CN101603024A - Vaccines of Mycoplasma Pneumonia and Actinobacillus Pleuropneumoniae Serum Type 1 Genetic Engineering Strains and Their Application - Google Patents

Abstract

The invention belongs to the technical field of livestock infectious diseases and relates to the technical field of bacterial genetic engineering. In particular, it relates to the construction, vaccine preparation and application of a recombinant Actinobacillus pleuropneumoniae serotype 1 and mycoplasma pneumonia bivalent genetic engineering strain. The present invention is characterized in that a strain of recombinant Actinobacillus pleuropneumoniae (Actinobacilluspleuropneumoniae) serotype 1 attenuated strain SLW05 is constructed, and the recombinant strain is preserved in the China Center for Type Culture Collection, and its preservation number is CCTCC NO: M209068, which is The mycoplasma pneumoniae antigenic P36 gene fragment is inserted into the chromosome of Actinobacillus pleuropneumoniae serotype 1 attenuated strain SLW03, and the nucleotide sequence of the P36 gene fragment is shown in SEQ ID NO: 1 in the sequence table. The invention also discloses the bivalent genetic engineering vaccine prepared by the recombinant strain and its application.

Description

Porcine mycoplasmal pneumonia and porcine contagious pleuropneumonia actinobacillus serum 1 type gene engineering strain vaccine and application

Technical field

The invention belongs to the livestock contagious disease technical field, relevant with the bacterial gene field of engineering technology.Be specifically related to a kind of structure, vaccine production and application of recombinate actinobacillus pleuropneumoniae serum 1 type and the strain of porcine mycoplasmal pneumonia bivalent gene engineering.

Background technology

Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae is called for short App) is once called as the pleuropneumonia influenzae, and (Haemophiluspleuropneumoniae HP), can cause that acute and chronic infection takes place the pig of each age level.Actinobacillus pleuropneumoniae belongs to pasteurellosis bacillus section (Pasteurellaceae), Actinobacillus (Actinobacillus), is the little coccobacillus of a kind of Gram-negative, pod membrane and pili are arranged, do not form gemma, can produce toxin, discover also amphitrichous of this bacterium recently.According to the dependency of APP growth, APP is divided into biological I type and two biotypes of biological II type to Reduced nicotinamide-adenine dinucleotide (Nicotinamide Adenine Dinucleotide, NAD claims the V factor again).The growth of biological I type bacterial strain relies on NAD, and the growth of biological I type bacterial strain does not rely on NAD, but product is with assisting growth (Nielsen R before needing other specific purine or purine, Andresen L O, Plambeck T, Nielsen J P, Krarup L T, JorsalS E.Serological characterization of Actinobacillus pleuropneumoniae biotype 2 strains isolatedfrom pigs in two Danish herds.Vet Microbiol, 1997,54 (1): 35-46).

The actinobacillus pleuropneumoniae major antigen is antigens such as capsular polysaccharide, lipopolysaccharides, outer membrane protein, hemolysin, adhesin (Wang Chun comes etc., 2001).Simultaneously, APP's is a kind of many virulence factors cause of disease, and its virulence factor is a lot.Have now found that with the pathogenic relevant virulence factor of APP and comprise capsular polysaccharide (CP), lipopolysaccharides (LPS), outer membrane protein (OMP), change iron-binding protein (TBP), haemolysis extracellular toxin (Apx), proteolytic enzyme, permeability factor, adhesion factor, pili, urease and bacterium and utilize ability etc. what Transferrins,iron complexes carried iron.

Porcine mycoplasmal pneumonia (Macoplasmal pneumoniae of swine, MPS) be by mycoplasma hyopneumoniae (Mycoplasmahyopneumoniae, Mhp) a kind of long-term hazards pig industry that causes chronic, contagious disease, and be distributed widely in countries in the world (J.Vicca etc., Evaluation of Virulence of Mycoplasma hyopneumoniae Field Isolation[J] .VeterinaryMicrobilogy, 97 (2003): 177-190.A.D.Leman, Diseases of Swine, Eighth ed.Iowa State UniversityPress, Ames, Iowa, U.S.A.1999.).The sickness rate of same swinery is up to 100%; particularly at high-density rearing's large-scale pig farm; just be difficult to control in case break out this disease; cause serious economy loss (Ross.R.F.Mycoplasmal disease; In.Straw; B.E.; Proceedingsof the Eighth Diseases of Swine[J] .Iowa State University Press; Ames, IA, 1999; p:495-510.TungdaHsu etc.; Molecular analysis of the P97 cilium adhesin operon of Mycoplasmahyopneumoniae[J] .Gene, 1998,214:13-23.).No matter be by Mhp and APP infects separately or polyinfection all is the one of the main reasons that cause present respiratory complication, cause serious harm (Kobisch to pig industry, M., 1993.Pathologie pulmonaire duporcine modele experimental associant Mycoplasma hyopneumoniae and Actinobacilluspleuropneumoniae; Gottschalk, M., Taylor, T., 2006.Actinobacillus pleuropneumoniae.In Straw, B.E., Zimmerman, J.J., D ' Allaire, S., Taylor, D.J. (Eds.), Diseases of Swine).

Lannet and Bornfors (1957) find, has strong immunizing power after suffering from the pig rehabilitation of MPS, and this just means that this can prevent this disease by immunization.Therefore, be undoubtedly a kind of comparatively rational and effective approach by the anti-system of immunization MPS.The prevention porcine mycoplasmal pneumonia adopts deactivation vaccine, subunit vaccine and attenuated vaccine more at present, has brought into play positive effect in this disease of control; The commercialized vaccine of prevention porcine contagious pleuropneumonia mainly is traditional bacteria inactivation vaccine and subunit vaccine, and inactivated vaccine can be induced the generation immune response, avoids the generation of attacking and stop chronic pleuropneumonia once more of APP but can not watch for animals; Though subunit vaccine has immune effect preferably, because the albumen relevant with this sick immunoprotection is more, and subunit vaccine only contains single or several immunogens, thereby immune protective efficiency is also very limited.The porcine contagious pleuropneumonia attenuated vaccine is a kind of new generation vaccine; it can excite the good humoral immunity; cellular immunization and mucosal immunity; has the cross protection that good antibody response and different serotypes bacterium infect; and it is easy to use; cost is low; be focus (the Prideaux C T etc. of current domestic and international research; Vaccination and protection of pigs against pleuropneumonia with a vaccine strain ofActinobacillus pleuropneumoniae produced by site-specific mutagenesis of the ApxII operon.Infect Immun; 1999; 67 (4): 1962-1966.Tonpitak W etc.; Construction of an Actinobacilluspleuropneumoniae serotype 2 prototype live negative-marker vaccine.Infect Immun; 2002; 70 (12): 7120-7125.Liwen L etc.; Construction and immunogencity of a Δ apxIC/ Δ apxIIC double mutant ofActinobacillus pleuropneumoniae serovarl.Micobiological Societies.2007; 274 (1): 55-62.JinlinL etc.; Potentialuse an Actinobacillus pleuropneumoniaedouble mutant Strain apxIIC apxIVA as livevaccine that allows serological differentiation between vaccinated and infected animals.Vaccine.2007,25 (44): 7696-7705.).

Infect simultaneously or the present situation of secondary infection at these two kinds of cause of diseases in clinical, can cause pig stress increase only and inoculate independent vaccine respectively with aquaculture cost, so that weak actinobacillus pleuropneumoniae is expressed the proteic mutant strain of MHP major antigen, and development efficient gene engineered vaccine, be the strategy of these two kinds of respiratory infectious diseases of prevention.Except medicinal application with improve the feeding and management condition, the immunization of vaccine is that global pig industry is prevented this two kinds of measures that disease is mainly taked at present.Single vaccine at PCP and MPS, prevention effect is better, but needs repeatedly immunization, and research can prevent that many sick divalence even multivalent genetic engineered vaccines are imperative by a pin.

Summary of the invention

The present invention seeks to overcome the defective of prior art, prepare a kind of recombinate actinobacillus pleuropneumoniae serum 1 type and the strain of porcine mycoplasmal pneumonia bivalent gene engineering, utilize the bivalent gene engineering strain that makes up to prepare actinobacillus pleuropneumoniae serum 1 type and porcine mycoplasmal pneumonia bivalent gene vaccine and application

The present invention implements by the following technical programs:

The applicant has made up a kind of actinobacillus pleuropneumoniae, and (Actinobacillus pleuropneumoniae, APP) test number of serum 1 type low virulent strain is SLW05 (APPapxIC ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺), this reorganization low virulent strain is delivered Chinese typical culture collection center (CCTCC) preservation in the Wuhan University of Chinese Wuhan City, Hubei Province on April 10th, 2009, this is by the classification of the low virulent strain of preservation name and be numbered (Actinobacillus pleuropneumoniae) SLW05, its deposit number is CCTCC NO:M209068, this low virulent strain is to insert porcine mycoplasmal pneumonia antigenic gene fragment in actinobacillus pleuropneumoniae serum 1 type low virulent strain SLW03 karyomit(e), and the nucleotide sequence of its gene fragment is shown in sequence table SEQ ID NO:1.

A kind of actinobacillus pleuropneumoniae serum 1 type less-virulent strain SLW05 of the present invention, its engineering strain is derived from the dual-gene disappearance strain of actinobacillus pleuropneumoniae serum 1 type SLW03 (the Liwen L that the applicant openly reported before the applying date, Weicheng B, Yonggang S, etal.Construction and immunogencity of a Δ apxIC/ Δ apxIIC double mutant of Actinobacilluspleuropneumoniae serovarl.Micobiological Societies.2007,274 (1): 55-62.).Described reorganization actinobacillus pleuropneumoniae serum 1 type low virulent strain SLW05 is on two main virulence gene incitant apxIC of disappearance actinobacillus pleuropneumoniae serum 1 type, apxIIC basis, insert the mycoplasma hyopneumoniae P 36 immunogenic protein in urase karyomit(e), and disappearance 1889bp urase structure gene.This low virulent strain SLW05 no longer has cytotoxicity and hemolytic activity, thereby has very high security.The simultaneous mutation bacterial strain can also be expressed the immunogenic protein of mycoplasma hyopneumoniae except expressing avirulent ApxIA and ApxIIA albumen, and has the ability of good immunoprotection porcine contagious pleuropneumonia and two kinds of transmissible diseases of porcine mycoplasmal pneumonia.

Step of the present invention is as follows:

1, be that target gene makes up homology arm with porcine contagious pleuropneumonia actinobacillus less-virulent strain SLW03 urase structure gene (is the gene order of U89957 with reference to the GenBank accession number), the fragment of 518bp of increasing respectively makes up the homology upper arm, and the fragment of amplification 770bp makes up the homology underarm.Respectively homology arm is connected with suicide plasmid pEMOC2 (U.S. Princeton University Dr.Greg professor Smith is so kind as to give) and makes up intermediate transfer plasmid pEIU (attached see shown in Figure 1).In the process that makes up, at first choose Sal I and Not I double enzyme site the homology underarm is inserted, and then the homology upper arm is inserted, finally make up intermediate transfer plasmid pEIU with Sma I and Sal I double digestion.

2, the structure of intermediate transfer plasmid pUCN36: amplification strong promoter sequence Ner (is the gene order of AJ391256 with reference to the GenBank accession number), be connected to the EcoRI of cloning vector pUC18 (available from the precious biotechnology in Dalian company limited) and Kpn I multiple clone site, through restriction enzyme site EcoRI and Kpn I enzyme cut identify errorless after, the immunogenic gene lactate dehydrogenase P 36 of mycoplasma is connected to Kpn I and the Sal I multiple clone site of the cloning vector pUC18 that contains the strong initiating sequence of Ner, through enzyme cut identify errorless after final name intermediate transfer plasmid be pUCN36 (attached see shown in Figure 2).

3, the structure of intermediate transfer plasmid pEUN36: intermediate transfer plasmid pEIU is reclaimed after with Sal I single endonuclease digestion and dephosphorylation, and intermediate transfer plasmid pUCN36 reclaims little fragment after with Xho I and Sal I double digestion.With T4 link enzyme the fragment that reclaims is connected again, cut through enzyme and identify that it is pEUN36 (attached see shown in Figure 3) that the intermediate transfer plasmid is named in errorless back.

4, the structure of recombination bacillus coli X7213/pEUN36: the described intermediate transfer plasmid of step 3 pEUN36 is converted into intestinal bacteria X7213 (is so kind as to give by Dr.Roy Curtiss professor III of Washington, DC university; Edwards, R.A., L.H.Keller, and D.M.Schifferli.1998.Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression.Gene 207:149-157) competent cell obtains the positive colony bacterial strain through chlorampenicol resistant screening and PCR method detection.

5, with obtaining the reorganization bacterium that single cross is changed: toadstool (SLW03) overnight incubation respectively a little less than positive intestinal bacteria that step 4 is obtained and actinobacillus pleuropneumoniae 1 type in conjunction with the method that shifts, collect thalline with aseptic phosphoric acid buffer (PBS, available from Invitrogen company) wash twice, adjust cell concentration to OD ₆₀₀Be 0.8.Respectively getting 100 μ l bacteria suspensions mixes; aseptic nitrocellulose is affixed on niacinamide-containing adenine dinucleotide (Nicotinamide Adenine Dinucleotide; NAD; claim the V factor again; available from Chinese Shanghai chemical reagents corporation) and DAP (2; the 6-diaminopimelic acid; available from Sigma company) TSA (be Trypsin soy agar substratum; available from U.S. GIBCO company; with this substratum is basal component, and additional is 1% Reduced nicotinamide-adenine dinucleotide and 10% calf serum by volume) on the solid plate, with the mixed bacterium drop on filter membrane; 37 ℃ of overnight incubation are done the contrast of donor and acceptor simultaneously.Wash bacterium liquid on the filter membrane, aseptic PBS washes twice, and coating contains the TSA plate of TSA, paraxin, and weak toadstool SLW03 and intestinal bacteria X7213 can not grow, reorganization bacterium SLW05 (APPapxIC ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) can grow.

6, further obtain the reorganization bacterium of non-resistant mark with SacB negative sense screening and chlorampenicol resistant screening: the reorganization bacterium that step (5) is obtained carry out further that the SacB negative sense screens and paraxin (paraxin is Cm, available from Invitrogen company) the responsive screening of resistance (reference: Tonpitak W etc., Construction of an Actinobacillus pleuropneumoniae serotype 2 prototype live negative-markervaccine.Infect Immun, 2002,70 (12): 7120-7125.), contain the disappearance of the immunogenic gene of porcine mycoplasmal pneumonia and urase gene through PCR checking after, obtain the mutant strain SLW05 (APPapxIC of non-resistant mark ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺).

7, stability test: the mutant strain SLW05 continuous passage that step (6) is obtained the non-resistant mark cultivated for 20 generations, obtained stable mutant strain through the PCR method checking.

8, animal safety test: with 5.8 * 10 ⁸CFU/ml, 5.8 * 10 ⁷CFU/ml, 5.8 * 10 ⁶CFU/ml, 5.8 * 10 ⁵The reorganization porcine contagious pleuropneumonia actinobacillus less-virulent strain SLW05 (APPapxIC of the parent's less-virulent strain SLW03 of CFU/ml and expression mycoplasma hyopneumoniae P 36 gene ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) distinguish 8 5-6 of abdominal cavity inoculation Balb/C mouse in age in week, write down death time and quantity respectively, one week of continuous recording.

9, protection of animal test: according to the test-results of 8 gained with 1.8 * 10 ⁷The Balb/C mouse in 6 ages in week of the reorganization porcine contagious pleuropneumonia actinobacillus less-virulent strain SLW05 of the expression mycoplasma hyopneumoniae P 36 gene that does not contain resistance marker of CFU/ml (is the gene order of X67286 with reference to the GenBank accession number) immunity, if mycoplasma hyopneumoniae attenuated vaccine immunity control group and TSB (are Trypsin soy agar substratum, available from U.S. GIBCO company, with this substratum is basal component, additional is 1% Reduced nicotinamide-adenine dinucleotide and 10% calf serum by volume) blank immune control group, detect mycoplasma pneumonia and special P36 antibody horizontal.

Beneficial effect of the present invention:

1, one of beneficial effect of the present invention is to utilize actinobacillus pleuropneumoniae as the living vaccine carrier.The present invention is as carrier with App serum 1 type low virulent strain (SLW03), also be a kind of of bacteria carrier, and the bacterial vaccine carrier has following advantage: 1. its production is cheap relatively, no matter in developed country or developing country all is applicable to immunization on a large scale; 2. bacteria carrier vaccine great majority are to antibiotic sensitive, if when other beyond thought acute reactions occurring when immunization, can utilize microbiotic to be controlled; 3. can be by oral and nasal feeding inoculation, method is simple; 4. can carry bigger gene fragment, be easy to make up polyvalent vaccine; 5. the effect that has immunological adjuvant not only can stimulate the general immunity of body, and can stimulate the mucosa-immune of body; 6. the inducing action site is clearer and more definite, and is more safe and reliable.

2, two of beneficial effect of the present invention is biological safety height.The present invention adopts the P36 gene that will have the strong promoter sequence to be inserted in the urase structure gene, makes up the intermediate transfer carrier pEUN36 that contains antibiotic marker and negative sense selection markers.With pEUN36 transformed into escherichia coli X7213 competent cell, carry out the conjugal transfer test with pleuropneumonia 1 type less-virulent strain SLW03 again, by homologous recombination, utilize auxotrophy sieve method and negative sense sieve method, obtain the APP mutant strain SLW05 (APPapxIC that the P36 gene inserts the urase gene ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺), this mutant strain does not contain antibiotics resistance gene.

3, three of the beneficial effect of the present invention BIVALENT VACCINE FOR NEONTAL strains that provide porcine contagious pleuropneumonia and porcine mycoplasmal pneumonia.Independent infection of China's mycoplasma hyopneumoniae and actinobacillus pleuropneumoniae at present or polyinfection are the one of the main reasons that causes present respiratory complication, cause serious harm to pig industry.And infect simultaneously at these two kinds of cause of diseases in clinical at present or the present situation of secondary infection, can cause pig stress increase only and inoculate independent vaccine respectively with aquaculture cost, so that weak actinobacillus pleuropneumoniae is expressed the proteic mutant strain of MHP major antigen, and development efficient gene engineered vaccine, be the strategy of these two kinds of respiratory infectious diseases of prevention.

Description of drawings

Sequence table SEQ ID NO:1 is the nucleotide sequence of the porcine mycoplasmal pneumonia antigenic gene inserted in actinobacillus pleuropneumoniae of the present invention (Actinobacillus pleuropneumoniae) the serum 1 type low virulent strain SLW03 karyomit(e).

Fig. 1: shown to be used to make up porcine mycoplasmal pneumonia and actinobacillus pleuropneumoniae intermediate transfer plasmid pEIU physical map and to make up schema.

Fig. 2: shown to be used to make up porcine mycoplasmal pneumonia and actinobacillus pleuropneumoniae intermediate transfer plasmid pUCN36 physical map and to make up schema.

Fig. 3: shown to be used to make up porcine mycoplasmal pneumonia and actinobacillus pleuropneumoniae intermediate transfer plasmid pEUN36 physical map and to make up schema.

Fig. 4: homology arm urase gene amplification fragment (P5/P6, P7/P8), M:DNA marker among the figure (DL2,000); 1 is the Ure underarm; 2 is the Ure upper arm; 3 negative contrasts;

Fig. 5: P36 gene amplification fragment (P3/P4), M:DNA marker among the figure (DL2,000); 1 is the P36 gene; 2 negative contrasts;

Fig. 6: the amplified fragments figure (P1/P2) of Pner gene; M:DNA marker among the figure (DL2,000); 1 is the Pner gene; 2 negative contrasts;

Fig. 7: the enzyme of transferring plasmid pEIU and pUNC36 is cut evaluation figure, M1:DNA marker among the figure (DL 15,000); M2:DNAmarker (DL2,000); 1:pEIU/SmaI+NotI; 2:pEIU/NotI+SalI; 3:pEIU/SalI+SmaI; 4:pUNC36/XhoI+SalI; 5:pUNC36/KpnI+SalI; 6:pUNC36/XhoI+KpnI.

Fig. 8: the enzyme of transferring plasmid pEUN36 is cut evaluation figure, M1:DNAmarker among the figure (DL 15,000); M2:DNAmarker (DL 2,000); 1:pEUN36/NotI+SmaI; 2:pEUN36/SmaI+SalI.

Fig. 9: do not contain the PCR evaluation figure (P9/P10) of the contagious pleuropneumonia less-virulent strain SLW05UreBC genetically deficient mutant strain of resistance marker, M1:DNA marker among the figure (DL 2,000); 1 is recombinant bacterial strain; 2 are parent strain SLW03 contrast.

Figure 10: the genetic stability experimental result of reorganization porcine contagious pleuropneumonia actinobacillus less-virulent strain SLW05 that does not contain the expression mycoplasma hyopneumoniae P 36 gene of resistance marker.Be illustrated as the PCR qualification result of P36 gene fragment in the reorganization bacterium, M:DNA marker among the figure (DL 2,000); 1 promptly contrasts for parent strain SLW03; 2-11 is that 1-20 is for recombinant bacterial strain

Figure 11: be to detect the P36 expression of gene with the SDS-PAGE method, M is protein marker among the figure; 1 is parent strain SLW03 contrast; 2 is recombinant bacterial strain SLW05; 3 are prokaryotic expression P36 contrast; Arrow is depicted as amalgamation and expression albumen P36.

Figure 12: be to detect the P36 expression of gene with the Western-blot method, 1 is recombinant bacterial strain SLW05 among the figure; 2 promptly contrast for parent strain SLW03.

Figure 13: the Western-blot analytical results that detects the different incubation time P36 of the reorganization porcine contagious pleuropneumonia actinobacillus less-virulent strain SLW05 gene expression amount of the expression mycoplasma hyopneumoniae P 36 gene that does not contain resistance marker.M is protein marker among the figure; 1-4 is respectively the thalline of getting after 3h, 5h, 7h, 9h cultivate; 5 promptly contrast for parent strain SLW03.

Figure 14: be not contain the reorganization porcine contagious pleuropneumonia actinobacillus less-virulent strain SLW05 of expression mycoplasma hyopneumoniae P 36 gene of resistance marker and the comparison diagram of parent strain SLW03 growth curve.

Figure 15: be pMID-18T plasmid map available from precious biotechnology (Dalian) company limited.

Figure 16: be plasmid pMD-18T-P36 restriction enzyme mapping, M:DNA marker among the figure (DL2,000) and DNA marker (DL15,000); 1,2:pMD-18T-P36/BamHI+HindIII.

Figure 17: be pGEX-KG plasmid map available from precious biotechnology (Dalian) company limited.

Figure 18: be that plasmid pGEX-KG-P36 enzyme is cut evaluation figure, M:DNA marker among the figure (DL15,000) and DNA marker (DL2,000); 1,2:pGEX-KG-P36/BamHI+HindIII.

Embodiment

Embodiment 1 makes up the intermediate transfer plasmid

1. primer design (being used for gene clone shown in the table 1 and Molecular Detection)

Table 1:PCR primer

In the table in the primer sequence underscore partly be corresponding restriction enzyme site, the primer sequence back underscore of P36 gene design be respectively restriction enzyme site and ribosome bind site, the primer of table 1 is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

2. the amplification of urase dna homolog arm, strong promoter sequence and P36 gene

With the genome of toadstool (SLW03) a little less than the APP serum 1 type is template with primer to P5 and P6, P7 and the P8 homology arm up and down that increases respectively.The pcr amplification reaction system is: 10 * Taq Buffer5.0 μ l (contains mg ²⁺), 2.5mmol/L dNTPs 4 μ l, each 1.0 μ l of 20 μ mol/L upstream and downstream primers, DMSO 5.0ul, TaqDNA polysaccharase 0.5 μ l, template 2 μ l, aseptic double-distilled water adds to 50 μ l.The PCR reaction conditions: 94 ℃ of sex change 4min, enter 30 circulations (72 ℃ are extended 1min for 94 ℃ of sex change 45sec, 58 renaturation 45sec), last 72 ℃ are extended 10min.Amplification PCR products is through 0.8% agarose gel electrophoresis analysis, and a big or small 518bp band is the homology upper arm, and a size is that the 770bp band is the homology underarm.

With the plasmid pMIDG301 (being so kind as to give by Britain Imperial university Dr.Langford professor PR) that contains Ner strong promoter sequence is template, with primer to P1 and P2 amplification strong promoter sequence.The pcr amplification reaction system is: 10 * Taq Buffer5.0 μ l (contains mg ²⁺), 2.5mmol/LdNTPs 4 μ l, each 1.0 μ l of 20 μ mol/L upstream and downstream primers, DMSO 5.0ul, PrimerSTAR HS archaeal dna polymerase 1.0 μ l, template 2 μ l, aseptic double-distilled water adds to 50 μ l.The PCR reaction conditions: 94 ℃ of sex change 4min, enter 30 circulations (72 ℃ are extended 1min for 98 ℃ of sex change 15sec, 57 renaturation 30sec), last 72 ℃ are extended 10min.Amplification PCR products is through 0.8% agarose gel electrophoresis analysis, and a big or small 351bp band starts by force for Ner.

With the porcine mycoplasmal pneumonia living vaccine (available from Nanjing Tianbang Bio-industry Co., Ltd..Authentication code: veterinary drug new word (2006) 100991026, network address: genome http://home.njtb.com/Article/cpsj/cpsj/200608/214.html) is a template with primer to P3 and P4 amplification immunogenicity P36 gene, and the pcr amplification reaction system is: 10 * Taq Buffer5.0 μ l (contains mg ²⁺), 2.5mmol/L dNTPs 4 μ l, each 1.0 μ l of 20 μ mol/L upstream and downstream primers, DMSO 5.0ul, TaqDNA polysaccharase 0.5 μ l, template 2 μ l, aseptic double-distilled water adds to 50 μ l.The PCR reaction conditions: 94 ℃ of sex change 4min, enter 30 circulations (72 ℃ are extended 1min for 94 ℃ of sex change 45sec, 58 renaturation 45sec), last 72 ℃ are extended 10min.Amplification PCR products is through 0.8% agarose gel electrophoresis analysis, and a big or small 948bp band is P36.

Embodiment 2 intermediate transfer plasmid pEIU, pUCN36 and pEUN36 make up

After the PCR product reclaimed purifying, with Sal I and Not I double digestion homology right arm and pEMOC2 carrier, connect after reclaiming purifying, transformed into escherichia coli DH5 α, screening positive clone extracts plasmid in a small amount, with the restriction restriction endonuclease recombinant plasmid is carried out enzyme and cuts evaluation, through 0.8% agarose gel electrophoresis, confirm that its size meets with expection; Again with Sma I and Sal I double digestion homology left arm and the pEMOC2 that contains the homology right arm, connect after reclaiming purifying, transformed into escherichia coli DH5 α, screening positive clone, extract plasmid in a small amount, with the restriction restriction endonuclease recombinant plasmid is carried out enzyme and cut evaluation,, confirm that its size meets with expection through 0.8% agarose gel electrophoresis; The no base that confirms homology right arm and homology left arm through order-checking mismatches, and this recombinant plasmid is named as pEIU.

After the PCR product reclaimed purifying, also be connected with the pUC18 carrier with Kpn I double digestion Ner strong promoter sequence with EcoR I, transformed into escherichia coli DH5 α, screening positive clone, extract plasmid in a small amount, with the restriction restriction endonuclease recombinant plasmid is carried out enzyme and cut evaluation,, confirm that its size meets with expection through 0.8% agarose gel electrophoresis; Again with Kpn I and Sal I double digestion P36 and the pUC18 carrier that contains the Ner sequence, connect after reclaiming purifying, transformed into escherichia coli DH5 α, screening positive clone, extract plasmid in a small amount, with the restriction restriction endonuclease recombinant plasmid is carried out enzyme and cut evaluation,, confirm that its size meets with expection through 0.8% agarose gel electrophoresis; Confirm that through order-checking no base mismatches this recombinant plasmid called after pUCN36.

Middle transferring plasmid pEIU is reclaimed purifying after with SalI single endonuclease digestion and dephosphorylation, and intermediate transfer plasmid pUCN36 reclaims the little fragment of purifying after with Xho I and SalI double digestion.With T4 link enzyme the fragment that reclaims is connected again, transformed into escherichia coli X7213, screening positive clone, extract plasmid in a small amount, with restriction enzyme digestion recombinant plasmid is carried out enzyme and cut evaluation, through 0.8% agarose gel electrophoresis, confirm that its size meets with expection, with this recombinant plasmid called after pEUN36.

Embodiment 3 conjugal transfers

With the recombinant plasmid pEUN36 that makes up adopt the heat shock method (Huang Peitang etc. translate. Sa nurse Brooker J, Russell D W work. molecular cloning experiment guide (third edition), Beijing: Science Press, 2002) transformed into escherichia coli X7213, positive intestinal bacteria that obtain and actinobacillus pleuropneumoniae 1 type less-virulent strain SLW03 parent strain be overnight incubation respectively, collect thalline and wash twice, adjust bacteria concentration to OD with aseptic PBS ₆₀₀Be 0.8.Respectively get 100 μ l bacteria suspensions and mix, aseptic nitrocellulose is affixed on the TSA solid plate that contains NAD and DAP, on filter membrane, 37 ℃ of overnight incubation are done the contrast of donor and acceptor simultaneously with the mixed bacterium drop.Wash bacterium liquid on the filter membrane, aseptic PBS washes twice, and coating contains the TSA plate of TSA, Cm, and SLW03 parent plant and intestinal bacteria X7213 can not grow, and the reorganization bacterium can grow; The bacterium of will recombinating further carries out the screening of SacB negative sense, with the responsive screening of chlorampenicol resistant (reference: Tonpitak W etc., Construction of an Actinobacillus pleuropneumoniae serotype2 prototype live negative-marker vaccine.Infect Immun, 2002,70 (12): 7120-7125.), obtain the recombination mutation strain SLW05 of non-resistant mark.

The evaluation of embodiment 4 recombination mutation strains

Extract the reorganization porcine contagious pleuropneumonia actinobacillus less-virulent strain SLW05 (APPapxIC of the expression mycoplasma hyopneumoniae P 36 gene that does not contain resistance marker ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) genome, be template with this genome, with primer P3/P4 is increased, observe the 948bp band that whether occurs expecting; With primer P9/P10 is increased simultaneously, carry out pcr amplification and identify mutant strain urase genetically deficient fragment.The pcr amplification reaction system is: 10 * Taq Buffer5.0 μ l (contains mg ²⁺), 2.5mmol/L dNTPs 4 μ l, each 1.0 μ l of 20 μ mol/L upstream and downstream primers, DMSO 5.0ul, TaqDNA polysaccharase 0.5 μ l, template 2 μ l, aseptic double-distilled water adds to 50 μ l.The PCR reaction conditions: 94 ℃ of sex change 4min, enter 30 circulations (72 ℃ are extended 2min for 94 ℃ of sex change 45sec, 57 renaturation 45sec), last 72 ℃ are extended 10min.Agarose gel electrophoresis through 0.8% detects the band of seeing the 1889bp size that whether occurs expecting.Contain the band of 948bp size in its qualification result, and the bacterial strain that does not have a 1889bp size strip is a mutant strain.

Embodiment 5 expresses the biological characteristics of the recombinant bacterial strain SLW05 of P36 gene fragment

1, the preparation of P36 gene fragment e. coli expression product guinea pig antiserum

With P36 fragment among EcoR I and the Hind III double digestion plasmid pMD18-P36 (seeing Figure 15,16) and pGEX-KG (, seeing Figure 17), use T behind the recovery purifying available from precious biotechnology (Dalian) company ₄DNA ligase connects, transformed into escherichia coli DH5.The competence bacterium is cultivated 12h for 37 ℃ on solid LB flat board, prepare plasmid acquisition expression plasmid pGEX-KG-P36 (enzyme is cut evaluation and seen Figure 18) in a small amount thereby choose bacterium.The pGEX-KG-P36 plasmid is carried out sequencing, after confirming that the clone is correct, transformed into escherichia coli BL21 (available from precious biotechnology (Dalian) company limited), containing penbritin (Amp, final concentration 50 μ g/mL) the picking positive transformant is to liquid LB substratum on the solid LB flat board, and 37 ℃, 200r/min are cultured to logarithmic phase (light absorption value OD ₆₀₀=0.6～1.0) add IPTG (final concentration 1mM) and carry out abduction delivering 4h, use gst fusion protein to extract test kit Glutathione Sepharose 4B Kit (available from U.S. Qiagen company), expression product is carried out purifying, obtaining concentration is the amalgamation and expression albumen of 171 μ g/mL, called after GST-P36.With 100 μ g expression product GST-P36 and equal-volume Freund's complete adjuvant (available from U.S. Sigma company) uniform mixing, subcutaneous injection cavy (available from Hubei Province Preventive Medicine Academy's Experimental Animal Center), respectively at 2 weeks (two exempt from) back booster immunizations 1 time (using Freund's incomplete adjuvant) available from U.S. Sigma company.Exempt from back 14d blood sampling 2, extract serum, 0.22 μ m membrane filtration degerming, put-20 ℃ standby.

2, SLW05 (APPapxIC ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) the expression characterization analysis of recombinant bacterial strain

Picking recombinant bacterial strain SLW05 (APPapxIC ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) single bacterium colony in the TSB liquid nutrient medium, 37 ℃, 200r/min are cultivated 16h, 8, the centrifugal collection thalline of 000r/min carries out polyacrylamide gel electrophoresis (SDS-PAGE), and uses mouse-anti GST-P36 serum to carry out Western-blotting and analyze.The result shows SLW05 (APPapxIC ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) to express size be recombinant protein about 36kDa, and the recombinant protein of expressing has good immunological response originality (seeing Figure 12).

3, SLW05 (APPapxIC ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) evaluation of urease activity of recombinant bacterial strain

With SLW05 (APPapxIC ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) recombinant bacterial strain and parent plant SLW03 (APPapxIC ^-/ apxIIC ^-) inoculation contains the TSB substratum of NAD, use the plain detection kit of urea (available from U.S. Sigma company, according to the specification sheets operation of this test kit) then, the result shows that recombinant bacterial strain SLW05 detected result is negative, and parent strain SLW03 is positive, proves successfully to have made up urase deletion mutantion strain.

4, SLW05 (APPapxIC ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) the growth characteristics analysis of recombinant bacterial strain

Compare (as Figure 14) by growth curve to parent bacterium SLW03 and recombination mutation strain SLW05.The parent strain SLW03 of incubated overnight and the reorganization porcine contagious pleuropneumonia actinobacillus less-virulent strain SLW05 that do not contain the expression mycoplasma hyopneumoniae P 36 gene of resistance marker are inoculated into 100ml TSB substratum with 1: 1000 ratio respectively, the inoculation back was every sampling in 1.5 hours, get altogether 6 times, measure viable count, relatively two strain bacterium growing abilities.The speed of growth of finding them does not have very big difference, illustrates that recombination mutation strain SLW05 is not subjected to remarkably influenced at external energy for growth.

5, SLW05 (APPapxIC ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) the genetic stability test of recombinant bacterial strain

SLW05 (APPapxIC with the present invention's preparation ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) recombinant bacterial strain is streak culture on the TSA solid plate, picking list bacterium colony is in the TSB substratum, 37 ℃, 200r/min are cultivated 16h, by volume 1: 1,000 ratio is transferred in the TSB substratum and cultivates 12h, by volume 1: 1 once more, 000 ratio was transferred in the TSB liquid nutrient medium, carried out 10 switchings continuously.Carry out pcr amplification with primer P3/P4, the hereditary situation of amplification P36 in the reorganization bacterium seen Figure 10.Figure 10 shows that each amplification does not all have observable difference, shows that the SLW05 recombinant bacterial strain that the present invention prepares can genetic stability.

Embodiment 6Western blot analyzes

Collect the thalline of the reorganization porcine contagious pleuropneumonia actinobacillus less-virulent strain SLW05 of the expression mycoplasma hyopneumoniae P 36 gene that does not contain resistance marker, after boiling sex change, run SDS-PAGE, and establish contrast of P36 albumen and parent strain SLW03 contrast.

(1) changes film: after the SDS-PAGE electrophoresis finishes, adopt half-dried transfer method transfer printing nitrocellulose filter, change general 2h of film time.After changeing the film end, ponceau is dyeed and protein band occurs to film, marks as the proteic position of the reference of molecular weight standard with soft pencil, uses the rinsed with deionized water nitrocellulose filter then, changes water number therebetween.

(2) sealing: nitrocellulose filter is put into the hybridization bag that can add heat sealing, with filter membrane area 0.1 ~ 0.15mL/cm ²Amount add confining liquid (containing the TBST that 1% bovine serum albumin is BSA), get rid of airtight sack behind the bubble as far as possible, lie against room temperature incubation 0.5-1h on the shaking table.

(3) with anti-a joint: nitrocellulose filter is transferred in the new hybridization bag, by 0.1 ~ 0.15mL/cm ²Amount, add mouse-anti P36 serum with TBST dilution (as one anti-, multispecific antibody is long-pending than being 1: 100) respectively, seal behind the eliminating bubble, lie against room temperature incubation 1h on the shaking table.TBST washes 3 times, each 5 ~ 10min.

(4) with two anti-joints: filter membrane is transferred in the new hybridization bag, by 0.1 ~ 0.15ml/cm ²Amount to add with TBST be the sheep anti-mouse antibody of 1: 2500 (DAB the is a substrate) horseradish peroxidase-labeled of diluting with volume ratio, room temperature incubation 0.5 ~ 1h.TBST washes 3 times, each 5 ~ 10min.Wash 2 times each 5 ~ 10min again with TBS.

(5) colour developing: with DAB is substrate, then nitrocellulose filter is put into the 10mL substrate solution, and colour developing 1 ~ 15min in case protein band occurs, stops with deionized water immediately, takes a picture.

The test of embodiment 8 animal safeties

With 5.8 * 10 ⁸CFU/ml, 5.8 * 10 ⁷CFU/ml, 5.8 * 10 ⁶CFU/ml, 5.8 * 10 ⁵Parent strain SLW03 (the APPapxIC of CFU/ml ^-/ apxIIC ^-) and do not contain the reorganization porcine contagious pleuropneumonia actinobacillus less-virulent strain SLW05 (APPapxIC of the expression mycoplasma hyopneumoniae P 36 gene of resistance marker ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) 8 Balb/C mouse of difference abdominal cavity inoculation, write down death time and quantity respectively, one week of continuous recording.Test-results sees Table 2.

Table 2: poison test of recombiant vaccine bacterial strain SLW05 of the present invention and parent plant SLW03 force rate

Embodiment 9 recombinant bacterial strain SLW05 (APPlapxIC of the present invention ^-/ apxIIC ^-/ UreBC ^-/ p36 ⁺) detect at the intravital immune efficacy of mouse

1, the immune programme for children of mouse:

The BALB/c mouse of using 5-6 age in week is divided into 3 groups as the immune efficacy evaluation according to test requirements document, is respectively the SLW05 (APPapxIC of the present invention's preparation ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) recombinant bacterial strain vaccine group, porcine mycoplasmal pneumonia living vaccine (available from sky, Nanjing biotechnology company of nation) group and non-immune blank group.Immunization route is that intramuscular injection 0.2mL (contains 1.8 * 10 ⁷CFU viable bacteria amount) bacterium liquid or TSB substratum, booster immunization is 1 time after 14 days.Exempt to detect in back 14 days, 28 days mycoplasma hyopneumoniae serum antibody (with the MHP antibody assay kit of IDEXX company) respectively at preceding 0 day of immunity, head.GST fusion expressed product GST-P36 with preparation among the embodiment 5 is that antigen (256ng/ hole) bag is by elisa plate simultaneously, press indirect ELISA method (with reference to Liu Zhonghui etc., immunology common experimental technology, Beijing: Science Press, 2002) detection P36 gene recombinant protein antibody horizontal.

2, immune mouse humoral immunization antibody horizontal detects

Blood sampling before the mouse immune, second and third time blood sampling exempt to carry out in back 14,28 days at head respectively, choose 10 through the blood sampling of tail vein negative pressure for every group, and separation of serum detects anti-mycoplasma hyopneumoniae serum antibody, P36 specific antibody titres respectively, averages.The results are shown in Table 3.As can be seen from Table 3, head exempts from back the 2nd all SLW05 (APPapxIC of the present invention ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) reorganization bacteria vaccine group and porcine mycoplasmal pneumonia living vaccine (available from sky, Nanjing biotechnology company of nation) group mycoplasma hyopneumoniae antibody is 1: 20 and 1: 160, two exempt from 2 week backs (they being that head exempted from back 28 days), SLW05 (APPapxIC of the present invention ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) reorganization bacteria vaccine group and porcine mycoplasmal pneumonia living vaccine (available from sky, Nanjing biotechnology company of nation) group mycoplasma hyopneumoniae antibody rises to 1: 40 and be higher than 1: 1280.Head exempts from 2 all SLW05 (APPapxIC of the present invention ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) reorganization bacteria vaccine group and porcine mycoplasmal pneumonia living vaccine (available from sky, Nanjing biotechnology company of nation) group P36 specific antibody titres is 1: 160 and 1: 320, two exempt from 2 weeks of back, SLW05 (APPapxIC of the present invention ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) reorganization bacteria vaccine group and porcine mycoplasmal pneumonia living vaccine (available from sky, Nanjing biotechnology company of nation) group P36 specific antibody titres rose to respectively 1: 320 and 1: 640.And the PBS that carries out synchronously contrast all negative (＜1: 10,＜1: 20).Two exempt from 2 weeks of back (being that head exempts from 4 weeks of back), the SLW05 (APPapxIC of the present invention's preparation ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) the ELISA antibody that produces of reorganization bacteria vaccine group and porcine mycoplasmal pneumonia living vaccine (available from sky, Nanjing biotechnology company of nation) group all has rising to a certain degree, though the SLW05 reorganization bacterium of the present invention's preparation is compared with porcine mycoplasmal pneumonia living vaccine (available from sky, Nanjing biotechnology company of nation) group, it is all relative on the low side that the antibody of mycoplasma hyopneumoniae and P36 specific antibody are tired, but the SLW05 (APPapxIC of the present invention's preparation ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) reorganization bacteria vaccine group all can produce mycoplasma hyopneumoniae and P36 specific antibody, the antibody titer mean level (ML) was respectively 1: 40 and 1: 320, PBS control group antibody test still negative (＜1: 10,＜1: 20).The above results shows the SLW05 (APPapxIC that the present invention prepares ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) can induce body to produce the humoral immunoresponse(HI) of specific anti-mycoplasma hyopneumoniae behind the reorganization bacteria vaccine immune mouse.

The reorganization bacterium SLW05 (APPapxIC of table 3 the present invention preparation ^-/ apxIIC ^-/ UreBC ^-/ P36 ⁺) vaccine immune mouse Serum Antibody Detection (ELISA method)

Although content of the present invention is to describe in conjunction with present embodiment, can not think limitation of the scope of the invention, scope of the present invention is limited by appended claims.In addition, those skilled in the art carries out various changes or modification to the present invention in the appended claims restricted portion, and these changes or modified forms drop in the scope of the invention equally.

Sequence table

＜110〉Hua Zhong Agriculture University

＜120〉porcine mycoplasmal pneumonia and porcine contagious pleuropneumonia actinobacillus serum 1 type gene engineering strain vaccine and application

<130>

<141>2009-04-13

<160>2

<170>PatentIn?version?3.1

<210>1

<211>1394

<212>DNA

<213>Mycoplasma?hyopneumoniae

<220>

<221>gene

<222>(1)..(1394)

<223>

<220>

<221>CDS

<222>(327)..(1274)

<223>

<400>1

gttaacacgc?caccaggaat?tgatctaaaa?aaattagaac?taaataatgt?cgatggttat 60

gctcttggag?cttatattct?cattattatt?ttctcacttg?tttcttcaat?tggtttatgc 120

ccttttaaag?gaaccaaatc?cagaatataa?aaaattatta?aaaatacgta?gtttttctga 180

aattgaacga?atcaaaaaat?aaaaaaaatt?tcatttttga?ttcgttttaa?ttaaaaataa 240

gaaaaaaatt?atcattactt?gtttttaatt?aaaaaaaatt?aaatctatca?atgaaaaaat 300

ttaacaaaaa?aggagaaatc?aaactt?atg?aaa?cct?att?aaa?ata?gct?cta?att 353

Met?Lys?Pro?Ile?Lys?Ile?Ala?Leu?Ile

1 5

ggt?gct?gga?aat?gtc?gga?aat?tcc?ttc?ctt?tat?gca?gca?atg?aat?caa 401

Gly?Ala?Gly?Asn?Val?Gly?Asn?Ser?Phe?Leu?Tyr?Ala?Ala?Met?Asn?Gln

10 15 20 25

gga?ctt?gca?tcc?gag?tat?gga?att?att?gat?att?aat?cct?gat?ttt?gcc 449

Gly?Leu?Ala?Ser?Glu?Tyr?Gly?Ile?Ile?Asp?Ile?Asn?Pro?Asp?Phe?Ala

30 35 40

gat?ggt?aat?gct?ttt?gat?ttt?gaa?gat?gcc?tca?gct?tct?ttg?cct?ttt 497

Asp?Gly?Asn?Ala?Phe?Asp?Phe?Glu?Asp?Ala?Ser?Ala?Ser?Leu?Pro?Phe

45 50 55

ccg?att?agt?gtc?tcc?cgt?tat?gaa?tat?aaa?gat?cta?aaa?gat?gct?gat 545

Pro?Ile?Ser?Val?Ser?Arg?Tyr?Glu?Tyr?Lys?Asp?Leu?Lys?Asp?Ala?Asp

60 65 70

ttt?att?gta?att?aca?gcg?gga?aga?cca?caa?aaa?ccg?ggt?gaa?act?cgg 593

Phe?Ile?Val?Ile?Thr?Ala?Gly?Arg?Pro?Gln?Lys?Pro?Gly?Glu?Thr?Arg

75 80 85

ctt?gaa?tta?gta?gct?gat?aac?atc?cga?att?atc?cgg?gaa?att?gca?cta 641

Leu?Glu?Leu?Val?Ala?Asp?Asn?Ile?Arg?Ile?Ile?Arg?Glu?Ile?Ala?Leu

90 95 100 105

aaa?gtc?aaa?gaa?agt?ggc?ttt?agt?gga?ata?agt?att?att?gtt?gct?aat 689

Lys?Val?Lys?Glu?Ser?Gly?Phe?Ser?Gly?Ile?Ser?Ile?Ile?Val?Ala?Asn

110 115 120

cct?gtt?gat?ata?att?aca?agg?gct?tac?cgg?gat?gca?tct?gga?ttt?tcc 737

Pro?Val?Asp?Ile?Ile?Thr?Arg?Ala?Tyr?Arg?Asp?Ala?Ser?Gly?Phe?Ser

125 130 135

gat?caa?aaa?gtt?atc?ggt?agt?gga?act?gtt?tta?gat?aca?gca?agg?ctt 785

Asp?Gln?Lys?Val?Ile?Gly?Ser?Gly?Thr?Val?Leu?Asp?Thr?Ala?Arg?Leu

140 145 150

caa?ttt?gca?atc?gca?aaa?aga?gca?aaa?gta?tcg?cct?aat?tcg?gtt?cag 833

Gln?Phe?Ala?Ile?Ala?Lys?Arg?Ala?Lys?Val?Ser?Pro?Asn?Ser?Val?Gln

155 160 165

gcc?tac?gtg?atg?ggt?gaa?cat?ggt?gat?tca?tct?ttt?gtt?gct?tat?tca 881

Ala?Tyr?Val?Met?Gly?Glu?His?Gly?Asp?Ser?Ser?Phe?Val?Ala?Tyr?Ser

170 175 180 185

aat?att?aaa?att?gcc?ggt?gaa?tgt?ttc?tgt?gct?tat?tct?aaa?cta?acc 929

Asn?Ile?Lys?Ile?Ala?Gly?Glu?Cys?Phe?Cys?Ala?Tyr?Ser?Lys?Leu?Thr

190 195 200

gga?att?gat?agc?tca?aat?tac?gaa?aaa?gaa?ctt?gaa?tat?cca?gtt?tct 977

Gly?Ile?Asp?Ser?Ser?Asn?Tyr?Glu?Lys?Glu?Leu?Glu?Tyr?Pro?Val?Ser

205 210 215

cgc?cgg?gct?tat?gaa?att?att?aat?cgt?aaa?agg?gca?aca?ttt?tat?gga 1025

Arg?Arg?Ala?Tyr?Glu?Ile?Ile?Asn?Arg?Lys?Arg?Ala?Thr?Phe?Tyr?Gly

220 225 230

att?ggt?gca?gct?att?gcc?aaa?ata?gtt?tct?aat?att?atc?aaa?gat?aca 1073

Ile?Gly?Ala?Ala?Ile?Ala?Lys?Ile?Val?Ser?Asn?Ile?Ile?Lys?Asp?Thr

235 240 245

aaa?aat?att?atg?att?gcc?gga?gca?aat?tta?cga?gga?gaa?tac?gga?ttt 112l

Lys?Asn?Ile?Met?Ile?Ala?Gly?Ala?Asn?Leu?Arg?Gly?Glu?Tyr?Gly?Phe

250 255 260 265

cac?gga?gta?aat?atc?gga?gtt?cca?gtt?gtt?tta?gga?gca?aac?gga?att 1169

His?Gly?Val?Asn?Ile?Gly?Val?Pro?Val?Val?Leu?Gly?Ala?Asn?Gly?Ile

270 275 280

gaa?aaa?att?att?gag?att?agt?ctt?aat?gat?aaa?gaa?aaa?gaa?aaa?ttt 1217

Glu?Lys?Ile?Ile?Glu?Ile?Ser?Leu?Asn?Asp?Lys?Glu?Lys?Glu?Lys?Phe

285 290 295

gcc?aaa?tca?gtt?gca?atc?att?gat?aaa?att?tat?cag?gat?gca?att?aaa 1265

Ala?Lys?Ser?Val?Ala?Ile?Ile?Asp?Lys?Ile?Tyr?Gln?Asp?Ala?Ile?Lys

300 305 310

aat?att?taa?ttttttagga?aaaacagttt?aaaaatctct?ctataaattt 1314

Asn?Ile

315

aatattttta?tcctatgatt?ttaaaaaagt?actataaatt tatagtactt?ttttaatttt?1374

tagcaaaaaa?attgaagctt 1394

<210>2

<211>315

<212>PRT

<213>Mycoplasma?hyopneumoniae

<400>2

Met?Lys?Pro?Ile?Lys?Ile?Ala?Leu?Ile?Gly?Ala?Gly?Asn?Val?Gly?Asn

1 5 10 15

Ser?Phe?Leu?Tyr?Ala?Ala?Met?Asn?Gln?Gly?Leu?Ala?Ser?Glu?Tyr?Gly

20 25 30

Ile?Ile?Asp?Ile?Asn?Pro?Asp?Phe?Ala?Asp?Gly?Asn?Ala?Phe?Asp?Phe

35 40 45

Glu?Asp?Ala?Ser?Ala?Ser?Leu?Pro?Phe?Pro?Ile?Ser?Val?Ser?Arg?Tyr

50 55 60

Glu?Tyr?Lys?Asp?Leu?Lys?Asp?Ala?Asp?Phe?Ile?Val?Ile?Thr?Ala?Gly

65 70 75 80

Arg?Pro?Gln?Lys?Pro?Gly?Glu?Thr?Arg?Leu?Glu?Leu?Val?Ala?Asp?Asn

85 90 95

Ile?Arg?Ile?Ile?Arg?Glu?Ile?Ala?Leu?Lys?Val?Lys?Glu?Ser?Gly?Phe

100 105 110

Ser?Gly?Ile?Ser?Ile?Ile?Val?Ala?Asn?Pro?Val?Asp?Ile?Ile?Thr?Arg

115 120 125

Ala?Tyr?Arg?Asp?Ala?Ser?Gly?Phe?Ser?Asp?Gln?Lys?Val?Ile?Gly?Ser

130 135 140

Gly?Thr?Val?Leu?Asp?Thr?Ala?Arg?Leu?Gln?Phe?Ala?Ile?Ala?Lys?Arg

145 150 155 160

Ala?Lys?Val?Ser?Pro?Asn?Ser?Val?Gln?Ala?Tyr?Val?Met?Gly?Glu?His

165 170 175

Gly?Asp?Ser?Ser?Phe?Val?Ala?Tyr?Ser?Asn?Ile?Lys?Ile?Ala?Gly?Glu

180 185 190

Cys?Phe?Cys?Ala?Tyr?Ser?Lys?Leu?Thr?Gly?Ile?Asp?Ser?Ser?Asn?Tyr

195 200 205

Glu?Lys?Glu?Leu?Glu?Tyr?Pro?Val?Ser?Arg?Arg?Ala?Tyr?Glu?Ile?Ile

210 215 220

Asn?Arg?Lys?Arg?Ala?Thr?Phe?Tyr?Gly?Ile?Gly?Ala?Ala?Ile?Ala?Lys

225 230 235 240

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

245 250 255

Ala?Asn?Leu?Arg?Gly?Glu?Tyr?Gly?Phe?His?Gly?Val?Asn?Ile?Gly?Val

260 265 270

Pro?Val?Val?Leu?Gly?Ala?Asn?Gly?Ile?Glu?Lys?Ile?Ile?Glu?Ile?Ser

275 280 285

Leu?Asn?Asp?Lys?Glu?Lys?Glu?Lys?Phe?Ala?Lys?Ser?Val?Ala?Ile?Ile

290 295 300

Asp?Lys?Ile?Tyr?Gln?Asp?Ala?Ile?Lys?Asn?Ile

305 310 315

Claims (4)

1. A recombinant Actinobacillus pleuropneumoniae serotype 1 attenuated strain SLW05, preserved in the China Center for Type Culture Collection, its preservation number is CCTCC NO: M209068, the attenuated strain is produced in Actinobacillus pleuropneumoniae The serotype 1 attenuated strain SLW03 is inserted into the chromosome of the Mycoplasma porcine pneumonia antigenic P36 gene fragment, and the nucleotide sequence of the P36 gene fragment is shown in the sequence table SEQ ID NO: 1. 2. A bivalent genetically engineered vaccine for porcine infectious pleuropneumonia and mycoplasma swine pneumonia prepared from the attenuated strain SLW05 of Actinobacillus pleuropneumoniae serotype 1 with the preservation number of CCTCC NO: M209068 as claimed in claim 1. 3. The application of the attenuated strain SLW05 according to claim 1 in the preparation of a bivalent genetic engineering vaccine of Actinobacillus pleuropneumoniae serotype 1 and Mycoplasma hyopneumoniae. 4. The application of the bivalent genetic engineering vaccine according to claim 2 in the prevention and control of Actinobacillus pleuropneumoniae serotype 1 and Mycoplasma hyopneumoniae.

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