CN105087616A - Fusion gene of mycoplasma hyopneumoniae and preparation method of fusion gene - Google Patents
Fusion gene of mycoplasma hyopneumoniae and preparation method of fusion gene Download PDFInfo
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Abstract
The invention discloses a fusion gene of mycoplasma hyopneumoniae and a preparation method of the fusion gene, and belongs to the field of genetic engineering technology. P46 and P65 genes adopted in the invention can directly design specific primers containing corresponding restriction sites according to a coding region sequence released in NCBI GeneBank. A PCR product of the P46 gene is subject to double enzyme digestion and connection with a prokaryotic expression vector pET-28a, so as to construct a recombinant expression vector pET-28a-P46; and the recombinant expression vector pET-28a-P46 is subject to double enzyme digestion and connection with a PCR product of the P65 gene, so as to directly construct a recombinant expression vector pET-28a-P46-P65. The fusion gene and the preparation method have the following advantages: instead of Linker connection, the P46 and P65 genes are directly subject to double enzyme digestion connection so as to prepare the recombinant expression vector pET-28a-P46-P65; and compared with a process of connecting genes by virtue of Linker and then connecting to a prokaryotic expression vector, the recombinant vector construction process is simple and is easy to prepare the recombinant expression vector, and consumed time is significantly shortened.
Description
Technical field
The invention belongs to gene engineering technology field, relate to the research to animal bacteria sexually transmitted disease gene vaccine, specifically a kind of fusion gene and preparation method thereof of mycoplasma hyopneumoniae.
Background technology
Mycoplasma hyopneumoniae (Mycoplasmahyopneumoniae, Mhp) can cause the bronchopneumonia of pig, is commonly called as swine enzootic pneumonia.The harm of this disease be Mhp can with porcine respiratory epithelial cilium generation specific adhesion, thus destroy animal body respiratory mucosa-cilium barrier, cause the infection of other pathogenic agent, respiratory tract disease is increased the weight of, then PRDC (porcine respiratory disease complex) (Porcinerespiratorydiseasecomplex, PRDC) is caused.
Mhp requires very high to culture condition, more difficult cultivation, and at present institute study what report is gene from 4 kinds of pathotype mycoplasmas, is 168 strains, J strain, 7448 types and 232 types respectively.Swine enzootic pneumonia is chronic disease, and spread extensively, mortality ratio is not high but carrying rate is very high, is difficult to thorough radical cure, and is often seasonal generation.Due to domestic at present for the cultivation of mycoplasma hyopneumoniae and isolation technique very imperfect, this just more increases the difficulty to mycoplasma hyopneumoniae research.Current China to the control of this disease mainly to put prevention first, but due to the exploitation for this bacterial vaccine almost complete in adjuvated full cell preparation or film preparation, there is no one and prove fully effective.Research shows, the specific binding of adhesion protein and respiratory mucosa cilium is the requisite paathogenic factor causing porcine mycoplasmal pneumonia.Studies have reported that the adhesion protein of Mhp and the combination of porcine respiratory mucociliary, is the requisite paathogenic factor causing porcine mycoplasmal pneumonia.Adhesion protein mainly cytoplasm protein P36 in Mhp, membranin P46, P65 and P74, adhesion factor P97, P102, P216, P159, P116, Mhp271, Mhp107, Mhp684 and heat shock protein(HSP) DnaK.According to bibliographical information, P97, P102, P216, P159, P116 are Vitrum AB associated proteins, mainly through the Vitrum AB protein binding in extracellular matrix on respiratory mucosa cilium, cause immune response; P36 has the cytoplasmic protein of the activity of serum lactic dehydrogenase, and cause immune response in late period, immunogenicity is lower slightly; P46 albumen is Cell membrane antigens, and early immune can be caused to react; P65 is one of heat shock protein A subunit family member, has immunostimulation, has esterase activity simultaneously; Dnak is the function that heat shock protein(HSP) has immunological adjuvant, and its N end structure has the function of molecular chaperones, and C end has main immunogenicity.Because current China lacks the effective vaccine of prevention mycoplasma hyopneumoniae infection, in order to more effective control and minimizing Mhp are to the harm of livestock industry, the immunogenicity of research mycoplasma hyopneumoniae fusion rotein P65-DnaKc is put forth effort in this experiment.Adhesion protein P65 immunogenicity level selected by experiment is high, can cause early immune reaction, between its kind conservative property and specificity high, be often used for the exploitation of Mhp recombinant vaccine and subunit vaccine as immunodominant proteins at present; Adhesion protein DnaKc is the C-terminal fragment of heat shock protein(HSP) DnaK, and immunogenicity is good, and has the function of immunological adjuvant, and induction body produces strong humoral immune reaction and cell immune response; This experiment constructs the expression vector of p65, dnaKc and p65-dnaKc gene respectively, by prokaryotic expression, purifying, using the albumen of acquisition as recombinant vaccine immune mouse, utilize the antibody titer in ELISA detection serum, find that P65, DnaKc and P65-DnaKc albumen all has good immunogenicity, wherein the immunogenicity of fusion rotein P65-DnaKc is obviously better than single albumen P65 and DnaKc.Challenge test finds that fusion rotein P65-DnaKc can cause stronger immune response in Mice Body, and protection ratio is higher.Therefore, utilize the development of this fusion gene, develop the recombinant vaccine preventing and treating mycoplasma hyopneumoniae infection safely and efficiently, will have great potentialities and can be.
Summary of the invention
The present invention is few in order to overcome present mycoplasma hyopneumoniae market effective vaccine, and the defect of vaccine preparation process, devise fusion gene of a kind of mycoplasma hyopneumoniae and preparation method thereof, the gene P46 of mycoplasma hyopneumoniae is connected with P65 gene tandem, thus the better fusion gene P46-P65 of adaptive immune originality.
The technical solution used in the present invention is: a kind of fusion gene of mycoplasma hyopneumoniae, and its fusion gene is P46-P65, and its nucleotide sequence is as shown in sequence table SEQ IDNO:1.
Described fusion gene is prepared by the following method:
1) by the PCR primer of P46 gene and prokaryotic expression plasmid pET-28a double digestion respectively, and connection of spending the night, build recombinant expression vector pET28a-P46;
2) by the PCR primer of P65 gene and recombinant expression vector pET28a-P46 double digestion, and connection of spending the night, build the recombinant expression vector pET28a-P46-P65 containing fusion gene;
3) by external mutating technology, change the TGA codon of three in P46 gene into TGG codon, thus build the recombinant expression vector pET28a-P46-P65 that can carry out expressing in prokaryotic cell prokaryocyte;
4) cut glue by race 1% agarose gel electrophoresis reclaim the pET28a in removal recombinant expression vector pET28a-P46-P65 thus obtain the fusion gene P46-P65 that can carry out expressing in prokaryotic cell prokaryocyte;
Wherein, the nucleotide sequence of P46 gene is as shown in SEQIDNO:2;
The nucleotide sequence of P65 gene is as shown in SEQIDNO:3.
Step 1) described in double digestion to use respectively be restriction enzyme BamH I and Sal I, in 37 DEG C of double digestion 3h.
Step 1) with step 2) described in spend the night and be connected spending the night of using T4DNA ligase enzyme to carry out and connect.
Step 2) described in double digestion use restriction enzyme Sal I and Hind III respectively, in 37 DEG C of double digestion 3h.
Step 4) described in leakage of electricity swimming cut glue to be mass concentration be 1% ~ 5% sepharose.
The invention has the beneficial effects as follows:
The immunogenicity of the fusion gene P46-P65 1, utilizing preparation method of the present invention to prepare apparently higher than the immunogenicity of individual gene, for development of new mycoplasma hyopneumoniae gene vaccine pad has descended solid basis;
2, fusion gene P46-P65 is lower-cost genetic resources, and the subunit vaccine expression amount based on P46-P65 fusion gene is higher, is easy to purifying and preparation, and to compare preparation cost lower with inactivated vaccine and the attenuated vaccine of routine;
3, P46 and the P65 gene used in the present invention, directly according to the coding region sequence issued in NCBIGeneBank, the Auele Specific Primer of design containing corresponding restriction enzyme site.The PCR primer of P46 gene is with prokaryotic expression carrier pET-28a double digestion and be connected, and builds recombinant expression vector pET-28a-P46; Make the PCR primer double digestion of recombinant expression vector pET-28a-P46 and P65 gene again and be connected, direct construction recombinant expression vector pET-28a-P46-P65.Advantage does not need to use Linker to connect P46 and P65 gene, directly connects through double digestion and just can obtain recombinant expression vector pET-28a-P46-P65.Construction of recombinant vector process compared to, use Linker to connect gene and is connected with prokaryotic expression carrier, process is simple and be easy to obtain, and expend time in obviously minimizing;
4, the purge process of P46-P65 fusion gene uses His-Ni
2+carry out purifying, the flushing of foreign protein and the acquisition of target protein, only need the imidazole solution adding different solubility to rinse, principle is according to ion-exchange chromatography.More be easily understood compared to AKTAFPLC (AmershamBiosciencesUPC-900) process operation step and principle, and it is less to expend financial resources, time consumption is less, only needs about 4h;
5, tested by indirect ELISA, result proves, the immunogenicity expressing the fusion rotein P46-P65 of also purifying through the present invention is higher than in P97R1-Linker-P36-Linker-P46 fusion rotein 100-1000 doubly;
6 and the present invention has also carried out challenge test demonstrates, the protection ratio of the subunit vaccine of P46-P65 fusion gene is higher.Lung tissue's significant protective effect is had to mouse.
Accompanying drawing explanation
Fig. 1 is P65 gene PCR product schematic diagram;
Fig. 2 is P46 gene PCR product schematic diagram;
Fig. 3 is pET-28a-P46-P65 double digestion checking schematic diagram;
Fig. 4 is supernatant liquor and the precipitation race glue schematic diagram of induction escherichia coli P46-P65 protein expression;
Fig. 5 is Western-blotting and SDS-PAGE detection figure;
Fig. 6 is that challenge viral dosage is without vaccine protection group mouse lung transmission electron microscope picture;
Fig. 7 is challenge viral dosage vaccine protection group mouse lung transmission electron microscope picture.
Wherein, in accompanying drawing, M represents protein molecular quality standard, and 1 represents unloaded expression precipitates, 2 represent unloaded expression supernatant, 3 representative induction 6h recombinant expression vector precipitations, 4 representative induction 6h recombinant expression vector supernatants, 5 represent P65 albumen, 6 represent P46 albumen, 7 represent P46-P65 albumen, and A represents the Western-blotting analysis chart of recombinant protein, and B represents the SDS-PAGE analysis chart of recombinant protein.
Embodiment
Fusion gene of the present invention obtains as follows: first according on Genebank announce P46 and P65 gene, design primer go forward side by side performing PCR amplification.And the PCR primer of P46 gene and prokaryotic expression plasmid pET-28a are used restriction enzyme BamH I and Sal I respectively, in 37 DEG C of double digestion 3h, and use T4DNA ligase enzyme to spend the night connection.Build recombinant expression vector pET28a-P46.The PCR primer of P65 gene and recombinant expression vector pET28a-P46 are used restriction enzyme Sal I and Hind III respectively, in 37 DEG C of double digestion 3h, and use T4DNA ligase enzyme to spend the night connection, build the recombinant expression vector pET28a-P46-P65 containing fusion gene, and by external mutating technology, change the TGA codon of three in P46 gene into TGG codon, thus build the recombinant expression vector that can carry out expressing in prokaryotic cell prokaryocyte.The present invention also comprises this and melts gene and preparing the application in mycoplasma hyopneumoniae recombinant vaccine.
One, below the structure of P46-P65 fusion gene is described in detail:
1, the design of primer, as shown in table 1:
Table 1
Have underscore part to be restriction enzyme site in table 1 in primer sequence, primer has Shanghai biotechnology company limited to synthesize.
2, the amplification of P46 and P65 gene
With mycoplasma hyopneumoniae 168 low virulent strain for template, utilize Auele Specific Primer in table 1, carry out the specific amplification of P46 (ID:16186839) and P65 (ID:16186824) gene.PCR system is as shown in table 2, and PCR primer as depicted in figs. 1 and 2.
Table 2
The amplification program of P46 is: 94 DEG C of 4min; 94 DEG C of 30s, 64.1 DEG C of 30s, 70min1min, 25 circulations; 72 DEG C extend 10min.Sepharose through 1% detects, and obtains the fragment of 1260bp length.The amplification program of P65 is: 94 DEG C of 4min; 94 DEG C of 30s, 65 DEG C of 30s, 70min1min, 25 circulations; 72 DEG C extend 10min.Sepharose through 1% detects, and obtains the fragment of 963bp length.
3, the structure of recombinant expression vector pET-28a-P46-P65
According on Genebank announce P46 and P65 gene, design primer go forward side by side performing PCR amplification.And the PCR primer of P46 gene and prokaryotic expression plasmid pET-28a are used restriction enzyme BamH I and Sal I double digestion respectively, and connection of spending the night.Build recombinant expression vector pET28a-P46.The PCR primer of P65 gene and recombinant expression vector pET-28a-P46 are used respectively restriction enzyme Sal I and Hind III double digestion, and connection of spending the night.Build the recombinant expression vector pET-28a-P46-P65 containing fusion gene, and by external mutating technology, change the TGA codon of three in P46 gene into TGG codon, thus build the recombinant expression vector that can carry out expressing in prokaryotic cell prokaryocyte.Thus obtain the fusion gene P46-P65 that can carry out expressing in prokaryotic cell prokaryocyte.Through PCR checking, double digestion and sequencing analysis checking, prove that recombinant expression vector pET-28a-P46-P65 successfully constructs, see Fig. 3, pET-28a-P46-P65 double digestion checking schematic diagram.
The structure (negative control group in immunization experiment) of recombinant expression vector pET-28a-P46 and pET-28a-P65
According on Genebank announce P46 and P65 gene, design primer go forward side by side performing PCR amplification.The amplification program of P46 is: 94 DEG C of 4min; 94 DEG C of 30s, 64.1 DEG C of 30s, 70min1min, 25 circulations; 72 DEG C extend 10min.The amplification program of P65 is: 94 DEG C of 4min; 94 DEG C of 30s, 65 DEG C of 30s, 70min1min, 25 circulations; 72 DEG C extend 10min.And the PCR primer of P46 gene and prokaryotic expression plasmid pET-28a are used restriction enzyme BamH I and Sal I double digestion respectively, and connection of spending the night.Build recombinant expression vector pET28a-P46.And by external mutating technology, change the TGA codon of three in P46 gene into TGG codon, thus build the pET-28a-P46 recombinant expression vector that can carry out expressing in prokaryotic cell prokaryocyte.The PCR primer of P65 gene and recombinant expression vector pET-28a are used respectively restriction enzyme Sal I and Hind III double digestion, and connection of spending the night, build the recombinant expression vector pET-28a-P65 containing fusion gene.Respectively through PCR checking, double digestion and sequencing analysis checking, prove that recombinant expression vector pET28a-P46 and pET28a-P65 successfully constructs.
Two, the prokaryotic expression of recombinant expression vector pET-28a-P46-P65
1, the conversion of plasmid
By expression vector pET-28a-P46-P65 (card receive chloramphenicol resistance) transformation of E. coli BL21 (DE3) cell containing mycoplasma hyopneumoniae P46-P65 fusion gene.Concrete steps are as follows:
1) get the recombinant expression plasmid pET-28a-P46-P65 of 50 μ L, join in the e. coli bl21 competent cell of 100 μ L, flick mixing.Leave standstill 30min on ice.
2) heat shock 30s in 42 DEG C of water-baths.
3) ice bath leaves standstill 2min.
4) 37 DEG C of 180rpm spread cultivation 1h.
5) get 100 μ L spread cultivation after cell coat on the LB solid plate containing KN chloramphenicol resistance.Cultivate 12-16h for 37 DEG C.
2, prokaryotic expression
Picking colony spreads cultivation in LB substratum, uses plasmid extraction kit (Quan Shijin Bioisystech Co., Ltd) to extract plasmid, and verifies.The correct bacterial strain of checking is got 10 μ L and carries out spreading cultivation 12-16h, the bacterium liquid that spreads cultivation getting 1mL joins in the LB substratum of 100mL, and cultivate 3h for 37 DEG C, until OD600 reaches 0.4, the IPTG adding 1mmol/L cultivates 22h under 16 DEG C of conditions.By the thalline after induction under 8000rpm4 DEG C of condition, centrifugal 10min, abandons supernatant, collects thalline.Use the ice-cold physiological saline of 5mL resuspended by collecting thalline, and carry out ultrasonication, break-up frequency is work 8s, interval 10s.Until thalline presents the liquid of clear.By broken liquid centrifugal 10min under 12000rpm4 DEG C of condition, cleer and peaceful precipitation in collection.Prove that fusion rotein has carried out solubility expression by SDS-PAGE and Westernning-blotting checking.Result as shown in Figure 4.
Three, the purifying of P46-P65 fusion rotein
With the P46-P65 fusion rotein that soluble form is expressed, use His-Ni
2+post carries out purifying.Because pET-28a is with histidine-tagged prokaryotic expression carrier, after recombinant expression vector pET-28a-P46-P65 expresses, fusion rotein P46-P65, also with histidine-tagged, can use His-Ni
2+post carries out purifying.Purification step is as follows:
1) just broken liquid centrifugal 10min under 12000rpm4 DEG C of condition, collects supernatant.
2) supernatant is placed in His-Ni
2+in post, hatch 3h on ice; Release whole protein liquid,
3) use the Washbuffer wash buffer nickel post of 25mmol/L imidazole concentration, rinse 4 times;
4) finally use the ElutionBuffer damping fluid that imidazole concentration is 500mmol/L to hatch 10min, and collect washing fluid, ElutionBuffer washing fluid is purifying P46-P65 fusion rotein solution.
5) purification step of single albumen P46 and P65 is with fusion rotein P46-P65.Result is verified through SDS-PAGE and Westernning-blotting, as shown in Figure 5.
Four, the biological experiment of subunit vaccine of the present invention and control group vaccine on mouse immune efficacy
1, the immune programme for children of BALB/C mice
Balb/c female experimental mouse body weight being about 25g is divided into three groups, fusion rotein is divided into inject experimental group, single protein injection experimental group and control group, often organize each 50, experimental group abdominal injection recombination fusion protein and each 100 μ L of single albumen (recombinant protein total content is 50 μ g), control group intraperitoneal injection of saline 100 μ L.The target protein calculated and adjuvant 1:1 (volume ratio) are mixed emulsification and inject.The immunity cycle is 7d, and immunity three times, lasts 21d altogether.One exempts to use Freund's complete adjuvant, and two exempt to exempt from three to use Freund's incomplete adjuvant.Three exempted from end after one week, blood is got in mouse docking, use the recombinant protein of purifying as antigen, mice serum is as primary antibodie, HRP horseradish peroxidase resists as two, carry out indirect ELISA experiment and detect mice serum antibody titer, experimental group data/control group data (P/N) are greater than 2 for positive; Otherwise be then negative.
The mice group experiment of table 3 mycoplasma hyopneumoniae recombinant subunit vaccine
2, mouse antibodies level
Use indirect ELISA is tested, and measures mice serum antibody titer.Use P46, P65 and P46-P65 of purifying to spend the night 4 DEG C as antigen to hatch, and use mice serum to hatch 1h as primary antibodie 37 DEG C, HRP horseradish peroxidase resists 37 DEG C as two and hatches 1h, uses nitrite ion TMB colour developing 0.5h.Use spectrophotometer analysis experimental result.Result is as shown in table 4.
Table 4P46-P65 recombinant protein immunity ELISA result
3, the determination of toxic agent amount is attacked
Mycoplasma special culture media is used to carry out gradient dilution (0 ~ 1 × 10 Mhp168 low virulent strain bacterium liquid
10cCU/mL), get healthy mouse in addition, be divided into 7 groups, often organize 10, each dilution gradient gets 600 μ L bacterium liquid intraperitoneal injection of mice.The disease symptom of mouse after inoculation Mhp168 low virulent strain bacterium liquid: occur poor appetite after inoculation 7d, the hair of wing of nose both sides drops, movable slowly, spirit is not good etc., after dissecting, lung tissue's obvious fibrosis and without color.Record the healthy state of experimental group and control group (physiological saline) mouse in inoculation after one week and in conjunction with anatomical results, determine to attack toxic agent amount.As shown in table 5.
Table 5Mhp168 low virulent strain causes a disease Dose Results
4, challenge test
Experimental mice three is exempted to terminate latter one week, according to ELISA experimental result, selects 50 mouse that antibody titer is higher, with maximum pathogenic dosage 1 × 10
10the Mhp168 low virulent strain bacterium liquid injection mouse of CCU/mL, every mouse peritoneal is injected 600 μ L and is carried out challenge test; Choose healthy group and injecting normal saline group in addition as negative control, often organize 50, every Mhp168 low virulent strain bacterium liquid only injecting 600 μ L.Inoculate the incidence of record mouse after 1 week and in conjunction with anatomical results, calculate protection ratio.As shown in table 6.
The protection ratio of table 6P46-P65 antigen-4 fusion protein gene engineered vaccine
5, the electron microscopic observation of mouse lung tissue slice
Choose the mouse of inoculation subunit vaccine, belly is dissected, and gets lung tissue and makes ultrathin section(ing).Shown in Electronic Speculum result Fig. 6, Fig. 7.Fig. 6 be challenge viral dosage without vaccine protection group mouse lung transmission electron microscope picture, Fig. 7 is challenge viral dosage vaccine protection group mouse lung transmission electron microscope picture.Step is as follows:
Front fixing: fixing more than 2-4h before 4% glutaraldehyde.
(1) phosphoric acid buffer of 1/15M embathes 2 times, each 20min.
1-2h fixed by (2) 1% osmic acids.
(3) phosphoric acid buffer of 1/15M embathes 2 times, each 20min.
(4) acetone dewaters step by step, 50%, 70%, 80%, 90%, 100% (2 times), each 10-15min, and room temperature is got back in the dehydration of second time 100% acetone, and other steps are carried out at 4 DEG C of refrigerators.
(5) soak into: 1:1 (pure acetone: embedding medium): 1h, 37 DEG C.
1:3 (pure acetone: embedding medium): more than 3h or spend the night, 37 DEG C.
Pure embedding medium soaks into more than 5h, 37 DEG C.
(6) embed
(7) be polymerized: 37 DEG C of incubator 12h, 60 DEG C of 36-48h (add dry ball after 4 DEG C of preservations).
(8) ultrathin section(ing): thickness about 50nm.
(9) dye: uranyl acetate and the dual dye method of lead plumbate.
(10) electron microscopic observation is projected.
Claims (6)
1. a fusion gene for mycoplasma hyopneumoniae, is characterized in that: its fusion gene is P46-P65, and its nucleotide sequence is as shown in sequence table SEQ IDNO:1.
2. the fusion gene of a kind of mycoplasma hyopneumoniae according to claim 1, is characterized in that: described fusion gene is prepared by the following method:
1) by the PCR primer of P46 gene and prokaryotic expression plasmid pET-28a double digestion respectively, and connection of spending the night, build recombinant expression vector pET28a-P46;
2) by the PCR primer of P65 gene and recombinant expression vector pET28a-P46 double digestion, and connection of spending the night, build the recombinant expression vector pET28a-P46-P65 containing fusion gene;
3) by external mutating technology, change the TGA codon of three in P46 gene into TGG codon, thus build the recombinant expression vector pET28a-P46-P65 that can carry out expressing in prokaryotic cell prokaryocyte;
4) cut glue by leakage of electricity swimming reclaim the pET28a in removal recombinant expression vector pET28a-P46-P65 thus obtain the fusion gene P46-P65 that can carry out expressing in prokaryotic cell prokaryocyte;
Wherein, the nucleotide sequence of P46 gene is as shown in SEQIDNO:2;
The nucleotide sequence of P65 gene is as shown in SEQIDNO:3.
3. the fusion gene of a kind of mycoplasma hyopneumoniae according to claim 2, is characterized in that: step 1) described in double digestion use restriction enzyme BamH I and Sal I respectively, in 37 DEG C of double digestion 3h.
4. the fusion gene of a kind of mycoplasma hyopneumoniae according to claim 2, is characterized in that: step 1) with step 2) described in spend the night and be connected spending the night of using T4DNA ligase enzyme to carry out and connect.
5. the fusion gene of a kind of mycoplasma hyopneumoniae according to claim 2, is characterized in that: step 2) described in double digestion use restriction enzyme Sal I and Hind III respectively, in 37 DEG C of double digestion 3h.
6. the fusion gene of a kind of mycoplasma hyopneumoniae according to claim 2, is characterized in that: step 4) described in leakage of electricity swimming cut glue to be mass concentration be 1% ~ 5% sepharose.
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| CN108101969A (en) * | 2017-11-13 | 2018-06-01 | 南京大爻网络科技有限公司 | A kind of and the relevant antigen of porcine mycoplasmal pneumonia and its application |
| CN109678968A (en) * | 2018-12-20 | 2019-04-26 | 武汉科前生物股份有限公司 | Mycoplasma hyopneumoniae subunit vaccine and the preparation method and application thereof |
| CN109678968B (en) * | 2018-12-20 | 2022-06-28 | 武汉科前生物股份有限公司 | Mycoplasma hyopneumoniae subunit vaccine and preparation method and application thereof |
| CN113181356A (en) * | 2021-03-19 | 2021-07-30 | 河北师范大学 | Preparation of pig breeding and respiratory obstruction syndrome and mycoplasma hyopneumoniae bigeminy multi-epitope vaccine |
| CN113181356B (en) * | 2021-03-19 | 2022-08-02 | 河北师范大学 | Preparation of pig breeding and respiratory obstruction syndrome and mycoplasma hyopneumoniae bigeminy multi-epitope vaccine |
| CN114853910A (en) * | 2022-05-19 | 2022-08-05 | 福建省农业科学院畜牧兽医研究所 | Immunogenic mycoplasma ovipneumoniae HSP70-P113 fusion protein |
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