CN102127533B - Preparation method of recombinant porcine circovirus type 2 Cap antigen - Google Patents

Abstract

The invention discloses a preparation method of a recombinant porcine circovirus type 2 Cap antigen, which comprises the following steps: using a yeast expression system to amplify the PCV2-ORF2 gene; then, constructing an expression engineering bacterium: inserting an amplification sequence into a yeast expression vector to form a recombinant expression vector, linearizing, and transforming into a pichia pastoris host cell so as to finish the construction of the expression engineering bacterium; and finally, carrying out secretory expression on the recombinant microzyme to obtain the recombinant porcine circovirus type 2 Cap antigen protein. The yeast expression system can carry out processing, folding and posttranslational modification on the expressed protein, so that the expressed protein has biological activity. Compared with Escherichia coli, the yeast expression system has more advantages; and compared with a mammal cell expression system, the yeast expression system is more convenient to operate. Compared with Saccharomyces cerevisiae, the pichia pastoris can not be easily subjected to hyperglycosylation, thereby facilitating the separation and purification steps. By using the yeast expression system, the invention has the advantages of simple operation process, high expression quantity and low production cost, can implement large-scale production, and is beneficial to the popularization and application of the porcine circovirus subunit vaccine.

Description

The antigenic preparation method of reorganization porcine circovirus 2 type Cap

Technical field

The present invention relates to biological technical field, be specifically related to the vaccine of a kind of Cap antigen of recombinate antigenic preparation method of porcine circovirus 2 type Cap and yeast expression thereof for the basis preparation.

Background technology

PCV2 is one of minimum virus of newfound infected pigs in recent years; Mainly show as clinically become thin, palor, poor growth, expiratory dyspnea, dermatitis diarrhoea, jaundice etc.; Since the nineties in 20th century Canada reported first; This disease worldwide is widely current, and has caused tremendous loss for the countries in the world pig industry, and this big country of raising pigs of China also deeply hurts.There are some researches prove that pig circular ring virus 2 viral disease and multiple factor acting in conjunction cause disease.When this disease and other cause of diseases are invaded body jointly, help PCV2 and in target cell, duplicate propagation, thereby cause immunosuppression.The specifics of not treating PCV2 at present; Vaccinoprophylaxis is still the first-selected measure of prevention and control PCV2, subunit vaccine safety, effective, the existing abroad good commercialization subunit vaccine of immune effect; Domestic still do not have a subunit vaccine registration listing, and prices are rather stiff for the subunit vaccine of import.

The environment resistibility is stronger to external world for circovurus type 2, in sour environment and in the chloroform, can survive the long period.Genome is the sub-thread cyclic DNA, and total length is 1,767bp or 1, and 768bp contains 11 open frames of reading, and wherein ORF1, ORF2, ORF3 study often, and ORF2 is by 702 based compositions.Its major antigen is transcribed by the viral DNA complementary strand, and coding contains 234 in amino acid, the nucleocapsid protein (Cap albumen) of the about 27.8ku of size.The 65th～87 in PCV2 cap albumen, 113～139 and 193～207 amino acids are PCV2 specific antigens epi-position.

PCV2 Cap albumen does not have intersecting protective as main protective immunity albumen with PCV1 Cap albumen, and the researchist serves as the research focus to give expression to Cap albumen both at home and abroad.Explored various phraseologies at present; Like escherichia coli expression, baculovirus expression, recombinant adenoviral vector etc., comparatively successful example has baculovirus expression system, the Cap albumen that recombinant baculovirus goes out in expressed in insect cells; Molecular weight is about 30KD; Under Electronic Speculum, observe, find that the virus nucleocapsid albumen that obtains with separation and purification is consistent, show that Cap albumen oneself in the external source expression system is assembled into the viroid particle; Evidence can induce behind the immune animal to produce high-level specific antibody and cause specific immune response.External existing commercial subunit vaccine listing can produce protective immunological reaction, significantly reduces viremia.The method that domestic part colleges and universities and scientific research institution are used to prepare the porcine circovirus 2 type subunit vaccine has the recombinant adenovirus of structure and recombinant baculovirus expression Cap albumen, and the former immune effect is still to be tested, and latter's production cost is higher.

Yeast expression system has been one of comparatively sophisticated heterogenous expression system, and it is simple to operation, and success ratio is high, has both satisfied the prokaryotic expression high-density growth, has been prone to the advantage of cultivating, and possesses the function of eukaryotic expression system posttranslational modification again.Expressed proteins output is high, may reside in the cell, also can secrete to born of the same parents.The albumen that self produces is few, and does not have toxicity, in recent years; The expression of in yeast expression system, succeeding of existing large number of biological goods, the professional etiquette of going forward side by side modelling fermentative prodn is low as production cost; The heterogenous expression system that quantum of output is high, yeast expression system has advantage very much.Transform goal gene according to the inclined to one side preferendum of yeast codon, continue to optimize expression condition, the huge space of improving expression amount is arranged.

Summary of the invention

The object of the present invention is to provide a kind of preparation method of porcine circovirus 2 type subunit antigen.

Another object of the present invention provides the vaccine that comprises according to above-mentioned preparing method's gained porcine circovirus 2 type subunit antigen.

A kind of preparation method of porcine circovirus 2 type subunit antigen is to adopt the yeast expression system preparation.

Concrete steps are following:

(1) amplification PCV2-ORF2 gene;

(2) construction expression engineering bacteria: extension increasing sequence inserts Yeast expression carrier, forms recombinant expression vector, transforms pichia spp host cell, construction expression engineering bacteria after the linearizing;

(3) recombination microzyme secreting, expressing.

PCV2-ORF2 gene described in the step (1) is full gene of PCV2-ORF2 or the PCV2-ORF2 gene transformed according to the inclined to one side preferendum of yeast codon.

The primer of amplification PCV2-ORF2 gene is SEQ ID NO:1 and SEQ ID NO:2.

Yeast expression carrier described in the step (2) is pGAPZ α A, pGAPZ α B or pGAPZ α C.

The pichia spp host cell is yeast X33, SMD1168 described in the step (2).

A kind of pig circular ring virus subunit vaccine is to comprise above-mentioned preparing method's gained antigen.Antigen effective constituent concentration is 200～350 μ g/mL in the vaccine.

The above-mentioned preparation method who states vaccine is that the gained antigenic solution is mixed with sanitas, stablizer, antimicrobial drug, adds adjuvant, and regulating pH is 7.0～7.5, promptly obtains subunit vaccine.

Wherein sanitas is for preparation vaccine sanitas commonly used, like LL37 antibacterial peptide, formaldehyde, phenol, phenylcarbinol, nisin, parabens, borax etc.Stablizer is an EDTA Disodium.Antibacterials comprise like GT, mycillin etc.

Above-mentioned adjuvant is aluminium glue, card uncle ripple 971, saponin, liposome, CpG-ODN, nanometer adjuvant or oil emulsion adjuvant.

Compared with prior art, the present invention has following beneficial effect:

The employing yeast expression system is simple to operate, expression amount is higher, and production cost is low, can carry out scale operation.Behind the construction recombination plasmid, transform, utilize the Z resistance can filter out positive transformant, improve Zeocin through electroporation ^TMResistance concentration can be screened the transformant of high copy amount.It is simple that experimental implementation obtains the recombination yeast engineering bacteria, and success ratio is high, when this expression system plasmid transforms, is that plasmid integration is arrived the yeast genes group, comparatively stable, avoids goal gene to lose, heredity that can be stable.Yeast expression system and insect cell expression system, mammalian cell expression system have the function of processing folding and posttranslational modification to expressed proteins equally; Thereby make the expressed proteins biologically active; More have superiority compared with intestinal bacteria, but easier than the operation of mammalian cell expression system.And compare with yeast saccharomyces cerevisiae, pichia spp is difficult for excessive glycosylation.This virus mainly causes immuning tissue's pathology, is the door of other bacteriums, poisoning intrusion, and the security of the pig circular ring virus deactivation vaccine that exists at present is still waiting checking; The PCV2-Cap that insect cell expression system is expressed has been prepared into commercialized vaccine; And clinical effectiveness is good, since complex manufacturing, the reason of production cost; Cause it to cost an arm and a leg, be difficult to promote.Pichia yeast expression system can adopt the basic medium fermentation; Only provide the yeast growth desired nutritional to get final product high density fermentation; Dried cell weight can reach 100g/l; Simultaneously yeast cell self excretory albumen seldom, separation and purification often needs comparatively complicated technology, and the separation and purification of yeast expression system expressed proteins only need separate supernatant, micro-filtration, concentrate 3 steps of desalination and get final product.Under the culture condition after the optimization, expression amount is high, and production cost is low, helps promoting the use of of pig circular ring virus subunit vaccine.

Description of drawings

Fig. 1: the technological line figure that makes up the recombination yeast engineering bacteria;

The SDS-PAGE electrophorogram that Fig. 2: PCV2-Cap expresses, M is for dye the molecular weight of albumen standard in advance, and 1 is to express supernatant without concentrated pGAPZ α A-ORF2-X33, and 2 is the expression supernatant of pGAPZ α A-X33 empty carrier;

Fig. 3: detect the immunogenic immunoblotting assay figure of PCV2-Cap.M is for dye the molecular weight of albumen standard in advance, and 1-2 expresses supernatant without concentrated pGAPZ α A-ORF2-X33, and 3 is the expression supernatant of pGAPZ α A-X33 empty carrier.

Embodiment

The biomaterial that uses among the following embodiment is: Zeocin ^TMResistance, expression vector pGAPZ α A, Pchia pastorisRecipient bacterium X33, SMD1168 purchase the company in Invitrogen; RTapArchaeal dna polymerase, T4 DNA Ligase, restriction enzyme XhoI, NotI, AvrII is the TaKaRa Company products; E.coliDH5 α is preserved by Agricultural University Of South China's veterinary college Infectious Diseases Lab.Plasmid extraction kit, PCR purification kit, glue reclaim test kit available from PROMEGA company.All the other biochemical reagents are purchased the biological ltd in ancient cooking vessel state.Viral source is that the ELISA detection is the sick pig lungs of serology male.

Embodiment 1

With plasmid pGAPZ α A is Yeast expression carrier, is the expressive host bacterium with yeast X33, and the preparation method may further comprise the steps:

1. from circovurus type 2 infected pigs lungs isolated viral, whole genome amplification obtains goal gene ORF2.Upstream primer is SEQ ID NO:1, and downstream primer is SEQ ID NO:2, and reaction conditions is: 94 ℃ of preparatory sex change 5min, begin 30 circulations then, and 90 ℃ of 30S, 50 ℃ of 40S, 72 ℃ of 45S, last 72 ℃ are extended 10min.

2.PCV2 the ORF2 gene is connected and conversion with the pMD18-T cloning vector; With the goal gene ORF2 4.5 μ L that obtain of amplification and Solution I 5.0 μ L in pMD18-T carrier 0.5 μ L and the step 1 gently behind the mixing; 16 ℃ of connections are spent the night, and connect product and are labeled as pMD18-ORF2.Connect the DH5 α competent cell 150 μ L that product 5 μ L transform preparation, the bacterium liquid of conversion is evenly spread LB flat board (penbritin=100 μ g/mL), and next day, picking list bacterium colony shook bacterium, send the order-checking of Invitrogen company, and the extracting plasmid, Xho IWith Not IAfter double digestion is identified successfully, plasmid be stored in-20 ℃ subsequent use.

3. Yeast expression carrier pGAPZ α A and pMD18-ORF2 are done Xho IWith Not IDouble digestion, through 1% agarose gel electrophoresis isolated genes fragment, glue reclaims expression vector and goal gene; Re-use the T4 dna ligase and connect, make up recombinant expression vector, be labeled as pGAPZ α A-ORF2; Transform mode transforms DH5 α competent cell, extracting recombinant plasmid in 2 set by step Xho IWith Not IAfter double digestion is identified successfully, recombinant plasmid be stored in-20 ℃ subsequent use.

4. recombinant expression vector pGAPZ α A-ORF2 transformed host cell pichia spp X33 makes up recombinant expressed engineering bacteria.With competence P.pastorisX33 (80 μ L) with The Avr IISingle endonuclease digestion pGAPZ α A-ORF2 (10 μ g) mixes mutually, 1.5 kV, 200 electric shocks, 5 ms.Yeast cell after transforming evenly is tiled in prepared fresh YPDS flat board (contains Zeocin ^TMResistance 100 μ g/mL), treat flat board,, in 30 ℃ of incubators, be cultured to single bacterium colony and (about 2 days) occur the flat board inversion liquid-absorbent (about 15min).Employing boils-freezes-and cooking method prepares bacterium liquid PCR template analysis P.pastorisTransformant, upstream primer are SEQ ID NO:1, and downstream primer is that SEQ ID NO:2 can amplify the positive transformant of single bacterium colony about 700 bp.Obtain recombination yeast engineering bacteria pGAPZ α A-ORF2-X33.

5.pGAPZ α A-ORF2-X33 does secreting, expressing with the YPDS nutrient solution.As seed liquor, it is muddy to cultivate about 18h bacterium liquid in 4mL YPD nutrient solution for white single bacterium colony of picking smooth surface, neat in edge, and draw 1mL and be resuspended in (loading amount is 10%) in the 50mLYPDS substratum, 220r/min, 30 ℃ of concussions are cultivated.Behind the 72h the centrifugal collection culture supernatant of 5000r/min * 10min make SDS-PAGE or be stored in-80 ℃ subsequent use.The SDS-PAGE electrophorogram that PCV2-Cap expresses is seen Fig. 2, a specific band is arranged near 26KDa, and empty carrier does not have, and shows PCV2 ORF2 gene successful secreting, expressing in yeast X33.Detect the immunogenic immunoblotting assay figure of PCV2-Cap and see Fig. 3, anti-as one with PCV2 infected pigs serum, horseradish peroxidase-labeled goat-anti pig IgG is anti-as two, through DAB-H ₂O ₂After the colour developing, a specific band near 26KDa, occurs, empty carrier does not occur.Show that the Cap albumen that PCV2 ORF2 gene is expressed has certain antigenicity in yeast.

PGAPZ α A carrier is the composing type strong promoter with pGAP, under the situation that glucose exists, can induce yeast to get into the protein expression state, promptly in the YPDS substratum; Yeast cell growth; Express target protein simultaneously, do not need methanol induction to express, methyl alcohol is a kind of colourless, transparent, inflammable, volatile toxic liquid; Hazardous in transportation, preservation process, expression product also are difficult to avoid methyl alcohol to pollute.Culture medium carbon source can disposable in batches interpolation, also can suitably reduce by primary carbon source, adds carbon source through stream about 6-10h, and the advantage that stream adds carbon source is to improve the utilization of carbon source rate of yeast cell.High copy number needn't obtain the high expression level amount surely; Single copy is lower than 2 copy and 3 copy; Reason possibly be that the sub-translation efficiency of high copy is also high; Nutrition supply is not enough, can not carry out in order with posttranslational modification expressed proteins processing is folding, and selecting suitable multiple copied also is the emphasis that improves expression amount.It is 6.5 that this experiment can be selected phosphate buffered saline buffer control expression process pH value of solution value.Improve dissolved oxygen amount as much as possible, evidence, expression amount increases along with dissolved oxygen amount and raises.Bacterium liquid initial OD values should reach more than 1.5 during expression, and generally under well-fed situation, bacterium liquid density is high more, and expression amount is high more.Cultivate recombination microzyme to OD=2～6 with the YPD nutrient solution earlier, draw bacterium liquid and be resuspended in the YPDS nutrient solution, make OD >=1.5; Maybe when reaching OD=2～6, the centrifugal collection thalline of 5000r/min is resuspended in the YPDS nutrient solution; Expression time can be 3～5 days, in the culturing process, guarantee competent oxygen dissolving value (DO); Express temperature and be controlled between 28 ℃～30 ℃, be higher than 30 ℃, be unfavorable for yeast cell growth.The temperature initial stage can be controlled in 28～30 ℃, along with bacterial concentration increases, suitably reduces culture temperature, alleviate the yeast cell growth excessive velocities, and hydrolase of proteolysis is too high, thereby influences the output of target protein.Will notice in the expression process preventing that excess foam from producing, can influence the DO value, the culture bacteria weight in wet base can reach 40g/L when collecting the expression supernatant, even 100 g/L, even 150 g/L, even higher.The process of expressing is adjustable, can optimize, and makes the expression amount very large space of having risen violently, and this needs constantly grope in process of production.

Embodiment 2

With plasmid pGAPZ α A is Yeast expression carrier, is the expressive host bacterium with yeast SMD1168, and the preparation method may further comprise the steps:

1. from circovurus type 2 infected pigs lungs isolated viral, whole genome amplification obtains goal gene ORF2.Upstream primer is SEQ ID NO:1, and downstream primer is SEQ ID NO:2, and reaction conditions is: 94 ℃ of preparatory sex change 5min, begin 30 circulations then, and 90 ℃ of 30S, 50 ℃ of 40S, 72 ℃ of 45S, last 72 ℃ are extended 10min.Transform the ORF2 gene according to the inclined to one side preferendum of yeast codon, send the full gene of Sangon Biotech (Shanghai) Co., Ltd. the synthetic goal gene that obtains.Recombinant plasmid is labeled as pBluescript II SK ⁺-ORF2.Target gene sequences is total to 734bp shown in SEQ ID NO:3.

2. pBluescript II SK ⁺-ORF2 plasmid 2 μ L transform the DH5 α competent cell 150 μ L of preparation, and the bacterium liquid of conversion is evenly spread LB flat board (penbritin=100 μ g/mL), and next day, picking list bacterium colony shook bacterium, the extracting plasmid, Xho IWith Not IAfter double digestion is identified successfully, plasmid is stored in-20 ℃ subsequent use.

3. said with Yeast expression carrier pGAPZ α A and the recombinant plasmid pBluescript II SK that contains goal gene like embodiment 1 step 3 ⁺-ORF2 does Xho IWith Not IDouble digestion, through 1% agarose gel electrophoresis isolated genes fragment, glue reclaims expression vector and goal gene, re-uses the T4 dna ligase and connects, and makes up recombinant expression vector pGAPZ α A-ORF2.PGAPZ α A-ORF2 transforms DH5 α competent cell, the extracting recombinant plasmid, Xho IWith Not IAfter double digestion is identified successfully, recombinant plasmid be stored in-20 ℃ subsequent use.

4. recombinant expression vector pGAPZ α A-ORF2 transformed host cell pichia spp SMD1168 makes up recombinant expressed engineering bacteria.With competence P.pastorisSMD1168 (80 μ L) with The Avr IISingle endonuclease digestion pGAPZ α A-ORF2 (10 μ g) mixes mutually, 1.5 kV, 200 electric shocks, 5 ms.Yeast cell after transforming evenly is tiled in prepared fresh YPDS flat board (contains Zeocin ^TMResistance 100 μ g/mL), treat flat board,, in 30 ℃ of incubators, be cultured to single bacterium colony and (about 2 days) occur the flat board inversion liquid-absorbent.Employing boils-freezes-and cooking method prepares bacterium liquid PCR template analysis P.pastorisTransformant, upstream primer are SEQ ID NO:1, and downstream primer is that SEQ ID NO:2 can amplify the positive transformant of single bacterium colony about 700 bp.Obtain recombination yeast engineering bacteria pGAPZ α A-ORF2-SMD1168.

5.pGAPZ α A-ORF2-SMD1168 does secreting, expressing with the YPDS nutrient solution.As seed liquor, it is muddy to cultivate about 24h bacterium liquid in the 4mLYPD nutrient solution for white single bacterium colony of picking smooth surface, neat in edge, draws 1mL and is resuspended in (loading amount is 10%) in the 50mLYPDS substratum, 220r/min, 30 ℃ of shaking culture.The centrifugal 10min of 5000r/min behind the 72h, collect culture supernatant make SDS-PAGE or be stored in-80 ℃ subsequent use.

PGAPZ α A carrier is a strong promoter with pGAP, in substratum, adds glucose and can induce yeast to get into the protein expression state, in the YPDS substratum; Yeast cell growth; Express target protein simultaneously, culture medium carbon source can disposable in batches interpolation, also can suitably reduce by primary carbon source; Add carbon source through stream about 12h, flow the utilization of carbon source rate that the advantage that adds carbon source is to improve yeast cell.Temperature is controlled at 28～30 ℃, and the SMD1168 speed of growth is comparatively slow, but also has superiority, and is unlikely to make the bacterium excessively fast passage, and the deficient protein enzyme reduces excretory proteolytic degradation rate, thereby improves the expression amount of target protein.Bacterium liquid density should reach the OD value more than 1.5 during expression, and under well-fed situation, bacterium liquid density is high more in the certain limit, and expression amount is high more.Earlier cultivate recombination microzyme to OD=2～6, draw bacterium liquid and be resuspended in the YPDS nutrient solution, make OD >=1.5, maybe when reaching OD=2～6 with the YPD nutrient solution; The centrifugal collection thalline of 5000r/min is resuspended in the YPDS nutrient solution, and expression time can be 3～5 days; But in the culturing process, guarantee competent oxygen dissolving value (DO), expressing pH is 6.0; 6.5 more excellent, 7.0 is more excellent, can not surpass 8.0.To notice in the expression process preventing that nutrient solution from producing excess foam and influencing the DO value, cultivate bacterial concentration and can reach 40g/L, even 100g/L, even 150g/L, even higher.

When expressing, nutrient solution should be regulated and control in the needs scope, and adds phosphoric acid buffer, keeps the stable of pH, helps the stable existence of target protein, and away from the proteic iso-electric point pI of Cap, in this experiment, can not be lower than pH6.0.An amount of interpolation proteinase inhibitor and protease substrate also are desirable, like EDTA, amino acid, peptone etc., are principle not influence yeast cell growth propagation.To consider the osmotic pressure of yeast cell in nutrient solution simultaneously.

Separate and purifying:

After express to accomplish separating supernatant, supernatant be stored in-80 ℃ for use, or directly carry out the analysis of SDS-PAGE testing goal protein expression without concentrating; Do contrast with the empty carrier expression; Foreign protein is few, during high-visible target protein band, can carry out purifying protein and be used to prepare subunit vaccine.Purification process is more, and a small amount of purifying Cap albumen can be with yeast together with supernatant 5; Centrifugal 10 minutes of 000r/min; Supernatant with molecular weight 10,000 dialysis tubings in pH7.4 sterilization PBS damping fluid dialysis until balance, during change damping fluid 5～7 times; Have ready conditions and to use mechanical stirring, accelerate dialysis speed.Concentrate the dialysis product with PEG8000 again, the product of will dialysing is retained in dialysis tubing and is built in the PEG8000 powder, reaches requirement up to concentrating amount.A large amount of separation and purification, it is concentrated and purified that suggestion takes centrifugal collection supernatant-ceramic membrane micro-filtration-nf membrane to desalt, also can selective membrane filtering separation supernatant, supernatant carries out ultrafiltration and concentration, again the nanofiltration desalination.This experiment is expressed in bio-reactor, and total protein concentration is measured with Bradford method A595nm place in culture supernatant dialysis back, and calculating expressed albumen can be up to 0.08～1g/L; Gel thin-layer scanner scanning, recombinant protein account for total protein 40%～80%, expressing quantity and depend on the control that bacterial classification is preserved situation and expression condition; When fouled by microzyme or vigor reduction; Possibly secrete more foreign protein, and the target protein secretion is less or degraded, so answer strict control aseptic to the whole process of secreting, expressing target protein from making up the screening positive transformant; Otherwise directly have a strong impact on expression amount, even do not express.Because not contaminated ideal situation is expressed down, foreign protein is few, so downstream of the present invention are machined to 3 steps that needed more; Can keep protein-active, simple, expression amount is high; Production cost is lower, makes widespread usage verify that the subunit vaccine of its validity becomes possibility.

The preparation of embodiment 3. subunit vaccines

One or more compositions such as sanitas, protection liquid, stablizer, antimicrobial drug are mixed with the Cap albumen of separation and purification; Adding adjuvant mixes with saline water or PBS; Mixture pH value is transferred to physiological value, and promptly the pig circular ring virus subunit vaccine is formed in pH7.0～7.5.Adjuvant can be selected aluminium glue, card uncle ripple 971, saponin, liposome, CpG-ODN, nanometer adjuvant, oil emulsion adjuvant etc. for use; Can select to add cytokine such as Interferon, rabbit, interleukin-etc.; Wherein the proteic effective constituent concentration of Cap should reach 200-350 μ g/mL, every dosage 200-350 μ g.

Test-results: with the Cap albumen of the embodiment of the invention 1 preparation according to a small amount of separation and purification protein process purifying described herein after, measure the Cap protein concentration, using the saline water dilution is that 1000 μ g/mL are as antigen.The effective antigen amount of this subunit vaccine is 200 μ g/mL, and the whole content of LL37 antibacterial peptide is 100U/mL, adds the former liquid hold-up of aluminium glue and be about 20%, requires to carry out the check of finished product vaccine according to the veterinary biologics rules, carries out clinical trial with qualified product.Choose 20 of 25 healthy age in days weanling pigs of performance, weigh during 25 ages in days, it is not remarkable that each organizes piglet body weight difference, is divided into 4 groups at random, and serology detects the negative piglet of PCV2 has 10.14 days booster immunizations behind the initial immunity through 2 weeks, use porcine circovirus 2 type virulent strain virus liquid (10 ⁴TCID ₅₀/ mL) 2mL attacks poison to A, B, C, D group collunarium.Carry out immunity test according to following table 1:

Table 1

Test group	The positive number of serotype/total number	Inoculum	Dosage
				The A group	3/4	Cap albumen (aluminium glue adjuvant)	2 parts
The B group	2/4	Subunit vaccine (Bo Linge)	2 parts
				The C group	2/4	Empty carrier albumen	2 parts
The D group	3/4	PBS	2 parts
				The E group	0/4	/	/

The result: 53 ages in days are weighed before promptly attacking poison, do significance of difference analysis, four groups and the comparison of E group, and difference is not remarkable, and empty carrier and E group otherness are not remarkable.Explain that the non-target protein of recombination yeast engineering bacteria excretory does not influence the healthy and growth velocity of piglet.Weigh in 81 age in days piglets, make the significance of difference relatively with the E group.A, B group and E group difference are not remarkable, and C, D group and E group difference are extremely remarkable.All about 10 days, heating paresthesia occurs after attacking poison, A, B group fever time are about 2～5 days, disappear subsequently, and be no abnormal with E group contrast expression.C, 8/8 front and back of D group engender slight PWMS (multisystemic exhaustion syndrome) symptom to moderate, performance poor appetite, the depressed and growthing lag of spirit.Test-results shows that Cap albumen can produce the protective effect of anti-circovurus type 2 invasion and attack to piglet, infers that thus the porcine circovirus 2 type subunit vaccine of Cap protein Preparation is the infection and the relative disease of prevention and control porcine circovirus 2 type effectively.

SEQUENCE?LISTING

 

< 110>Agricultural University Of South China

 

< 120>the antigenic preparation method of reorganization porcine circovirus 2 type Cap

 

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Claims (3)

1. the antigenic preparation method of reorganization porcine circovirus 2 type Cap is to adopt yeast expression system, and preparation process is following:

(1) amplification PCV2-ORF2 gene;

(2) construction expression engineering bacteria: extension increasing sequence inserts Yeast expression carrier, forms recombinant expression vector, transforms pichia spp host cell, construction expression engineering bacteria after the linearizing;

(3) recombination microzyme secreting, expressing, porcine circovirus 2 type Cap antigen protein obtains recombinating;

PCV2-ORF2 gene described in the step (1) is the PCV2-ORF2 gene of transforming according to the inclined to one side preferendum of yeast codon, and its nucleotide sequence is shown in SEQ ID NO:3;

The primer nucleotides sequence of amplification PCV2-ORF2 gene is classified SEQ ID NO:1 and SEQ ID NO:2 as.

2. preparation method according to claim 1 is characterized in that Yeast expression carrier described in the step (2) is pGAPZ α A, pGAPZ α B or pGAPZ α C.

3. preparation method according to claim 1 is characterized in that pichia spp host cell described in the step (2) is yeast X33 or yeast SMD1168.

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