CN105820216A - Porcine parvovirus VP2 protein - Google Patents

Abstract

The invention aims at providing porcine parvovirus VP2 protein which is used for preparing a subunit vaccine so as to overcome deficiencies in the prior art .The amino acid sequence of the porcine parvovirus VP2 protein is SEQ ID NO:1 .The invention provides novel VP2 protein which lays a firm foundation for industrialized production of the porcine parvovirus subunit vaccine; the vaccine prepared through the protein is high in antigenic stability, high in purity, high in specificity and high in sensitivity, no other uncorrelated antibody is produced, an immune effect detection method is convenient and accurate, and production is very easy .Production is conducted without using animal bodies or embryoid bodies, tissue residues do not exist, and the safety is high.

Description

A kind of PPV VP 2 protein

Technical field

The invention belongs to vaccine antigen protein triage techniques field, particularly to a kind of PPV VP 2 protein.

Background technology

Pig parvoviral (Porcineparvovirus, PPV) is one of important pathogen causing sow breeding difficulty, may result in Sow abortion, premature labor, product stillborn fetus, mummy tire, weak son and sow is sterile and newborn piglet mortality.China is separated to PPV from various places after the eighties in 20th century in succession, further investigation reveals that the Seropositive rates of this disease is up to more than 85% in some swinerys.PPV often exists in inapparent infection, and the especially phenomenon of low dosage persistent infection often occurs.This virus removes the breeding difficulty causing sow, also can cause multiple disease with other pathogen synergism, cause the biggest economic loss to pig industry.Pig parvoviral serotype is single, have higher immunogenicity, and vaccination is to control the effective measures that PPV infects.

Complete pathogen contains much antigen, but also not all antigen can produce protective response by stimulation of host.Some of which antigen can also cause allergy, immunosuppressant and other side effect, and this is the defect using complete pathogen to make vaccine.And it is loaded down with trivial details that Process of Antigen is prepared in the inactivated vaccine production of domestic application at present, and attenuated vaccine is the most also with the presence of vaccination incidence.This problem then can be solved by subunit vaccine.But subunit vaccine needs the antigen protein providing immunogenicity the most maybe can resist variation source of disease.

Summary of the invention

It is an object of the invention to provide a kind of PPV VP 2 protein, and be used for preparing subunit vaccine, thus make up the deficiencies in the prior art.

The PPV VP 2 protein of the present invention, its aminoacid sequence is SEQIDNO:1；

Encoding the gene of above-mentioned PPV VP 2 protein, its a kind of nucleotide sequence is SEQIDNO:2；

Above-mentioned PPV VP 2 protein can be used for preparing vaccine；

Another aspect of the invention is to provide a kind of recombinant bacterial strain for recombinant expressed above-mentioned PPV VP 2 protein；It is the yeast expression vector of the gene containing nucleotide sequence described above to be converted Pichia yeast obtain, express the pichia pastoris phaff X33-VP2 (PichiapastorisX33-VP2) of PPV VP 2 protein, the China typical culture collection center being preserved in Wuhan on March 9th, 2016, deposit number is that X33-VP2 bacterial strain is made PPV VP 2 protein obtain high efficient expression under the effect of methanol by CCTCCNO:M2016098；Described derivant is the methanol of final concentration 0.55%.

Another aspect of the present invention is to provide a kind of subunit vaccine, and its antigen is above-mentioned PPV VP 2 protein.

The present invention provides a kind of novel VP2 albumen, has established solid foundation for industrialized production pig parvoviral subunit vaccine；Vaccine prepared by this albumen is high except having Antigen Stability, and purity is high, high specificity, and sensitivity is high, does not produce other uncorrelated antibody, outside immune effect detection method is the most accurate, is also easy to produce.Produce without animal body or idiosome, will not be with tissue residue thing, but safety is high.

Accompanying drawing explanation

Fig. 1 is the amplification qualification figure of embodiment of the present invention PPV VP 2 protein gene PCR,

Fig. 2 is the blast comparison diagram of the VP2 albumen of the present invention；

Fig. 3 is embodiment of the present invention recombinant bacterium fermentation inducement expression product SDS-PAGE electrophoresis rating diagram.

Detailed description of the invention

Below in conjunction with the accompanying drawings embodiments of the invention are specifically described.

Embodiment 1: the screening of pig parvoviral (PPV) VP2 gene

Within 2010, in Shandong Province, multiple plants occur in that the symptom of porcine parvovirus, and have injected existing parvovirus vaccine before Affected individuals pig, thus it is speculated that the virus of infection there occurs variation；Therefore from Affected individuals, carry out the screening of pig parvoviral；Finishing screen have selected pig parvoviral PPV1.

Antigenicity for the virus of checking screening, the Strain of 5 separate sources employing the PPV1 strain comprising screening prepares vaccine as antigen, challenge viral dosage is carried out with the virus liquid of screening after immunity SPF pig, result shows compared to other swine parvovirus vaccine, the vaccine itself prepared has more preferable immune effect (p ＜ 0.05), it is thus determined that it there occurs the variation in heredity.

Antigenic characteristic according to PPV VP 2 protein and aminoacid sequence, design synthesis pair of primers, primer1:5'-GCGGTACCATGTCTGAAAATGTTGAAGAAC-3', containing KpnI site；

Primer2:5'-GCGGCCGCCTAATATAATTTTCTTGGAAT-3', containing NotI site.Taking PPV strain virus gene as template, PCR expands purpose fragment, and product reclaims and connects pMD18-T carrier, converts and screening positive clone pMD18-T-VP2, through sequencing the amplification qualification figure of pig parvoviral VP2 gene PCR (Fig. 1 be)；Its nucleotides sequence is classified as SEQIDNO:2, and the sequence of the albumen of coding is SEQIDNO:1.Compared with the pig parvoviral VP2 gene disclosed in NCBI, the highest homology is 96%；In the case of existence 579 is amino acid whose, show that the PPV VP 2 protein of the present invention exists no less than 23 amino acid whose differences (Fig. 2) with published albumen.

Embodiment 2: the preparation of recombination porcine parvovirus VP2 albumen

Comprise the following steps: a. construction of expression vector；B. construction expression bacterial strain；C. the induction of recombinant VP 2 albumen and extraction purification.Specific as follows: to use KpnI and NotI double digestion product respectively after 1.2% agarose gel electrophoresis positive colony plasmid pMD18-T-VP2 and expression vector pPICZ α carrier, reclaim test kit with DNA gel to reclaim, obtain about 1.7kb and 3.3kb fragment respectively, connect 16 DEG C of orientations and build pPICZ α-VP2 expression vector, after enzyme action is identified correctly；After plasmid linearization, electricity is transformed into Pichia sp. competent cell, build Pichiapastoris expression strain X33-VP2, and extract expression strain X33-VP2 genome, use primer primer1 and primer2 to carry out PCR and identify correct, being preserved in China typical culture collection center on March 9th, 2016, deposit number is CCTCCM2016098；Carrying out abduction delivering, the picking monoclonal colony inoculation containing X33-VP2 is in BMGY fluid medium, and 30 DEG C of shaken cultivation overnight, are centrifuged and collect thalline, after suspending with appropriate BMMY, adds 0.55% methanol, induces 96 hours for 30 DEG C.4 DEG C, 9000rpm is centrifuged 5min, retains supernatant, and after adding the ammonium sulfate precipitation of 40%, 12000rpm is centrifuged 5min and collects albumen precipitation, with the PBS molten albumen of weight.Add protein electrophoresis sample-loading buffer, after boiling water boiling 8 minutes, separation gel with 12% carry out SDS-PAGE qualification (Fig. 3 is the SDS-PAGE electrophoresis rating diagram of recombinant bacterium fermentation inducement expression product, in figure 1,2, precipitate for VP2 protein expressioning product, M is molecular weight marker proteins matter).

Embodiment 3: the preparation of vaccine

The Pichiapastoris expression strain X33-VP2 strain producing PPV VP 2 protein is preserved in China typical culture collection center, and deposit number is CCTCCM2016098.

1. X33-VP2 strain is inoculated in the YPD fluid medium containing bleomycin by the preparation of seedling bacterium solution, 30 DEG C of shaken cultivation 16～18 hours.Then streak inoculation is in the YPD solid medium added with bleomycin, choose colonies typical 2～3 be mixed in a small amount of YPD fluid medium, put shaken cultivation 18 hours in 30 DEG C of shaking tables, quantitative separating, after purely inspection, as first order seed.Take first order seed to be inoculated in BMGY fluid medium, 30 DEG C of shaken cultivation 16～18 hours, after microscopy, put 2～8 DEG C of preservations, as secondary seed.

2. fermenter volume 60% (V/V) the addition incomplete fluid medium of BMGY is pressed in the preparation of seedling albumen, add defoamer by culture medium 0.1% (V/V) simultaneously, it is passed through high-temp steam sterilizing 30 minutes, treat that culture medium temperature is down to 32 DEG C, add YNB and biotin, Pigs Inoculated parvovirus VP2 protein production Pichia sp. X33-VP2 secondary seed solution, fermentation tank parameter arranges and is respectively mixing speed 800r/min, temperature 30 DEG C, maintains DO value (dissolved oxygen amount) 20%.Bacterium solution after cultivating 24 hours adds methanol, and the speed of adding of methanol is that 2ml/h/L abduction delivering is cultivated, according to above ferment control parameter and technique abduction delivering 120 hours；Fermentation culture bacterium solution is centrifuged 30 minutes through tube centrifuge 10000r/min, the supernatant of results, after adding ammonium sulfate precipitated protein, is centrifuged 30 minutes results precipitations by 12000r/min, adds appropriate physiological saline solution albumen precipitation.

3., in protein liquid is placed in inactivation bottle by inactivation, metering adds 10% formalin, and with adding with shaking so that it is be sufficiently mixed, the ultimate density of formalin is 0.1%.Pour into after adding formalin in another inactivation bottle, contact inactivator to avoid the virus adhered near bottleneck to fail.37 DEG C inactivation 16 hours after take out, put 2～8 DEG C of preservations.

4. the inspection of semifinished product

(1) steriling test carries out steriling test by existing " Chinese veterinary pharmacopoeia " annex.

(2) determining the protein quantity presses Bradford method detection protein content.

(3) protein liquid after inactivation is taken a small amount of inoculation YPD solid medium by inactivation inspection, is placed in 30 DEG C and continues to cultivate 72 hours.Observe without colony growth, sentence inactivation inspection qualified.

Embodiment 2: the preparation of subunit inactivated vaccine

Semi-finished product VP2 proteantigen through after the assay was approved carries out vaccine and prepares (in following preparation, each liquid component is counted by volume).

(1) oil phase preparation takes white oil for animals 95 parts, aluminium stearate 1 part, is placed in after being heated to 80 DEG C in oil phase preparation tank, then Jia Siben-805 parts, to temperature reach 115 DEG C time, maintain 30min, standby after cooling.

(2) PPV VP 2 protein is used normal saline dilution to become 150 μ g/0.1ml by aqueous phase preparation.Take 5 parts of tween 80s after sterilizing, add in Agitation Tank, be simultaneously introduced seedling protein liquid 95 parts, stir 20～30min, make tween 80 be completely dissolved.

(3) emulsifying takes oil phase 2 parts and is put in high-speed shearing machine, starts the stirring of motor slow rotation, slowly adds aqueous phase 1 part, with 10000r/min, emulsifying 5 minutes simultaneously.After emulsifying, taking 10ml, be centrifuged 15 minutes with 3000r/min, the aqueous phase separated out at the bottom of pipe should be less than 0.5ml.

Embodiment 3, vaccine product inspection

(1) character

Outward appearance vaccine should be milky Emulsion, free from admixture and outer package should be qualified.

Dosage form is water-in-oil type.Take a cleaning suction pipe, draw a small amount of vaccine and instill in cold water, in addition to the 1st, all should indiffusion.

Stability is drawn vaccine 10ml and is added in centrifuge tube, is centrifuged 15 minutes with 3000r/min, and the aqueous phase separated out at the bottom of pipe should be less than 0.5ml.

Viscosity is carried out by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.

(2) loading quantity inspection is carried out by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.

(3) steriling test is carried out by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.

(4) safety verification 50 age in days piglet 5, every cervical region subcutaneous injection vaccine 1.0ml, sets comparison 5 simultaneously, raises at identical conditions, Continuous Observation 14 days, and record test pig is searched for food, drunk water and clinical setting.Any locally and systemically untoward reaction caused by vaccine should be occurred without.

(5) efficacy test first farrowing sow 5, every cervical region subcutaneous injection vaccine, 0.5ml/ only, separately takes 5 and compares as the most immune with age in days first farrowing sow.After insemination of sows, conceived to counteracting toxic substances when 38-40 days, result is not separated to virus from the blood plasma of the sow of inoculation subunit vaccine, compares and be then separated to virus in the blood plasma of sow.Counteracting toxic substances was slaughtered after 40 days, and vaccination of sows cherishes 65 the first-born pigs altogether, is not the most separated to virus, and compares sow and cherish 60 the first-born pigs altogether, wherein have the internal organs of 31 to be separated to virus from their internal organs.Result shows, can resist the attack of virus with PPV VP 2 protein subunit vaccine immunity sow, occur without clinical symptoms, and tire pig virus purification is feminine gender., there is slight clinical symptoms in matched group, and tire pig virus purification positive rate, more than 50%, shows that pig parvoviral subunit inactivated vaccine can provide and is effectively protected.

Contrast experiment shows, carries out immunity with the tiny disease vaccine of pig sold in the market, then carries out challenge viral dosage with the PPV1 strain of present invention screening；Result shows that the immune effect of the vaccine sold in the market is far below the effect of the VP2 protein vaccine of the present invention；It is presumably due to the VP2 protein vaccine of PPV1 strain morph and cause the immune effect of current vaccine bad.

Claims (4)

1. a PPV VP 2 protein, it is characterised in that its described PPV VP 2 protein includes:

1) aminoacid sequence is the albumen of SEQIDNO:1；

2) by 1) in protein delation, replace, insert or add one or several aminoacid, and have 1) in the albumen of protein active.

2. a gene, it is characterised in that the described PPV VP 2 protein described in gene code claim 1.

3. gene as claimed in claim 2, it is characterised in that the nucleotides sequence of described gene is classified as SEQIDNO:2.

4. the application in preparing vaccine of the PPV VP 2 protein described in claim 1.