CN107828810B - A kind of fusion and its preparing the application in pneumovax - Google Patents

A kind of fusion and its preparing the application in pneumovax Download PDF

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CN107828810B
CN107828810B CN201711016027.XA CN201711016027A CN107828810B CN 107828810 B CN107828810 B CN 107828810B CN 201711016027 A CN201711016027 A CN 201711016027A CN 107828810 B CN107828810 B CN 107828810B
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ply
linker
mycoplasma pneumoniae
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fusion
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CN107828810A (en
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李晓菊
文良均
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Jiaxing Maiwei Metabolic Biotechnology Co., Ltd.
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Wuhan Mai Tver Biological Technology Co Ltd
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Abstract

The amalgamation and expression of the present invention dominant antigen region P1a-P30a of mycoplasma pneumoniae P1 and P30, and utilize the portion gene amalgamation and expression PLY-P1a-P30a of the gene of pneumolysin PLY and mycoplasma pneumoniae P1 and P30, the immunogenicity of PLY-P1a-P30a albumen is significantly higher than the immunogenicity of P1a-P30a, not only increase the immunogenicity of mycoplasma pneumoniae subunit P1a-P30a, and the immunization with streptococcus pneumonia, the toxicity to mouse without clinical observation.Fusion PLY-P1a-P30a is lower-cost genetic resources, the subunit vaccine expression quantity of PLY-P1a-P30a fusion is high, it is easy to purify and prepare, 2 years or more stability and immunocompetence can be kept at -80 DEG C, preparation cost significantly reduces compared with conventional inactivated vaccine and attenuated vaccine, lays the foundation for the research and development of mycoplasma pneumoniae and Streptococcus pneumoniae vaccine.

Description

A kind of fusion and its preparing the application in pneumovax
Technical field
The present invention relates to technical field of pharmaceutical biotechnology, and in particular to a kind of streptococcus pneumonia is melted with mycoplasma pneumoniae gene Close the preparation method and application of gene, pneumolysin and its preparation and application as immunopotentiator.
Background technique
Pneumonia is to occur in terminal air flue, the inflammation of alveolar and interstitial lung, is common respiratory tract infectious disease, generally By failing thoroughly to be treated after the infection of the upper respiratory tract or patients with low immunity, caused by causing inflammation downlink to spread.WHO statistics Data show (GURGEL R Q, BEZERRA P G, DUARTE MDO C, et al.Relative frequency, Possible risk factors, viral codetection rates, and seasonality of respiratory syncytial virus among children with lower respiratory tract infection in Northeastern Brazil [J] .Medicine (Baltimore), 2016,95 (15): e3090.), it is in recent years, acute to exhale The death rate for inhaling road infection has been only second to cardiovascular disease, occupies the 2nd, wherein lower respiratory tract infection, is led in world wide Cause children and the elderly's high incidence and high mortality principal element, the whole world every year because the children about 1,900,000 of pneumonia death~ 2200000 (progress [J] Chinese Sci-tech Journal Database (digest version) doctors of Lu Lan kingfisher Mycoplasma Pneumoniae Infection in Children treatment Medicine health, 2015, (4): 268-269).Common pathogen has streptococcus pneumonia, mycoplasma pneumoniae, the cheek in human respiratory infection Adenositis virus and Respiratory Syncytial Virus(RSV) etc..(SONG Q, XU B P, SHEN K L.Effects of bacterial and Viral co-infections of Mycoplasma pneumoniae in children:analysis report from Beijing Children's Hospital between2010and 2014 [J] .Int J Clin Exp Med, 2015,8 (9): 15666-15674), and most of viruses have had corresponding vaccine, but so far, mycoplasma pneumoniae (Mycoplasma pneumoniae, Mp) vaccine is still in conceptual phase.As Macrocyclolactone lactone kind medicine is answered extensively in clinical With having more and more antibody-resistant bacterium and generate, and have growing number of trend (SAKAI T, ISHIDA T, ARITA M, et al.A study on the clinical course and antimicrobial susceptibility of Mycoplasma pneumoniae in a community hospital [J] .Kansenshogaku Zasshi, 2015,89 (4): 458-464).Therefore, research and development mycoplasma pneumoniae and the vaccine of streptococcus pneumonia have prevention and control pneumonia critically important Effect.
Mycoplasma pneumoniae pneumonia accounts for the 5%~40% of community acquired pneumonia, and mainly teenager is caused to infect (Atkinson TP,Balish MF,Waites KB.Epidemiology,clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections[J] .FEMS Microbiol Rev,2008,32(6):956-973).The preventive effect of the inactivated vaccine of MP only has 36% at present, (LINCHEVSKI I, KLEMENT E, NIR-PAZ R.Mycoplasma pneumoniae vaccine protective efficacy and adverse reactions-systematic review and meta-analysis[J] .Vaccine, 2009,27 (18): 2437-2446).Since there are remaining virulence and pathogenic sexual factor not yet in people for attenuation seedling It is used on body, therefore, the direction mainly researched and developed at present is subunit vaccine.Since MP causes the first step of cellular damage to be to stick In host cell surface, therefore the emphasis of its vaccine research is that and inhibits sticking for pathogen.Stick relevant surface to MP Albumen P1 and P30 is particularly significant in its pathogenic course, and the encoding gene of the two adhesion proteins be in it is highly conserved Region, in the correlative study to both albumen, SCHURWANZ etc. chooses the strongest part of P1 immunogenicity and P30 fusion Express albumen, the effect of serum after being obtained after protein immunization cavy close to MP whole cell immune guinea pig.(SCHURWANZ N, JACOBS E, DUMKE R.Strategy to createchimeric proteins derived from functional adhesin regions of Mycoplasma pneumoniae for vaccine development[J].Infect Immun, 2009,77 (11): 5007-5015).Although the subunit vaccine safety for the research and development of P1 and P30 albumen is preferable, But it does not retain a more complete pathogenic particles, the poor situation of generally existing immunogenicity.
Pneumolysin (Pneumolysin, PLY) is streptococcus pneumonia (Streptococcus Pneumoniae, SP) secretion the important synocytotoxin of one kind.PLY is by the molecular weight that 471 amino acid form A kind of multifunctional protein of 53kDa.PLY while enhancing pathogen invasiveness, can complement activation classical pathway, induce macrophage Cell and monocyte etc. generate cell factor, activate body immune system, (Karmakar M, Katsnelson M, Malak HA,Greene NG,Howell SJ,Hise AG,Camilli A,Kadioglu A,Dubyak GR,Pearlman E.Neutrophil IL-1βprocessing induced by pneumolysin is mediated by the NLRP3/ ASC inflammasome and caspase-1activation and is dependent on K+efflux.The Journal of Immunology,2015,194(4):1763–1775).Therefore PLY is that the emphasis of Streptococcus pneumoniae vaccine is waited Antigen is selected, we also use it as the immunopotentiator of mycoplasma pneumoniae antigen, not only improve current mycoplasma pneumoniae vaccine Immunocompetence, and large range of prevention pneumonia.
Summary of the invention
The object of the present invention is to provide the fusions of a kind of effective prevention mycoplasma pneumoniae and streptococcus pneumoniae infection Preparation method and application.Using the antigenicity and Immune-enhancing effect of streptococcus pneumonia PLY albumen, in prevention streptococcus pneumonia The immune effect of mycoplasma pneumoniae can be enhanced simultaneously.
Object of the present invention is to what is be achieved through the following technical solutions:
A kind of fusion of mycoplasma pneumoniae and streptococcus pneumonia, it is characterised in that: fusion is mainly by PLY, P1a It is formed with P30a three parts gene order, PLY is pseudopneumolysin gene sequence, and P1a and P30a are respectively lung Section is immunized in the advantage of scorching mycoplasma P1 and P30 gene, and three is connected with each other using Linker and obtains mycoplasma pneumoniae and lung Scorching streptococcic fusion PLY-P1a-P30a.
The construction method of a kind of mycoplasma pneumoniae and the fusion of streptococcus pneumonia, it is characterised in that: pneumonia branch is former The portion gene P30a (331-792 bit base) of portion gene P1a (3478-4047 bit base) and the P30 of body P1 passes through Linker connection, then obtains PUC19-P1a-Linker-P30a and PUC19-PLY-Linker-P1a- with PLY Gene Fusion Linker-P30a.Respectively by carrier through NcoI and XhoI digestion, and it is connected respectively on pET30a carrier.Building is recombinated Plasmid pET30a-
P1a-Linker-P30a and pET30a-PLY-Linker-P1a-Linker-P30a.
Wherein:
The nucleotide sequence of P1a gene is as shown in SEQ ID NO:1；
The nucleotide sequence of P30a gene is as shown in SEQ ID NO:2；
The nucleotide sequence of PLY gene is as shown in SEQ ID NO:3；
The nucleotide sequence of P1a-P30a gene is as shown in SEQ ID NO:4；
The nucleotide sequence of PLY-P1a-P30a gene is as shown in SEQ ID NO:5；
The base sequence of above-mentioned Linker are as follows: GGCAGCGGCAGCGGCAGCGGCAGC.
The invention also includes above-mentioned PLY-P1a-P30a fusions to prepare mycoplasma pneumoniae and Streptococcus pneumoniae vaccine In application.
Beneficial effects of the present invention: the 1, present invention a kind of the P1a-P30a albumen and PLY-P1a-P30a, PLY- of fusion The immunogenicity of P1a-P30a albumen is significantly higher than the immunogenicity of P1a-P30a, not only increases mycoplasma pneumoniae subunit The immunogenicity of P1a-P30a, and the immunization with streptococcus pneumonia, the toxicity to mouse without clinical observation, to develop Novel mycoplasma pneumoniae and Streptococcus pneumoniae vaccine are laid a good foundation.2, fusion PLY-P1a-P30a is lower-cost base Because of resource, the subunit vaccine expression quantity based on PLY-P1a-P30a fusion is high (account for total protein 5%), is easy to purify With preparation (purification efficiency reaches 55%), preparation cost is significantly reduced compared with conventional inactivated vaccine and attenuated vaccine.3, melt Hop protein PLY-P1a-P30a can keep 2 years or more stability and immunocompetence at -80 DEG C.
Detailed description of the invention
Fig. 1: being techniqueflow block diagram of the invention；
Fig. 2: being the building schematic diagram of recombinant plasmid pET30a-P1a-Linker-P30a in the present invention；
Fig. 3: being the building schematic diagram of recombinant plasmid pET30a-PLY-Linker-P1a-Linker-P30a in the present invention；
Fig. 4: being the recombinant plasmid pET30a-P1a-Linker-P30a and pET30a-PLY-Linker- in the present invention The prokaryotic expression and digestion glue figure of P1a-Linker-P30a.Description of symbols in figure: M: molecular weight of albumen；1:pET30a-PLY- Linker-P1a-Linker-P30a (BL21) is not induced；37 DEG C of 2:pET30a-PLY-Linker-P1a-Linker-P30a IPTG induces 5h；3:pET30a-PLY-Linker-P1a-Linker-P30a digestion；4:pET30a-P1a-Linker-P30a37 DEG C IPTG induces 5h；37 DEG C of IPTG digestions of 5:pET30a-P1a-Linker-P30a；
Fig. 5: prepared by the method recombinant subunit vaccine: P1a-P30a and PLY-P1a-P30a and to phosphate The detection of antibody level after the immune BALB/c mouse of buffer (PBS) in serum.
Specific embodiment
Embodiment 1:
The building of 1.pET30a-P1a-Linker-P30a fusion
Using mycoplasma pneumoniae M129 bacterial strain as sequence template, P1a and P30a choose P1a-linker-P30 sequence respectively The 3478-4047 bit base of M129 bacterial strain P1 and the 331-792 bit base of P30, are connected by Linker, by raw work bioengineering Co., Ltd's synthesis is on PUC19-P1a-Linker-P30a carrier, NcoI and XhoI digestion, and is connected to pET30a carrier On.
PUC19-P1a-Linker-P30a digestion system is as follows:
PET30a carrier digestion system is as follows:
PUC19-P1a-Linker-P30a digestion system and pET30a carrier digestion system pass through 1% in 37 DEG C of reaction 2h Agarose gel electrophoresis detects digestion as a result, purification and recovery target fragment.Carrier is connected with target fragment with T4 ligase, Reaction system is as follows:
Carrier 1μL(50ng)
Target fragment 1μL(100ng)
Connect buffer 1μL
T4 ligase 0.5μL
Distilled water 6.5μL
Recombinant plasmid pET30a-P1a-Linker-P30a is obtained, structure chart is as shown in Figure 2.
2. the building of recombinant plasmid pET30a-PLY-Linker-P1a-Linker-P30a
P1a and P30a is using mycoplasma pneumoniae M129 bacterial strain as sequence mould in PLY-Linker-P1a-Linker-P30a sequence Plate, chooses the 3478-4047 bit base of M129 bacterial strain P1 and the 331-792 bit base of P30 respectively, and PLY chooses streptococcus pneumonia R6 bacterial strain is sequence template by Linker connection, by Sheng Gong bioengineering Co., Ltd gene chemical synthesis in PUC19-PLY- On Linker-P1a-Linker-P30a carrier, through NcoI and XhoI digestion, and on connection and pET30a carrier.
PUC19-PLY-Linker-P1a-Linker-P30a digestion system is as follows:
PET30a carrier digestion system is as follows:
PUC19-PLY-Linker-P1a-Linker-P30a digestion system and pET30a carrier digestion system are anti-in 37 DEG C 2h is answered, digestion is detected as a result, purification and recovery target fragment by 1% agarose gel electrophoresis.Carrier and target fragment T4 Ligase connection, reaction system are as follows:
Carrier 1μL(50ng)
Target fragment 1μL(100ng)
Connect buffer 1μL
T4 ligase 0.5μL
Distilled water 6.5μL
Recombinant plasmid pET30a--PLY-Linker-P1a-Linker-P30a is obtained, structure chart is as shown in Figure 3.
Case study on implementation 2: the prokaryotic expression of recombinant plasmid pET30a-P1a-Linker-P30a
The expression plasmid pET30a-P1a-Linker-P30a of P1a-P30a fusion (it is anti-to be blocked that by the conversion of plasmid Property) conversion e. coli bl21 competent cell.
Concrete operations are as follows:
1) it takes 50 μ L e. coli bl21 competent cell suspensions to be transferred in sterile 1.5ml EP pipe, 1 μ L is added and connects It practices midwifery object, gently rotates to mix content, place 30min on ice.
2) heat shock 90s (plasmid is allowed to enter competent cell) in 42 DEG C of water-baths.
3) it takes out and places 5min (competent cell is allowed to close) on ice.
4) every pipe adds 200 μ L LB culture mediums.Culture medium is heated up to 37 DEG C with water-bath, centrifuge tube is then transferred to 37 On DEG C shaking table, 60min is incubated, bacteria resuscitation is made.To reach effective conversion, revolving speed 180r/min.
5) competent cell for taking 100 μ L to convert is transferred on the LB agar plate containing corresponding antibiotic, with sterile elbow The cell of conversion is uniformly applied to agar plate surface by glass rod.
6) plate is set into 37 DEG C of cultures, until liquid is absorbed, is then inverted plate culture, 12~16h may occur in which bacterium colony. Case study on implementation 3: the prokaryotic expression of recombinant plasmid pET30a-PLY-Linker-P1a-Linker-P30a
The conversion of plasmid is by the expression plasmid pET30a-PLY-Linker-P1a- of PLY-P1a-P30a fusion Linker-P30a (blocking that resistance) converts e. coli bl21 competent cell.
Concrete operations are as follows:
1) it takes 50 μ L e. coli bl21 competent cell suspensions to be transferred in sterile 1.5ml EP pipe, 1 μ L is added and connects It practices midwifery object, gently rotates to mix content, place 30min on ice.
2) heat shock 90s (plasmid is allowed to enter competent cell) in 42 DEG C of water-baths.
3) it takes out and places 5min (competent cell is allowed to close) on ice.
4) every pipe adds 200 μ L LB culture mediums.Culture medium is heated up to 37 DEG C with water-bath, centrifuge tube is then transferred to 37 On DEG C shaking table, 60min is incubated, bacteria resuscitation is made.To reach effective conversion, revolving speed 180r/min.
5) competent cell for taking 100 μ L to convert is transferred on the LB agar plate containing corresponding antibiotic, with sterile elbow The cell of conversion is uniformly applied to agar plate surface by glass rod.
6) plate is set into 37 DEG C of cultures, until liquid is absorbed, is then inverted plate culture, 12~16h may occur in which bacterium colony.
Prokaryotic expression picking single colonie expands culture, and using small amount plasmid extraction kit, (Beijing Tiangeng biochemical technology has Limit company) it extracts the progress digestion identification of its plasmid and saves bacterium solution.After identification is correct, by the bacterium solution of preservation according to the body of 1:1000 Product ratio is recovered.Next day is induced according to the volume ratio of 1:100.In final concentration of 0.8mM IPTG, 37 DEG C and 180r/ It is induced under conditions of min.3h, 4h, 5h and 6h sample 1mL before induction and after induction respectively, and to convert pET-30a load The bacteria-induction product of the E.coli BL21 (DE3) of body is as blank control.After 12000r/min is centrifuged 1min, 100 μ L are added ddH2After O resuspended bacterium solution, 25 μ L 5 × Loading Buffer are added, 10min, ice bath 10min are boiled in 100 DEG C of boiling water Afterwards, sample analysis is carried out by 12%SDS-PAGE, determines whether the protokaryon albumen of recombination expresses.Carrying out SDS-PAGE detection When, before loading, all sample 10000r/min are centrifuged 1min, extract the sample loading for being located at sample liquid level upper layer.In addition, Western Blotting analysis is carried out using the monoclonal antibody of PLY as antibody, whether verifying recombinant protein expresses.Determining protokaryon weight After histone can stablize expression, 100mL recombinant bacterium is induced again, after being crushed by high pressure cracker, 12000r/min centrifugation Supernatant precipitating is boiled sample respectively, carries out 12%SDS-PAGE detection, determine the expression-form of recombinant protein by 10min.
PET30a-P1a-Linker-P30a and pET30a-PLY-Linker-P1a-Linker-P30a are with soluble form Expression is in supernatant.Test result is as shown in Figure 4.
Embodiment 4:pET30a-P1a-Linker-P30a and pET30a-PLY-Linker-P1a-Linker-P30a fusion The pET30a-P1a-Linker-P30a and pET30a-PLY-Linker-P1a- that the purifying of albumen is expressed with soluble form Linker-P30a fusion protein is purified with histidine tag, and purification step is as follows:
(1) by bacterium solution 500mL, 12000r/min the centrifugation 1min after induction 5h, supernatant is discarded, by precipitating Binding Buffer (1/10) is resuspended, and is placed in -80 DEG C of refrigerators, multigelation three times after, be crushed to super-pressure ripple it is limpid, 12000r/min is centrifuged 30min, takes supernatant, 0.22 μm of membrane filtration of supernatant is placed in spare on ice.
(2) 1ml nickel Ago-Gel prepacked column is taken, with 10ml equilibration buffer (the 50mM phosphate-buffered of PH7.4 Liquid NaCl containing 0.5M, 20mM imidazoles).
(3) sample of supernatant 10mL is taken, with the flow velocity loading of 0.5mL/min.
(4) unadsorbed sample, flow velocity 1mL/min are washed away with 15mL equilibration buffer.
(5) it is eluted with 5mL elution buffer (50mM phosphate buffer NaCl containing 0.5M, the 500mM imidazoles of PH7.4) Sample, flow velocity 1mL/min.
(6) it collects sample to dialyse in 4 DEG C, is freeze-dried pET30a-P1a-Linker-P30a and pET30a-PLY- Linker-P1a-Linker-P30a fusion protein, as a result such as Fig. 4.
Embodiment 5: the biological experiment of subunit vaccine and control vaccine of the invention to mouse immune effect
1,30 5-6 week old female BAl BIc/c mouse of the immune programme of BALB/c mouse are divided into 3 groups, respectively PBS (PBS, Formula: 8g NaCl, 0.2g KCl, 1.44g Na are weighed2HPO4、0.24g KH2PO4, it is dissolved in 800mL ddH2In O, pH is adjusted extremely 7.2, it is settled to 1L, autoclaving, room temperature preservation is spare) control group, P1a-P30a and PLY-P1a-P30a immune group, often Group 10, intramuscular injection is immune, is immunized according to shown in table 3, is immunized 2 times altogether, and progress two is exempted from after head exempts from 21d.Exempt from for the first time 2,3,5,6 arteries and veins negative pressure hemostix after epidemic disease, separates serum, and detection Serum Antibody is horizontal.
Table 3: the mouse experiment grouping of mycoplasma hyopneumoniae recombinant subunit vaccine
Group Ingredient Dosage/only Mouse quantity
Group 1 PBS 100 μ L+15% aluminium glue adjuvants 5
Group 2 P1a-P30a 50 μ g+15% aluminium glue adjuvants 5
Group 3 PLY-P1a-P30a 50 μ g+15% aluminium glue adjuvants 5
2, antibody level in mice serum
Using mycoplasma pneumoniae and streptococcus pneumonia whole bacterial protein coated elisa plate (4 μ g/mL), first immunisation is detected respectively The the 2nd, 3,5,6 week Serum Antibody is horizontal afterwards, the results showed that PLY-P1a-P30a immune group be within the 3rd week after the first exemption can produce compared with There is mycoplasma pneumoniae specific antibody in 4th week in high-caliber mycoplasma pneumoniae specific antibody, P1a-P30a.Reinforcement is exempted from After epidemic disease, in addition to phosphate buffer group, other immune group antibody levels are all in significant ascendant trend, PLY-P1a-P30a immune group The specific antibody of generation rises rapidly, reaches higher level, difference extremely significant (P < 0.01, the t- compared with P1a-P30a test).PLY-P1a-P30a immune group have still have it is stronger
In addition, the results showed that PLY-P1a-P30a immune group can produce the pneumonia chain of higher level on the 3rd week after the first exemption Coccus specific antibody, PBS group and P1a-P30a do not occur antibody, after booster immunization, what PLY-P1a-P30a immune group generated Specific antibody rises rapidly, reaches higher level, test result is as shown in Figure 5.
<110>Li Xiaoju
<120>a kind of fusion and its application in pneumovax is being prepared
<160> 5
<170> Patent In version 3.1
<210> 1
<211> 570
<212> DNA
<213>mycoplasma pneumoniae (Mycoplasma pneumoniae, Mp)
<400> 1
gcggcctttc gtggcagttg ggtcaaccgg ttgggccgcg tggagagtgt gtgggatttg 60
aagggggtgt gggcggatca agctcagtcc gactcgcaag gatctaccac caccgcaaca 120
aggaacgcct taccggagca cccgaatgct ttggcctttc aggtgagtgt ggtggaagcg 180
agtgcttaca agccaaacac gagctccggc caaacccaat ccactaacag ttccccctac 240
ctgcacttgg tgaagcctaa gaaagttacc caatccgaca agttagacga cgatcttaaa 300
aacctgttgg accccaacca ggttcgcacc aagctgcgcc aaagctttgg tacagaccat 360
tccacccagc cccagcccca atcgctcaaa acaacgacac cggtatttgg gacgagtagt 420
ggtaacctca gtagtgtgct tagtggtggg ggtgctggag ggggttcttc aggctcaggt 480
caatctggcg tggatctctc ccccgttgaa aaagtgagtg ggtggcttgt ggggcagtta 540
ccaagcacga gtgacggaaa cacctcctcc 570
<210> 2
<211> 462
<212> DNA
<213>mycoplasma pneumoniae (Mycoplasma pneumoniae, Mp)
<400> 2
Aaggaacgcc aagaacagtt agcggaacag ctacaacgca tttctgccca acaagaagag 60
caacaagcgt tagaacaaca agcagctgct gaagcccatg ctgaagcgga agttgaacca 120
gcaccacaac cagtaccagt accacctcaa ccccaagtcc aaattaactt cggtccccgt 180
actggtttcc cacctcaacc cggtatggcg cctcgtccag gtatgccgcc acaccccggt 240
atggctccaa gacctggttt cccacctcaa cccggtatgg cgcctcgtcc aggtatgccg 300
ccacaccccg gtatggctcc aagacctggt ttcccacctc aacccggtat ggcgcctcgt 360
ccaggcatgc cgccacaccc cggtatggct ccaagacctg gtttcccacc tcaacccggt 420
atggcgcctc gtcccggaat gcaaccacca cgtcctggca tg 462
<210> 3
<211> 1416
<212> DNA
<213>streptococcus pneumonia (Streptococcus pneumoniae, SP)
<400> 3
atggctaaca aggctgttaa cgacttcatc cttgctatga actacgacaa gaagaagctt 60
cttactcacc agggtgagag tatcgagaac cgtttcatca aggagggtaa ccagcttcct 120
gacgagttcg ttgttatcga gcgtaagaag cgtagtctta gtactaacac tagtgacatc 180
agtgttactg ctactaacga cagtcgtctt taccctggtg ctcttcttgt tgttgacgag 240
actcttcttg agaacaaccc tactcttctt gctgttgacc gtgctcctat gacttacagt 300
atcgaccttc ctggtcttgc tagtagtgac agtttccttc aggttgagga ccctagtaac 360
agtagtgttc gtggtgctgt taacgacctt cttgctaagt ggcaccagga ctacggtcag 420
gttaacaacg ttcctgctcg tatgcagtac gagaagatca ctgctcacag tatggagcag 480
cttaaggtta agttcggtag tgacttcgag aagactggta acagtcttga catcgacttc 540
aacagtgttc acagtggtga gaagcagatc cagatcgtta acttcaagca gatctactac 600
actgttagtg ttgacgctgt taagaaccct ggtgacgttt tccaggacac tgttactgtt 660
gaggacctta agcagcgtgg tatcagtgct gagcgtcctc ttgtttacat cagtagtgtt 720
gcttacggtc gtcaggttta ccttaagctt gagactacta gtaagagtga cgaggttgag 780
gctgctttcg aggctcttat caagggtgtt aaggttgctc ctcagactga gtggaagcag 840
atccttgaca acactgaggt taaggctgtt atccttggtg gtgaccctag tagtggtgct 900
cgtgttgtta ctggtaaggt tgacatggtt gaggacctta tccaggaggg tagtcgtttc 960
actgctgacc accctggtct tcctatcagt tacactacta gtttccttcg tgacaacgtt 1020
gttgctactt tccagaacag tactgactac gttgagacta aggttactgc ttaccgtaac 1080
ggtgaccttc ttcttgacca cagtggtgct tacgttgctc agtactacat cacttggaac 1140
gagcttagtt acgaccacca gggtaaggag gttcttactc ctaaggcttg ggaccgtaac 1200
ggtcaggacc ttactgctca cttcactact agtatccctc ttaagggtaa cgttcgtaac 1260
cttagtgtta agatccgtga gtgcactggt cttgcttggg agtggtggcg tactgtttac 1320
gagaagactg accttcctct tgttcgtaag cgtactatca gtatctgggg tactactctt 1380
taccctcagg ttgaggacaa ggttgagaac gactaa 1416
<210> 4
<211> 1062
<212> DNA
<213>artificial sequence
<220>
<221> gene
<222>(1) .. (1062)
<223>
<400> 4
atggctataa atccccgtct caccccgtgg acatatcgta atacctcctt ttcctccttc 60
cccttcaccg gcgagaatcc cggcgcctgg gccttcgtca gcgataattc cgccaagggc 120
attaccgccg gctccggctc ccagcagacc acctatgatc ccaccagcac cgaggccgcc 180
ttcaccgcct ccaccacctt tgccttcagc agctatgatt tcgccggcag cgccttctat 240
gatttcgatt tttccaagtt caatccccag acccccacca gcgatcagac cggccagatt 300
acctttaatc cctttggcgg ctttggcttc tccggcgccg ccccccagca gtggaatgag 360
gtcaagaata aggtccccgt cgaggtcgcc caggatccct ccaatcccta tagctttgcc 420
gtcttcttcg tccccagctc cgtcgtctat tatgagcagt tccagagcgg cttcggcttc 480
ccccagcaga gcaccgagtc cggccagaat acctccacca ccggcgccat gtttggcttc 540
aaggtcaaga atgccgaggc cgataccgcc aagggcagcg gcagcggcag cggcagcttt 600
caacgtatac gtgctcaaca agaagaacaa caagcttttg aacaacaagc tgctgctgaa 660
gctcatgctg aagctgaagt tgaacctgct cctcaacctg ttcctgttcc tcctcaacct 720
caagttcaaa taaattttgg tcctcgtact ggttttcctc ctcaacctgg tatggctcct 780
cgtcctggta tgcctcctca tcctggtatg gctcctcgtc ctggttttcc tcctcaacct 840
ggtatggctc ctcgtcctgg tatgcctcct catcctggta tggctcctcg tcctggtttt 900
cctcctcaac ctggtatggc tcctcgtcct ggtatgcctc ctcatcctgg tatggctcct 960
cgtcctggtt ttcctcctca acctggtatg gctcctcgtc ctggtatgca acctcctcgt 1020
cctggtatgc ctcctcaacc tggttttcct cctaaacgtt aa 1062
<210> 5
<211> 2496
<212> DNA
<213>artificial sequence
<220>
<221> gene
<222>(1) .. (2496)
<223>
<400> 5
atggctaaca aggctgttaa cgacttcatc cttgctatga actacgacaa gaagaagctt 60
cttactcacc agggtgagag tatcgagaac cgtttcatca aggagggtaa ccagcttcct 120
gacgagttcg ttgttatcga gcgtaagaag cgtagtctta gtactaacac tagtgacatc 180
agtgttactg ctactaacga cagtcgtctt taccctggtg ctcttcttgt tgttgacgag 240
actcttcttg agaacaaccc tactcttctt gctgttgacc gtgctcctat gacttacagt 300
atcgaccttc ctggtcttgc tagtagtgac agtttccttc aggttgagga ccctagtaac 360
agtagtgttc gtggtgctgt taacgacctt cttgctaagt ggcaccagga ctacggtcag 420
gttaacaacg ttcctgctcg tatgcagtac gagaagatca ctgctcacag tatggagcag 480
cttaaggtta agttcggtag tgacttcgag aagactggta acagtcttga catcgacttc 540
aacagtgttc acagtggtga gaagcagatc cagatcgtta acttcaagca gatctactac 600
actgttagtg ttgacgctgt taagaaccct ggtgacgttt tccaggacac tgttactgtt 660
gaggacctta agcagcgtgg tatcagtgct gagcgtcctc ttgtttacat cagtagtgtt 720
gcttacggtc gtcaggttta ccttaagctt gagactacta gtaagagtga cgaggttgag 780
gctgctttcg aggctcttat caagggtgtt aaggttgctc ctcagactga gtggaagcag 840
atccttgaca acactgaggt taaggctgtt atccttggtg gtgaccctag tagtggtgct 900
cgtgttgtta ctggtaaggt tgacatggtt gaggacctta tccaggaggg tagtcgtttc 960
actgctgacc accctggtct tcctatcagt tacactacta gtttccttcg tgacaacgtt 1020
gttgctactt tccagaacag tactgactac gttgagacta aggttactgc ttaccgtaac 1080
ggtgaccttc ttcttgacca cagtggtgct tacgttgctc agtactacat cacttggaac 1140
gagcttagtt acgaccacca gggtaaggag gttcttactc ctaaggcttg ggaccgtaac 1200
ggtcaggacc ttactgctca cttcactact agtatccctc ttaagggtaa cgttcgtaac 1260
cttagtgtta agatccgtga gtgcactggt cttgcttggg agtggtggcg tactgtttac 1320
gagaagactg accttcctct tgttcgtaag cgtactatca gtatctgggg tactactctt 1380
taccctcagg ttgaggacaa ggttgagaac gacggcagcg gcagcggcag cggcagcgcg 1440
gcctttcgtg gcagttgggt caaccggttg ggccgcgtgg agagtgtgtg ggatttgaag 1500
ggggtgtggg cggatcaagc tcagtccgac tcgcaaggat ctaccaccac cgcaacaagg 1560
aacgccttac cggagcaccc gaatgctttg gcctttcagg tgagtgtggt ggaagcgagt 1620
gcttacaagc caaacacgag ctccggccaa acccaatcca ctaacagttc cccctacctg 1680
cacttggtga agcctaagaa agttacccaa tccgacaagt tagacgacga tcttaaaaac 1740
ctgttggacc ccaaccaggt tcgcaccaag ctgcgccaaa gctttggtac agaccattcc 1800
acccagcccc agccccaatc gctcaaaaca acgacaccgg tatttgggac gagtagtggt 1860
aacctcagta gtgtgcttag tggtgggggt gctggagggg gttcttcagg ctcaggtcaa 1920
tctggcgtgg atctctcccc cgttgaaaaa gtgagtgggt ggcttgtggg gcagttacca 1980
agcacgagtg acggaaacac ctcctccggc agcggcagcg gcagcggcag caaggaacgc 2040
caagaacagt tagcggaaca gctacaacgc atttctgccc aacaagaaga gcaacaagcg 2100
ttagaacaac aagcagctgc tgaagcccat gctgaagcgg aagttgaacc agcaccacaa 2160
ccagtaccag taccacctca accccaagtc caaattaact tcggtccccg tactggtttc 2220
ccacctcaac ccggtatggc gcctcgtcca ggtatgccgc cacaccccgg tatggctcca 2280
agacctggtt tcccacctca acccggtatg gcgcctcgtc caggtatgcc gccacacccc 2340
ggtatggctc caagacctgg tttcccacct caacccggta tggcgcctcg tccaggcatg 2400
ccgccacacc ccggtatggc tccaagacct ggtttcccac ctcaacccgg tatggcgcct 2460
cgtcccggaa tgcaaccacc acgtcctggc atgtaa 2496

Claims (3)

1. the fusion of a kind of mycoplasma pneumoniae and streptococcus pneumonia, which is characterized in that its nucleotide sequence such as SEQ ID NO : shown in 5.
2. the construction method of mycoplasma pneumoniae as described in claim 1 and the fusion of streptococcus pneumonia, it is characterised in that:
It is P30a base by the 331-792 bit base that the 3478-4047 bit base of mycoplasma pneumoniae P1 is P1a gene and P30 Because then obtaining PUC19-P1a-Linker-P30a and PUC19-PLY- with PLY Gene Fusion by Linker connection Linker-P1a-Linker-P30a；Respectively by carrier through NcoI and XhoI digestion, and it is connected respectively on pET30a carrier, structure It builds to obtain recombinant plasmid pET30a-P1a-Linker-P30a and pET30a-PLY-Linker-P1a-Linker-P30a,
Wherein:
The nucleotide sequence of P1a gene is as shown in SEQ ID NO: 1；The nucleotide sequence of P30a gene such as SEQ ID Shown in NO: 2；
The nucleotide sequence of PLY gene is as shown in SEQ ID NO: 3；
The nucleotide sequence of P1a-P30a gene is as shown in SEQ ID NO: 4；The nucleotides sequence of PLY-P1a-P30a gene Column are as shown in SEQ ID NO: 5；
The base sequence of above-mentioned Linker are as follows: GGCAGCGGCAGCGGCAGCGGCAGC.
3. fusion described in claim 1 is preparing the application in mycoplasma pneumoniae and Streptococcus pneumoniae vaccine.
CN201711016027.XA 2017-10-25 2017-10-25 A kind of fusion and its preparing the application in pneumovax Active CN107828810B (en)

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CN104293816A (en) * 2014-07-04 2015-01-21 华中农业大学 Mycoplasma hyopneumoniae fusion gene and application
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
CN105884902A (en) * 2015-11-09 2016-08-24 李越希 Fusion protein of mycoplasma pneumonia protein epitope and preparation and application thereof

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CN104293816A (en) * 2014-07-04 2015-01-21 华中农业大学 Mycoplasma hyopneumoniae fusion gene and application
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
CN105884902A (en) * 2015-11-09 2016-08-24 李越希 Fusion protein of mycoplasma pneumonia protein epitope and preparation and application thereof

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