaggactac 480
aacgacaagg ctaagacctt catcaaggac ggagaccaga acatgactat ctacaagcca 540
gacaaggtcc tgggcaaggt ggccgtcgaa gtgctgcgcg tgctgatcgc taagaagaac 600
aaggcctccc gtagcgaagt cgagaacgaa ctgaaggcta agctgcctaa catctccttc 660
aagtacgaca accagaccta caaggtgcag ggcaagaaca tcaacaccat cctggtctct 720
ccagtcatcg tgactaaggc taacgtggac aaccctgacg ccggaggttc aggaatcgag 780
gctgccccaa ggggactgcc tcagatcgaa gtctctttct caatcgacgt gaacggtatc 840
accactgtct ccgctaagga caagaagact ggcaaggagc agaccatcac tatcaagaac 900
acctctactc tgtcagagga agagatcaac aagatgatcc aggaggctga agagaacagg 960
gaagccgacg ctctgaagaa ggacaagatc gagaccactg tgagagccga aggactgatc 1020
aaccagctgg agaagagcat caccgaccag ggtgaaaaga tcgaccctaa gcagaaggag 1080
ctgctggaaa agcagatcca ggagctgaag gacctgctga aggaagagaa gactgacgag 1140
ctgaagctga agctggacca gatcgaagct gctgctcaat ctttcgctca ggctactgct 1200
cagcaggcta acacttccga gagcgacccc aaggctgacg acagcaacac catcgacgcc 1260
gaaatcaagc aggacggtga cactggactg tctaagaacc caatcgagtt ggtggaggga 1320
tggttttctt cttggaaatc atctattgct tcattttttt tcattatcgg tctgattatt 1380
ggactgttcc tggtgctgcg cgtgggtatt catctgtgca ttaaactgaa gcacactaag 1440
aagcgccaga tctatactga tattgaaatg aacagactgg gaaagcatca tcaccaccac 1500
cactaaaagc tt 1512
<210> 3
<211> 289
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Met Val Ser Ala Ile Val Leu Thr Val Leu Leu Ala Ala Ala Ala His
1 5 10 15
Ser Ala Pro Ala Ala Gly Ile Pro Ala Thr Ala Leu Ser Ala Thr Pro
20 25 30
Gly Thr Thr Val Leu Leu Thr Thr Val Ala Thr Pro Ser Thr Leu Val
35 40 45
Ala Met Met Ala Pro Ala Ile Ala Ala Pro Leu Pro Pro Gly Gly Gly
50 55 60
Ser Ala Pro Leu Ser Val Pro Pro Gly Thr Thr Ala Ile Ala Leu Val
65 70 75 80
Leu Val Gly Pro Thr Pro Cys Ser Pro Ile Thr Gly Gly Ala Ala Gly
85 90 95
Val Gly Ser Ser Ala Val Ile Leu Ala Ala Ala Pro Val Thr Leu Ala
100 105 110
Thr Ala Leu Thr Thr Ala Pro Thr Val Ala Thr Ser Ser Ala His Thr
115 120 125
Ile Thr Gly Pro Pro Ser Thr His Ser Ala Thr Pro Thr Pro Leu Pro
130 135 140
Val Leu Ala Ser Thr Ile Ala Thr Pro Gly Pro Ala Ala Leu Ala Ala
145 150 155 160
Gly Leu Thr Leu Ala Leu Gly Thr Thr Gly Ala Val Ala His Val Gly
165 170 175
Leu Gly Thr Ala Pro Gly Ala Ser Ile Thr Ala Gly Gly Thr Ala Ile
180 185 190
Ala Val Thr Met Thr Val Gly Pro Ala Gly Pro Ala Leu Leu Ala Pro
195 200 205
Pro Leu Ala Pro Leu Gly Ala Thr Gly Leu Ser Leu Ala Pro Ile Gly
210 215 220
Leu Val Gly Gly Thr Pro Ser Ser Thr Leu Ser Ser Ile Ala Ser Pro
225 230 235 240
Pro Pro Ile Ile Gly Leu Ile Ile Gly Leu Pro Leu Val Leu Ala Val
245 250 255
Gly Ile His Leu Cys Ile Leu Leu Leu His Thr Leu Leu Ala Gly Ile
260 265 270
Thr Thr Ala Ile Gly Met Ala Ala Leu Gly Leu His His His His His
275 280 285
His
<210> 4
<211> 499
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Met Val Ser Ala Ile Val Leu Thr Val Leu Leu Ala Ala Ala Ala His
1 5 10 15
Ser Ala Pro Ala Gly Gly Leu Ala Gly Gly Val Ala Leu Leu Val Leu
20 25 30
Gly Leu Ala Ala Leu Ile Leu Gly Ile Leu Pro Gly Pro Pro Ala Ala
35 40 45
Leu Pro Gly Ala Ala Leu Pro Val Ala Ala Leu Pro Gly Ala Ala Leu
50 55 60
Pro Val Ala Ala Leu Pro Gly Ala Ala Leu Pro Val Ala Ala Leu Pro
65 70 75 80
Gly Ala Ala Leu Pro Val Ala Ala Leu Pro Gly Ala Ala Leu Pro Gly
85 90 95
Ala Ala Leu Pro Val Ala Thr Ala Thr Ala Thr Ala Thr Gly Pro Ser
100 105 110
Leu Thr Ala Leu Pro Leu Gly Ala Thr Pro Pro Met Ala Pro Ser Thr
115 120 125
Leu Leu Gly Thr Thr Ala Gly Ala Leu Leu Ser Leu Leu Thr Pro Gly
130 135 140
Ile Ala Val Pro Leu Gly Leu Gly Gly Ser Gly Gly Ala Thr Ala Ala
145 150 155 160
Leu Ala Leu Thr Pro Ile Leu Ala Gly Ala Gly Ala Met Thr Ile Thr
165 170 175
Leu Pro Ala Leu Val Leu Gly Leu Val Ala Val Gly Val Leu Ala Val
180 185 190
Leu Ile Ala Leu Leu Ala Leu Ala Ser Ala Ser Gly Val Gly Ala Gly
195 200 205
Leu Leu Ala Leu Leu Pro Ala Ile Ser Pro Leu Thr Ala Ala Gly Thr
210 215 220
Thr Leu Val Gly Gly Leu Ala Ile Ala Thr Ile Leu Val Ser Pro Val
225 230 235 240
Ile Val Thr Leu Ala Ala Val Ala Ala Pro Ala Ala Gly Gly Ser Gly
245 250 255
Ile Gly Ala Ala Pro Ala Gly Leu Pro Gly Ile Gly Val Ser Pro Ser
260 265 270
Ile Ala Val Ala Gly Ile Thr Thr Val Ser Ala Leu Ala Leu Leu Thr
275 280 285
Gly Leu Gly Gly Thr Ile Thr Ile Leu Ala Thr Ser Thr Leu Ser Gly
290 295 300
Gly Gly Ile Ala Leu Met Ile Gly Gly Ala Gly Gly Ala Ala Gly Ala
305 310 315 320
Ala Ala Leu Leu Leu Ala Leu Ile Gly Thr Thr Val Ala Ala Gly Gly
325 330 335
Leu Ile Ala Gly Leu Gly Leu Ser Ile Thr Ala Gly Gly Gly Leu Ile
340 345 350
Ala Pro Leu Gly Leu Gly Leu Leu Gly Leu Gly Ile Gly Gly Leu Leu
355 360 365
Ala Leu Leu Leu Gly Gly Leu Thr Ala Gly Leu Leu Leu Leu Leu Ala
370 375 380
Gly Ile Gly Ala Ala Ala Gly Ser Pro Ala Gly Ala Thr Ala Gly Gly
385 390 395 400
Ala Ala Thr Ser Gly Ser Ala Pro Leu Ala Ala Ala Ser Ala Thr Ile
405 410 415
Ala Ala Gly Ile Leu Gly Ala Gly Ala Thr Gly Leu Ser Leu Ala Pro
420 425 430
Ile Gly Leu Val Gly Gly Thr Pro Ser Ser Thr Leu Ser Ser Ile Ala
435 440 445
Ser Pro Pro Pro Ile Ile Gly Leu Ile Ile Gly Leu Pro Leu Val Leu
450 455 460
Ala Val Gly Ile His Leu Cys Ile Leu Leu Leu His Thr Leu Leu Ala
465 470 475 480
Gly Ile Thr Thr Ala Ile Gly Met Ala Ala Leu Gly Leu His His His
485 490 495
His His His
Claims (8)
1. a kind of nucleic acid, gene order is as shown in SEQ ID NO.1 or its gene order is as shown in SEQ ID NO.2；Or
Person contains the nucleic acid as shown in SEQ ID NO.1 and the nucleic acid as shown in SEQ ID NO.2 simultaneously.
2. a kind of recombination bacillary viral vector, which is characterized in that contain the gene as shown in SEQ ID NO.1；Or containing such as
Gene shown in SEQ ID NO.2；Or simultaneously containing the gene as shown in SEQ ID NO.1 and as shown in SEQ ID NO.2
Gene.
3. recombinant vector as claimed in claim 2, which is characterized in that the carrier of the channel genes is pFastBac
Dual。
4. a kind of recombinant baculovirus, which is characterized in that expression has amino acid sequence albumen as shown in SEQ ID NO.3, or
Person's expression has amino acid sequence albumen as shown in SEQ ID NO.4；Or expression simultaneously is containing as shown in SEQ ID NO.3
Albumen and the albumen as shown in SEQ ID NO.4.
5. a kind of porcine mycoplasmal pneumonia and porcine circovirus 2 type bigeminy vaccine, which is characterized in that the vaccine includes claim
Albumen described in 4.
6. bigeminy vaccine as claimed in claim 5, which is characterized in that further include vaccine adjuvant, suspending agent, surfactant,
One or more of antigens inactive agent or preservative.
7. the preparation method of vaccine as claimed in claim 5, which comprises the steps of:
Construct the expression vector containing nucleic acid described in claim 1；
The preparation of recombinant baculovirus；
Mouse is immunized in recombinant baculovirus and/or piglet verifies its immune effect.
8. nucleic acid described in claim 1, recombinant vector described in claim 2 or 3, recombination as claimed in claim 4 are rod-shaped
Vaccine viral, described in claim 5 or 6 prevents, treats in preparation, diagnoses porcine mycoplasmal pneumonia and/or porcine circovirus 2 type
Application in the drug of disease.
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| CN113913465A (en) * | 2021-09-17 | 2022-01-11 | 浙江洪晟生物科技股份有限公司 | Preparation method and application of swine mycoplasma pneumonia genetic engineering subunit vaccine carrying swine GMCSF molecular adjuvant |
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Effective date of registration: 20231122 Address after: Room B502, 5th Floor, Building B, Science and Technology Innovation Park, No. 398 Mahuan Road, Lihai Street, Yuecheng District, Shaoxing City, Zhejiang Province, 312000 Patentee after: Zhejiang Hongsheng Biotechnology Co.,Ltd. Address before: 310018 room b2018, building 2, No. 452, Baiyang street, Hangzhou Economic and Technological Development Zone, Zhejiang Province Patentee before: HANGZHOU HONGSHENG BIOTECHNOLOGY CO.,LTD. Patentee before: ZHEJIANG SCI-TECH University Patentee before: HANGZHOU HONGQIAO ZHONGKE GENE TECHNOLOGY Co.,Ltd. |