CN104479012A - Preparation method of peste des petits ruminants virus yolk antibody - Google Patents
Preparation method of peste des petits ruminants virus yolk antibody Download PDFInfo
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Abstract
The invention provides a preparation method of a peste des petits ruminants virus (PPRV) yolk antibody. The preparation method of the PPRV yolk antibody comprises the following steps: (1) preparing a peste des petits ruminants virus antigen by using peste des petits ruminants viruses, inactivating the peste des petits ruminants virus antigen, mixing the inactivated peste des petits ruminants virus antigen with Freund's adjuvant, immunizing a laying hen with the mixture, collecting eggs, and preserving at the temperature of 4 DEG C; and (2) purifying by using octanoic acid to obtain the peste des petits ruminants virus yolk antibody. The peste des petits ruminants virus yolk antibody prepared by adopting the preparation method has the advantages of significant effect, high specificity, definite curative effect and low production cost, can take effect quickly, can effectively inhibit the attack of the peste des petits ruminants viruses and has a good application prospect.
Description
Technical field
The invention belongs to veterinary biologics field, specifically, relate to a kind of preparation method of PPR virus yolk antibody.
Background technology
PPR (Peste des Petits Ruminants, PPR), by PPR virus (Peste des Petits Ruminants Virus, PPRV) one caused is acute, deadly infectious disease, it is the notifiable infectious diseases that FAO/OIE specifies, this disease is considered to one of very important in the world deadly infectious disease, belong to paramyxovirus section (Paramyxoviriade) Morbillivirus (Morbollivirus), main infection goat, sheep and wild small ruminant.This disease with heating, stomatitis, diarrhoea for principal character.Nineteen forty-two, the Cote d'lvoire's report in the world first in West Africa there occurs PPR.Tibet Autonomous Region of China reported first PPR epidemic situation in 2007, in succession there is PPR epidemic situation again 2008,2010 Tibet regions subsequently, in succession report the generation of PPR epidemic situation in December, 2013 in Xinjiang Yili of China, Ba Yan Nor City, Inner Mongolia Autonomous Region in February, 2014.Because this disease has stronger propagated, therefore strengthen having important practical significance to China for the etiology of PPR and molecular biological characteristic research and epidemiology and diagnosis and prevention and control research.
The main generation adopting vaccine immunity to prevent this disease at present.Owing to also not having the specific medicament for virus disease, therefore on clinical treatment, mainly take symptomatic treatment and the generation by antibiotics complication prevention, result for the treatment of is undesirable.Therefore, urgently exploitation one is good for PPR virus specificity, result for the treatment of good and curative effect treats preparation rapidly.
Summary of the invention
The object of this invention is to provide a kind of preparation method of PPR virus yolk antibody.
In order to realize the object of the invention, the preparation method of a kind of PPR virus yolk antibody of the present invention, comprises the steps:
(1) get PPR virus and prepare PPR virus antigen, mix after antigens inactive with freund's adjuvant, immunization bird inlay, after inoculation, collect the preservation of 4 DEG C, egg;
(2) adopt sad method to purify and obtain PPR virus yolk antibody, specific as follows:
1. take 0.816g sodium acetate, measure 0.54mL Glacial acetic acid and 100mL distilled water, the acetate buffer 200mL of secure ph 4.5;
2. get the rear egg of immunity, isolate egg yolk liquid, get 100mL egg yolk liquid, by egg yolk liquid: the volume ratio mixing of acetate buffer=1:2-3 (preferred 1:2);
3. add 9-12mL (preferred 9mL) sad, shake up rear room temperature and leave standstill;
4. the centrifugal 30min of 3000r/min, collects supernatant liquor, and with 0.45 μm of membrane filtration, collect filtrate, namely freeze-drying obtains PPR virus yolk antibody.
Wherein, the method preparing PPR virus antigen is: be seeded in by PPR virus liquid on Vero (African green monkey kidney cell) monolayer cell, be placed in 37 DEG C, CO
2cultivate in incubator, when 75% cells showed cytopathic, receive poison ,-20 ~ 20 DEG C of multigelations 3 times, then 4 DEG C, the centrifugal 30min of 3000rpm, removing cell debris, collects supernatant liquor, is used as PPR virus antigen after density gradient centrifugation.
The method of inactivation antigen is as follows: PPR virus antigen is placed in 50 DEG C of water-baths 60 minutes.
Aforesaid method, after the antigen after deactivation being mixed by the volume ratio of 1:1 with freund's adjuvant in step (1), immunization bird inlay in 22 week age, initial immunity dosage is 0.3mg/, after immunity, 14d, 22d change incomplete Freund's adjuvant into and carry out 2 booster immunizations, immunizing dose be respectively 0.6mg/ only, 1.2mg/ only; After immunity, every day collects egg, measures PPR virus-specific antibodies in egg yolk liquid and tires, collect the yolk liquid that antibody titer is more than 1:128, for the preparation of PPR virus yolk antibody.
Aforesaid method, in step (2), freeze temperature is-38 DEG C of extremely complete freeze-drying, and in 4 DEG C of storages.
The PPR virus used in the present invention includes but not limited to Nigeria 75/1 strain.
The present invention also provides the PPR virus yolk antibody adopting aforesaid method to prepare.
The PPR virus yolk antibody adopting the inventive method to prepare has the advantages such as successful, specificity is high, result for the treatment of is rapid, curative effect is certain, production cost is low, PPR virus effectively can be stoped the attack of body, have a good application prospect.
Accompanying drawing explanation
Fig. 1 is the result adopting Western-blot method to detect yolk antibody reactionogenicity in the embodiment of the present invention 1; Wherein, A is the yolk antibody of the immune PPRV of preparation in the embodiment of the present invention 1, and B is the yolk antibody before immunity; M is Protein Marker, and 1 is restructuring PPRV-N albumen, and 2 is pET-32a (+) empty carrier, and 3 is restructuring PPRV-N albumen, and 4 is pET-32a (+) empty carrier.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The PPR virus used in following examples is Nigeria 75/1 strain.
Embodiment 1 PPR virus yolk antibody and preparation method thereof
1. the preparation of immunity vaccine
The preparation of 1.1 PPR virus propagation and antigen
PPR virus liquid is inoculated 3 bottles of Vero cells, every bottle of cell inoculation 1ml virus liquid, bottle floorage 25cm
2, build bottle cap, accumbency bottle pipe rotates gently, virus liquid is fully contacted with whole cell monolayer, adsorbs 40min, rock I time therebetween every 10min at 37 DEG C, abandoning virus liquid, directly adding the DMEM (Dulbecco containing 2% foetal calf serum without the need to inhaling; SModified Eagle Medium) cell maintenance culture solution 5ml, sets up cell controls, at 37 DEG C of CO simultaneously
2cultivate in incubator.Outwell maintenance medium after 24h to carry out changing liquid.Day by day observation of cell pathology, if 72h postoperative infection cytopathy is not obvious, then harvested cell suspension, after-20 ~ 20 DEG C of multigelations 3 times, at 4 DEG C, the centrifugal 30min of 3000rpm gets supernatant liquor and continues blind passage on cell, cytopathy is there is in blind passage to the s-generation, when 75% cells showed cytopathic, receive poison ,-20 ~ 20 DEG C of multigelations 3 times, then at 4 DEG C, the centrifugal 30min of 3000rpm, removing cell debris, collects supernatant liquid, is used as antigen after density gradient centrifugation.PPR virus antigen is placed in 50 DEG C of water-baths deactivation in 60 minutes.
The preparation of 1.2 vaccines
After deactivation, the malicious valency of PPR virus antigen is 1.0 × 10
6tCID
50.With freund's adjuvant press 1:1 (v/v) fully emulsified after, get bird inlay in 22 week age, through chicken double-vane, chest, belly and dorsal sc multi-point injection; Initial immunity dosage is 0.3mg/, changes incomplete Freund's adjuvant into and carry out 2 booster immunizations after 14d, 22d after immunity, and immunizing dose is respectively 0.6mg/, 1.2mg/; After immunity, every day collects egg, in 4 DEG C of preservations after numbering.
2. yolk antibody preparation
The results of 2.1 yolk and deactivation
2 exempt from rear beginning for 1 week collects through inspecting qualified immune egg by random samples, is sterilized by eggshell, takes craft or machinery to beat eggs.Abundant removing egg white (in vain), blastodisc and frenulum, collect yolk.Abundant stirring makes yolk be even paste, adds the distilled water that equal-volume also cools through 121 DEG C of 30min sterilizings, stirring and evenly mixing, 62.5 DEG C of heat inactivation 30min.
The sad method of 2.2 antibody is purified
Get the rear egg of immunity, isolate yolk liquid, get 100mL yolk liquid, by yolk liquid: the volume ratio mixing of damping fluid=1:2, fully mixes.Add sad (add 3% that sad amount is yolk liquid and damping fluid total amount) of 9mL, shake up rear room temperature and leave standstill.The centrifugal 30min of 3000r/min, collects supernatant liquor, with 0.45 μm of membrane filtration, collects filtrate, and measures its capacity.
The preparation of damping fluid: take 0.816g sodium acetate, measures 0.54mL Glacial acetic acid and 100mL distilled water, the acetate buffer 200mL of secure ph 4.5.
3. the yolk antibody freeze-drying of purifying
To purify and yolk antibody liquid through being up to the standards is sub-packed in cillin bottle in a small amount, on Freeze Drying Equipment, freeze-drying is spent the night, and in freeze-drying process, temperature is minimum is down to-38 DEG C, maintains 4h to complete freeze-drying, then returns to room temperature gradually, take out and be stored in 4 DEG C.
4. PPR virus yolk antibody bioactivity
Adopt indirect elisa method, using 1 μ g/mL PPR virus N protein as envelope antigen bag by 96 hole enzyme plates, 4 DEG C are spent the night; Take out rear PBST (the PBS damping fluid containing 0.05%Tween 20) buffer solution enzyme plate 3 times, again with PBST (the PBS damping fluid containing 0.01%Tween 20) the confining liquid 200 μ L/ hole sealase target containing 10% foetal calf serum, 37 DEG C of incubation 2h; Washing after taking out, to dry, the yolk antibody 100 μ L of every hole in addition antibody diluent doubling dilution, simultaneously using the yolk antibody before immunity as negative control, added diluted yolk antibody by N protein hole do blank, 37 DEG C of incubation 1h not wrap; The anti-chicken IgG of rabbit (by 1:5000 dilution) 100 μ L, 37 DEG C of incubation 1h of HRP mark are added after washing; After washing, every hole adds substrate TMB solution 100 μ L, and 37 DEG C of colour developing 15min, every hole adds 100 μ L 2mol/L H
2sO
4termination reaction, enzyme plate reads absorbancy (A) value that wavelength is 450nm.Result judges: measure hole A value > negative control hole average more than 2.1 times, can judge that yolk antibody prepared by the present invention is as the positive.
Adopt Western-blot method to detect the reactionogenicity of yolk antibody, concrete operations are as follows:
(1) the restructuring PPRV N protein of purifying is carried out SDS-PAGE electrophoresis, about 120V, 90min, after electrophoresis terminates, with wet robin, albumen is transferred on NC film, 350mA, 90min;
Wherein, the preparation method of PPRV N protein of recombinating is:
The primer of EcoR I and Not I restriction enzyme site is added in the PPRV N gene order design of announcing according to GenBank for a pair, is used for amplification PPRV N gene;
Upstream primer PPRV-N-F:5 '-CGGAATTCATGGCGACTCTTCTTAAAAGC-3 ' and downstream primer PPRV-N-R:5 '-TTGCGGCCGCTTAGCCGAGGAGATCCTTGTCGT-3 '
By PCR primer with after PCR primer Purification Kit, be connected with pMD-19T carrier.Product conversion will be connected to DH5 α competent cell, the solid agar be spread evenly across by bacterium liquid containing AMP is dull and stereotyped, be inverted in 37 DEG C of incubator overnight incubation, the single bacterium colony of picking white next day shakes bacterium and spends the night in LB (containing 100 μ g/mL penbritins) liquid nutrient medium, use alkaline lysis method of extracting plasmid, filter out positive plasmid.To identify that correct positive plasmid and empty carrier pET-32a (+) are respectively after EcoR I and Not I double digestion, reclaim digestion products, build 10 μ L linked systems, 16 DEG C of connections are converted into BL21 (DE3) competent cell after spending the night, and are inverted in overnight incubation in 37 DEG C of incubators.The single bacterium colony of picking white next day shakes bacterium and spends the night in LB (containing 100 μ g/mL penbritins) liquid nutrient medium, uses alkaline lysis method of extracting plasmid, filters out positive plasmid, called after pET-32a-PPRV-N.Get 2mL to be accredited as positive recombinant bacterium pET-32a-PPRV-N and to be inoculated in 200mL LB substratum (containing 100 μ g/mL penbritins), 37 DEG C, 200r/min shaking table is cultured to OD
600be about 0.6-0.8.In 28 DEG C, under 1mmol/LIPTG condition, the centrifugal 10min of abduction delivering 6h, 12000r/min collects thalline, with the resuspended thalline of PBS of original bacteria liquid 1/50 volume.By resuspended thalline in 4 DEG C of ultrasonications, until bacterium liquid becomes clarification, then in 4 DEG C, the centrifugal 30min of 12000r/min collects supernatant.Use the restructuring PPRV N protein in nickel sepharose FF protein purification column purification supernatant;
(2) after transfer printing terminates, rinse NC film with PBST, and NC film is placed in the PBST containing 5% skim-milk, close for 4 DEG C and spend the night;
(3) add the yolk antibody before the yolk antibody (namely in embodiment 1 preparation yolk antibody) of the immune PPRV doubly diluted by 1:500 and immunity, 37 DEG C, hatch 1h; PBST washs 3 times, 3min/ time;
(4) add the anti-chicken IgG of rabbit adding HRP mark doubly diluted by 1:5000,37 DEG C, hatch 1h; PBST washs 3 times, 3min/ time;
(5) with enhancement type HRP-DAB substrate colouring reagents box lucifuge colour developing 10min;
(6) ddH is used
2o color development stopping.
(7) the results are shown in Figure 1.
Fig. 1 result shows, yolk antibody and the PPRV Nigeria75/1 strain N protein of preparation react, and do not react with empty carrier; And blank and N protein are not reacted, show that the yolk antibody prepared contains PPRV antibody.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (6)
1. the preparation method of PPR virus yolk antibody, is characterized in that, comprises the steps:
(1) get PPR virus and prepare PPR virus antigen, mix after antigens inactive with freund's adjuvant, immunization bird inlay, after inoculation, collect the preservation of 4 DEG C, egg;
(2) adopt sad method to purify and obtain PPR virus yolk antibody, specific as follows:
1. take 0.816g sodium acetate, measure 0.54mL Glacial acetic acid and 100mL distilled water, the acetate buffer 200mL of secure ph 4.5;
2. get the rear egg of immunity, isolate egg yolk liquid, get 100mL egg yolk liquid, by egg yolk liquid: the volume ratio mixing of acetate buffer=1:2-3;
3. add 9-12mL sad, shake up rear room temperature and leave standstill;
4. the centrifugal 30min of 3000r/min, collects supernatant liquor, and with 0.45 μm of membrane filtration, collect filtrate, namely freeze-drying obtains PPR virus yolk antibody.
2. method according to claim 1, is characterized in that, the method preparing PPR virus antigen in step (1) is: be seeded on Vero monolayer cell by PPR virus liquid, be placed in 37 DEG C, CO
2cultivate in incubator, when 75% cells showed cytopathic, receive poison ,-20 ~ 20 DEG C of multigelations 3 times, then 4 DEG C, the centrifugal 30min of 3000rpm, removing cell debris, collects supernatant liquor, is used as PPR virus antigen after density gradient centrifugation.
3. method according to claim 1, is characterized in that, in step (1), the method for inactivation antigen is as follows: PPR virus antigen is placed in 50 DEG C of water-baths 60 minutes.
4. method according to claim 1, it is characterized in that, after the antigen after deactivation being mixed by the volume ratio of 1:1 with freund's adjuvant in step (1), immunization bird inlay in 22 week age, initial immunity dosage is 0.3mg/, after immunity, 14d, 22d change incomplete Freund's adjuvant into and carry out 2 booster immunizations, immunizing dose be respectively 0.6mg/ only, 1.2mg/ only; After immunity, every day collects egg, measures PPR virus-specific antibodies in egg yolk liquid and tires, collect the yolk liquid that antibody titer is more than 1:128, for the preparation of PPR virus yolk antibody.
5. method according to claim 1, is characterized in that, in step (2), freeze temperature is-38 DEG C of extremely complete freeze-drying.
6. the method according to any one of claim 1-5, is characterized in that, the PPR virus of use includes but not limited to Nigeria 75/1 strain.
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| CN105175539A (en) * | 2015-09-23 | 2015-12-23 | 天津瑞普生物技术股份有限公司 | Production method for refined yolk antibody for chicken infectious bursal disease |
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| CN105175539A (en) * | 2015-09-23 | 2015-12-23 | 天津瑞普生物技术股份有限公司 | Production method for refined yolk antibody for chicken infectious bursal disease |
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