CN102174086A - Porcine circovirus type 2 recombinant cap protein and subunit vaccine - Google Patents
Porcine circovirus type 2 recombinant cap protein and subunit vaccine Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology, and discloses a porcine circovirus type 2 recombinant cap protein and a subunit vaccine. The porcine circovirus type 2 cap protein expressed by recombinant Escherichia coli is obtained by steps of cloning a porcine circovirus type 2 cap protein in a nuclear localization signal area of which the N terminal is cut and which is rich in arginine into a prokaryotic expression vector to obtain a recombinant expression vector, transfecting the recombinant expression vector into Escherichia coli BL21(DE3), and expressing by using the recombinant Escherichia coli BL21(DE3). Tests prove that the constructed recombinant strain expresses a foreign protein stably. When the subunit vaccine is prepared from the expressed recombinant protein, an antigen has high purity and safety, does not have pathogenicity on animals such as pigs and the like, and passes safety evaluation easily.
Description
Technical field
The invention belongs to biology field, relate to a kind of porcine circovirus 2 type recombinant C ap albumen and subunit vaccine.
Technical background
Escherichia expression system is the expression system of present comparative maturity, have simple to operate, safety non-toxic, exogenous protein expression amount advantages of higher successfully has been used for the production of multiple protein.And Cap albumen is the capsid protein by about 30kD of the ORF2 genes encoding of porcine circovirus 2 type (PCV2), comprise a plurality of epitopes and in and epitope, be acknowledged as the main immune protective antigen of PCV2.Vaccine research mainly concentrates on different and expresses system expression Cap albumen, but also Cap albumen different fragments is not carried out the immunogenicity analysis at present, also prokaryotic expression Cap albumen is not carried out the report that vaccine research was studied and be used for to immanoprotection action.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, the PCV2 recombinant C ap albumen of the intestinal bacteria system expression with immanoprotection action is provided.
Another object of the present invention provides the proteic preparation method of this recombinant C ap.
Another purpose of the present invention provides a kind of proteic subunit vaccine of this recombinant C ap that comprises.
Purpose of the present invention is achieved through the following technical solutions:
A kind of porcine circovirus 2 type Cap albumen of expression of recombinant e. coli, this albumen system holds the porcine circovirus 2 type Cap albumen that is rich in arginic nuclear localization signal zone to be cloned into prokaryotic expression carrier amputation N and obtains recombinant expression vector, with this recombinant expression vector transfection Escherichia coli BL21 (DE3), express acquisition through this recombination bacillus coli BL21 (DE3).
The aminoacid sequence that described amputation N end is rich in the porcine circovirus 2 type Cap albumen (C) in arginic nuclear localization signal zone is SEQ ID NO.1, i.e. 51-234 amino-acid residue of the ORF2 of porcine circovirus 2 type (PCV2) coding.
The proteic nucleotides sequence of porcine circovirus 2 type Cap that the described amputation N end of encoding is rich in arginic nuclear localization signal zone is classified SEQ ID NO.2 as.
Described prokaryotic expression carrier is pET-32a.
The proteic screening of PCV2 recombinant C ap with intestinal bacteria system expression of immanoprotection action: hold the fragments of the different sizes of porcine circovirus 2 type Cap albumen that are rich in arginic nuclear localization signal zone to be cloned into prokaryotic expression carrier respectively amputation N and obtain recombinant expression vector; be converted into e. coli bl21 (DE3) respectively; obtain recombinant protein; by mouse immuning test, screening obtains to induce the recombinant C ap albumen (C) that produces high-caliber PCV2 specific antibody.
A kind of recombinant plasmid PET-32a-Cap-c, this plasmid are cloned into BamH I and two restriction enzyme sites of XhoI that prokaryotic expression carrier is pET-32a with the nucleotide fragments shown in the SEQ ID NO.2 to see gained.
A kind of recombination bacillus coli BL21-Cap-C contains described recombinant plasmid PET-32a-Cap-c.
A kind of proteic preparation method of porcine circovirus 2 type Cap of expression of recombinant e. coli of the present invention comprises the steps:
(1) according to PCV2SH pnca gene sequence (AY686763) design primer Cap51F (SEQ ID NO.3), Cap233R (SEQ ID NO.4), PK15 cell DNA with PCV2 SH virus strain infection is a template, the method of using PCR increases and obtains encoding the proteic gene fragment of Cap (SEQ ID NO.2), by BamH I and two restriction enzyme sites of XhoI, described gene fragment clone is gone in the prokaryotic expression carrier, the evaluation of cutting by enzyme and check order then obtains positive recombinant plasmid;
(2) step (1) is described through the correct recombinant plasmid transformed e. coli bl21 (DE3) of sequencing, obtain recombinant strains BL21-Cap-C (express recombinant Cap PROTEIN C fragment);
(3) cultivate described recombinant strains BL21-Cap-C, add IPTG and carry out abduction delivering, obtain described recombinant protein.
Wherein, described prokaryotic expression carrier is pET-32a (+).
Described recombinant strains BL21-Cap-C adds final concentration when being cultured to OD600 and reaching 0.6-0.8 be that the IPTG of 1mM carries out abduction delivering.
A kind of subunit vaccine is characterized in that comprising the porcine circovirus 2 type Cap albumen (C) and the adjuvant of the described expression of recombinant e. coli of claim 1.
Beneficial effect of the present invention:
The porcine circovirus 2 type Cap albumen of expression of recombinant e. coli of the present invention has the antigen purity height as subunit vaccine, and security is good, and immunogenicity is strong, and animals such as pig are not had pathogenic, the advantage by safety evaluation easily.
Evidence, the recombinant bacterial strain that the present invention makes up is expressed stable to the external source target protein.The vaccine that has proved this recombinant protein preparation by mouse immuning test can be induced the high-caliber PCV2 specific antibody of generation.The experiment of pig body confirms that further it has the good immune protection effect.
Therefore recombinant baculovirus expression Cap protein subunit vaccine of the present invention is compared with the inactivated vaccine of the PCV2 of present application, and this vaccine production process is simple relatively, and cost is lower, has more wide application prospect aspect the preventing and treating of PCV2.
Description of drawings
The carrier synoptic diagram is gone in Fig. 1 PCV2 Cap-a/b/c/d/e gene clone.
Fig. 2 PCV2 Cap-a/b/c/d/e gene PCR amplification,
The 1-5:PCR product, the gene fragment of corresponding respectively coding Cap-a/b/c/d/e; M:1kb plus ladder marker.
The double digestion of Fig. 3 PET-32a-Cap-a/b/c/d/e recombinant plasmid is identified
1-5: be respectively BamH I/Xho I double digestion recombinant plasmid PET-32a-Cap-a/b/c/d/e; M:1kb plus ladder marker.
Fig. 4 SDS-PAGE detects Recombinant Protein Expression,
1: albumen Marker; 2-6 is respectively recombinant protein c ap-A/B/C/D/E.
The Western blot of Fig. 5 recombinant protein identifies (His monoclonal antibody be one anti-)
1-5: recombinant protein c ap-A/B/C/D/E, 6: the empty carrier induced product.
Fig. 6 recombinant protein mouse immune antibody changes.
Fig. 7 recombinant protein c ap-C pig body experiment antibody changes.
Fig. 8 attacks poison back pig precursor virus mass formed by blood stasis and detects.
Fig. 9 attacks back 25 days lymphoglandula IHC detected results of poison.
Embodiment
The pcr amplification of embodiment 1 Cap albumen a-e gene fragment and the structure of prokaryotic expression plasmid
1.1 design of primers
According to PCV2 SH pnca gene sequence (AY686763) design primer, the upstream and downstream primer is respectively introduced BamH I and Xho I site, increase the respectively gene fragment of 51-150,51-200 among the ORF2,51-234,101-200 and 101-233aa, and called after Cap-a (SEQ ID NO.8), Cap-b (SEQ ID NO.9), Cap-c (SEQ ID NO.2), Cap-d (SEQ ID NO.10), Cap-e (SEQ ID NO.11).Primer sequence is as follows:
Cap51F AA
GGATCCCGCACCATCGGTTATAC(SEQ?ID?NO.3)
Cap101F?CC
GGATCCGTTAAGGTTGAATTCTG(SEQ?ID?NO.5)
Cap150R?TAT
CTCGAGTATGGTATGGCGGGAGG(SEQ?ID?NO.6)
Cap200R?TAA
CTCGAGTGCCGAGGCCTACA(SEQ?ID?NO.7)
Cap233R?CTT
CTCGAGTCACTTAGGGTTAAGTGGG(SEQ?ID?NO.4)
Above primer is synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.
1.2PCR amplification purpose fragment
(CGMCC NO.2389, preservation date: on March 4th, 2008) infect the PK15 cell, 72h gathers in the crops virus, adopts phenol: chloroform extraction method extracts DNA as the PCR reaction template with the PCV2SH strain.The PCR reaction system is: upstream, each 1.0 μ L of downstream primer (10pmol/L); MgCl
2(25mmol/L) 1.5 μ L; DNTP s (2.5mmol/L) 2.0 μ L; 10 * PCR buffer2.5 μ L; The dna profiling 2.0 μ L that extract; Taq enzyme (5U/ μ L) 0.2 μ L; The sterilization distilled water is mended to 25 μ L.Loop parameter: 95 ℃ of pre-sex change 5min; With 94 ℃ of 45s, 54 ℃ of 45s, 72 ℃ of 45s carry out 35 circulations; Last 72 ℃ are extended 10min.1% agarose gel electrophoresis is identified PCR product (the results are shown in Figure 2), reclaims the PCR product, and-20 ℃ of preservations are standby.
1.3 construction of recombinant plasmid and evaluation
With the goal gene that reclaims BamH I/Xho I double digestion, being cloned into same enzyme after the recovery purifying cuts among the expression vector plasmid pET-32a (Invitrogen) of processing, ligation is carried out at 16 ℃, to connect product Transformed E .coli DH5 α (NEB) competent cell afterwards, coating contains the LB flat board of penbritin, 37 ℃ of cultivations.Bacterium colony on the picking flat board is containing the LB culture medium culturing of penbritin, and the alkaline lysis method of extracting plasmid is identified (the results are shown in Figure 3) through PCR and BamH I/Xho I double digestion, and positive plasmid obtains the purpose band.Positive plasmid is served extra large Ying Jun company limited and is checked order.The recombinant plasmid that order-checking is correct is called after PET-32a-Cap-a, PET-32a-Cap-b, PET-32a-Cap-c, PET-32a-Cap-d, PET-32a-Cap-e respectively.
The structure of embodiment 2 recombinant strains BL21-Cap-a/b/c/d/e
2.1 transformed competence colibacillus intestinal bacteria BL-21
Among the embodiment 1 through the correct recombinant plasmid transformed intestinal bacteria BL-21 (NEB) of sequencing, obtain recombinant strains BL21-Cap-a, BL21-Cap-b, BL21-Cap-c, BL21-Cap-d, BL21-Cap-e respectively, simultaneously with empty plasmid pET-32a with method transformed competence colibacillus intestinal bacteria BL-21.
2.2 the optimization of expression of recombinant proteins condition
Picking recombinant strains BL21-Cap-a/b/c/d/e is dispersed in single colony inoculation in the LB liquid nutrient medium that contains penbritin with the intestinal bacteria BL-21 that contains empty plasmid pET-32a respectively, and 37 ℃ of joltings of spending the night are cultivated.
(1) the bacterium liquid 10 μ L that get incubated overnight are inoculated in 3ml and contain in the LB liquid nutrient medium of penbritin (50 μ g/ml), about 37 ℃ of 200rpm shaking culture 3h, make OD
600Reach 0.6～0.8, get the conduct in aseptic Eppendorf pipe of 100 μ L samples and induce preceding contrast;
(2) adding final concentration in above-mentioned bacterium liquid is the abduction delivering that the IPTG of 1.0mmol/L carries out recombinant protein, and after adding IPTG the 3rd, 4,5, the 6h sampling;
(3) 4 ℃ of bacteriums that the centrifugal 5min of 8000rpm collects abduction delivering, PBS (pH 7.2) is resuspended, repetitive scrubbing 2 times;
(4) abandon supernatant fully, with the resuspended bacterial precipitation of an amount of PBS;
(5) with bacterium liquid multigelation 3 times;
(6) ultrasonic treatment bacterium: power is selected 200W for use, on ice chest, operates, and ultrasonic degradation 5s, pause 5s becomes limpid until bacterium liquid;
(7) 4 ℃ of centrifugal 10min of 8000rpm, collect supernatant and precipitation respectively: will precipitate and use with the isopyknic PBS of supernatant resuspendedly, respectively to get 100 μ L standby with precipitation suspension for supernatant.
Finally choose OD
600Reach 0.6, adding final concentration is the IPTG of 1.0mmol/L, and inducing 4h is the optimum expression condition.
2.3SDS-PAGE electrophoretic analysis
Add 5 * protein sample sample-loading buffer (250mM Tris-HCl, pH6.8 in the sample; 10%SDS; 0.5% tetrabromophenol sulfonphthalein; 50% glycerine; The 5%2-mercaptoethanol), 100 ℃ are boiled 5min behind the abundant mixing, instantaneous centrifugal before the last sample, draw supernatant and carry out SDS-PAGE, the results are shown in Figure 4.
The great expression of embodiment 3 fusion roteins, purifying and antigenicity are identified
3.1 the great expression of fusion rotein, purifying
The best abduction delivering condition abduction delivering gene engineering recombinant bacterium 500ml that gropes by embodiment 2, centrifugal collection thalline, add the resuspended bacterial sediment of 5ml PBS by every 100ml bacterium liquid, centrifugal behind the ultrasonic degradation, cleer and peaceful inclusion body in the separation, inclusion body is resuspended with equal-volume PBS, and inclusion body is through inclusion body washing lotion washing purifying, and step is as follows:
(1) the centrifugal 15min of 8000rpm, inclusion body washing lotion I (50mM Tris-HCl; 100mM NaCl; 10mM EDTA; 1%Triton X-100) resuspended precipitation, 4 ℃ of 1-2h;
(2) the centrifugal 15min of 8000rpm, inclusion body washing lotion II (50mM Tris-HCl; 100mM NaCl; 10mM EDTA; 0.5%Triton X-100) resuspended precipitation, 4 ℃ of 1-2h;
(3) above step repeats 1-2 time;
(4) the centrifugal 15min of 8000rpm, the resuspended precipitation of 8M urea, 4 ℃ of 12-24h fully dissolve albumen;
(5) the centrifugal 15min of 8000rpm draws supernatant to new Eppendorf pipe, the spectrophotometric determination protein concentration, and 4 ℃ or-20 ℃ of preservations are standby.
(6) protein concentration is adjusted to about 0.3mg/ml, in the dialysis tubing of packing into, in dialyzate, progressively added PBS, make urea concentration be reduced to 6M, 5M, 4M, 3.5M, 3M, 2.5M, 2M, 1.5M, 1M makes protein renaturation.Each gradient is put 4 ℃ and is kept 6-8h, carries out under agitation condition.
(7) the centrifugal 15-20min of 8000rpm draws supernatant, and 0.45 μ m membrane filtration redeterminates protein concentration, 4 ℃ or-20 ℃ preservations of packing.
3.2 the Western blot of recombinant protein identifies
(1)SDS-PAGE；
(2) transfer printing: electrophoresis takes off gel and transfer device (half-dried transfer printing) is installed in the following order promptly after finishing
+ the utmost point: blank-sponge-NC film-gel-sponge-blackboard :-the utmost point, the transfer printing condition is 20V, 30min;
(3) transfer printing is taken off the NC film after finishing, and it is dropped into the 30s that dyes in the 5% ponceau staining fluid, marks the Position Approximate of Marker band and target protein band with pencil, and observes the transfer printing situation;
(4) with rinsed with deionized water NC film, until flush away ponceau staining fluid;
(5) the NC film is sealed spend the night (room temperature jog) with the PBST confining liquid that contains 10% skimming milk;
(6) (Abmart, Shanghai China) with after confining liquid (or 1: 100) dilution in 1: 1000, join on the NC film room temperature jog 2h with His-Tag Mouse McAb;
(7) with PBST washing 5 times, 5min/ time;
(8) add 1: 20000 the dilution sheep anti-mouse igg-HRP, room temperature jog 1.5h;
(9) with PBST washing 5 times, 5min/ time;
(10) with chemical luminescence reagent kit colour developing (Supersignal West Pico Trial Kit), the X exposure is developed, and the results are shown in Figure 5, and as can be seen from Figure 5 five recombinant proteins of Cap-A-E are correctly expressed, and size conforms to expection.Western blot result shows that the recombinant protein of expression can react with the His monoclonal antibody, has antigenicity preferably.
The mouse immuning test of embodiment 4 recombinant proteins
Recombinant protein c ap-A/B/C/D/E concentration is adjusted into 0.5mg/mL, mixes with Freund's complete adjuvant or Freund equal-volume respectively, emulsification prepares vaccine.Choose and clean level ICR mouse, 10 every group 5 ages in week.Head exempts from the vaccine 0.2mL of every subcutaneous multi-point injection in back with the Freund's complete adjuvant preparation, exempts from back 14d in head and carries out booster immunization, booster immunization Freund's incomplete adjuvant emulsification, every subcutaneous multi-point injection 0.2mL in back.Exempt from back 14d, 28d in head and get 5 eyeball blood sampling separation of serum, ELISA detects the special antibody titers of PCV2: with antigen coated liquid dilution recombinant antigen to 5 μ g/mL, every hole 100 μ L, 4 ℃ are spent the night behind 37 ℃ of 2h, abandon coating buffer, with 37 ℃ of sealings of 5% skimming milk 2h, PBST washing 3 times, serum to be checked is done 1: 200 times of dilution, and every hole adds 100 μ L, hatches 1.5h for 37 ℃; PBST washing 3 times adds the HRP-goat anti-mouse igg that dilutes at 1: 20000, hatches 1h for 37 ℃; PBST washing 3 times, every hole add TMB colour developing liquid 100 μ L, 2M H
2SO
4On microplate reader, read OD after the termination reaction
450Value.The results are shown in Figure 6.The result shows, all can induce body to produce antibody response after five fragment immunity, and each immune group antibody horizontal difference is not remarkable when 14d; During 28d, each is organized antibody titer and all rises to higher level, and it is the highest to compare the Cap-C slice groups, and the Cap-D group is the poorest.Therefore Cap-C demonstrates better immunogenicity, can be used as the candidate molecules of subunit vaccine.
The pig body protection test of embodiment 5 recombinant subunit vaccines
5.1 test design
20 28 age in days weanling pigs, through PCR and RT-PCR detect PCV2 and PRRSV negative, ELISA detects negative antibody.Be divided into 5 groups at random, 5 every group.The first winding kind PCV2 subunit vaccine (Cap-C, 0.5mg/mL), second winding kind PCV2 inactivated vaccine (iPCV2,10
5TCID
50/ mL, the 2mL/ head), third and fourth group is not inoculated respectively and is contrasted (CC) and blank (NC) as attacking poison, and one exempts from two groups of difference booster immunizations before back 14 days, and dosage method is with for the first time.
Attack malicious 2mL (PCV2 SH, CGMCC NO.2389,10 the 28th day every pig
5TCID
50/ mL, collunarium, each nostril 1mL), attack the 4th, 7 day every pig oxter, poison back and 4 injections of buttocks through Freund's incomplete adjuvant emulsive keyhole hemocyanin (1mg/mL), while abdominal injection 10mL thioglycollate medium, the 11st, 19 day abdominal injection 10mL thioglycollate medium.All pigs were all cutd open and to kill attacking poison in back 25 days.
Attack poison back isolated rearing, every morning is measured every pig rectal temperature, and twice observation morning and afternoon has no abnormal clinical manifestation (spirit is depressed, cough, expiratory dyspnea, skin by hair, appetite stimulator), and (the 0-6 branch of giving a mark, 0 fragrant expression is asymptomatic, and the high more expression symptom of score value is obvious more).Head exempted from back 14 days, 28 days, attacked the blood sampling respectively in 4,7,11,19 and 25 days of poison back, and separation of serum is used for the detection of antibody horizontal and viremia.Every pig is weighed respectively when attacking the poison same day and cuing open extremely, is used to calculate relative day weight gain (body weight when RDWG=(cuing open body weight-body weight when attacking poison when killing)/25/ attacks poison).Cut open and observe every pig pathology substantially when killing, get lymphoglandula, lungs are fixed in 10% buffered formalin, be used for tissue slice and make and immunohistochemical experiment.
5.2 result
5.2.1 clinical manifestation
After the immunity, each experimental group pig no abnormality seen reaction.After attacking poison, attack malicious control group rose at the 9th day performance spirit depressed, flock together etc., also have the jaundice performance, it is poor slightly that immune group has only the 1-2 head spirit of short period of time to occur; Body temperature is attacked the poison group above 39.5 ℃ of fates and is higher than all the other each immune group, significant difference.It is normal that blank group keeps during whole test.Calculate the weightening finish situation, attack malicious control group RDWG and be lower than all the other each groups, immune group and blank group no significant difference.
Table 1 is attacked poison back clinical manifestation and RDWG sums up
Different letter representation significant differences
5.2.2 antibody changes
The serum sample of (53 days) carries out ELISA experiment detection antibody titer when getting immunity back 14 days, 28 days and cuing open extremely.Immunity back each immune group serum of 2 week all change sun, and the antibody of existing higher level produces, and further raise to 28 days antibody horizontals; Attack and respectively organize antibody behind the poison and slightly raise, attack malicious control group porcine blood serum and all change sun, also reach higher level.And blank group still negative (Fig. 7).
5.2.3 viremia
All are attacked malicious pig and all produce viremia, attack malicious papova in the time of 7 days and reach higher level, attack the poison group to 11 and 19 days and reach higher level, apparently higher than immune group.Immune group is in lower level at 7d and 19d, and (Fig. 8) slightly raises during 11d.Though this explanation vaccine immunity can not stop the generation of viremia fully, can delay the time that viremia produces, and reduces the intensity of viremia.
5.2.4 cardinal principle/micro-pathology
Experiment finishes to cut open all pigs extremely, carries out the observation of cardinal principle pathology, and each organizes the pig lungs in various degree consolidation, oedema, and hemorrhage performance is arranged individually; Lymphoglandula is observed normal substantially, only has indivedual lymphs to have oedema, hemorrhage performance; Kidney, spleen liver are all normal.The pulmonary lesion of attacking malicious control group is apparently higher than all the other each groups.
Get lungs, lymphoglandula and make section and carry out the performance of the visible significantly interstitial pneumonia of pathological study pathology severe patient, interstitial proliferation, alveolus wall thickens, and alveolar space dwindles, and has hemorrhage and inflammatory exudate in alveolar space or the organ.The lungs of immune group are then normal substantially, and a matter is thinner, and alveolar is clear or slight interstitial proliferation is only arranged, and alveolus wall thickens.Attack poison group lymphoglandula and show lymphocyte disappearance, lymphatic nodule structure deteriorate even disappearance, the lymph node pathological change degree of immune group is obviously slight.This explanation vaccine immunity can alleviate the pathology damage of PCV2 to tissue.
The micro-pathology marking of table 2 lungs lymphoglandula situation
Different letter representation significant differences.
5.2.5?IHC
Adopt the positive porcine blood serum of PCV2 that PCV2 antigen in all lymphoglandula samples is carried out immunohistochemical methods and detect, adopt the DAB colour developing, positive cell is pale brown look.Attack malicious control group and all be judged to the positive for 5 parts, and positive cell number is more; Lymphocyte disappearance, lymph follicle obscurity boundary or disappearance are more obvious.Immune group is all negative, and the lymphocyte disappearance is also arranged, the lymph follicle obscurity boundary, but than the former degree slight (Fig. 9).This shows that vaccine immunity can alleviate the pathology damage of virus to lymphoglandula to a certain extent, reduces the content of virus in the lymphoglandula.
Claims (10)
1. the porcine circovirus 2 type Cap albumen of an expression of recombinant e. coli, it is characterized in that this albumen is to hold the porcine circovirus 2 type Cap albumen that is rich in arginic nuclear localization signal zone to be cloned into prokaryotic expression carrier amputation N to obtain recombinant expression vector, with this recombinant expression vector transfection Escherichia coli BL21 (DE3), express acquisition through this recombination bacillus coli BL21 (DE3).
2. the porcine circovirus 2 type Cap albumen of expression of recombinant e. coli according to claim 1 is characterized in that the proteic aminoacid sequence of porcine circovirus 2 type Cap that described amputation N end is rich in arginic nuclear localization signal zone is SEQ IDNO.1.
3. the porcine circovirus 2 type Cap albumen of expression of recombinant e. coli according to claim 1 and 2, the proteic nucleotides sequence of porcine circovirus 2 type Cap that the described amputation N end that it is characterized in that encoding is rich in arginic nuclear localization signal zone is classified SEQ ID NO.2 as.
4. the porcine circovirus 2 type Cap albumen of expression of recombinant e. coli according to claim 1 is characterized in that described prokaryotic expression carrier is pET-32a.
5. a recombinant plasmid PET-32a-Cap-c is characterized in that this plasmid is the nucleotide fragments shown in the SEQ ID NO.2 to be cloned into BamH I and two restriction enzyme sites of XhoI that prokaryotic expression carrier is pET-32a see gained.
6. a recombination bacillus coli BL21-Cap-C is characterized in that containing the described recombinant plasmid PET-32a-Cap-c of claim 5.
7. the proteic preparation method of porcine circovirus 2 type Cap of the described expression of recombinant e. coli of claim 1 is characterized in that comprising the steps:
(1) according to PCV2 SH pnca gene sequence A Y686763 design primer Cap51F:SEQ ID NO.3, Cap233R:SEQ ID NO.4, PK15 cell DNA with PCV2 SH virus strain infection is a template, the method of using PCR increases and obtains encoding the proteic gene fragment SEQ ID of Cap NO.2, by BamH I and two restriction enzyme sites of XhoI, the proteic gene fragment clone of described coding Cap is gone in the prokaryotic expression carrier, the evaluation of cutting by enzyme and check order then obtains positive recombinant plasmid;
(2) step (1) is described through the correct recombinant plasmid transformed e. coli bl21 (DE3) of sequencing, obtain recombinant strains BL21-Cap-C;
(3) cultivate described recombinant strains BL21-Cap-C, add IPTG and carry out abduction delivering, obtain described recombinant protein.
8. the proteic preparation method of porcine circovirus 2 type Cap of expression of recombinant e. coli according to claim 5 is characterized in that described prokaryotic expression carrier is pET-32a.
9. the proteic preparation method of porcine circovirus 2 type Cap of expression of recombinant e. coli according to claim 5, the IPTG that it is characterized in that adding when described recombinant strains BL21-Cap-C is cultured to OD600 and reaches 0.6-0.8 final concentration and be 1mM carries out abduction delivering.
10. subunit vaccine is characterized in that comprising the porcine circovirus 2 type Cap albumen and the adjuvant of the described expression of recombinant e. coli of claim 1.
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| CN102839189A (en) * | 2012-07-18 | 2012-12-26 | 华南农业大学 | Method for high efficiency expression of PCV 2 Cap protein by pCold-sumo expression vector |
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| US9717785B2 (en) | 2013-01-25 | 2017-08-01 | Fundação De Amparo À Pesquisa Do Estado De Mg-Fapemig | Recombinant antigens of porcine circovirus 2 (PCV-2) for vaccine formulations, diagnostic kit and use thereof |
| CN105087624A (en) * | 2015-08-18 | 2015-11-25 | 肇庆大华农生物药品有限公司 | Expression vector of Cap protein of porcine circovirus (PCV) 2 as well as construction method and application thereof |
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| CN107446028A (en) * | 2017-07-21 | 2017-12-08 | 江苏南农高科技股份有限公司 | Artificial reconstructed PCV2 Cap proteins, recombinant virus and its application |
| CN108159409A (en) * | 2017-12-25 | 2018-06-15 | 南京大爻网络科技有限公司 | A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application |
| CN108611359A (en) * | 2018-05-04 | 2018-10-02 | 武汉科前生物股份有限公司 | The preparation method and applications of 3 virus-like particle of pig circular ring virus |
| CN108611359B (en) * | 2018-05-04 | 2021-11-19 | 武汉科前生物股份有限公司 | Preparation method and application of porcine circovirus type 3 virus-like particles |
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