CN102174086B - 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 current comparative maturity, have simple to operate, safety non-toxic, exogenous protein expression amount advantages of higher, successfully 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 expression systems and expresses Cap albumen, but also Cap albumen different fragments is not carried out to Analysis of Immunogenicity at present, also prokaryotic expression Cap albumen is not carried out to immanoprotection action research and for the report of vaccine research.
Summary of the invention
The object of the invention is for 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 is to provide the preparation method of this recombinant C ap albumen.
Another object of the present invention is to provide a kind of subunit vaccine that comprises this recombinant C ap albumen.
Object of the present invention is achieved through the following technical solutions:
A kind of carrying Cap gene of porcine circovirus type 2 of expression of recombinant e. coli, the carrying Cap gene of porcine circovirus type 2 that arginic nuclear localization signal region is rich in by amputation N end in this albumen system is cloned into prokaryotic expression carrier and obtains recombinant expression vector, by this recombinant expression vector transfection Escherichia coli BL21 (DE3), through this recombination bacillus coli BL21 (DE3), express and obtain.
The aminoacid sequence that described amputation N end is rich in the carrying Cap gene of porcine circovirus type 2 (C) in arginic nuclear localization signal region is SEQ ID NO.1, i.e. 51-234 amino-acid residue of the ORF2 of porcine circovirus 2 type (PCV2) coding.
The described amputation N of coding holds the nucleotides sequence of the carrying Cap gene of porcine circovirus type 2 that is rich in arginic nuclear localization signal region to classify SEQ ID NO.2 as.
Described prokaryotic expression carrier is pET-32a.
There is the screening of PCV2 recombinant C ap albumen of the intestinal bacteria system expression of immanoprotection action: the fragments that amputation N end is rich in to the different sizes of carrying Cap gene of porcine circovirus type 2 in arginic nuclear localization signal region are cloned into respectively prokaryotic expression carrier and obtain recombinant expression vector; be converted into respectively e. coli bl21 (DE3); obtain recombinant protein; by mouse immuning test, screening obtains can induce the recombinant C ap albumen (C) that produces high-caliber PCV2 specific antibody.
A recombinant plasmid PET-32a-Cap-c, this plasmid is the nucleotide fragments shown in 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.
A recombination bacillus coli BL21-Cap-C, contains described recombinant plasmid PET-32a-Cap-c.
A preparation method for the carrying Cap gene of porcine circovirus type 2 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), the PK15 cell DNA of PCV2 SH virus strain infection of take is template, the method amplification of application PCR obtain the encoding gene fragment (SEQ ID NO.2) of Cap albumen, by BamH I and two restriction enzyme sites of XhoI, described gene fragment clone is entered in prokaryotic expression carrier, then the evaluation of cutting by enzyme and check order, obtains positive recombinant plasmid;
(2) by step (1) Suo Shu through the correct recombinant plasmid transformed e. coli bl21 (DE3) of sequencing, obtain recombinant strains BL21-Cap-C (express recombinant C ap PROTEIN C fragment);
(3) cultivate described recombinant strains BL21-Cap-C, add IPTG to carry out abduction delivering, obtain described recombinant protein.
Wherein, described prokaryotic expression carrier is pET-32a (+).
Described recombinant strains BL21-Cap-C is cultured to that when OD600 reaches 0.6-0.8, to add final concentration be that the IPTG of 1mM carries out abduction delivering.
A subunit vaccine, is characterized in that the carrying Cap gene of porcine circovirus type 2 (C) and the adjuvant that comprise expression of recombinant e. coli claimed in claim 1.
Beneficial effect of the present invention:
It is high that the carrying Cap gene of porcine circovirus type 2 of expression of recombinant e. coli of the present invention has antigen purity as subunit vaccine, and security is good, and immunogenicity is strong, and the animals such as pig are not had pathogenic, easy of the advantage of safety evaluation.
Evidence, the recombinant bacterial strain that the present invention builds is expressed stable to external source target protein.By mouse immuning test, proved that vaccine prepared by this recombinant protein can be induced and produced high-caliber PCV2 specific antibody.The experiment of pig body further confirms that it has good immune protective effect.
Therefore recombinant baculovirus expression Cap protein subunit vaccine of the present invention is compared with the inactivated vaccine of the PCV2 of current application, and this vaccine preparation process is relatively simple, and cost is lower, has more wide application prospect aspect the preventing and treating of PCV2.
Accompanying drawing explanation
Fig. 1 PCV2 Cap-a/b/c/d/e gene clone enters carrier schematic diagram.
Fig. 2 PCV2 Cap-a/b/c/d/e gene PCR amplification,
1-5:PCR product, the respectively gene fragment of corresponding 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 the expression of recombinant protein,
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 is primary antibodie)
1-5: recombinant protein c ap-A/B/C/D/E, 6: empty carrier induced product.
Fig. 6 recombinant protein mouse immune antibody changes.
Fig. 7 recombinant protein c ap-C pig body experiment antibody changes.
After Fig. 8 attacks poison, pig precursor virus mass formed by blood stasis detects.
Fig. 9 attacks latter 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, upstream and downstream primer is respectively introduced BamH I and Xho I site, increase the respectively gene fragment of 51-150,51-200 in 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 object fragment
With PCV2SH strain (CGMCC NO.2389, preservation date: on March 4th, 2008) infect PK15 cell, 72h gathers in the crops virus, adopts phenol: chloroform extraction method extracts DNA as PCR reaction template.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; Sterilizing distilled water is mended to 25 μ L.Loop parameter: 95 ℃ of denaturation 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 PCR product, and-20 ℃ save backup.
The structure of 1.3 recombinant plasmids and evaluation
By BamH I/Xho I double digestion for the goal gene reclaiming, after recovery purifying, being cloned into same enzyme cuts in the expression vector plasmid pET-32a (Invitrogen) of processing, ligation is carried out at 16 ℃, to connect afterwards product Transformed E .coli DH5 α (NEB) competent cell, coating is dull and stereotyped containing the LB of penbritin, 37 ℃ of cultivations.Bacterium colony on picking flat board, in the LB culture medium culturing containing penbritin, alkaline lysis method of extracting plasmid, identifies (the results are shown in Figure 3) through PCR and BamH I/Xho I double digestion, positive plasmid obtains object band.Positive plasmid is served Hai Ying fine horse company limited and is checked order.By order-checking correct recombinant plasmid difference called after PET-32a-Cap-a, PET-32a-Cap-b, PET-32a-Cap-c, PET-32a-Cap-d, PET-32a-Cap-e.
The structure of embodiment 2 recombinant strains BL21-Cap-a/b/c/d/e
2.1 transformed competence colibacillus intestinal bacteria BL-21
In embodiment 1 through the correct recombinant plasmid transformed intestinal bacteria BL-21 (NEB) of sequencing, obtain respectively recombinant strains BL21-Cap-a, BL21-Cap-b, BL21-Cap-c, BL21-Cap-d, BL21-Cap-e, simultaneously by empty plasmid pET-32a with method transformed competence colibacillus intestinal bacteria BL-21.
The optimization of 2.2 expression of recombinant proteins conditions
Picking recombinant strains BL21-Cap-a/b/c/d/e and be dispersed in single colony inoculation in the LB liquid nutrient medium that contains penbritin containing the intestinal bacteria BL-21 of empty plasmid pET-32a respectively, 37 ℃ of joltings of spending the night are cultivated.
(1) the bacterium liquid 10 μ L that get incubated overnight are inoculated in the LB liquid nutrient medium that 3ml contains penbritin (50 μ g/ml), and 37 ℃ of 200rpm shaking culture 3h left and right, make OD
600reach 0.6～0.8, get 100 μ L samples and as induction is front, contrast in aseptic Eppendorf pipe;
(2) to adding final concentration in above-mentioned bacterium liquid, be the abduction delivering that the IPTG of 1.0mmol/L carries out recombinant protein, and after adding IPTG the 3rd, 4,5,6h 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 completely, with the resuspended bacterial precipitation of appropriate PBS;
(5) by bacterium liquid multigelation 3 times;
(6) ultrasonic treatment bacterium: power is selected 200W operates on ice chest, ultrasonic degradation 5s, and pause 5s, until bacterium liquid becomes limpid;
(7) 4 ℃ of centrifugal 10min of 8000rpm, collect respectively supernatant and precipitation: precipitation is used with the isopyknic PBS of supernatant resuspended, 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 induction 4h is optimum condition of the expression.
2.3SDS-PAGE electrophoretic analysis
In sample, add 5 * protein sample sample-loading buffer (250mM Tris-HCl, pH6.8; 10%SDS; 0.5% tetrabromophenol sulfonphthalein; 50% glycerine; 5%2-mercaptoethanol), fully mix latter 100 ℃ and boil 5min, instantaneous centrifugal before loading, 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
The great expression of 3.1 fusion roteins, purifying
The best abduction delivering condition expression profile engineering recombinant bacterium 500ml groping by embodiment 2, centrifugal collection thalline, by every 100ml bacterium liquid, add the resuspended bacterial sediment of 5ml PBS, centrifugal after ultrasonic degradation, cleer and peaceful inclusion body in 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, spectrophotometric determination protein concentration, and 4 ℃ or-20 ℃ save backup.
(6) protein concentration is adjusted to 0.3mg/ml left and right, packed in dialysis tubing, in dialyzate, progressively add 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 maintained 6-8h, under agitation condition, carries out.
(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.
The Western blot of 3.2 recombinant proteins identifies
(1)SDS-PAGE；
(2) transfer printing: after electrophoresis finishes, take off gel and transfer device (half-dried transfer printing) is installed in the following order
+ the utmost point: blank-sponge-NC film-gel-sponge-blackboard :-the utmost point, transfer printing condition is 20V, 30min;
(3) NC film is taken off in transfer printing after finishing, and is dropped into the 30s that dyes in 5% ponceau staining fluid, marks the Position Approximate of Marker band and target protein band, and observe transfer printing situation with pencil;
(4) with rinsed with deionized water NC film, until wash away ponceau staining fluid;
(5) NC film is sealed to spend the night (room temperature jog) with the PBST confining liquid containing 10% skimming milk;
(6) after His-Tag Mouse McAb (Abmart, Shanghai, China) is diluted with confining liquid 1: 1000 (or 1: 100), join on NC film room temperature jog 2h;
(7) with PBST washing 5 times, 5min/ time;
(8) add the sheep anti-mouse igg-HRP of dilution in 1: 20000, 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), 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 obtain correction, and size conforms to expection.Western blot result shows that the recombinant protein of expressing can react with His monoclonal antibody, has good antigenicity.
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 respectively with Freund's complete adjuvant or Freund's incomplete adjuvant equal-volume, vaccine is prepared in emulsification.Choose clean level ICR mouse in 5 week age, 10 every group.Head exempts from the vaccine 0.2mL that every subcutaneous multi-point injection in back is prepared with Freund's complete adjuvant, exempts from rear 14d and carries out booster immunization, booster immunization Freund's incomplete adjuvant emulsification, every subcutaneous multi-point injection 0.2mL in back in head.In head, exempt from rear 14d, 28d 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, after 37 ℃ of 2h, 4 ℃ are spent the night, and abandon coating buffer, with 37 ℃ of sealing 2h of 5% skimming milk, PBST washing 3 times, serum to be checked is done to 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 diluting at 1: 20000, hatches 1h for 37 ℃; PBST washing 3 times, every hole adds TMB nitrite ion 100 μ L, 2M H
2sO
4after termination reaction, in microplate reader, read OD
450value.The results are shown in Figure 6.Result shows, five fragment immunity all can induce body to produce antibody response afterwards, and when 14d, each immune group antibody horizontal difference is not remarkable; During 28d, each is organized antibody titer and all rises to higher level, compares Cap-C slice groups the highest, and Cap-D group is the poorest.Therefore Cap-C demonstrates good 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 at random 5 groups, 5 every group.First group of inoculation PCV2 subunit vaccine (Cap-C, 0.5mg/mL), second group of inoculation PCV2 inactivated vaccine (iPCV2,10
5tCID
50/ mL, 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 latter 14 days, and dosage method is with for the first time.
The 28th day every pig, attack malicious 2mL (PCV2 SH, CGMCC NO.2389,10
5tCID
50/ mL, collunarium, each nostril 1mL), attack after poison the 4th, 7 days every pig oxters and 4 injections of buttocks through the keyhole hemocyanin (1mg/mL) of Freund's incomplete adjuvant emulsification, while abdominal injection 10mL thioglycollate medium, the 11st, 19 days abdominal injection 10mL thioglycollate mediums.All pigs attack poison within latter 25 days, all cut open and kill.
Attack the rear isolated rearing of poison, every morning is measured every pig rectal temperature, twice observation morning and afternoon has or not abnormal clinical manifestation (depressed, the cough of spirit, expiratory dyspnea, skin are by hair, appetite stimulator), and give a mark that (0-6 divides, 0 fragrant expression is asymptomatic, and the higher expression symptom of score value is more obvious).Head exempts from latter 14 days, 28 days, attacks poison blood sampling respectively in latter 4,7,11,19 and 25 days, and separation of serum, for the detection of antibody horizontal and viremia.Attack the poison same day and cut open every pig while killing and weigh respectively, for calculating relative day weight gain (body weight when RDWG=(body weight while cuing open body weight while killing-attack poison)/25/ attacks poison).Cut open and while killing, observe every pig pathology substantially, get lymphoglandula, lungs are fixed in 10% buffered formalin, for tissue slice, make and immunohistochemical experiment.
5.2 result
5.2.1 clinical manifestation
After immunity, each experimental group pig no abnormality seen reaction.Attack after poison, attack malicious control group at the 9th day, rise performance spirit depressed, flock together etc., also have jaundice performance, immune group only has 1-2 head to occur that the spirit of short period of time is slightly poor; Body temperature surpasses 39.5 ℃ of number of days and attacks malicious group higher than all the other each immune group, significant difference.Blank group keeps normal at whole duration of test.Calculate Gain weight, attack malicious control group RDWG lower than all the other each groups, immune group and blank group no significant difference.
Table 1 is attacked the rear clinical manifestation of poison and RDWG sums up
Different letter representation significant differences
5.2.2 antibody changes
The serum sample of getting immune latter 14 days, 28 days and cut open (53 days) while killing carries out ELISA experiment and detects antibody titer.After immunity, 2 weeks each immune group serum all turns sun, and the antibody generation of existing higher level further raises to 28 days antibody horizontals; Attack and respectively organize antibody after poison and slightly raise, attack malicious control group porcine blood serum and all turn sun, also reach higher level.And blank group still negative (Fig. 7).
5.2.3 viremia
Allly attack malicious pig and all produce viremia, in the time of 7 days, attack malicious papova and reach higher level, to 11 and 19 days, attack malicious group and reach higher level, apparently higher than immune group.Immune group is at 7d and 19d in lower level, and (Fig. 8) slightly raises during 11d.Though this explanation vaccine immunity can not stop the generation of viremia completely, 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 to kill all pigs, carries out cardinal principle pathology and observes, and each organizes pig lungs consolidation, oedema in various degree, has individually hemorrhage performance; Lymphoglandula is observed normal, only has indivedual lymphs to have oedema, hemorrhage performance; Kidney, spleen liver are all normal.Attack the pulmonary lesion of malicious control group 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 organ.The lungs of immune group are normal, and interstitial is thinner, and alveolar is clear or only have slight interstitial proliferation, and alveolus wall thickens.Attack that poison group lymphoglandula shows Lymphocyte depletion, lymphatic nodule structure deteriorate even disappears, 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 to carry out immunohistochemical methods detection to PCV2 antigen in all lymphoglandula samples, adopt DAB colour developing, positive cell is brown color.Attack 5 parts of malicious control groups and be all judged to the positive, and positive cell number is more; Lymphocyte depletion, lymph follicle obscurity boundary or disappearance are more obvious.Immune group is all negative, also has Lymphocyte depletion, 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 lymphoglandula.
Claims (7)
1. the carrying Cap gene of porcine circovirus type 2 of an expression of recombinant e. coli, it is characterized in that carrying Cap gene of porcine circovirus type 2 that arginic nuclear localization signal region is rich in by amputation N end in this albumen system is cloned into prokaryotic expression carrier and obtains recombinant expression vector, by this recombinant expression vector transfection Escherichia coli BL21 (DE3), through this recombination bacillus coli BL21 (DE3), express and obtain; The aminoacid sequence that described amputation N end is rich in the carrying Cap gene of porcine circovirus type 2 in arginic nuclear localization signal region is SEQ ID NO.1.
2. a recombinant plasmid PET-32a-Cap-c, is characterized in that this plasmid is that the nucleotide fragments shown in SEQ ID NO.2 is cloned into prokaryotic expression carrier is pET-32a's
bamHi and
xhogained between two restriction enzyme sites of I.
3. a recombination bacillus coli BL21-Cap-C, is characterized in that containing recombinant plasmid PET-32a-Cap-c claimed in claim 2.
4. a preparation method for the carrying Cap gene of porcine circovirus type 2 of expression of recombinant e. coli claimed in 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, the PK15 cell DNA of PCV2 SH virus strain infection of take is template, the method amplification of application PCR obtain the encoding gene fragment SEQ ID NO.2 of Cap albumen, by
bamHi and
xhotwo restriction enzyme sites of I, enter the gene fragment clone of described coding Cap albumen in prokaryotic expression carrier, and the evaluation of then cutting by enzyme and check order, obtains positive recombinant plasmid;
(2) by step (1) Suo Shu 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 to carry out abduction delivering, obtain described recombinant protein.
5. the preparation method of the carrying Cap gene of porcine circovirus type 2 of expression of recombinant e. coli according to claim 4, is characterized in that described prokaryotic expression carrier is pET-32a.
6. the preparation method of the carrying Cap gene of porcine circovirus type 2 of expression of recombinant e. coli according to claim 4, is characterized in that described recombinant strains BL21-Cap-C is cultured to that when OD600 reaches 0.6-0.8, to add final concentration be that the IPTG of 1mM carries out abduction delivering.
7. a subunit vaccine, is characterized in that the carrying Cap gene of porcine circovirus type 2 and the adjuvant that comprise expression of recombinant e. coli claimed in claim 1.
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| CN114214337A (en) * | 2021-12-29 | 2022-03-22 | 广州格雷特生物科技有限公司 | Preparation method and application of porcine circovirus type 2a Cap protein |
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| CN101948849A (en) * | 2010-09-02 | 2011-01-19 | 洛阳普莱柯生物工程有限公司 | Preparation and use of truncated Cap protein of porcine circovirus type 2 |
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