CN114853910A - Immunogenic mycoplasma ovipneumoniae HSP70-P113 fusion protein - Google Patents
Immunogenic mycoplasma ovipneumoniae HSP70-P113 fusion protein Download PDFInfo
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Abstract
The invention discloses a mycoplasma ovipneumoniae HSP70-P113 fusion protein with immunogenicity, which is prepared from mycoplasma ovipneumoniaeBody heat shock protein geneHSP70And mycoplasma ovipneumoniae adhesin geneP113The amino acid sequence of the fusion gene coded by the spliced fusion gene is the sequence shown in SQE ID NO. 2. Western Blot tests prove that the mycoplasma ovipneumoniae HSP70-P113 fusion protein has immunogenicity and can be used for preparing mycoplasma ovipneumoniae subunit vaccines.
Description
Technical Field
The invention belongs to the technical field of recombinant proteins, and particularly relates to a mycoplasma ovipneumoniae HSP70-P113 fusion protein with immunogenicity.
Background
Mycoplasma ovipneumoniae (Mycoplasma ovipneumoniaeMovi) is one of the main pathogens causing mycoplasma pneumonia of sheep and goats, is distributed worldwide, and brings huge economic loss to the world sheep breeding industry. The treatment of the mycoplasma ovipneumoniae mainly comprises antibiotics, however, the treatment effect of the mycoplasma ovipneumoniae is not ideal due to drug resistance caused by the antibiotics, so the mycoplasma ovipneumoniae is mainly prevented. However, at present, the Movi vaccine in China is mainly an inactivated vaccine, and mainly inactivates a whole bacterium antigen. However, mycoplasma epidemic in different places has genetic diversity, cross protection among strains is low, in addition, the yield of Movi in vitro culture is relatively low, time is consumed, and the whole bacterial antigen has potential safety hazard, so that the development of efficient and safe Movi novel vaccines is a trend in recent years. The subunit vaccine has the advantages of high safety, no nucleic acid, no toxicity dissipation, no reinforcment, single antigen component, high purity, high vaccine yield and the like, and becomes the development trend of the vaccine. Mycoplasma ovipneumoniae (Movi) has not yet been marketed as a subunit vaccine. Many studies are currently focused on screening proteins with good immunogenicity for the preparation of subunit vaccines by epitope identification. P113 is adhesin, adhesin is an important virulence factor of mycoplasma and also the most important immunogen, and is the main protein causing the organism to generate immune response, heat shock protein HSP70 is a highly conserved biomolecule in the organism and is also an important membrane protein of Movi, and the protein has immunogenicity and immune adjuvant effect, has obvious advantages of C-terminal antigenicity, and can induce the organism to generate humoral immunity and cellular immune response. The invention passes through screeningHSP70Gene and of the MO ATCC 29419 isolateP113Dominant antigen regions in the gene, performing rare codon optimization on the gene sequences of the two dominant antigen regions, and performing rare codon optimization on the two dominant antigen regionsA linker (GGGGSGGGGS) is added in the middle of the sequence of each gene and then copied to a pET-30a (+) prokaryotic expression vector, which is named HSP70-P113-pET-30a (+). Through induced expression, protein purification and mouse immunization, Western Blot tests prove that the HSP70-P113 recombinant protein has immunogenicity and can provide a basis for development of a mycoplasma ovipneumoniae subunit vaccine.
Disclosure of Invention
The invention aims to provide an immunogenic fusion protein of Mycoplasma ovipneumoniae HSP 70-P113.
In order to achieve the purpose, the invention adopts the following technical scheme:
an immunogenic fusion protein of Mycoplasma ovipneumoniae HSP70-P113, wherein the amino acid sequence of the fusion protein of Mycoplasma ovipneumoniae HSP70-P113 is a sequence shown in SQE ID NO. 2.
The fusion gene is formed by splicing mycoplasma ovipneumoniae heat shock protein gene HSP70 and mycoplasma ovipneumoniae adhesin gene P113 by using a linker (GGGGSGGGGS), and the nucleotide sequence of the fusion gene is a sequence shown as SQE ID NO. 1.
An E.coli expression plasmid vector constructed by inserting the above fusion gene between Nde I and BamH I sites of plasmid pET-30a, E.coli BL21(DE 3).
An Escherichia coli strain for producing the Mycoplasma ovipneumoniae HSP70-P113 fusion protein, wherein the Escherichia coli strain contains the plasmid vector, and the Escherichia coli strain is BL21(DE 3).
A method for preparing the Mycoplasma ovipneumoniae HSP70-P113 fusion protein comprises the following steps:
1) inserting a fusion gene with a sequence shown as SQE ID NO.1 between Nde I and BamH I sites of an Escherichia coli plasmid pET-30a, transforming the Escherichia coli with the plasmid to obtain a strain containing HSP70-P113-pET-30a (+), inoculating and culturing, and performing IPTG induced expression;
2) centrifugally cracking the expressed thallus, harvesting an inclusion body, carrying out heavy suspension by using PBS buffer solution, centrifuging, taking supernatant, carrying out Ni-NTA affinity chromatography, and obtaining mycoplasma ovipneumoniae HSP70-P113 fusion protein;
wherein, the Escherichia coli is BL21(DE 3).
The Mycoplasma ovipneumoniae HSP70-P113 fusion protein is applied to the preparation of Mycoplasma ovipneumoniae subunit vaccines.
The invention has the following remarkable advantages: according to the invention, HSP70 with immunogenicity and immune adjuvant effect and adhesin P113 with good immunogenicity are selected for the first time, codon optimization is carried out by selecting the dominant antigen regions of the two genes, HSP70-P113 recombinant protein is successfully expressed, and the protein has strong immunogenicity, so that a basis is provided for development of mycoplasma ovipneumoniae subunit vaccines. In addition, the HSP70-P113 recombinant protein has immunogenicity, can also react with positive serum of mycoplasma ovipneumoniae, provides antigen for establishment of serological methods such as an indirect ELISA method of mycoplasma ovipneumoniae and the like, and is the most core component of an ELISA kit.
Drawings
FIG. 1 is a drawing ofHSP70-P113Plasmid map of pET-30a (+).
FIG. 2 shows soluble expression and inclusion body expression of HSP70-P113 fusion protein. Lane M: protein marker; lane PC 1 : BSA (1. mu.g); lane PC 2 : BSA (2 μ g); lane NC: an uninduced whole cell sample; lane 1: inducing whole cell samples for 16h at 15 ℃; lane 2: whole cell samples were induced at 37 ℃ for 4 h; lane NC 1 : uninduced cell lysis supernatant samples; lane 3: inducing cell lysis supernatant samples for 16h at 15 ℃; lane 4: inducing cell lysis supernatant samples for 4 h at 37 ℃; lane NC 2 : (ii) an uninduced cytolytic pellet sample; lane 5: inducing the cell lysis sediment sample for 16h at 15 ℃; lane 6: the cell lysis pellet samples were induced at 37 ℃ for 4 h.
FIG. 3 shows the result of SDDS-PAGE gel electrophoresis analysis of purified HSP70-P113 fusion protein. Lane M: protein marker; lane 1: purified HSP70-P113 fusion protein.
FIG. 4 shows the results of immunogenicity analysis of HSP70-P113 fusion protein. Lane M: protein marker; a: the primary antibody is a mouse anti-HSP 70-P113 polyclonal antibody; b: the primary antibody is a mouse anti-Movi polyclonal antibody; c: primary antibody was normal control mouse serum (blank).
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1HSP70-P113Fusion gene design and synthesis
Analysis of Heat shock protein Gene of Mycoplasma ovipneumoniae FJ-01CL isolate Using Protean software in DNAStar7.0 softwareHSP70And the adhesin gene of the Mycoplasma ovipneumoniae ATCC 29419 isolateP113The gene sequences of the two dominant antigen regions are spliced after rare codon optimization, and a GGGGSGGGGS sequence linker is added in the middle to obtain the dominant antigen regionHSP70-P113A fusion gene.HSP70-P113The total length of the fusion gene is 1392bp, and the fusion gene has a nucleotide sequence shown as SQE ID NO.1 and an amino acid sequence shown as SEQ ID NO. 2.HSP70-P113The fusion gene synthesis was completed by Nanjing Kinshire Co.
Example 2HSP70-P113Construction of (E) -pET-30a (+) recombinant plasmid
By gene cloningHSP70-P113The fusion gene is inserted into an escherichia coli plasmid pET-30aNde IAndBamH Iconstructing recombinant plasmid between sitesHSP70-P113-pET-30a(+)。HSP70-P113The map of the-pET-30 a (+) plasmid is shown in FIG. 1.
Example 3HSP70-P113Prokaryotic expression of (E) -pET-30a +
The preserved BL21(DE3) was placed on ice for 30 min, and 100 ng of recombinant plasmid was addedHSP70-P113-pET-30a (+), gently blowing and sucking, fully mixing, and reacting on ice for 30 min; heat shock at 42 ℃ for 90S; standing on ice for 3 min after the heat shock is finished; then 100 mL of LB liquid medium was added; shaking and culturing at 37 deg.C and 200 rpm for 60 min in shaking bed; the bacterial liquid is evenly mixed and coated on a kanamycin-resistant plate with the density of 50 mu g/mL, and the plate is placed upside downThe culture was carried out overnight at 37 ℃. The next day, 3 single clones were picked, inoculated into test tubes containing LB liquid medium containing 50. mu.g/mL kanamycin, respectively, and cultured in a shaker at 37 ℃; when the culture solution OD 600 When the concentration reaches 0.6-0.8, adding 0.5 mM IPTG into 2 test tubes respectively, culturing at 15 ℃ for 16h and at 37 ℃ for 4 h respectively, and taking the last test tube as a negative reference; samples were prepared and analyzed by SDS-PAGE to detect protein expression.
Sample preparation for SDS-PAGE analysis:
1) the pellet was centrifuged at 450. mu.L and resuspended in 300. mu.L of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5% glycerol, pH 8.0) and sonicated for 1 min.
2) The whole bacteria sample: mu.L of lysate was mixed with 20. mu.L of SDS-PAGE protein loading buffer (5X) (Biyuntian Biotech Co., Ltd.), heated at 100 ℃ for 10min, centrifuged at 15000rpm for 5min, and the supernatant was left to discard the precipitate for loading.
3) Supernatant and inclusion body samples: 200 μ L of lysate was centrifuged at 15000rpm for 10min, and the supernatant and pellet were separated. mu.L of SDS-PAGE protein loading buffer (5X) was mixed with 160. mu.L of the supernatant to prepare a supernatant sample. The pellet was resuspended in 150. mu.L of 5 SDS-PAGE protein loading buffer (5X) and used as an inclusion body sample. The supernatant sample and the inclusion body sample are respectively heated at 100 ℃ for 10min, then centrifuged at 15000rpm for 5min, and the supernatant is left to discard the precipitate for loading.
SDS-PAGE results showed (FIG. 2), that the size of the expressed HSP70-P113 fusion protein is about 51 KD; when the induction temperature is 15 ℃, HSP70-P113 fusion protein expressions are respectively carried out in the supernatant (soluble expression) and the sediment (inclusion body) of the bacterial liquid; HSP70-P113 fusion protein is expressed predominantly in the form of inclusion bodies at an induction temperature of 37 ℃.
Example 3 purification of HSP70-P113 fusion protein
Placing the preserved BL21(DE3) on ice for 30 min, adding 100 ng recombinant plasmid HSP70-P113-pET-30a (+), gently blowing and sucking, fully mixing uniformly, and reacting on ice for 30 min; heat shock at 42 ℃ for 90S; standing on ice for 3 min after the heat shock is finished; then 100 mL of LB liquid medium was added; shaking and culturing at 37 deg.C and 200 rpm for 60 min in shaking bed; will be provided withThe mixture was spread onto a 50. mu.g/mL kanamycin-resistant plate, which was then incubated overnight at 37 ℃. The next day, a single clone was selected and inoculated into LB liquid medium containing 50. mu.g/mL kanamycin, and cultured in a shaker at 37 ℃; when the culture solution OD 600 When the temperature reaches 0.6-0.8, adding 0.5 mM IPTG into the mixture, and inducing the mixture for 16 hours at the temperature of 15 ℃; after the induction, centrifuging at 10000 rpm and 4 ℃ for 10min, discarding the supernatant, and resuspending the precipitate with PBS buffer (0.01M, pH7.2); centrifuging at 10000 rpm and 4 deg.C for 10min, discarding supernatant, and resuspending the precipitate with PBS buffer (0.01M, pH7.2); centrifuging at 10000 rpm at 4 deg.C for 10min, discarding supernatant, resuspending the precipitate with PBS buffer (0.01M, pH7.2), ultrasonic treating in ice bath for 20 min, and crushing thallus; centrifuging at 12000 rpm and 4 ℃ for 10min, collecting supernatant, and purifying according to the specification of protein iso Ni-NTA Resin to obtain the target protein. The result of the SDDS-PAGE gel electrophoresis analysis of the purified protein is shown in FIG. 3, and the fusion protein has obvious bands at corresponding positions, and the 51KD HSP70-P113 fusion protein is obtained, and the result is matched with the expected result.
Example 4 preparation of Mycoplasma ovipneumoniae HSP70-P113 fusion protein polyclonal antibody and immunogenicity analysis
Preparation of polyclonal antibody of Mycoplasma ovipneumoniae HSP70-P113 fusion protein: mixing the purified HSP70-P113 fusion protein with equivalent Freund complete adjuvant, performing ultrasonic emulsification, and performing subcutaneous immune injection on Balb/c mice for 3 times. The 15 th day and the 30 th day after the first immunization are respectively the second immunization period and the third immunization period. For the first immunization, HSP70-P113 fusion protein is mixed with equal volume of Freund complete adjuvant for immunization injection, and the injection dose is 50 mu g of fusion protein per mouse; the HSP70-P113 fusion protein for the second immunization and the HSP70-P113 fusion protein for the third immunization are mixed with an equal volume of Freund incomplete adjuvant and then injected with a booster immunization, and the injection dose is 50 mu g of fusion protein per mouse. On day 9 after the third immunization, mice were tailed, blood was collected, serum was separated, the antibody level was measured, and the antibody titer was measured. Serum from preimmunized mice was used as a negative control. After the antibody titer meets the requirement (the antibody titer is more than 1: 5000), the whole blood of the mouse is collected by adopting an orbital bleeding method, serum is separated, and the antigen-antibody affinity purification is carried out on the serum to obtain the mycoplasma ovipneumoniae HSP70-P113 fusion protein polyclonal antibody.
Immunogenicity analysis of HSP70-P113 fusion protein: the purified HSP70-P113 fusion protein is subjected to SDS-PAGE analysis, and then transferred to a nitrocellulose membrane by a semidry method, and Western-blot tests are carried out by respectively using a polyclonal antibody of the mycoplasma ovipneumoniae HSP70-P113 fusion protein, a polyclonal antibody of the mycoplasma ovipneumoniae and a negative control mouse serum as primary antibodies and using Goat anti-mouse IgG (H + L), HRP conjugate, ELISA and WB as secondary antibodies (using the concentration of 1: 5000). Western-blot analysis results show (figure 4) that the HSP70-P113 fusion protein polyclonal antibody can perform specific reaction with the HSP70-P113 fusion protein antigen to form a band with the size of 51 KD; the mycoplasma ovipneumoniae polyclonal antibody and the HSP70-P113 fusion protein antigen have specific reaction, and a strip with the size of 51KD appears; no specific band appears between HSP70-P113 fusion protein and the serum of a negative control mouse. The above results indicate that the HSP70-P113 fusion protein has immunogenicity.
Example 5 configuration of a kit for detecting Mycoplasma ovipneumoniae antibodies and methods of use thereof
Coating an enzyme label plate: diluting the purified HSP70-P113 fusion protein to 5 mu g/mL by using a coating buffer solution, adding a 96-well enzyme label plate with 100 mu L/well, setting the 100 mu L/well coating buffer solution as a control, and coating overnight at 4 ℃; wash the plate 3 times with PBST, pat dry; adding blocking liquid to block the ELISA plate, incubating at 37 deg.C for 45min, washing the plate with PBST for 3 times, and drying.
The negative standard serum was a mouse serum negative for mycoplasma ovipneumoniae antibodies.
The positive standard serum is the serum of mice positive for mycoplasma ovipneumoniae antibodies.
The coating buffer is a carbonate buffer with pH 9.6 and is prepared from Na 2 CO 3 1.59g，NaHCO 3 2.93g, adding double distilled water to reach the constant volume of 1000 mL.
The main component of the sealing liquid is 3% bovine serum albumin.
The enzyme-labeled secondary antibody is donkey anti-sheep IgG (H + L) labeled by peroxidase.
The diluent mainly comprises 0.5% bovine serum albumin.
The substrate solution was a TMB developing solution (Kyoho Biotech, Guangzhou).
The final solution was prepared from 22.2mL of concentrated sulfuric acid and 177.8mL of distilled water.
When in use, 100 mu L of diluted sample to be detected is taken and added into a detection plate; 3 holes are respectively arranged for yin-yang contrast, and each hole is 100 mu L; gently shaking the sample in the well (without overflow), and incubating at 37 deg.C for 45 min; the solution in the plate hole is thrown off, the plate is washed 5 times by PBST, and then the plate is patted dry on clean absorbent paper; adding 100 mu L of peroxidase-labeled donkey anti-sheep IgG (H + L) diluted by 1:10000 to each well, and incubating for 45min at 37 ℃; the solution in the plate hole is thrown off, the plate is washed 5 times by PBST, and then the plate is patted dry on clean absorbent paper; adding 100 mu L of TMB developing solution into each hole, and incubating for 10min at 37 ℃; add 50. mu.L of stop solution to each well, measure the result within 10 minutes (gently shake on shaker before detection), and measure OD with microplate reader 450nm 。
The condition for determining the test to be established is the OD of the sample 450nm Value and negative control OD 450nm The value ratio is greater than or equal to 2.1, and the result is judged to be positive; otherwise, the result is judged to be negative.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> animal husbandry and veterinary institute of agricultural academy of sciences of Fujian province
<120> an immunogenic Mycoplasma ovipneumoniae HSP70-P113 fusion protein
<130>
<160> 2
<170> PatentIn version 3.3
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atttatgaac aggtggttag cttcgttaaa gaaagcacca tcctgattgc ggagttcaac 840
tttagcctgc agaagattac cagccgtctg aaccagcaag tgcaaaacct gatcagcgcg 900
cgtaccgcga actttgcgga ccagaacagc gcgaccagca acccgaccga tccgagcacc 960
attcgtccgg tggacttcca acacgatctg aacaaggcga aagacagcaa agaatttggc 1020
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Met His His His His His His Thr Ala Glu Asp Asn Gln Thr Glu Val
1 5 10 15
Thr Ile Ser Val Val Gln Gly Glu Arg Gln Leu Ala Ala Asp Asn Lys
20 25 30
Met Leu Gly Arg Phe Asn Leu Ser Gly Ile Glu Pro Ala Pro Arg Gly
35 40 45
Leu Pro Gln Ile Glu Val Ser Phe Ser Ile Asp Val Asn Gly Ile Thr
50 55 60
Thr Val Ser Ala Lys Asp Lys Lys Thr Gly Lys Glu Gln Thr Ile Thr
65 70 75 80
Ile Lys Asn Thr Ser Thr Leu Ser Glu Glu Glu Ile Asn Arg Met Ile
85 90 95
Gln Glu Ala Glu Glu Asn Arg Glu Ala Asp Ala Ile Lys Lys Asp Lys
100 105 110
Ile Glu Thr Thr Val Arg Ala Glu Gly Leu Ile Asn Gln Leu Glu Lys
115 120 125
Ser Ile Thr Asp Gln Gly Glu Lys Ile Asp Pro Lys Gln Lys Glu Leu
130 135 140
Leu Glu Lys Gln Ile Gln Glu Leu Lys Asp Leu Val Lys Glu Glu Lys
145 150 155 160
Ile Asp Glu Leu Lys Thr Lys Leu Asp Gln Ile Glu Gln Ala Ala Gln
165 170 175
Ala Phe Ala Gln Ala Ser Ala Gln Gln Ala Asn Asn Ala Ser Glu Thr
180 185 190
Asp Ser Gln Asp Ser Asn Thr Ile Asp Ala Glu Ile Lys Gln Asn Gly
195 200 205
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Phe Ser Asp Ala Lys Ile
210 215 220
Ser Tyr Gln Ile Leu Glu Ile Leu Pro Asp Asp Ala Asn Gln Asn Phe
225 230 235 240
Lys Val Lys Phe Gln Ala Ser Gln Lys Leu Ala Asn Gly Asp Ile Ala
245 250 255
Lys Ser Asp Ile Tyr Glu Gln Val Val Ser Phe Val Lys Glu Ser Thr
260 265 270
Ile Leu Ile Ala Glu Phe Asn Phe Ser Leu Gln Lys Ile Thr Ser Arg
275 280 285
Leu Asn Gln Gln Val Gln Asn Leu Ile Ser Ala Arg Thr Ala Asn Phe
290 295 300
Ala Asp Gln Asn Ser Ala Thr Ser Asn Pro Thr Asp Pro Ser Thr Ile
305 310 315 320
Arg Pro Val Asp Phe Gln His Asp Leu Asn Lys Ala Lys Asp Ser Lys
325 330 335
Glu Phe Gly Glu Lys Ile Ser Ser Tyr Phe Pro Ser Leu Asn Asn Leu
340 345 350
Leu Ala Ser Leu Lys Asn Ser Gln Glu Asn Lys Leu Pro Asn Ser Met
355 360 365
Asp Thr Ile Phe Asn Phe Asp Phe Ala Arg Asp Lys Ser Thr Gly Gln
370 375 380
Phe Val Ser Leu Gln Asn Gln Val Pro Ser Phe Phe Leu Glu Ala Asn
385 390 395 400
Leu Thr Ser Ser Ala Arg Gln Met Leu Asp Ser Ser Ser Ser Phe Ser
405 410 415
Thr Val Val Asn Ala Ile Lys Leu Glu Lys Asn Asp Lys Ser Ser Tyr
420 425 430
Phe Leu Asn Tyr Ser Asp Phe Phe Asp Asn Leu Ser Leu Lys Asn Leu
435 440 445
Thr Lys Thr Asp Phe Lys Ser Asp Met Gly Asp
450 455
Claims (10)
1. An immunogenic Mycoplasma ovipneumoniae HSP70-P113 fusion protein, which is characterized in that: the amino acid sequence of the Mycoplasma ovipneumoniae HSP70-P113 fusion protein is a sequence shown in SQE ID NO. 2.
2. A fusion gene encoding mycoplasma ovipneumoniae HSP70-P113 fusion protein of claim 1, wherein: the nucleotide sequence of the fusion gene is a sequence shown in SQE ID NO. 1.
3. An escherichia coli expression plasmid vector, characterized in that: the plasmid vector is constructed by the fusion gene of claim 2 inserted into the plasmid pET-30a between Nde I and BamH I sites.
4. The E.coli expression plasmid vector of claim 3, wherein: the Escherichia coli is BL21(DE 3).
5. An E.coli strain for producing Mycoplasma ovipneumoniae HSP70-P113 fusion protein of claim 1, characterized in that: the Escherichia coli strain contains the plasmid vector of claim 3.
6. The Escherichia coli strain according to claim 5, wherein: the Escherichia coli is BL21(DE 3).
7. A method of making mycoplasma ovipneumoniae HSP70-P113 fusion protein of claim 1, characterized in that: the method comprises the following steps:
inserting the fusion gene of claim 2 between Nde I and BamH I sites of E.coli plasmid pET-30a, transforming E.coli with the plasmid to obtain a strain containing HSP70-P113-pET-30a (+), inoculating and culturing, and inducing expression with IPTG;
and (3) after centrifugally cracking the expressed thallus, harvesting an inclusion body, carrying out heavy suspension by using PBS buffer solution, centrifuging, taking supernatant, and carrying out Ni-NTA affinity chromatography to obtain the mycoplasma ovipneumoniae HSP70-P113 fusion protein.
8. The method of claim 7, wherein: the Escherichia coli is BL21(DE 3).
9. A kit for detecting mycoplasma ovipneumoniae antibodies, comprising mycoplasma ovipneumoniae HSP70-P113 fusion protein according to claim 1.
10. The use of a Mycoplasma ovipneumoniae HSP70-P113 fusion protein as claimed in claim 1 in the preparation of a Mycoplasma ovipneumoniae subunit vaccine.
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| CN102242140A (en) * | 2011-04-19 | 2011-11-16 | 宁夏大学 | Mycoplasma ovipneumoniae Hsp70 (DnaK) C terminal gene recombinant plasmid |
| CN105087616A (en) * | 2015-09-10 | 2015-11-25 | 河北师范大学 | Fusion gene of mycoplasma hyopneumoniae and preparation method of fusion gene |
| US20160264631A1 (en) * | 2013-11-21 | 2016-09-15 | Agricultural Technology Research Institute | Composition for preventing mycoplasma spp. infection |
| CN111925452A (en) * | 2020-10-19 | 2020-11-13 | 苏州世诺生物技术有限公司 | Mycoplasma hyopneumoniae genetic engineering subunit vaccine, and preparation method and application thereof |
| CN113430214A (en) * | 2021-06-22 | 2021-09-24 | 贵州大学 | Construction method of multi-pathogen mycoplasma ovis pneumonia nucleic acid vaccine |
| CN113444743A (en) * | 2021-06-22 | 2021-09-28 | 贵州大学 | Construction method of sheep mycoplasma pneumonia bivalent nucleic acid vaccine containing adjuvant gene |
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|---|---|---|---|---|
| CN102242140A (en) * | 2011-04-19 | 2011-11-16 | 宁夏大学 | Mycoplasma ovipneumoniae Hsp70 (DnaK) C terminal gene recombinant plasmid |
| US20160264631A1 (en) * | 2013-11-21 | 2016-09-15 | Agricultural Technology Research Institute | Composition for preventing mycoplasma spp. infection |
| CN105087616A (en) * | 2015-09-10 | 2015-11-25 | 河北师范大学 | Fusion gene of mycoplasma hyopneumoniae and preparation method of fusion gene |
| CN111925452A (en) * | 2020-10-19 | 2020-11-13 | 苏州世诺生物技术有限公司 | Mycoplasma hyopneumoniae genetic engineering subunit vaccine, and preparation method and application thereof |
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| CN113444743A (en) * | 2021-06-22 | 2021-09-28 | 贵州大学 | Construction method of sheep mycoplasma pneumonia bivalent nucleic acid vaccine containing adjuvant gene |
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