CN105349562B - Express recombinant vector, recombinant bacterium and its application of PPV VP 2 protein - Google Patents
Express recombinant vector, recombinant bacterium and its application of PPV VP 2 protein Download PDFInfo
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Abstract
The present invention provides recombinant vector, recombinant bacterium and its application of expression PPV VP 2 protein, belongs to field of biotechnology.The recombinant vector for expressing PPV VP 2 protein is obtained after PPV VP 2 protein encoding gene is inserted into prokaryotic expression carrier pEASY-blunt E1.It the present invention also provides the recombinant bacterium of expression PPV VP 2 protein, is obtained after the recombinant vector is imported Escherichia coli.The present invention expresses the recombinant vector of PPV VP 2 protein, after importing Escherichia coli, can solubility expression VP2 albumen, which has coagulation and excellent antigenicity, is expected to applied to preparing swine parvovirus vaccine.PPV VP 2 protein can efficiently be prepared using recombinant bacterium of the present invention, production cost is low, it is easy to operate, do not operate pig parvoviral with better biological safety.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to express recombinant vector, the recombinant bacterium of PPV VP 2 protein
And its application.
Background technique
Pig parvoviral (porcine parvovirus, PPV) is one of the main pathogen for causing sow breeding difficulty,
Especially first farrowing sow is fallen ill even more serious under conditions of not immune, with stillborn foetus, the mummification of fetus, stillbirth, Sow abortion, is prolonged
Phase heat and production are weak young for main feature.After Cartwright isolates PPV for the first time within 1967, the virus is worldwide
Wide-scale distribution and prevalence.After China's eighties in last century reform and opening-up, as large-scale pig farm gradually increases, PPV is in China
Infection it is more serious, positive rate has brought tremendous economic losses up to 90% or more to the pig raising industry in China.PPV
Belong to Parvoviridae, parvovirus category, PPV genome encoding there are 3 kinds of structure eggs: VP1, VP2 and VP3, and 3 kinds non-structural
Albumen: NS1, NS2 and NS3.Wherein VP2 albumen is the major structural protein for constituting PPV viral capsid, and is most important
Immune protective antigen.
Many researchs and clinical practice show that vaccine immunity is an effectively method for controlling PPV prevalence.Pig body is exempted from
After epidemic disease, the protection for resisting PPV infection mainly is obtained by generating humoral immune response.China is used to prevent the epidemic disease of PPV
Seedling is mainly inactivated vaccine, i.e., fastens PPV in the pigs such as ST cell source passage small cell and cultivate, separation removal cell impurities
Obtaining has infectious virus, is mixed and made into vaccine with chemical reagent inactivation, adjuvant later.PPV inactivated vaccine has safety
Well, the advantages that cryo-conservation is not needed.But inactivated vaccine is there is also production cost valuableness, time-consuming for production, needs a large amount of labor
Power and the unstable disadvantage of immune effect；In addition, the chemical reagent and residual viral DNA of inactivation may deposit immune swine body
In risk.Novel PPV subunit vaccine obtains extensive research, and VP2 not only has good immune after expressing in vitro
Originality, and can induce and generate strong immunization protectiveness.Currently used for studying the table of pig parvoviral subunit vaccine antigenic
It is mainly the insect cell expression system of baculovirus infection up to system, expression albumen has high dissolubility and coagulation, energy
Virus-like particle is generated, immune protective effect is preferable, but its production cost ratio PPV inactivated vaccine is higher by several times, in production operation
Aspect is increasingly complex, higher to personnel qualifications.Under normal conditions, prokaryotic expression system is compared to eukaryotic system, albumen
Expression quantity is high, production cost is low, and production operation is more simple, also lower to personnel qualifications, but in the prior art, it uses
The VP2 albumen of prokaryotic expression system preparation is without soluble and coagulation inclusion body, and immunogenicity is poor, is exempted from after immune
The not handy general HI test of epidemic disease index is evaluated.
Summary of the invention
It is an object of the present invention to provide the recombinant vectors of expression PPV VP 2 protein, which is imported large intestine
After bacillus, can solubility expression VP2 albumen, the VP2 albumen have coagulation and excellent antigenicity.
It is a further object of the present invention to provide the recombinant bacteriums for preparing PPV VP 2 protein, being capable of solubility expression VP2
Albumen, the VP2 albumen have coagulation and excellent antigenicity, can be used for preparing swine parvovirus vaccine.
Another object of the present invention is to provide the method for preparing PPV VP 2 protein, and this method can be prepared efficiently
VP2 albumen, production cost are low, easy to operate, do not operate pig parvoviral with better biological safety.From actual operation
Consider, is expected to replace inactivated vaccine antigen for vaccine.
The purpose of the present invention adopts the following technical scheme that realization.
The recombinant vector for expressing PPV VP 2 protein is that PPV VP 2 protein encoding gene is inserted into protokaryon
It is obtained after expression vector pEASY-blunt E1.
In the present invention, the PPV VP 2 protein encoding gene derives from pig parvoviral PPV-JS plants.
It is that the recombinant vector is imported into large intestine bar the present invention also provides the recombinant bacterium of expression PPV VP 2 protein
It is obtained after bacterium.
The Escherichia coli are BL-21.
The present invention also provides application of the recombinant bacterium in terms of preparing PPV VP 2 protein.
In the present invention, the application includes the steps that the induction recombinant bacterium expression PPV VP 2 protein.
Preferred technical solution exists, when recombinant bacterium culture OD600 reaches 0.4-0.6, using IPTG inducing expression pig
Parvovirus VP2 albumen.
In preferred technical solution, IPTG is added to the final concentration of 0.05-0.2mM in recombinant bacterium culture.
The present invention expresses the recombinant vector of PPV VP 2 protein, being capable of solubility expression after importing Escherichia coli
VP2 albumen, the VP2 albumen have coagulation and excellent antigenicity, are expected to be applied to prepare swine parvovirus vaccine.Using this
Invention recombinant bacterium can efficiently prepare PPV VP 2 protein, production cost is low, it is easy to operate, do not operate pig parvoviral
With better biological safety.
Detailed description of the invention
Fig. 1 shows the amplification of VP2 protein coding gene, and wherein swimming lane M is 2 000 Marker of DL, remaining swimming
Road is pcr amplification product.
Fig. 2 shows the double digestion qualification result of recombinant plasmid p-PPV-VP2, and wherein swimming lane M is DL 2 000
Marker, swimming lane 1 are positive recombinant plasmid p-PPV-VP2.
Fig. 3 shows expression of the VP2 albumen in BL-21-VP2 and control bacterium BL-21-p.Marker:Thermo
Scientific PageRuler pre-dyed Ladder；1: control bacterium BL-21-p lysate supernatant；On 2:BL-21-VP2 lysate
Clearly；3: control bacterium BL-21-p cracks liquid precipitate；4:BL-21-VP2 cracks liquid precipitate.
Fig. 4 is the coagulation testing result of each sample, and every row left side shows the number in hole, every row sample concentration from left to right
Successively reduce.
Fig. 5 show OD600 be 0.4,0.5 and 0.6 when induction after, the coagulation testing result of cellular lysate liquid supernatant,
Leftmost one label shows the OD600 value before every row sample induction, and the right provides the hemagglutinative titer of every row sample.
Fig. 6 using PPV-JS or BL-21-VP2 4 unit antigens detect serum to be checked HI antibody titer as a result, its
The text on the middle left side points out that the serum of every row detection, the text on the right point out the number in hole.
Specific embodiment
The recombinant bacterium of the building expression VP2 albumen of embodiment 1
One building recombinant expression carrier
1. design of primers
Referring to PPV- NADL2 pnca gene sequence (KF049424.1), set using 5.0 software of Primer premier
Primer PPV-VP2-kpn i and PPV-VP2-bamh i is counted, (is abbreviated as PPV-JS for expanding PPV-JS plants of pig parvoviral
Strain, is disclosed in CN102965345A, and China Committee for Culture Collection of Microorganisms's common micro-organisms center's deposit number is CGMCC
NO.6605) VP2 protein coding gene full sequence.
The nucleotide sequence (SEQ ID NO:2) of PPV-VP2-kpn i is as follows:
The nucleotide sequence (SEQ ID NO:3) of ggtaccATGAGTGAAAATGTGGAACAAG, PPV-VP2-bamh i are as follows:
GgatccCTAGTATAATTTTCTTGGTAT, by Shanghai, English fine horse biology Co., Ltd is synthesized.Wherein PPV-VP2-kpn i is carried
I restriction enzyme site of Kpn, PPV-VP2-bamh i carry I restriction enzyme site of Bamh.
The extraction of 2.PPV-JS pnca gene group DNA
(1) the SDS solution that 50 μ l are added in 450 μ l of PPV-JS strain virus sample, concentration is 10% is taken, 3 μ l Proteinase Ks are added；
(2) 30min is incubated under the conditions of concussion in 56 DEG C of water-baths；
(3) the TRIS- phenol of 500 μ l is added, mixes, after five minutes, is centrifuged 10 under the conditions of 4 DEG C, 10000-12000r/min
Minute；
(4) take supernatant, 1:1(V/V be added) the 500 μ l of TRIS- phenol and chloroform mixture of mixing, effect is after five minutes
10000-12000r/min is centrifuged 10 minutes；
(6) supernatant is taken, the chloroform of 500 μ l is added, is acted on 5 minutes, 10000-12000r/min is centrifuged 10 minutes；
(7) supernatant is taken, in addition 2 times of clear volume of dehydrated alcohol and 10%(concentration expressed in percentage by volume) sodium acetate aqueous solution；
(8) mixture obtained by step (7) is placed at -20 DEG C 30 minutes or more；
(9) sample placed at -20 DEG C is taken to be centrifuged 10 minutes in 4 DEG C, 10000r/min；
(10) abandon supernatant, in precipitating be added 75%(concentration expressed in percentage by volume) ethanol water, in 4 DEG C, 6500r/min
Centrifugation 5 minutes, discards supernatant；It repeats 2 times；
(11) blot or dry ethyl alcohol；
(12) ddH of 30 μ l is added2O dissolution, obtains PPV-JS pnca gene group DNA, is placed at -20 DEG C and saves.
The amplification and purification and recovery of 3.VP2 protein coding gene
(1) reaction system is set as 50 μ L systems: 0.5 μ L PrimerSTAR high fidelity enzyme, 10 5 × PS of μ L
Buffer, 4 μ L dNTPs, each 1 μ L(20 μm ol/L of primer PPV-VP2-kpn i and PPV-VP2-bamh i), 2 μ L PPV-
JS pnca gene group DNA, 28.5 μ L ddH2O。
(2) reaction condition: 98 DEG C of 2min；98 DEG C of 15s, 60 DEG C of 30 s, 72 DEG C of 2min, 30 circulations；72℃
5min.Product is saved at 4 DEG C.
(3) using the genetic fragment of 1% agarose gel electrophoresis recycling amplification, using 2 000 Marker of DL as control.
As a result as shown in Figure 1, occurring the VP2 protein coding gene band of specificity at about 1.7kb.Using OMEGA DNA gel
QIAquick Gel Extraction Kit recycles VP2 protein coding gene.Specification is shown in concrete operations.
The nucleotide sequence of PPV-JS plants of VP2 protein coding genes of pig parvoviral is as shown in SEQ ID NO:1.
The reagent used during PCR is purchased from TAKARA.
4. the building of expression plasmid
(1) VP2 protein coding gene is inserted into pEASY-blunt E1 prokaryotic expression carrier (purchased from the full formula gold biology in Beijing
Technology Co., Ltd.), construction recombination plasmid p-PPV-VP2.Linked system is 5 μ L systems: 2 μ L VP2 protein coding gene glue
Recovery product, 1 μ L pEASY-blunt E1 carrier, 2 μ L ddH2O.24 DEG C of connection 20min.
(2) connection product is added in the DH5 α competent cell of 100 μ L, 30 min of ice bath, later 42 DEG C of heat shocks
90 s, 5 min of ice bath.1mL LB culture medium is added, 180 rpm cultivate 1 h on 37 DEG C of shaking tables, 10000g centrifugation 1 after taking-up
Min abandons 800 μ L supernatants, stays 200 μ L supernatants that thallus is resuspended, and all coating has on the LB plate of ammonia benzyl (Amp) resistance, and 37
DEG C overnight incubation.
(3) single colonie on the above-mentioned LB plate of random picking, is inoculated in the LB liquid medium with ammonia benzyl resistance, 37
It DEG C is incubated overnight.
5. the extraction of plasmid
Plasmid in the recombinant bacterium selected in the present embodiment step 4 is extracted using ancient cooking vessel state small amount plasmid extraction kit,
Concrete operation step is shown in kit specification.
6. the identification of recombinant plasmid
10 μ L double digestion identification systems: 0.5 μ L BamH I, 0.5 μ L Kpn I, 1 μ 10 × K of L Buffer, 4 μ L plasmids, 4
μL ddH2O, 37 DEG C of 1 h of digestion.The electrophoretograms of digestion products as shown in Fig. 2, plasmid identified through BamH I and I digestion of Kpn it is correct,
For positive recombinant plasmid, it is named as p-PPV-VP2.Recombinant plasmid p-PPV-VP2 Song Ying fine horse company is sequenced, sequencing is correct.
Two building recombinant bacteriums
1. recombinant plasmid p-PPV-VP2 conversion expression bacterium
(1) conventionally, by the competent cell of p-PPV-VP2 recombinant plasmid transformed Escherichia coli BL-21, then
It is coated on containing 50 μ g/mL Amp(ampicillins) on the LB plate of resistance, 37 DEG C of overnight incubations.
(2) single colonie on the above-mentioned Amp resistance LB plate of picking, is named as recombinant bacterium BL-21-VP2, is inoculated in containing 50 μ
In the LB fluid nutrient medium of g/mL Amp resistance, shaken cultivation is stayed overnight under the conditions of 37 DEG C, 200rpm.
The competent cell of plasmid pEASY-blunt E1 conversion BL-21 is obtained into control bacterium according to above-mentioned same procedure
BL-21-p。
2. recombinant bacterium inducing expression and SDS-PAGE identification
(1) BL-21-VP2 and control bacterium BL-21-p culture solution are pressed into 1:100(volume ratio respectively) it is inoculated with containing 50 μ g/ml
The LB liquid medium of Amp.
(2) 2h, at this time OD under being cultivated under the conditions of 37 DEG C, 200rpm600About 0.4.
(3) IPTG of final concentration of 0.1 mM, the shaken cultivation 4 hours under the conditions of 37 DEG C, 200rpm is added.
(4) after cultivating, 5ml BL-21-VP2 and control bacterium BL-21-p bacterium solution are taken respectively, after ultrasonication processing,
It is centrifugated cellular lysate liquid supernatant precipitating, carries out SDS-PAGE electrophoresis.As a result as shown in figure 3, on BL-21-VP2 lysate
There are purpose bands in clear, and supernatant has higher hemagglutination activity, and HA potency reaches 29, recombinant bacterium BL-21-VP2 lysate is heavy
There are the inclusion body of destination protein in shallow lake, hemagglutination activity is very low, and HA potency is only 25, illustrate that VP2 albumen is real in BL-21-VP2
Solubility expression is showed, and the VP2 albumen hemagglutinative titer expressed is higher.The detection method of HA potency is shown in embodiment 2.
The coagulation identification of 2 VP2 albumen of embodiment and HA-HI test (HA) measurement
1. the preparation of 1% guinea pig red blood cells liquid
(1) anti-coagulants (4% sodium citrate solution) 1mL is drawn with syringe；At least 1 SPF cavy is taken, take a blood sample about 4mL,
It is mixed with 1mL anti-coagulants, is then slowly added into 10mL centrifuge tube and mixes.
(2) guinea pig red blood cells are washed: by the blood mixture fluid in step (1) in 10mL centrifuge tube under the conditions of 1500 rpm
Centrifugation 10min, abandoning supernatant, taking precipitate, addition 5ml PBS buffer solution (pH 7.2 is the aqueous solution containing following compositions:
NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO410mmol/L, KH2PO42mmol/L, similarly hereinafter), it is gently mixed,
It is centrifuged 10min through 1500 rpm again, suction pipe removes the leucocyte film on supernatant precipitating red blood cell upper layer.Washing process repeats 2
After secondary, 5mL PBS buffer solution (pH 7.2) is added and is gently mixed red blood cell, 4 DEG C save backup, and use in 5 days.
(3) 20% guinea pig red blood cells liquid: the red blood cell obtained after taking step (2) to handle, in taper graduated centrifuge tube
1500rpm is centrifuged 10 minutes, is discarded clearly, is observed erythrocyte volume in centrifuge tube, the PBS buffer solution of 4 times of volumes is added
(pH7.2), it is uniformly mixed, obtains 20% guinea pig red blood cells liquid.
(4) 10% guinea pig red blood cells liquid: the red blood cell obtained after taking step (2) to handle, in taper graduated centrifuge tube
1500rpm is centrifuged 10 minutes, is discarded clearly, is observed erythrocyte volume in centrifuge tube, the PBS buffer solution (pH of 9 times of volumes is added
7.2) it, is uniformly mixed, obtains 10% guinea pig red blood cells liquid.
(5) 1% guinea pig red blood cells liquid: taking 10% guinea pig red blood cells liquid, 1 mL, is added 9mL PBS buffer solution (pH 7.2), mixes
It closes uniformly, obtains 1% guinea pig red blood cells liquid.
2. the coagulation identification of VP2 albumen and blood clotting (HA) titration
The BL-21-VP2 lysate supernatant and precipitating that in 1 title two of Example prepared by step 2, to verify the present invention
Method preparation VP2 albumen it is coagulation.By the BL-21-VP2 cracking liquid precipitate PBS buffer solution isometric using supernatant
(pH7.2) it is resuspended, obtains VP2 inclusion bodies of protein re-suspension liquid.
Different samples is detected in the 96 holes every row hole of V-type hemagglutination plate, and number is that PBS blank control, the PPV positive are right respectively
According to, control bacterium BL-21-p negative control, sample well 1(VP2 inclusion bodies of protein re-suspension liquid) and sample well 2(BL-21-VP2 crack
Liquid supernatant).The PBS buffer solution (pH7.2) of 25 μ L is added in every Kong Zhongxian, and sample then is added in first hole of every round
Product are added 25 μ L VP2 inclusion bodies of protein re-suspension liquids in sample well 1, are added on 25 μ L BL-21-VP2 lysates in sample well 2
25 μ L PBS buffer solution (pH7.2) are added in PBS blank control in clear liquid, and it is 2 that 25 μ L HA-HI tests are added in PPV positive control9's
PPV-JS strain virus antigen liquid (is obtained) using ST cell amplification PPV-JS strain virus antigen, compares bacterium BL-21-p negative control
25 μ L of middle addition compare ultrasonic lysate before the full bacterium induction of bacterium BL-21-p.Carry out 2 times of doubling dilutions respectively to each sample, then
The PBS buffer solution (pH7.2) of 25 μ L is added in every hole, 1% guinea pig red blood cells liquid, 25 μ L is finally added in every hole, at 37 DEG C
After acting on 2h, result is observed.
As a result such as Fig. 4, the soluble VP2 albumen of BL-21-VP2 expression has coagulation, and hemagglutinative titer is up to 29, with
The HA-HI test of PPV-JS strain virus antigen liquid is similar；The hemagglutinative titer (HA) of VP2 inclusion bodies of protein is lower, and only only 25。
3 VP2 protein preparation method condition optimizing of embodiment
1. the most suitable bacteria concentration optimization before recombinant bacterium BL-21-VP2 induction
It (1) is by volume that 1:100 is seeded to the LB liquid containing 50 μ g/ml ampicillins by BL-21-VP2 bacterium solution
In body culture medium.
(2) 2 ~ 3h is cultivated under the conditions of 37 DEG C, 200rpm, selecting OD600 (somatic cells density) is 0.4,0.5 and 0.6
When bacterium solution, be separately added into the IPTG of final concentration of 0.1 mM, then shaken cultivation 4 hours under the conditions of 37 DEG C, 200rpm, into
Row inducing expression.
(3) after cultivating, take 5ml bacterium solution ultrasonic, 12000rpm centrifugation takes lysate supernatant, according to side in embodiment 2
Method detects hemagglutinative titer, to judge the expression quantity of solubility VP2 albumen.
As a result as shown in figure 5, when OD600 is 0.4,0.5 and 0.6 after induction, the hemagglutinative titer point of cellular lysate liquid supernatant
Do not reach 29、210With 211.It is induced in cell concentration OD600=0.6, the hemagglutinative titer of VP2 albumen pronucleus expression is most
Height reaches 211。
4 VP2 albumen of embodiment makes 4 unit antigens and is applied to hemagglutination-inhibition test (HI)
1. the VP2 albumen of prokaryotic expression makes 4 unit antigens
Taking hemagglutinative titer is 29PPV-JS strain virus liquid (using ST cell amplification obtain) using PBS buffer solution (
PH7.2 4 times of antigens) are diluted to, i.e., PPV-JS strain virus liquid are subjected to 2 times of doubling dilutions, until the hemagglutinative titer of dilution is
22, obtain the 4 unit antigens of PPV-JS.It is 2 by hemagglutinative titer in embodiment 39BL-21-VP2 lysate supernatant carry out 2 times times
Than dilution, taking hemagglutinative titer is 224 unit antigens of the dilution as BL-21-VP2.
2. pig PPV negative serum, positive serum are handled
The positive serum after 200 μ L pig PPV negative serums and PPV vaccine immunity is taken to be handled as follows respectively: by blood
400 μ L, 25%(mass percentage concentration are added in the inactivation treatment 30min clearly in 56 DEG C of water-baths) white pottery soil suspension, mix rear chamber
Temperature places 30 min, and 4000rpm is centrifuged 10min, takes supernatant, is added 200 μ L, 20% guinea pig red blood cells liquid, oscillation mix after
37 DEG C of 1 h of effect；4000rpm is centrifuged 10min, supernatant is drawn, as the diluted blood serum sample of 1:4.
3. hemagglutination-inhibition test
Be respectively adopted BL-21-VP2, PPV-JS 4 unit antigens come the pig PPV positive serum after detection processing HI it is anti-
Body potency, to investigate the antigenicity of the VP2 albumen of BL-21-VP2 expression.Control bacterium BL-21-p negative control (control is set simultaneously
Ultrasonic lysate before the full bacterium induction of bacterium BL-21-p).
The PBS buffer solution (pH7.2) of 25 μ L, every row hole detection are all added in 96 hole each hole of V-shaped hemagglutination plate of standard
Different samples, number is BL-21-VP2 antigen hole, PPV-JS antigen hole and control bacterium BL-21-p negative control respectively.Every row
In be added 25 μ L in the 1st hole treated serum to be checked, mix, take out 25 μ L and add to the 2nd hole, and so on to treated
Serum to be checked carries out 2 times of doubling dilutions, until the 12nd hole, abandons 25 μ L.In BL-21-VP2 antigen hole, BL-21-VP2 is added in every hole
4 unit antigen 25 μ L；In PPV-JS antigen hole, the 4 unit antigen 25 μ L of PPV-JS are added in every hole；Compare bacterium BL-21-p yin
Property control in, ultrasonic lysate before the full bacterium induction of control bacterium BL-21-p is added in every hole.37 DEG C of 1 h of effect, 1% globefish is added in every hole
25 μ L of Mice red cell liquid, oscillation mix, and set 37 DEG C of 2 h of effect, observe result.
As a result such as Fig. 6, it is seen that with the HI antibody for the serum to be checked that the 4 unit antigens of PPV-JS or BL-21-VP2 detect
Potency size is identical, is 27, show that VP2 albumen prepared by the present invention and PPV-JS strain virus have same antigen, antigen
Epitope is similar to provirus, this is characteristic not available for the VP2 albumen of prokaryotic expression in the prior art, therefore prepared by the present invention
VP2 albumen has the prospect applied to vaccine.The above results explanation, VP2 albumen prepared by the present invention are not only sick with PPV-JS plants
Poison agglutination guinea pig red blood cells having the same are coagulation, and can react with PPV specific antibody in serum, inhibit red thin
Born of the same parents' agglutination.
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>recombinant vector, recombinant bacterium and its application of PPV VP 2 protein are expressed
<130> 20151218
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1740
<212> DNA
<213>pig parvoviral PPV-JS plants
<400> 1
atgagtgaaa atgtggaaca acacaaccct attaatgcag gcactgaatt gtctgcaaca 60
ggaaatgaat ctgggggtgg gggcggcggt ggcgggggta ggggtgctgg gggggttggt 120
gtgtctacag gtactttcaa taatcaaaca gagtttcaat acttggggga gggcttggtt 180
agaatcactg cacacgcatc aagactcata catctaaata tgccagaaca cgaaacatac 240
aaaagaatac atgtactaaa ttcagaatca ggggtggcgg gacaaatggt acaagacgat 300
gcacacacac aaatggtaac accttggtca ctaatagatg ctaacgcatg gggagtgtgg 360
ttcaatccag cggactggca gttaatatcc aacaacatga cagaaataaa ctgggttagt 420
tttgaacaag aaatattcta tgtagtactt aaaacaatta cagaatcagc aacctcacca 480
ccaaccaaac tatataataa tgatcgaact gcaagcttaa tggtcgcact agacaccaat 540
aacacacttc catacacacc agcagcacct agaagtgaaa cacttggttt ttatccatgg 600
ttacctacaa aaccaactca atacagatat tacctatcat gcatcagaaa cctaaatcca 660
ccaacataca ctggacaatc acaacaaata acagactcaa tacaaacagg actacacagt 720
gacattatgt tctacacaat agaaaatgca gtaccaattc atcttctaag aacaggagat 780
gaattctcca caggaatata tcactttgac acaaaaccac taaaactaac tcactcatgg 840
caaacaaata gatctctagg actgcctcca aaactactaa ctgaacctac cacagaagga 900
gaccaatacc caggaacact accagcagct aacacaagaa aaggttatca ccaaacaact 960
aataatagct acacagaagc aacagcaatt aggccagctc aggtaggata taatacacca 1020
tacatgaatt ttgaatactc caatggtgga ccatttctaa ctcctatagt accaacagca 1080
gacacacaat ataatgatga tgaaccaaat ggtgctataa gatttacaat gggttaccaa 1140
catggacaat taaccacatc ttcacaagag ctaggaagat acacattcaa tccacaaagt 1200
aaatgtggaa gagctccaaa tcaacaattt aaccaacagg caccactaaa cctagaaaat 1260
acaaataatg gaacacgttt accttcagat ccaataggag ggaaatctaa catgcatttc 1320
atgaatacac tcaatacata tggaccatta acagcactaa acaatactgc acctgtattt 1380
ccaaatggtc aaatatggga taaagaactt gatacagatc taaaacctag actacatgtt 1440
acagctccat ttgtttgtaa aaacaatcca ccaggacaac tatttgtaaa aatagcacca 1500
aacctaacag atgatttcaa tgctgactct cctcaacaac ctagaataat aacttattca 1560
aacttttggt ggaaaggaac actaacattc acagcaaaaa tgagatccag taatatgtgg 1620
aaccctatta gacaacacac aacaacagca gaaaacattg gtaaatatat tcctacaaat 1680
attggtggca tgaaaatgtt tccagaatat tcacaactta taccaagaaa attatactag 1740
<210> 2
<211> 28
<212> DNA
<213> artificial
<220>
<223>primer PPV-VP2-kpn i
<400> 2
ggtaccatga gtgaaaatgt ggaacaag 28
<210> 3
<211> 27
<212> DNA
<213> artificial
<220>
<223> PPV-VP2-bamh i
<400> 3
ggatccctag tataattttc ttggtat 27
Claims (7)
1. expressing the recombinant vector of PPV VP 2 protein, it is characterised in that insert PPV VP 2 protein encoding gene
It is obtained after entering prokaryotic expression carrier pEASY-blunt E1, the PPV VP 2 protein encoding gene is tiny from pig
It is PPV-JS plants viral.
It 2. expressing the recombinant bacterium of PPV VP 2 protein, is obtained after recombinant vector described in claim 1 is imported Escherichia coli
?.
3. expressing the recombinant bacterium of PPV VP 2 protein according to claim 2, it is characterised in that the Escherichia coli are
BL-21。
4. application of the recombinant bacterium described in claim 3 in terms of preparing PPV VP 2 protein.
5. applying according to claim 4, it is characterised in that including inducing the recombinant bacterium to express PPV VP 2 protein
The step of.
6. applying according to claim 5, it is characterised in that when recombinant bacterium culture OD600 reaches 0.4-0.6, use
IPTG inducing expression PPV VP 2 protein.
7. applying according to claim 6, it is characterised in that IPTG is added to the final concentration of 0.05- in recombinant bacterium culture
0.2mM。
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| CN105820216A (en) * | 2016-05-28 | 2016-08-03 | 青岛易邦生物工程有限公司 | Porcine parvovirus VP2 protein |
| CN105879024B (en) * | 2016-05-28 | 2020-11-27 | 青岛易邦生物工程有限公司 | Porcine parvovirus subunit vaccine |
| CN106148358A (en) * | 2016-07-15 | 2016-11-23 | 河南省农业科学院 | A kind of method utilizing escherichia expression system to prepare pig parvoviral virus sample particle subunits vaccine and application |
| CN107400676A (en) * | 2017-07-17 | 2017-11-28 | 福建省农业科学院畜牧兽医研究所 | Express the PEDV dual-gene prokaryotic fusion expression vectors of M+N and application |
| CN110845580A (en) * | 2019-11-05 | 2020-02-28 | 中国农业科学院兰州兽医研究所 | Method for assembling porcine parvovirus-like particles and identifying immunogenicity thereof |
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