CN104479012B - The preparation method of PPR virus Yolk antibody - Google Patents
The preparation method of PPR virus Yolk antibody Download PDFInfo
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- CN104479012B CN104479012B CN201410659205.0A CN201410659205A CN104479012B CN 104479012 B CN104479012 B CN 104479012B CN 201410659205 A CN201410659205 A CN 201410659205A CN 104479012 B CN104479012 B CN 104479012B
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Abstract
The present invention provides a kind of preparation method of PPR virus Yolk antibody, include the following steps: that (1) takes PPR virus to prepare PPR virus antigen, it will be mixed after antigens inactive with Freund's adjuvant, immunity inoculation bird inlay, 4 DEG C of egg preservations collected after inoculation；(2) it purifies to obtain PPR virus Yolk antibody using sad method.Have the advantages that effect is obvious, specificity is high, therapeutic effect is rapid, curative effect really, production cost are low etc. using PPR virus Yolk antibody prepared by the method for the present invention, it can effectively prevent PPR virus to the attack of body, have a good application prospect.
Description
Technical field
The invention belongs to veterinary biologics fields, specifically, being related to a kind of PPR virus Yolk antibody
Preparation method.
Background technique
Peste des petits ruminants (Peste des Petits Ruminants, PPR), is by PPR virus (Peste
Des Petits Ruminants Virus, PPRV) caused by one kind is acute, deadly infectious disease, be legal as defined in FAO/OIE
Reported communicable disease, the disease are considered as one of highly important deadly infectious disease in the world, belong to paramyxovirus section
(Paramyxoviriade) Morbillivirus (Morbollivirus), main infection goat, sheep and wild small ruminant.
The disease to generate heat, stomatitis, diarrhea is main feature.Nineteen forty-two, Cote d'lvoire's report for the first time in West Africa has occurred small in the world
Ruminate epizootic disease.Tibet Autonomous Region, China 2007 reports peste des petits ruminants epidemic situation for the first time, then in, Tibet in 2010 in 2008
Peste des petits ruminants epidemic situation occurs in succession again for area, and in December, 2013 is in Xinjiang Yili of China, 2 months 2014 Inner Mongolia Autonomous Region Bayan muirs
The generation of peste des petits ruminants epidemic situation is reported in succession in city.Since the disease has stronger propagated, aetology of the reinforcement to PPR
And molecular biological characteristic research and epidemiology and diagnosis are studied with prevention and control and are had important practical significance for China.
Mainly prevent the generation of this disease using vaccine immunity at present.Due to being directed to the spy of viral disease not yet
Drug is imitated, therefore mainly takes symptomatic treatment and the generation with antibiotics prevention complication, treatment on clinical treatment
Effect is undesirable.It would therefore be highly desirable to develop a kind of for PPR virus specificity is good, therapeutic effect is good and curative effect is rapid
Treat preparation.
Summary of the invention
The object of the present invention is to provide a kind of preparation methods of PPR virus Yolk antibody.
In order to achieve the object of the present invention, the preparation method of a kind of PPR virus Yolk antibody of the invention, including
Following steps:
(1) it takes PPR virus to prepare PPR virus antigen, will be mixed after antigens inactive with Freund's adjuvant,
Immunity inoculation bird inlay collects 4 DEG C of egg preservations after inoculation；
(2) it purifies to obtain PPR virus Yolk antibody using sad method, specific as follows:
1. weighing 0.816g sodium acetate, 0.54mL glacial acetic acid and 100mL distilled water, the acetate of secure ph 4.5 are measured
Buffer 200mL；
2. taking immune rear egg, egg yolk liquid is isolated, 100mL egg yolk liquid is taken, by egg yolk liquid: acetate buffer=1:2-
The volume ratio of 3 (preferably 1:2) mixes；
3. 9-12mL (preferably 9mL) octanoic acid is added, it is stored at room temperature after shaking up；
4. 3000r/min is centrifuged 30min, supernatant is collected, with 0.45 μm of membrane filtration, filtrate is collected, is lyophilized up to small
Ruminate epizootic disease virus Yolk antibody.
Wherein, the method for PPR virus antigen is prepared are as follows: PPR virus liquid is seeded in the (Africa Vero
Green monkey kidney cell) on cell monolayer, 37 DEG C are placed in, CO2It is cultivated in incubator, poison is received when 75% cells showed cytopathic ,-
20~20 DEG C multigelation 3 times, then 4 DEG C, 3000rpm be centrifuged 30min, remove cell fragment, collect supernatant, density gradient
It is used as PPR virus antigen after centrifugation.
The method of inactivation antigen is as follows: PPR virus antigen is placed in 50 DEG C of water-baths 60 minutes.
Method above-mentioned after mixing the antigen after inactivation by the volume ratio of 1:1 with Freund's adjuvant in step (1), is immunized
Be inoculated with 22 week old bird inlays, initial immunity dosage be 0.3mg/ only, after immune 14d, 22d be changed to incomplete Freund's adjuvant into
2 booster immunizations of row, immunizing dose are respectively 0.6mg/, 1.2mg/；It is daily after immune to collect egg, measure egg yolk
Peste des petits ruminants virus-specific antibodies potency in liquid collects the yolk liquid that antibody titer is 1:128 or more, is used to prepare small ruminate
Epizootic disease virus Yolk antibody.
Method above-mentioned, freeze temperature is -38 DEG C to complete freeze-drying in step (2), and is stored in 4 DEG C.
PPR virus used in the present invention includes but is not limited to 75/1 plant of Nigeria.
The present invention also provides the PPR virus Yolk antibodies using above method preparation.
Using the PPR virus Yolk antibody of the method for the present invention preparation is obvious with effect, specificity is high, treatment
The advantages such as effect is rapidly, curative effect really, production cost be low, can effectively prevent PPR virus to the attack of body, have
Good application prospect.
Detailed description of the invention
Fig. 1 is the result for detecting Yolk antibody reactionogenicity in the embodiment of the present invention 1 using Western-blot method；Its
In, A is the Yolk antibody of the immune PPRV prepared in the embodiment of the present invention 1, and B is the Yolk antibody before being immunized；M is protein molecular
Amount standard, 1 is recombination PPRV-N albumen, and 2 be pET-32a (+) empty carrier, and 3 be recombination PPRV-N albumen, and 4 be pET-32a (+)
Empty carrier.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
PPR virus used in the following embodiment is 75/1 plant of Nigeria.
1 PPR virus Yolk antibody of embodiment and preparation method thereof
1. the preparation of immune vaccine
The preparation of 1.1 PPR virus proliferation and antigen
PPR virus liquid is inoculated with 3 bottles of Vero cells, every bottle of cell inoculation 1ml virus liquid, bottom of bottle area
25cm2, bottle cap is covered, accumbency bottle pipe gently rotates, and comes into full contact with virus liquid with entire cell monolayer, adsorbs at 37 DEG C
40min shakes I times every 10min therebetween, abandons virus liquid without inhaling, directly adds the DMEM (Dulbecco containing 2% fetal calf serum；
S Modified Eagle Medium) cell maintenance culture solution 5ml, while cell controls are set up, in 37 DEG C of CO2In incubator
Culture.Maintaining liquid is outwelled afterwards for 24 hours to carry out changing liquid.Day by day cytopathy is observed, if 72h postoperative infection cytopathy is unobvious, is received
Cell suspension is obtained, 3000rpm is centrifuged 30min supernatant is taken to continue in cell at 4 DEG C after multigelation 3 times at -20~20 DEG C
There is cytopathy in upper blind passage, blind passage to the second generation, poison are received when 75% cells showed cytopathic, -20~20 DEG C freeze repeatedly
Melt 3 times, then at 4 DEG C, 3000rpm is centrifuged 30min, removes cell fragment, collects supernatant liquor, uses after density gradient centrifugation
Make antigen.PPR virus antigen is placed in 60 minutes in 50 DEG C of water-baths and is inactivated.
The preparation of 1.2 vaccines
The malicious valence of PPR virus antigen is 1.0 × 10 after inactivation6TCID50.It is filled with Freund's adjuvant by 1:1 (v/v)
After dividing emulsification, 22 week old bird inlays are taken, through chicken double-vane, chest, abdomen and dorsal sc multi-point injection；Initial immunity dosage is
0.3mg/ only, is changed to incomplete Freund's adjuvant and carries out 2 booster immunizations, immunizing dose is respectively after 14d, 22d after immune
0.6mg/, 1.2mg/；It is daily after immune to collect egg, it is saved after number in 4 DEG C.
2. prepared by Yolk antibody
The harvest and inactivation of 2.1 yolk
2 exempt to start within latter 1 week to collect the immunity eggs qualified through sampling observation, and eggshell is sterilized, takes and either manually or mechanically beats eggs.It fills
Divide and remove egg white (white), blastodisc and frenulum, collects yolk.Being sufficiently stirred makes yolk in uniform paste, is added in equal volume through 121 DEG C
The distilled water that 30min sterilizes and cools down, stirs and evenly mixs, 62.5 DEG C of heat inactivation 30min.
The sad method of 2.2 antibody purifies
Immune rear egg is taken, yolk liquid is isolated, takes 100mL yolk liquid, by yolk liquid: buffer=1:2 volume ratio
Mixing, mixes well.The octanoic acid (amount that octanoic acid is added is the 3% of yolk liquid and buffer total amount) of 9mL is added, shakes up rear room temperature
It stands.3000r/min is centrifuged 30min, collects supernatant, with 0.45 μm of membrane filtration, collects filtrate, and measure its capacity.
The preparation of buffer: weighing 0.816g sodium acetate, measures 0.54mL glacial acetic acid and 100mL distilled water, secure ph
4.5 acetate buffer 200mL.
3. the Yolk antibody freeze-drying of purification
By purification and through examining qualified Yolk antibody liquid to be sub-packed in cillin bottle in a small amount, in being lyophilized on freeze dryer overnight, freeze
Temperature is minimum during dry is down to -38 DEG C, maintains 4h to complete freeze-drying, is then gradually brought to room temperature, taking-up is stored in 4 DEG C.
4. PPR virus Yolk antibody bioactivity
Using indirect elisa method, 96 hole enzyme marks are coated with using 1 μ g/mL PPR virus N protein as envelope antigen
Plate, 4 DEG C overnight；It is washed ELISA Plate 3 times after taking-up with PBST (PBS buffer solution containing 0.05%Tween 20) buffer, then with
200 hole μ L/ of PBST (PBS buffer solution containing 0.01%Tween 20) confining liquid closing ELISA Plate containing 10% fetal calf serum, 37
DEG C incubate 2h；It washs, dry after taking-up, every hole is subject to the 100 μ L of Yolk antibody of antibody diluent doubling dilution, while with immune
Preceding Yolk antibody does blank control, 37 DEG C of temperature as negative control, not to be coated with the N protein hole diluted Yolk antibody of addition
Educate 1h；The rabbit anti-chicken IgG that HRP label is added after washing (is diluted) 100 μ L, 37 DEG C of incubation 1h by 1:5000；Every hole adds after washing
Enter 100 μ L of substrate TMB solution, 37 DEG C of colour developing 15min, every hole adds 100 μ L 2mol/L H2SO4Reaction is terminated, is read on ELISA Plate
Taking wavelength is absorbance (A) value of 450nm.As a result judge: measurement hole A value > 2.1 times of negative control hole mean value or more can determine that
Yolk antibody prepared by the present invention is the positive.
Using the reactionogenicity of Western-blot method detection Yolk antibody, concrete operations are as follows:
(1) the recombination PPRV N protein of purifying is subjected to SDS-PAGE electrophoresis, 120V, 90min or so, after electrophoresis,
Albumen is transferred on NC film with wet robin, 350mA, 90min；
Wherein, recombinate PPRV N protein the preparation method comprises the following steps:
According to a pair of of addition EcoR I of PPRV N gene order design of GenBank announcement and drawing for I restriction enzyme site of Not
Object, for expanding PPRV N gene；
Upstream primer PPRV-N-F:5 '-CGGAATTCATGGCGACTCTTCTTAAAAGC-3 ' and downstream primer PPRV-N-
R:5 '-TTGCGGCCGCTTAGCCGAGGAGATCCTTGTCGT-3 '
After PCR product PCR product Purification Kit, it is connect with pMD-19T carrier.By connection product convert to
Bacterium solution is spread evenly across the solid agar plate containing AMP, is inverted in 37 DEG C of incubator overnight incubations by DH5 α competent cell,
Next day picking white single bacterium, which is fallen within, shakes bacterium in LB (containing 100 μ g/mL ampicillins) fluid nutrient medium and stays overnight, and is mentioned with alkaline lysis
Plasmid is taken, positive plasmid is filtered out.Correct positive plasmid and empty carrier pET-32a (+) will be identified respectively through EcoR I and Not
After I double digestion, digestion products are recycled, construct 10 μ L linked systems, conversion is thin to BL21 (DE3) competence after 16 DEG C of connections overnight
Born of the same parents are inverted in overnight incubation in 37 DEG C of incubators.Next day picking white single bacterium falls within LB (containing 100 μ g/mL ampicillins) liquid
Bacterium is shaken in body culture medium to stay overnight, and with alkaline lysis method of extracting plasmid, is filtered out positive plasmid, is named as pET-32a-PPRV-N.It takes
2mL is accredited as positive recombinant bacterium pET-32a-PPRV-N and is inoculated in 200mL LB culture medium (containing 100 μ g/mL ampicillins)
In, 37 DEG C, 200r/min shaking table culture to OD600About 0.6-0.8.In 28 DEG C, inducing expression 6h under the conditions of 1mmol/LIPTG,
12000r/min is centrifuged 10min and collects thallus, and thallus is resuspended with the PBS of 1/50 volume of original bacteria liquid.Thallus will be resuspended in 4 DEG C of ultrasounds
Broken, until bacterium solution becomes clarifying, then in 4 DEG C, 12000r/min is centrifuged 30min and collects supernatant.Use nickel Ago-Gel
Recombination PPRV N protein in FF protein purification column purification supernatant；
(2) after transferring, NC film is rinsed with PBST, and NC film is placed in the PBST containing 5% skimmed milk power, 4 DEG C of envelopes
It closes overnight；
(3) be added by 1:500 times of diluted immune PPRV Yolk antibody (Yolk antibody prepared in embodiment 1) and
Yolk antibody before immune, is incubated for 1h by 37 DEG C；PBST is washed 3 times, 3min/ times；
(4) it is added by the 1:5000 times of diluted rabbit anti-chicken IgG that HRP label is added, 37 DEG C, is incubated for 1h；PBST washing 3
It is secondary, 3min/ times；
(5) colour developing 10min is protected from light with enhanced HRP-DAB substrate colour reagent box；
(6) ddH is used2O color development stopping.
(7) the result is shown in Figure 1.
Fig. 1 the result shows that, Nigeria75/1 plants of N proteins of Yolk antibody and PPRV of preparation react, with empty carrier
It does not react；And blank control is not reacted with N protein, shows that the Yolk antibody of preparation contains PPRV antibody.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (2)
1. PPR virus Yolk antibody, which is characterized in that the preparation method of the Yolk antibody includes the following steps:
(1) it takes PPR virus to prepare PPR virus antigen, will be mixed after antigens inactive with Freund's adjuvant, is immunized
It is inoculated with bird inlay, 4 DEG C of egg preservations are collected after inoculation；
(2) it purifies to obtain PPR virus Yolk antibody using sad method, specific as follows:
1. weighing 0.816g sodium acetate, 0.54mL glacial acetic acid and 100mL distilled water, the acetate salt buffer of secure ph 4.5 are measured
Liquid 200mL；
2. taking immune rear egg, egg yolk liquid is isolated, 100mL egg yolk liquid is taken, by egg yolk liquid: acetate buffer=1:2 body
Product is than mixing；
3. 9mL octanoic acid is added, it is stored at room temperature after shaking up；
4. 3000r/min is centrifuged 30min, supernatant is collected, with 0.45 μm of membrane filtration, filtrate is collected, is lyophilized and is ruminated up to small
Epizootic disease virus Yolk antibody:
The method of PPR virus antigen is prepared in step (1) are as follows: PPR virus liquid is seeded in Vero single layer
On cell, 37 DEG C are placed in, CO2It is cultivated in incubator, poison is received when 75% cells showed cytopathic, -20~20 DEG C freeze repeatedly
Melt 3 times, then 4 DEG C, 3000rpm is centrifuged 30min, removes cell fragment, collects supernatant, is used as after density gradient centrifugation small anti-
Hay epizootic disease viral antigen；
The method of inactivation antigen is as follows in step (1): PPR virus antigen is placed in 50 DEG C of water-baths 60 minutes；
After mixing the antigen after inactivation by the volume ratio of 1:1 with Freund's adjuvant in step (1), 22 week old of immunity inoculation is laid eggs mother
Chicken, initial immunity dosage are 0.3mg/, and 14d, 22d are changed to incomplete Freund's adjuvant and carry out 2 booster immunizations after immune, exempt from
Epidemic disease dosage is respectively 0.6mg/, 1.2mg/；It is daily after immune to collect egg, measure peste des petits ruminants disease in egg yolk liquid
Malicious specific antibody potency collects the yolk liquid that antibody titer is 1:128 or more, it is anti-to be used to prepare PPR virus yolk
Body；
The PPR virus is 75/1 plant of Nigeria.
2. Yolk antibody according to claim 1, which is characterized in that freeze temperature freezes for -38 DEG C to complete in step (2)
It is dry.
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| CN201410659205.0A CN104479012B (en) | 2014-11-18 | 2014-11-18 | The preparation method of PPR virus Yolk antibody |
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