CN102533629B - Preparation method of avian encephalomyelitis virus VP1 protein subunit vaccine - Google Patents
Preparation method of avian encephalomyelitis virus VP1 protein subunit vaccine Download PDFInfo
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Abstract
The invention relates to a preparation method of an avian encephalomyelitis virus VP1 protein subunit vaccine. In the invention, a primer of the avian encephalomyelitis VP1 is synthesized, and a strain of recombinant bacteria of an efficient expression vector with the gene is established and obtained so as to realize efficient expression of the avian encephalomyelitis VP1 protein in escherichia coli; the expression quantity of soluble protein accounts for about 60% of the total protein of the bacteria; and the expression product is soluble protein in cytosol and has the antigen activity of natural VP1 protein. The avian encephalomyelitis VP1 subunit vaccine prepared by taking the recombinant bacteria as the production strain can effectively protect the poultry from the attack of avian encephalomyelitis virus.
Description
Technical field
The present invention relates to the preparation method of avian encephalomyclitis virus VP1 protein subunit vaccine, belong to the veterinary biologics field.
Background technology
Avian encephalomyelitis claims epidemic tremor again, be by avian encephalomyclitis virus (Avian Encephalomyelitis Virus, AEV) transmissible disease of a kind of main infringement chick that causes, its characteristic pathological change is the apyetous encephalomyelitis, classical symptom is an ataxia, neck tremble and gradual paralysis (Zhao Zhenhua, Yu Wangsheng, Hao Xianpu etc. the generation of avian encephalomyelitis and diagnosis. Inner Mongol herding science .1994,3:34-37).The area of the nearly all raising commercial chicken in the world all took place should disease, and China has broken out this disease in various places since the eighties in last century successively, brings bigger financial loss to aviculture.Because this disease has unexpected appearance, be difficult to predict the extended period, can be by characteristics such as level contact and vertical transmissions, so very serious to the harm of aviculture, unique effective way is by kind of the chicken inoculation vaccine of laying eggs being prevented the morbidity of offspring chick.Domestic research to avian encephalomyelitis vaccine at present concentrates on development of Inactivated Vaccine mostly, abroad is the research report of attenuated live vaccines.
AEV is the little RNA enterovirus genus member of section, genome is strand underlying stock RNA (Calnek, B.W., Luginbuhl, R.E., Helmboldt, C.F., 1997.Avian encephalomyelitis diseases of Poultry.Iowa State University Press, Ames, IA, pp.571-581).AEV only has a translation starting point, coding produces a polyprotein, and polyprotein is under the effect of the activated protein of encoding viral, and cascade is hydrolyzed into functional protein, VP1 is a kind of in four kinds of structural protein of virus, with the intrusion of virus and to participate in constituting the antigenic component of virus relevant.The Li Wei (2008) of Singapore report recently can protect chicken to avoid the infection of AEV with the VP1 subunit vaccine of cell expressing effectively.They are with the major antigen albumen VP1 of AEV, VP0, VP3 expresses in cell SF9 respectively, purifying protein then, make subunit vaccine, and to VP1, VP0 and VP3 biological activity detect, found that: VP1 is main antigen protein, can protect chicken to avoid infection (the Wei L of AEV effectively, Chee LL, Wei T et al.The VP1 protein of avian encephalomyelitis virus is a major host-protective immunogen that serves as diagnostic potential.J Virol Methods.2008Apr; 149 (1): 56-62.).
Complete pathogenic agent contains many antigens, but is not that all antigens can both produce protective response by stimulation of host.Wherein some antigen can also cause allergy, immunosuppression and other side effects, and this is to use complete pathogenic agent to do the defective of vaccine.And it is loaded down with trivial details that the deactivation vaccine production of present domestic application prepares the antigen process, and attenuated vaccine now also has the vaccination incidence to exist.Then can address this problem with subunit vaccine, especially this vaccine is except having the Antigen Stability height, the purity height, high specificity, the susceptibility height does not produce other uncorrelated antibody, the immune effect detection method convenient accurately outside, also very be easy to produce.Without animal body or idiosome production, can not have the tissue residue thing.And it is do not need deactivation, but safe.
By avian encephalomyclitis virus VP1 cDNA complete sequence is screened and analyzes, chosen wherein one section cDNA sequence that epitope enriches, can efficiently express in prokaryotic cell prokaryocyte, make up prokaryotic expression carrier, carried out prokaryotic expression, and successfully given expression to soluble recombining albumen.
Utilize the present invention can produce the avian encephalomyelitis subunit vaccine, have commercial use value.
Summary of the invention
The purpose of this invention is to provide that the proteic gene of a kind of avian encephalomyclitis virus VP1 is synthetic, the method for making of expression vector establishment and product, in the hope of in large-scale industrial production, producing efficient, safe, inexpensive avian encephalomyclitis virus VP1 protein subunit vaccine.
Realize that technical scheme of the present invention is:
1. make up a kind of proteic genetic engineering Escherichia coli of avian encephalomyelitis VP1 of expressing and to contain to some extent that avian encephalomyelitis VP1 protein gene (sequence 1) is connected into cloning vector PM-18T, and change colon bacillus DH5 α over to; With identifying correct positive plasmid pMD18-T-VP1, behind ECORI and SalI double digestion,, be recovered to the VP1 gene through 1.2% agarose gel electrophoresis; The VP1 gene that reclaims is connected into pET-32a, be built into the expression vector pET-32a-VP1 of avian encephalomyelitis VP1 protein gene, transform expression type intestinal bacteria (Escherichia coli) Rossetta, acquisition can be expressed the proteic genetic engineering Escherichia coli pET-32a-VP1/Rossetta of avian encephalomyelitis VP1 strain.
2. the avian encephalomyclitis virus VP1 protein subunit vaccine utilization of preparation makes up and obtains to express the proteic genetic engineering Escherichia coli of avian encephalomyelitis VP1 (Escherichia coli) pET-32a-VP1/Rossetta strain as producing bacterial strain, and preparation encephalomyelitis VP1 albumen and conventional mineral oil adjuvant form avian encephalomyclitis virus VP1 protein subunit vaccine.
3. the avian encephalomyclitis virus VP1 protein subunit vaccine that the present invention relates to is that the recombinant escherichia coli pET-32a-VP1/Rossetta that will obtain among the present invention is inoculated on the LB flat board that contains penbritin and paraxin, picking colony after 37 ℃ of overnight incubation, (prescription is: Tryptones 1% at the LB nutrient solution that contains penbritin and paraxin and colon bacillus substratum, yeast extract 0.5%, sodium-chlor 1%) be cultured to bacterium liquid OD in
600Be 0.6～0.8 o'clock, adding final concentration is the lactose of 0.02M, carries out inducing culture 5～7h, centrifugal collection thalline; The thalline of collecting is suspended with physiological saline, put the ultrasonic disruption thalline; Avian encephalomyelitis VP1 albumen after ammonium sulfate extraction, dialysis; Mineral oil adjuvant inactivated vaccine preparation method is prepared into vaccine routinely again.
The specific embodiment of the invention
One, seedling bacterial classification
1. synthetic, the expression vector establishment of the proteic gene of avian encephalomyclitis virus VP1
The present invention designs to have synthesized and can produce the proteic gene of avian encephalomyelitis VP1, and this gene contains following nucleotide sequence (sequence 1):
5′-ATGGGGAAAG?AGGATGAAGG?AGGATTTTCC?AGTGTGCCTG?AAGTGGAGCA?ACATGTTGTT?GAGGACAAGG?AACCACAGGG?ACCTTTGCAC?GTGACACCTT?TTGGCGCTGT?TAAAGCCATG?GAGGACCCTC?AGTTGGCGAG?GAAAACACCT?GGTACATTTC?CTGAATTAGC?TCCTGGTAAA?CCTCGACACA?CAGTAGACCA?TATGGATCTG?TATAAGTTTA?TGGGGCGTGC?CCATTACTTG?TGGGGACATA?AATTTACCAA?AACTGATATG?CAGTACACAT?TCCAGATACC?ATTGAGTCCC?ATCAAGGAGG?GTTTTGTGAC?GGGAACGCTT?AGGTGGTTTT?TAAGTCTTTT?CCAATTGTAT?CGTGGTTCTC?TCGACATTAC?CATGACATTC?GCAGGAAAGA?CTAATGTAGA?CGGCATTGTA?TACTTTGTGC?CTGAGGGTGT?TGCGATAGAG?ACTGAGAGGA?AGGAACAGAC?CCCTCTGCTC?ACGTTGAACT?ACAAAACATC?GGTAGGTGCC?ATTAGGTTTA?ATACTGGACA?AACTACAAAT?GTCCAGTTTA?GGATCCCTTT?CTACACGCCA?CTGGAGCACA?TCGCAACCCA?TTCTAAAAAC?GCGATGGACT?CAGTCTTGGG?GGCAATTACA?ACTCAGATCA?CCAATTATAG?TGCTCAGGAT?GAGTACCTGC?AGGTCACCTA?CTATATCAGC?TTTAATGAAG?ATTCACAGTT?TTCTGTTCCC?AGAGCGGTAC?CAGTGGTTAG?CTCATTCACT?GACACATCTA?GCAAGACAGT?GATGAATACA?TATTGGCTCG?ATGATGACGA?GTTGGTGGAA?GGGTCCTCCC?ATTTTAGTTT?CGATGAAATT?GAGGAGGCCC?AATGTTAA-3′
Its aminoacid sequence is a sequence 2:
Met?Gly?Lys?Glu?Asp?Glu?Gly?Gly?Phe?Ser?Ser?Val?Pro?Glu?Val?Glu
Gln?His?Val?Val?Glu?Asp?Lys?Glu?Pro?Gln?Gly?Pro?Leu?His?Val?Thr
Pro?Phe?Gly?Ala?Val?Lys?Ala?Met?Glu?Asp?Pro?Gln?Leu?Ala?Arg?Lys
Thr?Pro?Gly?Thr?Phe?Pro?Glu?Leu?Asp?Pro?Gly?Lys?Pro?Arg?His?Thr
Val?Asp?His?MetAsp?Leu?Tyr?Lys?Phe?Met?Gly?Arg?Ala?His?Tys?Leu
Trp?Gly?His?Lys?Phe?Thr?Lys?Thr?Asp?Met?Gln?Tyr?The?Phe?Gln?Ile
Pro?Leu?Ser?Pro?Ile?Lys?Glu?Gly?Phe?Val?Thr?Gly?Thr?Leu?Arg?Trp
Phe?Leu?Ser?Leu?Phe?Gln?Leu?Tyr?Arg?Gly?Ser?Leu?Asp?Ile?Thr?Met
Thr?Phe?Ala?Gly?Lys?Thr?Asn?Val?Asp?Gly?Ile?Val?Tyr?Phe?Val?Pro
Glu?Gly?Val?Ala?Ile?Glu?Thr?Glu?Arg?Lys?Glu?Gln?Thr?Pro?Leu?Leu
Thr?Leu?Asn?Tyr?Lys?Thr?Ser?Val?Gly?Ala?Ile?Arg?Phe?Asn?Thr?Gly
Gln?Thr?Thr?Asn?Val?Gln?Phe?Arg?Ile?Pro?Phe?Tyr?Thr?Pro?Leu?Glu
His?Ile?Ala?Thr?His?Ser?Lys?Asn?Ala?Met?Asp?Ser?Val?Leu?Gly?Ala
Ile?Thr?Thr?Gln?Ile?Thr?Asn?Tyr?Ser?Ala?Gln?Asp?Glu?Tyr?Leu?Gln
Val?Thr?Tyr?Tyr?Ile?Ser?Phe?Asn?Glu?Asp?Ser?Gln?Phe?Ser?Val?Pro
Arg?Ala?Val?Pro?Val?Val?Ser?Ser?Phe?Thr?Asp?Thr?Ser?Ser?Lyr?Thr
Val?Met?Asn?Thr?Tyr?Trp?Leu?Asp?Asp?Asp?Glu?Leu?Val?Glu?Gly?Ser
Ser?His?Phe?Ser?Phe?Asp?Glu?Ile?Glu?Glu?Ala?Gln?Cys
Be the aforesaid gene that increases, the present invention has designed and synthesized following corresponding primer:
Primer 1:5 '-GGGGAATTCATGGGGAAAGAGGATGAAGGAGGA-3 ' (sequence 3)
Primer 2:5 '-GGGGTCGACTTAACATTGGGCCTCCTCAATTTC-3 ' (sequence 4)
To contain as mentioned above that the gene of nucleotide sequence (avian encephalomyelitis VP1 protein gene) is connected into cloning vector PM-18T, and change bacillus coli DH 5 alpha over to.After identifying that correct positive plasmid pMD18-T-VP1 is with ECORI and SalI double digestion,, be recovered to the VP1 gene through 1.2% agarose gel electrophoresis.The VP1 gene that reclaims is connected into pET-32a, is built into the expression vector pET-32a-VP1 of avian encephalomyelitis VP1 protein gene, transforms expression type intestinal bacteria Rossetta, and acquisition can be expressed the proteic gene engineering colibacillus of avian encephalomyelitis VP1.
Expression vector pET-32a (+) has the T7 promotor that efficiently expresses, and transcribed by force by phage t7 and translation signals is controlled, and can be induced by the t7 rna polymerase that the host bacterium provides under the effect of lactose.The avian encephalomyelitis VP1 protein expressing plasmid pET-32a-VP1 that makes up, (this bacterial strain and on December 13rd, 2011 send the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 China Committee for Culture Collection of Microorganisms of Chinese Academy of Sciences common micro-organisms centers, and deposit number is to be built into recombinant escherichia coli Rossetta/pET-32a-VP1 behind the transformed into escherichia coli conversion expression type colon bacillus Rossetta; CGMCC No.5581) inductor of adding 0.2mmol/L lactose solution makes avian encephalomyelitis VP1 albumen obtain to efficiently express.
2. the evaluation of expression product
With the thalline of PBS resuspension with ultrasonic treatment after, the centrifugal 15min of 12000r/min, get supernatant and add protein electrophoresis sample-loading buffer (available from the rich Deco skill Development Co., Ltd that steps in Beijing), after boiling water boils 8min, separation gel with 12% carries out the SDS-PAGE evaluation and (among Fig. 5, from left to right is respectively albumen marker, contrasts, full bacterium-1, full bacterium-2).Adopt ammonium sulfate precipitation (reset and add 5.6g ammonium sulfate on every 100ml, 4 ℃ are spent the night, the centrifugal 10min of 10000r/min then, collecting precipitation suspends with 10mlPBS (pH7.2)), Ni-NTA that supernatant liquor is carried out purifying (seeing note 1).To carry out SDS-PAGE with the product of Ni-NTA purifying and analyze (Fig. 6 from left to right is respectively the pipe of first under the elutriant wash-out, second pipe and the 3rd pipe).
Two, vaccine manufacturing
1. antigen prepd
(1) fermentation and abduction delivering
Recombination bacillus coli Rossetta/pET-32a-VP1 among the present invention is inoculated on the LB flat board that contains penbritin and paraxin 37 ℃ of overnight incubation.Picking colony is cultured to bacterium liquid OD in LB nutrient solution that contains penbritin and paraxin and colon bacillus substratum (prescription is: Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%)
600Be 0.6～0.8 o'clock, adding final concentration is the lactose of 0.02M, carries out inducing culture 5～7h, centrifugal collection thalline.
(2) purge process of expressing protein: the thalline of collecting is suspended with physiological saline, put the ultrasonic disruption thalline.Add solid ammonium sulfate to concentration and reach 10%, fully dissolve back 4 ℃ and deposit and spend the night.Centrifugal collecting precipitation.With the heavy molten precipitation of physiological saline, and with dialysis tubing (Beijing is auspicious to reach the production of permanent brightness development in science and technology company limited, interception: 14KD) dialyse.With the AGP method (Zhang Shuxia, Yang Zengqi. the development of avian encephalomyelitis agp antigen and application. animal medicine progress, 2001,22 (2): 59-60) measure the regenerant antigen titre.
2. the preparation of vaccine
The water preparation: 96 parts of the antigens that deactivation is good (it is 1: 16 that the fine jade expansion is tired), 4 parts of mixings of tween-80 are made water.
Oil phase preparation: 94 parts of white oils, Si Ben-80 6 part, 2 parts of mixings of aluminum stearate are made oil phase.
Emulsification: get oil phase and be put in the shears for 2 parts, start the motor slow rotation and stir, add 1 part of water simultaneously slowly, add the back with 10000r/min emulsification 5min.
Three, vaccine test
1, potency test
The immune Luo Man egg kind chicken of vaccine neck subcutaneous injection (0.5ml/ only) with preparation.With Ai Deshi avian encephalomyelitis antibody assay kit its antibody is detected, ELISA detected result S/P 〉=0.2 is positive.Collect the fertilization kind egg of immunity back laying hen, the inoculation strong malicious VR strain of avian encephalomyelitis (available from China Veterinery Drug Inspection Office) in the yolk sac, its dosage is 100EID
50, hatching, chick normally goes out shell.
Four, the vaccine involved in the present invention and the comparison of other vaccines
See Table 1.
Table 1 vaccine involved in the present invention compares with the vaccine that uses now
Microorganism involved in the present invention
Recombination bacillus coli (Escherichia coli) Rossetta/pET-32a-VP1 of the microorganism that the present invention relates to for making up, this bacterial strain send the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 China Committee for Culture Collection of Microorganisms of Chinese Academy of Sciences common micro-organisms centers on December 13rd, 2011, and deposit number is; CGMCC No.5581.
Positive effect of the present invention
The present invention relates to a kind of preparation method of avian encephalomyclitis virus VP1 protein subunit vaccine.The present invention is by having synthesized the primer of avian encephalomyelitis VP1 gene, made up and obtained the reorganization bacterium that a strain has the efficient expression vector of this gene, realized avian encephalomyelitis VP1 albumen efficiently expressing in intestinal bacteria, the soluble proteins expression amount accounts for 60% of bacterial protein, this expression product is a soluble proteins in the cytosol, has the proteic antigenic activity of natural VP1.Utilize this reorganization bacterium can protect bird to exempt from the attack of avian encephalomyclitis virus effectively as the avian encephalomyelitis VP1 subunit vaccine that the production bacterial strain is prepared into.
Description of drawings
Fig. 1 is embodiment of the invention avian encephalomyelitis VP1 albumen cDNA sequence and existing avian encephalomyclitis virus VR strain (available from China Veterinery Drug Inspection Office, YEBIO Bioengineering Co., Ltd of Qingdao preserves) nucleotide sequence contrast figure
Fig. 2 be embodiment of the invention avian encephalomyelitis VP1 protein gene PCR amplification evaluation figure wherein: 1 for VP1 PCR product, and M is DNA Maker 2000.
Fig. 3 is that the enzyme of embodiment of the invention pMD18T-VP1 is cut evaluation figure wherein: 1 is pMD18-T-VP1 double digestion product, and M is DNA Maker 2000.
Fig. 4 is the structure synoptic diagram of embodiment of the invention avian encephalomyelitis VP1 protein expressing plasmid
Fig. 5 be embodiment of the invention engineering bacterium fermentation abduction delivering product SDS-PAGE electrophoresis rating diagram wherein: from left to right be respectively albumen marker, contrast, full bacterium-1, full bacterium-2.
Fig. 6 be embodiment of the invention purifying avian encephalomyelitis VP1 Protein S DS-PAG electrophoresis rating diagram wherein: from left to right be respectively first under elutriant wash-out pipe, second pipe and the 3rd pipe.
Fig. 7 be embodiment of the invention avian encephalomyelitis VP1 protein product the agar diffusion test rating diagram wherein: 1,7 are the VP1 albumen of purifying; 2,4,6 are the rabbit anteserum after the immunity; 3,5 is supernatant negative control after the intestinal bacteria Rossetta fragmentation.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are specifically described:
Embodiment one,
1.AEV breeding
With AEV VR strain aseptic inoculation 6 age in days SPF chick embryo yolk sacs, 37 ℃ of hatchings are inoculated after the 9th day and the chicken embryo to be put into the brain that 4 ℃ of refrigerators are collected typical cytopathic chicken embryo, pancreas, gi tract and allantoic fluid; Handle pathological material of disease with collected allantoic fluid as diluent, through grinding, multigelation 3 times, the centrifugal 10min of 7000r/min gets viral suspension ,-80 ℃ of preservations.
2.PCR amplification VP1 gene, clone and sequencing (seeing Fig. 1, Fig. 2, Fig. 3)
According to delivering the synthetic a pair of primer P1 (sequence 3) of sequences Design; Primer P2 (sequence 4) contains the SalI site.The purified virus suspension adds Trizol cracking, chloroform extracting, isopropanol precipitating, diethylpyrocarbonate (DEPC) water dissolution.Downstream primer carries out reverse transcription for 42 ℃, obtains cDNA.Pcr amplification purpose fragment, and the product recovery is reclaimed test kit with dna gel reclaim (giving birth to worker's biotechnology company limited available from Shanghai).Reclaiming product is connected and transformed into escherichia coli DH5 α with pMD18-T carrier (the precious biotech firm in Dalian).Picking colony and screening positive clone pMD18-T-VP1.(Fig. 1 clones VP1 gene order and standard A EV VR strain sequence comparison diagram for the present invention.Fig. 2 is 1 for VP1 PCR product, and M is DNA Maker 2000.1 is pMD18-T-VP1 double digestion product among Fig. 3, and M is DNA Maker 2000.)
3.VP1 expression of gene (see figure 4)
Positive colony plasmid pMD18-T-VP1 and expression vector pET-32a carrier are used EcorI and SalI double digestion respectively, product is behind 1.2% agarose gel electrophoresis, reclaiming test kit with dna gel reclaims, collect the pET-32a fragment of VP1 and the 5.9kb of 0.85kb respectively, make up the pET-32a-VP1 expression plasmid 16 ℃ of directed connections, be converted into the bacillus coli DH 5 alpha competent cell, filter out PCR and identify correct positive colony.After sequence verification sequence and reading frame are errorless, positive plasmid pET-32a-VP1 is converted among the expression type intestinal bacteria Rossetta (available from Bo Maide company), construct recombination bacillus coli Rossetta/pET-32a-VP1, this bacterial strain send the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 China Committee for Culture Collection of Microorganisms of Chinese Academy of Sciences common micro-organisms centers on December 13rd, 2011, and deposit number is; CGMCC No.5581.The mono-clonal colony inoculation of picking fellatio character grain pET32a-VP1 is in containing penbritin and paraxin LB liquid nutrient medium, and 37 ℃ of shaking culture are spent the night.Gathering in the crops bacterium liquid next day is inoculated in the LB substratum that contains penbritin and paraxin once more and carries out enlarged culturing, 37 ℃ of shaking culture are pressed final concentration 0.02mmol/L and are added the 0.2mmol/L lactose to measuring A600=0.6～0.8 o'clock, induce 6h for 25～37 ℃, 4 ℃, the centrifugal 5min of 5000r/min.With PBS resuspension thalline.
4. express cracking and the protein electrophoresis (seeing Fig. 5, Fig. 6) of bacterium
With the thalline of PBS resuspension with ultrasonic treatment after, the centrifugal 15min of 12000r/min, get supernatant and add protein electrophoresis sample-loading buffer (available from the rich Deco skill Development Co., Ltd that steps in Beijing), after boiling water boils 8min, separation gel with 12% carries out the SDS-PAGE evaluation and (among Fig. 5, from left to right is respectively albumen marker, contrasts, full bacterium-1, full bacterium-2).Adopt ammonium sulfate precipitation (reset and add 5.6g ammonium sulfate on every 100ml, 4 ℃ are spent the night, the centrifugal 10min of 10000r/min then, collecting precipitation suspends with 10mlPBS (pH7.2)), Ni-NTA that supernatant liquor is carried out purifying (seeing note 1).To carry out SDS-PAGE with the product of Ni-NTA purifying and analyze (Fig. 6 from left to right is respectively the pipe of first under the elutriant wash-out, second pipe and the 3rd pipe).
5. the antigenic activity of recombinant protein detects (see figure 7)
The albumen supernatant that above-mentioned Ni-NTA affinitive layer purification is obtained joins preparation subunit vaccine in the freund's adjuvant (purchasing the company in sigma) of equivalent.Get the test rabbit multiple spot subcutaneous injection immunity of 1.5mL to 2 monthly ages, exempt from back 28d at head, the oil emulsion vaccine booster immunization that 56d uses Freund's incomplete adjuvant (purchasing the company in sigma) and equivalent purifying protein to make respectively, collect serum behind the immune for the third time 7d, detect sero-fast activity with the agar diffusion experiment.(among Fig. 7,1,7 is the VP1 albumen of purifying; 2,4,6 are the rabbit anteserum after the immunity; 3,5 is supernatant negative control after the intestinal bacteria Rossetta fragmentation)
Embodiment two
1. ferment and abduction delivering
Recombination bacillus coli Rossetta/pET-32a-VP1 is inoculated on the LB flat board of penbritin+paraxin, cultivates 10～12h for 37 ℃.10 bigger smooth colonies of picking are inoculated in 500mL and add in the LB nutrient solution of penbritin+paraxin.37 ℃ of concussion overnight incubation.Volume ratio by 2% transfers saturated bacterium liquid in 20, in the colon bacillus substratum of 000mL (prescription is Tryptones 1%, yeast extract 0.5%, sodium-chlor 1%).37 ℃, the OD value that 30% oxygen saturation is cultured to nutrient solution is 0.6～0.8 o'clock, and adding 0.2M lactose to final concentration is 0.02M, continues to cultivate 5～7h again.Centrifugal collection thalline.Add 4 parts of physiological saline by 1 part of thalline weight in wet base.Resuspended thalline ,-20 ℃ of preservations.
2, the purge process of expressing protein: frozen thalline suspension is thawed at 4 ℃.Add physiological saline to 1: 10 ratio.Put ultrasonic wave down to the bacterial cell disruption more than 95%.The saturation ratio that adds solid ammonium sulfate to 10% is fully dissolved back 4 ℃ and is deposited and spend the night.The centrifugal 20min of 12000r/min.Collecting precipitation.With the heavy molten precipitation of physiological saline, change in the dialysis tubing.Use normal saline dialysis 48h.Measure the regenerant antigen titre with the AGP method.Adding physiological saline, to be diluted to antigen titre be 1: 16.It is 1mg/ml that purified product is measured total protein content with the method for ultraviolet absorption, and measuring antigen titre with the AGP method then is 1: 512.Under the unit albumen weight AGP titre value be 1: 512.
3, the preparation of vaccine:
The water preparation: 96 parts of the antigens that deactivation is good, 4 parts of mixings of tween-80 are made water.
Oil phase preparation: 94 parts of white oils, Si Ben-80 6 part, 2 parts of mixings of aluminum stearate are made oil phase.
Emulsification: get oil phase and be put in the shears for 2 parts, start the motor slow rotation and stir, add 1 part of water simultaneously slowly, add the back with 10000r/min emulsification 5 minutes.
A collection of avian encephalomyelitis subunit inactivated vaccine is manufactured experimently in inspection after construction---laboratory, and lot number is 2010001.
1. proterties
Appearance milky white emulsion.
The formulation water-in-oil-type.Get a cleaning suction pipe, draw a small amount of vaccine and splash in the cold water, except that the 1st, all indiffusion.
Stability is drawn vaccine 10ml and is added in the centrifuge tube, and with 3000r/min centrifugal 15 minutes, the water that separate out at the pipe end is 0.1ml, and was up to specification.
Viscosity is undertaken by existing " Chinese veterinary drug allusion quotation " appendix, and k value is 46cP, and is up to specification.
2. steriling test carries out asepsis growth by the regulation of " Chinese veterinary drug allusion quotation ".
3. safety verification is with 10 of 7 age in days SPF chickens, and every muscle or neck subcutaneous injection vaccine 1ml observed 14, and test chicken is all strong as a result lives, and does not have any part and systemic adverse reactions.
4. the following method of efficacy test is appointed and is selected one.
1) serological method with 3 age in week 20 of SPF chickens, wherein 10 each muscle or neck subcutaneous injection vaccine 0.2ml, other 10 in contrast not immune, support with group feeding.Inoculate back 21 days, every chicken is taken a blood sample respectively, and separation of serum detects AEV antibody with ELISA.The results are shown in following table 2:
Table 2ELISA detected result
Annotate: the criterion of ELISA antibody positive, S/P 〉=0.2 are positive.
Embodiment three
With 10 of the subunit vaccine immunity Luo Man egg kind chickens in 14 age in week of preparation, 10 not immune comparing in addition.2,7,14,21,27,36,48, the 50 week blood sampling of immunity back is carried out antibody test with the avian encephalomyelitis antibody assay kit of Ai Deshi; 14,36,52 weeks of immunity back, respectively extract 30 pieces on kind egg under the chicken after the immunity at random, hatching is during to 6 ages in days, get each 20 pieces of fertilization embryos, the strong poison of AE (VR strain) of 10 times of dilutions of difference yolk sac inoculation, 0.2ml/ piece, hatch (dead person discards in the 48h, does not go into record) to normal hatching day for 37 ℃.Result's (seeing the following form 3) shows: it is positive that antibody is detected with Ai Deshi avian encephalomyelitis antibody assay kit after 2 weeks in the immunity back; Kind of the egg that produces, hatching back inoculation avian encephalomyelitis is poison by force, and chick normally goes out shell.Illustrate that this subunit vaccine effect is certain, immunity kind laying hen produces kind of (behind the egg incubation) and is highly resistant to the avian encephalomyelitis strong virus attack.
Table 3 egg kind chicken immune potency test result
Ni-NTA affinity chromatography method:
1.Ni-NTA His protein purification medium dress post, the MillQ washing medium surface of 3 times of medium volumes;
2. add 0.1mol/L NiSO4 and all become blue to medium, MillQ washs to effluent liquid colourless;
3.1 * Start Buffer balance purification media is carried out the target protein combination with the albumen supernatant by the post medium;
4.1 * Start Buffer washing is to there not being albumen to flow out (detecting OD with spectrophotometer judges);
5. carry out gradient elution with the damping fluid that contains different imidazole concentrations, be in charge of and collect till the extremely last no albumen outflow of the effluent liquid that plays protein peak (variation according to effluent liquid OD value is recorded on the ruling paper by self-balancing recorder) under each concentration.
Claims (3)
1. can express the proteic genetic engineering Escherichia coli of avian encephalomyelitis VP1 (Escherichiacoli) for one kind, it is characterized in that this project bacterium is that the avian encephalomyelitis VP1 protein gene of nucleotide sequence shown in sequence 1 is connected into cloning vector PM-18T, and change colon bacillus DH5 α over to; With identifying correct positive plasmid pMD18-T-VP1, behind EcoRI and SalI double digestion,, be recovered to the VP1 gene through 1.2% agarose gel electrophoresis; The VP1 gene that reclaims is connected into pET-32a, be built into the expression vector pET-32a-VP1 of avian encephalomyelitis VP1 protein gene, transform expression type intestinal bacteria Rossetta, acquisition can be expressed the proteic genetic engineering Escherichia coli of avian encephalomyelitis VP1 (Escherichia coli) pET-32a-VP1/Rossetta strain, this bacterial strain is delivered No. 3 China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 13rd, 2011, and deposit number is CGMCCNo.5581.
2. avian encephalomyclitis virus VP1 protein subunit vaccine, it is characterized in that this avian encephalomyclitis virus VP1 protein subunit vaccine by the proteic genetic engineering Escherichia coli preparation of the expressed avian encephalomyelitis VP1 of claim 1, this subunit vaccine contains encephalomyelitis VP1 albumen and the conventional mineral oil adjuvant that colon bacillus (Escherichia coli) CGMCCNo.5581 expresses.
3. the preparation method of the avian encephalomyclitis virus VP1 protein subunit vaccine of a claim 2, it is characterized in that: CGMCCNo.5581 is inoculated on the LB flat board that contains penbritin and paraxin with colon bacillus (Escherichia coli), picking colony after 37 ℃ of overnight incubation is cultured to bacterium liquid OD in LB nutrient solution that contains penbritin and paraxin and colon bacillus substratum
600Be 0.6～0.8 o'clock, adding final concentration is the lactose of 0.02M, carries out inducing culture 5～7h, centrifugal collection thalline; The thalline of collecting is suspended with physiological saline, put the ultrasonic disruption thalline; Avian encephalomyelitis VP1 albumen after ammonium sulfate extraction, dialysis; Mineral oil adjuvant inactivated vaccine preparation method is prepared into vaccine routinely again.
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| CN111000991B (en) * | 2019-12-20 | 2023-04-21 | 浙江普康生物技术股份有限公司 | Coxsackie virus A group 6 recombinant subunit protein vaccine and preparation method thereof |
| CN116836939B (en) * | 2023-07-05 | 2024-01-26 | 中国兽医药品监察所 | Anti-avian encephalomyelitis virus monoclonal antibody hybridoma cell strain, monoclonal antibody, reagent or kit and application thereof |
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| CN103710314A (en) * | 2014-01-02 | 2014-04-09 | 青岛易邦生物工程有限公司 | Avian Encephalomyelitis virus attenuated vaccine strain |
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