CN105087616B - A kind of fusion of mycoplasma hyopneumoniae and preparation method thereof - Google Patents
A kind of fusion of mycoplasma hyopneumoniae and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of fusions of mycoplasma hyopneumoniae and preparation method thereof, belong to gene engineering technology field, P46 and P65 genes used in the present invention, directly according to the coding region sequence issued in NCBI GeneBank, specific primer of the design containing corresponding restriction enzyme site.The PCR product and prokaryotic expression carrier pET 28a double digestions of P46 genes simultaneously connect, structure recombinant expression carrier pET 28a P46；The PCR product double digestion of recombinant expression carrier pET 28a P46 and P65 genes is enabled again and is connected, direct construction recombinant expression carrier pET 28a P46 P65.Advantage is need not to use Linker connection P46 and P65 genes, is directly over double digestion connection and recombinant expression carrier pET 28a P46 P65 just can be obtained.Construction of recombinant vector process is compared to using Linker linkers because being connect again with prokaryotic expression carrier, process is simple and readily available, and the consuming time significantly reduces.
Description
Technical field
The invention belongs to gene engineering technology field, it is related to the research to animal bacteria sexually transmitted disease gene vaccine, specifically
It is a kind of fusion of mycoplasma hyopneumoniae and preparation method thereof that ground, which is said,.
Background technology
The Bronchopneumonia that mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp) can cause pig, is commonly called as
Swine enzootic pneumonia.The harm of the disease is that with the cilium of porcine respiratory epithelial cell specific adhesion can occur for Mhp, dynamic to destroy
Object body respiratory mucosa-cilium barrier, causes the infection of other pathogens, breathing problem is made to aggravate, then pig is caused to exhale
Inhale tract disease syndrome (Porcine respiratory disease complex, PRDC).
Mhp requires condition of culture very high, it is more difficult to cultivate, study the former only from 4 kinds of pathological form branch of report at present
The gene of body is 168 plants respectively, J plants, 7448 types and 232 types.Swine enzootic pneumonia is chronic disease, spread extensively, the death rate it is not high but
Carrying rate is very high, it is difficult to thoroughly radical cure, and be often seasonality.Due to it is domestic at present for the culture of mycoplasma hyopneumoniae with
And isolation technics is not very perfect, this just more increases the difficulty studied mycoplasma hyopneumoniae.Current China is to the disease
Prevention mainly aim at prevention, but due to for the exploitation of the bacterial vaccine be almost entirely based on adjuvated full cell preparation or
Film preparation there is no one to be proved to be fully effective.Studies have shown that the specific binding of adhesion protein and respiratory mucosa cilium is
Cause the essential pathogenic factor of porcine mycoplasmal pneumonia.Studies have reported that the adhesion protein of Mhp is fine with porcine respiratory mucous membrane
The combination of hair, is the essential pathogenic factor for causing porcine mycoplasmal pneumonia.Adhesion protein is mainly cytoplasm protein in Mhp
P36, memebrane protein P46, P65 and P74, adhesion factor P97, P102, P216, P 159, P116, Mhp271, Mhp107, Mhp684
And heat shock protein DnaK.According to the literature, P97, P102, P216, P159, P116 are heparin binding proteins, mainly
By in the heparin protein binding to respiratory mucosa cilium in extracellular matrix, triggering an immune response；P36 has lactic dehydrogenase
Active cytoplasmic protein, cause late period immune response, immunogenicity is slightly lower；P46 albumen is Cell membrane antigens, Ke Yiyin
Play early stage immune response；P65 is one of heat shock protein A subunit family member, has immunostimulation, while having esterase
Activity；Dnak, which is heat shock protein, has the function of that immunologic adjuvant, N-terminal structure have the function of molecular chaperones, C-terminal to have main
Immunogenicity.Prevent the effective vaccine of mycoplasma hyopneumoniae infection since current China lacks, in order to more effectively control and
Harm of the Mhp to animal husbandry is reduced, the immunogene of research mycoplasma hyopneumoniae fusion protein P65-DnaKc is put forth effort in this experiment
Property.The selected adhesion protein P65 immunogenicities grade of experiment is high, can cause early immune reaction, inter-species conservative and spy
It is anisotropic high, the exploitation of Mhp recombinant vaccines and subunit vaccine is often used for as immunodominant proteins at present；Stick egg
White DnaKc is the C-terminal segment of heat shock protein DnaK, and immunogenicity is good, and has the function of immunologic adjuvant, induces machine
Body generates strong humoral immune reaction and cell immune response；This experiment constructs p65, dnaKc and p65-dnaKc base respectively
The expression vector of cause is immunized mouse using the albumen of acquisition as recombinant vaccine, utilizes ELISA by prokaryotic expression, purifying
The antibody titer in serum is detected, it is found that P65, DnaKc and P65-DnaKc albumen all have preferable immunogenicity, wherein melting
The immunogenicity of hop protein P65-DnaKc is substantially better than single albumen P65 and DnaKc.Challenge test finds fusion protein P65-
DnaKc can cause stronger immune response in Mice Body, and protective rate is higher.Therefore, using the fusion development,
The recombinant vaccine of the safe and efficient prevention mycoplasma hyopneumoniae infection of exploitation, will have great potentialities to be.
Invention content
The present invention in order to overcome present mycoplasma hyopneumoniae market effective vaccine few and the defect of vaccine preparation process,
A kind of fusion of mycoplasma hyopneumoniae and preparation method thereof is devised, by gene P46 and the P65 gene of mycoplasma hyopneumoniae
It is connected in series with, to the better fusion P46-P65 of adaptive immune originality.
The technical solution adopted by the present invention is：A kind of fusion of mycoplasma hyopneumoniae, fusion P46-
P65, nucleotide sequence such as sequence table SEQ ID NO：Shown in 1.
The fusion is prepared via a method which：
1) PCR product of P46 genes and prokaryotic expression plasmid pET-28a are distinguished into double digestion, and stays overnight connection, structure weight
Group expression vector pET28a-P46；
2) by the PCR product of P65 genes and recombinant expression carrier pET28a-P46 double digestions, and connection is stayed overnight, structure contains
There is the recombinant expression carrier pET28a-P46-P65 of fusion；
3) by external mutating technology, three TGA codons in P46 genes are changed into TGG codons, to build
The recombinant expression carrier pET28a-P46-P65 that can be expressed in prokaryotic cell；
4) it is removed in recombinant expression carrier pET28a-P46-P65 by running 1% agarose gel electrophoresis gel extraction
PET28a is to obtain the fusion P46-P65 that can be expressed in prokaryotic cell；
Wherein, the nucleotide sequence of P46 genes such as SEQ ID NO：Shown in 2；
The nucleotide sequence of P65 genes such as SEQ ID NO：Shown in 3.
It is restriction enzyme BamH I and Sal I that double digestion described in step 1) uses respectively, in 37 DEG C of double enzymes
Cut 3h.
Step 1) uses the overnight connection that T4DNA ligases carry out with the overnight connection described in step 2).
Double digestion described in step 2) uses restriction enzyme Sal I and Hind III respectively, in 37 DEG C of double digestions
3h。
What the race electrophoresis described in step 4) cut glue is the Ago-Gel that mass concentration is 1%~5%.
The beneficial effects of the invention are as follows：
1, it is apparently higher than individual gene using the immunogenicity of fusion P46-P65 prepared by preparation method of the present invention
Immunogenicity has descended solid foundation for development of new mycoplasma hyopneumoniae gene vaccine pad；
2, fusion P46-P65 is lower-cost genetic resources, the subunit vaccine based on P46-P65 fusions
Expression quantity is higher, is easy to purify and prepare, manufacturing cost is relatively low compared with conventional inactivated vaccine and attenuated vaccine；
3, P46 the and P65 genes used in the present invention, directly according to the code area sequence issued in NCBI GeneBank
Row, specific primer of the design containing corresponding restriction enzyme site.The PCR product and the bis- enzymes of prokaryotic expression carrier pET-28a of P46 genes
It cuts and connects, structure recombinant expression carrier pET-28a-P46；Recombinant expression carrier pET-28a-P46 and P65 gene is enabled again
PCR product double digestion simultaneously connects, direct construction recombinant expression carrier pET-28a-P46-P65.Advantage is need not to use
Linker connection P46 and P65 genes are directly over double digestion connection and recombinant expression carrier pET-28a-P46-P65 just can be obtained.
Construction of recombinant vector process is compared to using Linker linkers because being connect again with prokaryotic expression carrier, process is simple and easy
In obtaining, expending the time significantly reduces；
4, the purification process of P46-P65 fusions uses His-Ni2+It is purified, the flushing of foreign protein and purpose egg
White acquisition, it is only necessary to which the imidazole solution that different solubility are added is rinsed, and principle is according to ion-exchange chromatography.It compares
It is more easily understood, and consumes in AKTA FPLC (Amersham Biosciences UPC-900) process operating procedures and principle
Costly power is less, and time consumption is less, it is only necessary to 4h or so；
5, it is tested by indirect ELISA, the results show that the fusion protein P46-P65 for expressing and purifying by the present invention
Immunogenicity is higher than in 100-1000 times of P97R1-Linker-P36-Linker-P46 fusion proteins；
6 and the present invention also carried out challenge test and demonstrated, the protection of the subunit vaccine of P46-P65 fusions
Rate is higher.There is lung tissue's significant protective effect to mouse.
Description of the drawings
Fig. 1 is P65 gene PCR product schematic diagrames；
Fig. 2 is P46 gene PCR product schematic diagrames；
Fig. 3 is pET-28a-P46-P65 double digestions verification schematic diagram；
Fig. 4 is that the supernatant of escherichia coli P46-P65 protein expressions and precipitation is induced to run glue schematic diagram；
Fig. 5 is Western-blotting and SDS-PAGE detection figures；
Fig. 6 is 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.
Wherein, in attached drawing, M represents protein molecular quality standard, and 1 represents unloaded expression precipitation, and 2 represent in unloaded expression
Clearly, 3 induction 6h recombinant expression carriers precipitation is represented, 4 represent induction 6h recombinant expression carrier supernatants, and 5 represent P65 albumen, and 6 represent
P46 albumen, 7 represent P46-P65 albumen, and A represents the Western-blotting analysis charts of recombinant protein, and B represents recombinant protein
SDS-PAGE analysis charts.
Specific implementation mode
The fusion of the present invention obtains as follows：First according to the P46 and P65 announced on Genebank
Gene, design primer simultaneously carry out PCR amplification.And the PCR product of P46 genes and prokaryotic expression plasmid pET-28a are used respectively
Restriction enzyme BamH I and Sal I is connected overnight in 37 DEG C of double digestion 3h, and using T4DNA ligases.Structure recombinant expression
Carrier pET28a-P46.The PCR product of P65 genes and recombinant expression carrier pET28a-P46 are used into restriction enzyme respectively
Sal I and Hind III, it is connected overnight in 37 DEG C of double digestion 3h, and using T4DNA ligases, builds the recombination containing fusion
Expression vector pET28a-P46-P65, and by external mutating technology, three TGA codons in P46 genes are changed into TGG
Codon, the recombinant expression carrier that can be expressed in prokaryotic cell to structure.Melt gene the invention also includes this and exist
Prepare the application in mycoplasma hyopneumoniae recombinant vaccine.
One, the structure of P46-P65 fusions is described in detail below：
1, the design of primer, as shown in table 1：
Table 1
It is restriction enzyme site to have underscore part in table 1 in primer sequence, and primer is that have the limited public affairs of Shanghai biotechnology
Department's synthesis.
2, the amplification of P46 and P65 genes
Using 168 low virulent strain of mycoplasma hyopneumoniae as template, using specific primer in table 1, P46 (ID are carried out：
And P65 (ID 16186839)：16186824) specific amplification of gene.PCR system is as shown in table 2, PCR product such as Fig. 1 and figure
Shown in 2.
Table 2
The amplification program of P46 is：94℃4min；94 DEG C of 30s, 64.1 DEG C of 30s, 70min 1min, 25 cycles；72 DEG C are prolonged
Stretch 10min.It is detected by 1% Ago-Gel, obtains the segment of 1260bp length.The amplification program of P65 is：94℃
4min；94 DEG C of 30s, 65 DEG C of 30s, 70min 1min, 25 cycles；72 DEG C of extension 10min.It is examined by 1% Ago-Gel
It surveys, obtains the segment of 963bp length.
3, the structure of recombinant expression carrier pET-28a-P46-P65
According to P46 the and P65 genes announced on Genebank, design primer simultaneously carries out PCR amplification.And by P46 genes
PCR product and prokaryotic expression plasmid pET-28a use I double digestion of restriction enzyme BamH I and Sal respectively, and stay overnight connection.
Build recombinant expression carrier pET28a-P46.The PCR product of P65 genes and recombinant expression carrier pET-28a-P46 are made respectively
With III double digestion of restriction enzyme Sal I and Hind, and stay overnight connection.Build the recombinant expression carrier containing fusion
PET-28a-P46-P65, and by external mutating technology, three TGA codons in P46 genes are changed into TGG codons,
The recombinant expression carrier that can be expressed in prokaryotic cell to structure.Table can be carried out in prokaryotic cell to obtain
The fusion P46-P65 reached.By PCR verifications, double digestion and sequencing analysis verification, it was demonstrated that recombinant expression carrier pET-28a-
P46-P65 is built successfully, and referring to Fig. 3, pET-28a-P46-P65 double digestions verify schematic diagram.
The structure of recombinant expression carrier pET-28a-P46 and pET-28a-P65 are (for the negative control in immunization experiment
Group)
According to P46 the and P65 genes announced on Genebank, design primer simultaneously carries out PCR amplification.The amplification program of P46
For：94℃4min；94 DEG C of 30s, 64.1 DEG C of 30s, 70min 1min, 25 cycles；72 DEG C of extension 10min.The amplification program of P65
For：94℃4min；94 DEG C of 30s, 65 DEG C of 30s, 70min 1min, 25 cycles；72 DEG C of extension 10min.And by P46 genes
PCR product and prokaryotic expression plasmid pET-28a use I double digestion of restriction enzyme BamH I and Sal respectively, and stay overnight connection.
Build recombinant expression carrier pET28a-P46.And by external mutating technology, three TGA codons in P46 genes are changed
For TGG codons, the pET-28a-P46 recombinant expression carriers that can be expressed in prokaryotic cell to structure.By P65 bases
The PCR product and recombinant expression carrier pET-28a of cause use III double digestion of restriction enzyme Sal I and Hind respectively, and overnight
Connection builds the recombinant expression carrier pET-28a-P65 containing fusion.Pass through PCR verifications, double digestion and sequencing point respectively
Analysis verification, it was demonstrated that recombinant expression carrier pET28a-P46 and pET28a-P65 are built successfully.
Two, the prokaryotic expression of recombinant expression carrier pET-28a-P46-P65
1, the conversion of plasmid
By the expression vector pET-28a-P46-P65 containing mycoplasma hyopneumoniae P46-P65 fusions, (it is anti-that card receives mycin
Property) conversion e. coli bl21 (DE3) cell.It is as follows：
1) the recombinant expression plasmid pET-28a-P46-P65 for taking 50 μ L is added to the e. coli bl21 competence of 100 μ L
In cell, mixing is flicked.30min is stood on ice.
2) the heat shock 30s in 42 DEG C of water-baths.
3) ice bath stands 2min.
4) 37 DEG C of 180rpm spread cultivation 1h.
5) cell is coated on the LB solid plates containing KN chloramphenicol resistances after taking 100 μ L to spread cultivation.37 DEG C of culture 12-16h.
2, prokaryotic expression
Picking colony spreads cultivation in LB culture mediums, uses plasmid extraction kit (the complete limited public affairs of formula gold biotechnology
Department) extraction plasmid, and verified.Correct bacterial strain, which will be verified, takes 10 μ L to carry out the 12-16h that spreads cultivation, and the bacterium solution that spreads cultivation of 1mL is taken to add
Enter into the LB culture mediums of 100mL, the IPTG of 1mmol/L is added in 16 DEG C of conditions until OD600 reaches 0.4 in 37 DEG C of culture 3h
Lower culture 22h.By the thalline after induction under the conditions of 4 DEG C of 8000rpm, 10min is centrifuged, abandons supernatant, collects thalline.It will collect
Thalline is resuspended using the ice-cold physiological saline of 5mL, and carries out ultrasonication, and break-up frequency is work 8s, interval 10s.Until
The liquid of clear is presented in thalline.Broken liquid is centrifuged into 10min under the conditions of 4 DEG C of 12000rpm, collects supernatant precipitation.
Prove that fusion protein has carried out solubility expression by SDS-PAGE and Westernning-blotting verifications.As a result such as Fig. 4
It is shown.
Three, the purifying of P46-P65 fusion proteins
With the P46-P65 fusion proteins that soluble form is expressed, His-Ni is used2+Column is purified.Because pET-28a is band
There is the prokaryotic expression carrier of histidine tag, after recombinant expression carrier pET-28a-P46-P65 expression, fusion protein P46-P65
With histidine tag, His-Ni can be used2+Column is purified.Purification step is as follows：
1) broken liquid under the conditions of 4 DEG C of 12000rpm will be centrifuged 10min, and collects supernatant.
2) supernatant is placed in His-Ni2+In column, it is incubated 3h on ice；Shell egg white liquor is released,
3) the Wash buffer wash buffer nickel columns for using 25mmol/L imidazole concentrations, are rinsed 4 times；
4) it finally uses the Elution Buffer buffer solutions that imidazole concentration is 500mmol/L to be incubated 10min, and collects punching
Washing lotion, Elution Buffer flushing liquors are to purify P46-P65 fusion protein solution.
5) purification step of single albumen P46 and P65 are the same as fusion protein P46-P65.As a result through SDS-PAGE and
Westernning-blotting is verified, as shown in Figure 5.
Four, the biological experiment of subunit vaccine and control group vaccine on mouse immune efficacy of the present invention
1, the immune programme of BALB/C mice
The Balb/c female experimental mouse of weight about 25g are divided into three groups, are divided into fusion protein injection experimental group, single albumen
Experimental group and control group are injected, every group each 50, recombination fusion protein and each 100 μ L (recombinations of single albumen is injected intraperitoneally in experimental group
Albumen total content is 50 μ g), 100 μ L of control group intraperitoneal injection of saline.By the destination protein calculated and adjuvant 1：1 (body
Product ratio) mixing emulsifies and injects.The immune period is 7d, is immunized three times altogether, lasts 21d.One exempts to use Freund's complete adjuvant, and two exempt from
Exempt to use incomplete Freund's adjuvant with three.Three exempt from terminate after a week, mouse docking takes blood, using purifying recombinant protein as resist
It is anti-to carry out indirect ELISA experiment detection mice serum as secondary antibody as primary antibody, HRP horseradish peroxidases for original, mice serum
Body potency, experimental group data/control group data (P/N) are more than 2 for the positive；It is on the contrary then for feminine gender.
The mice group of 3 mycoplasma hyopneumoniae recombinant subunit vaccine of table is tested
2, mouse antibodies are horizontal
It is tested using indirect ELISA, measures mice serum antibody titer.Using purifying P46, P65 and P46-P65 as
Antigen 4 DEG C of incubations overnight, and use mice serum as 37 DEG C of incubation 1h of primary antibody, HRP horseradish peroxidases are as secondary antibody 37
DEG C it is incubated 1h, developing solution TMB is used to develop the color 0.5h.Use spectrophotometer analysis experimental result.The results are shown in Table 4.
ELISA results are immunized in 4 P46-P65 recombinant proteins of table
3, the determination of toxic dose is attacked
Mhp168 low virulent strain bacterium solutions are subjected to gradient dilution (0~1 × 10 using mycoplasma special culture media10CCU/mL),
In addition the mouse for taking health, is divided into 7 groups, every group 10, each dilution gradient takes 600 μ L bacterium solution intraperitoneal injection of mice.Mouse exists
The disease symptom being inoculated with after Mhp168 low virulent strain bacterium solutions：It loses the appetite after being inoculated with 7d, the hair of wing of nose both sides is fallen, living
Move slow, spirit is not good etc., after dissection, lung tissue's obvious fibrosis and without color.In inoculation record experiment after a week
Group and the health status of control group (physiological saline) mouse simultaneously combine anatomical results, and toxic dose is attacked in determination.As shown in table 5.
5 Mhp168 low virulent strains of table cause a disease Dose Results
4, challenge test
Experimental mice three exempt from after one week, according to ELISA experimental results, select antibody titer higher 50 small
Mouse, with maximum pathogenic dosage 1 × 1010The Mhp168 low virulent strain bacterium solutions of CCU/mL inject mouse, and every mouse peritoneal injects 600 μ L
Carry out challenge test；In addition health group and injecting normal saline group are chosen as negative control, every group 50, every is injected 600 μ
The Mhp168 low virulent strain bacterium solutions of L.The incidence of mouse is recorded after being inoculated with 1 week and combines anatomical results, calculates protective rate.Such as table
Shown in 6.
The protective rate of 6 P46-P65 antigen-4 fusion protein gene engineered vaccines of table
5, the Electronic Speculum observation of mouse lung histotomy
The mouse of inoculation subunit vaccine is chosen, abdomen dissection takes lung tissue to make ultra-thin section.Electronic Speculum result figure 6,
Shown in Fig. 7.Fig. 6 is challenge viral dosage without vaccine protection group mouse lung transmission electron microscope picture, and Fig. 7 is challenge viral dosage vaccine protection group
Mouse lung transmission electron microscope picture.Shown in steps are as follows：
Preceding fixation：2-4h or more is fixed before 4% glutaraldehyde.
(1) phosphate buffer of 1/15M embathes 2 times, each 20min.
(2) 1% osmic acids fix 1-2h.
(3) phosphate buffer of 1/15M embathes 2 times, each 20min.
(4) acetone is dehydrated step by step, 50%, 70%, 80%, 90%, 100% (2 times), each 10-15min, second
The dehydration of 100% acetone returns to room temperature, other steps are carried out in 4 DEG C of refrigerators.
(5) it is impregnated with：1:1 (pure acetone：Embedding medium)：1h, 37 DEG C.
1:3 (pure acetones：Embedding medium)：3h or more is stayed overnight, 37 DEG C.
Pure embedding medium is impregnated with 5h or more, 37 DEG C.
(6) it embeds
(7) it polymerize：37 DEG C of incubator 12h, 60 DEG C of 36-48h (adding 4 DEG C of preservations after dry ball).
(8) ultra-thin section：Thickness 50nm or so.
(9) it dyes：Uranium acetate and the dual dye method of lead plumbate.
(10) projection Electronic Speculum observation.
Claims (4)
1. a kind of i (mycoplasma hyopneumoniae) vaccine, it is characterised in that：The vaccine is fusion protein P46-P65, encoding fusion protein
Nucleotide sequence such as sequence table SEQ ID NO：Shown in 1, fusion protein P46-P65 uses His-Ni2+It is purified；It is described to melt
Hop protein is prepared via a method which：
1）The PCR product of P46 genes and prokaryotic expression plasmid pET-28a are distinguished into double digestion, and stay overnight connection, structure recombination table
Up to carrier pET28a-P46；
2）By the PCR product of P65 genes and recombinant expression carrier pET28a-P46 double digestions, and connection is stayed overnight, structure recombination table
Up to carrier pET28a-P46-P65；
3）By external mutating technology, three TGA codons in P46 genes are changed into TGG codons, to which structure can be with
The recombinant expression carrier pET28a-P46-P65 expressed in prokaryotic cell, and the expressed fusion protein in prokaryotic cell
P46-P65；
4）The prokaryotic cell thalline after expression is collected, the ice-cold physiological saline of the thalline of collection is resuspended, and it is broken to carry out ultrasound
It is broken, broken liquid is centrifuged into 10min under the conditions of 4 DEG C of 12000rpm, collects supernatant；Supernatant is placed in His-Ni2+In column, on ice
It is incubated 3h；Release shell egg white liquor；Using the Wash buffer wash buffer nickel columns that imidazole concentration is 25mmol/L, 4 are rinsed
It is secondary；It finally uses the Elution Buffer buffer solutions that imidazole concentration is 500mmol/L to be incubated 10min, and collects flushing liquor,
Elution Buffer flushing liquors are to purify P46-P65 fusion protein solution；
Wherein, the nucleotide sequence of P46 genes such as SEQ ID NO：Shown in 2；
The nucleotide sequence of P65 genes such as SEQ ID NO：Shown in 3；
The PCR amplification program of P46 is：94℃ 4min；94 DEG C of 30s, 64.1 DEG C of 30s, 70min 1min, 25 cycles；72℃
Extend 10min；
The PCR amplification program of P65 is：94℃ 4min；94 DEG C of 30s, 65 DEG C of 30s, 70min 1min, 25 cycles；72℃
Extend 10min.
2. a kind of i (mycoplasma hyopneumoniae) vaccine according to claim 1, it is characterised in that：Step 1）Described in double digestion
Restriction enzyme BamH I and Sal I are used respectively, in 37 DEG C of double digestion 3h.
3. a kind of i (mycoplasma hyopneumoniae) vaccine according to claim 1, it is characterised in that：Step 1）With step 2）Described in
Overnight connection use T4 DNA ligases carry out overnight connection.
4. a kind of i (mycoplasma hyopneumoniae) vaccine according to claim 1, it is characterised in that：Step 2）Described in double digestion
Restriction enzyme Sal I and Hind III are used respectively, in 37 DEG C of double digestion 3h.
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