CN110302370A - A kind of preparation method of Alfalfa plant vaccine - Google Patents
A kind of preparation method of Alfalfa plant vaccine Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of Alfalfa plant vaccine, are to solve the problem of existing RHDV is treated.Specific step is as follows: step 1 by the present invention, constructs intermediate transfer carrier；Step 2, the connection of exogenous dna fragment and intermediate transfer carrier；Step 3, the identification of recombinant plasmid；Step 4 prepares Alfalfa plant vaccine.Hepatitis E Virus Structural gene VP60 and rabbit IL-18 are transferred in Alfalfa by the present invention, toxicity test after the pressurized screening of the seedling of cultivation, identification, culture, concentration, the detection of immunogenicity, have many advantages, such as easy to produce, non-toxic diffusion, easy to operate, saving labour, save fund, reduce stress reaction, improve immune effect, has a extensive future.
Description
Technical field
The present invention relates to rabbit haemorrhagic disease therapy field, the preparation method of specifically a kind of Alfalfa plant vaccine.
Background technique
Rabbit haemorrhagic disease (Rabbit hemorrhagic disease, RHD) is by rabbit hemorrhagic disease virus (Rabbit
Hemorrhagic disease virus, RHDV) caused by acute, highly infectious, the high lethal of one kind disease, to exhale
Desorption system bleeding, organa parenchymatosum's oedema, extravasated blood and bleeding variation are characterized, and bring huge economic loss, Zeng Shi to rabbit keeping
A kind of devastating infectious disease of rabbit and be concerned.The disease was found for the first time in Jiangyin county in Jiangsu Province, China in 1984, it
Spread to national 25 provinces, municipalities and autonomous regions rapidly afterwards.So far the Korea, India and Lebanon in Asia, America Mexico, non-
The Cameroon in continent and Austria, the Belgium, Czech, Denmark, France, Germany, Greece, Luxembourg, Holland, Poland, west in Europe
Also there are generation in the states such as class's tooth, Sweden, Switzerland and Yugoslavia.The normal break out and spread of the disease, disease incidence and case fatality rate are high,
To susceptible animal pathogenicity rate up to 90%, case fatality rate is up to 100%, is to endanger one of disease of most serious of rabbit keeping.
Effective therapeutic agent there is no so far to RHDV infection, the applicable remedy measures of institute are only limitted to symptomatic treatment and support
Treatment.Currently, each state all increases the research dynamics of RHDV vaccine, so as to which RHDV infection is effectively prevented and controlled
It treats.The Attenuated vaccine used prevents the disease, but it is potential and dynamic to have the shortcomings that scattered poison, production cost are continuously increased etc.
The problems such as object welfare, so that researcher is dedicated to the development of recombinant vaccine to achieve the effect that prevent the disease.
Present RHDV vaccine preparation mainly with RHDV virus inoculation rabbit after, separate liver, grind filtration sterilization after, but
The in vitro culture for being virus is still problem, there are no suitable cultured cell in vitro is found, there is service life, expired rear vaccine loses
Effect.Now still cause scattered poison possible because inactivation has been not thorough, therefore it is necessary in virus structure with the liver inactivated vaccine of the rabbit that dies of illness
It is broken through on external permissive cell, developing gene vaccine is instantly or even the development trend in future.
Summary of the invention
The preparation method for being designed to provide a kind of Alfalfa plant vaccine of the embodiment of the present invention, to solve above-mentioned background
The problem of being proposed in technology.
To achieve the above object, the embodiment of the present invention provides the following technical solutions:
A kind of preparation method of Alfalfa plant vaccine, the specific steps are as follows:
Step 1 constructs intermediate transfer carrier:, will by the downstream of the structural proteins VP60 insertion combined promoter of RHDV
IL-18 gene is inserted into the downstream of Gene expression, constructs containing RHDV structural proteins VP60's and rabbit interleukins IL-18
Bird pox virus intermediate transfer carrier pUTAL-VP60-IL18；
Step 2, the connection of exogenous dna fragment and intermediate transfer carrier: the bird pox virus intermediate transfer carrier that will be prepared
It is added in 2 μ L 10 × connection buffers with exogenous dna fragment, adds water to 20 μ L thereto and be uniformly mixed, then add thereto
Enter T4DNA ligase and be uniformly mixed, then moment is centrifuged so that drop is gathered in tube bottom, and water bath processing obtains connection and produces
Object takes 3-4 μ L connection product to be converted into competent cell；
Step 3, the identification of recombinant plasmid: competent cell is cultivated, and obtains single bacterium colony, and single bacterium colony is existed
It is inoculated on solid medium containing antibiotic, and is gently dipped on the tube wall of the tubule of sterilizing, bacterium colony is made to be stained with tubule
It is cultivated on tube wall, obtains recombinant plasmid, recombinant plasmid is subjected to PCR (polymerase chain reaction) detection, test passes enter next
Step；
Step 4 prepares Alfalfa plant vaccine: fresh Agrobacterium EH105 competence is prepared using Calcium Chloride Method, it will
Transfected Recombinant Plasmid is into the positive Agrobacterium competent cell prepared, and ice bath 30 minutes, liquid nitrogen frozen 5 minutes, 37 water-baths 2
After minute, after 28 DEG C of 600 μ L LB liquid medium cultures being added 6 hours, it is applied to added with corresponding antibiotic culture dish
On, conversion spot carries out being accredited as positive plasmid by PCR.Positive Agrobacterium recombinant plasmid is obtained, clover grass seed is used and is disappeared
Venom processing, is inoculated on solid medium, the illumination cultivation in plant incubator, then connects positive Agrobacterium recombinant plasmid
Kind in the fluid nutrient medium containing antibiotic, collect thallus, remove supernatant, thallus is resuspended with fluid nutrient medium, dilute by centrifugation
It releases to original volume and is cultivated, after waiting the growth of Alfalfa seedling, Alfalfa seedling is moved into soil plantation, can be obtained into
Product.
As further embodiment of the embodiment of the present invention: the amount of the substance of exogenous dna fragment is bird pox virus in step 2
2-10 times of the amount of the substance of intermediate transfer carrier.
As further embodiment of the embodiment of the present invention: combined promoter is ATI-P7.5 × 20 in step 1, and series connection is opened
Mover is P7.5 × 16.
As further embodiment of the embodiment of the present invention: in step 2 water bath processing be 15-18 DEG C of holding 15-24 hours or
24-27 DEG C of person holding 0.8-2 hours.
As further embodiment of the embodiment of the present invention: connection buffering is added in bird pox virus intermediate transfer carrier in step 2
The preceding digestion filling-in using 100-200pmol and dephosphorization in liquid.
As further embodiment of the embodiment of the present invention: the revolving speed being centrifuged in step 2 is 1000rpm.
As further embodiment of the embodiment of the present invention: the temperature of thimerosal is 28-33 DEG C in step 4, solid medium
Using MS solid medium.
IL-18 is a kind of newly discovered cell factor, has multiple biological function.It by monocytes/macrophages and
Epithelial cell generates, and can activate NK cell, stimulates the T cell of activation to generate GM-CSF, IL-2, IFN-γ, inhibits the T of activation thin
Born of the same parents generate IL-10.The study found that IL-18 can improve the immune level of body by adjusting the expression of IFN, immunologic adjuvant is played
Effect, to reach control and prevention microorganism infection.Therefore, the structural proteins VP60 of IL-18 and RHDV is co-expressed, to rise
To the effect of gene adjuvant, the recombinant fowlpox vaccine virus of building coexpression structural proteins VP60, molecule adjuvant are effectively improved
It is inoculated with the cellular immunity and humoral immunity level of animal, prevents the generation and prevalence of the disease, reduces huge economic loss.
Compared with prior art, the beneficial effect of the embodiment of the present invention is:
The present invention constructs Alfalfa plant vaccine using gene recombination technology, prevents rabbit hemorrhagic syndrome epidemic disease, gram
It has taken that subunit vaccine immune effect is bad and recombinant viral vaccine dissipates the disadvantage of poison；
Hepatitis E Virus Structural gene VP60 and rabbit IL-18 are transferred in Alfalfa by the present invention, the seedling warp of cultivation
Toxicity test after pressurization screening, identification, culture, concentration, the detection of immunogenicity have easy to produce, non-toxic diffusion, easily behaviour
Make, save labour, save fund, reduce stress reaction, improve the advantages that immune effect, has a extensive future.
Specific embodiment
The technical solution of the patent is explained in further detail With reference to embodiment.
Embodiment 1
A kind of preparation method of Alfalfa plant vaccine, the specific steps are as follows:
Step 1 constructs intermediate transfer carrier: by the structural proteins VP60 of RHDV insertion combined promoter ATI-P7.5 ×
20 downstream, by the downstream of IL-18 gene insertion Gene expression P7.5 × 16, construct containing RHDV structural proteins VP60 and
The bird pox virus intermediate transfer carrier pUTAL-VP60-IL18 of rabbit interleukins IL-18；
The connection of exogenous dna fragment and intermediate transfer carrier: step 2 bird pox virus intermediate transfer carrier is used
The digestion filling-in of 150pmol and dephosphorization, the bird pox virus intermediate transfer carrier that obtains that treated will be in treated bird pox virus
Between transfer vector and exogenous dna fragment be added in 2 μ L 10 × connection buffers, the amount of the substance of exogenous dna fragment is fowl pos disease
6 times of the amount of the substance of malicious intermediate transfer carrier, then add water to 20 μ L thereto and be uniformly mixed, then be added thereto
It T4DNA ligase and is uniformly mixed, is then centrifuged with the revolving speed moment of 960rpm so that drop is gathered in tube bottom, water bath processing
Connection product is obtained, water bath processing is 16 DEG C and is kept for 24 hours or 25 DEG C being kept for 1 hour, and 3 μ L connection products is taken to be converted into sense
By state cell；
Step 3, the identification of recombinant plasmid: competent cell is cultivated, and obtains single bacterium colony, and single bacterium colony is existed
It is inoculated on solid medium containing antibiotic, and is gently dipped on the tube wall of the tubule of sterilizing, bacterium colony is made to be stained with tubule
It is cultivated on tube wall, obtains recombinant plasmid, recombinant plasmid is subjected to PCR (polymerase chain reaction) detection, test passes enter next
Step；
Step 4 prepares Alfalfa plant vaccine: fresh Agrobacterium EH105 competence is prepared using Calcium Chloride Method, it will
Transfected Recombinant Plasmid is into the positive Agrobacterium competent cell prepared, and ice bath 30 minutes, liquid nitrogen frozen 5 minutes, 37 water-baths 2
After minute, after 28 DEG C of 600 μ L LB liquid medium cultures being added 6 hours, it is applied to added with corresponding antibiotic culture dish
On, conversion spot carries out being accredited as positive plasmid by PCR, obtains positive Agrobacterium recombinant plasmid, and clover grass seed is used and is disappeared
Venom processing, is inoculated on solid medium, the illumination cultivation in plant incubator, then connects positive Agrobacterium recombinant plasmid
Kind in the fluid nutrient medium containing antibiotic, collect thallus, remove supernatant, thallus is resuspended with fluid nutrient medium, dilute by centrifugation
It releases to original volume and is cultivated, after waiting the growth of Alfalfa seedling, Alfalfa seedling is moved into soil plantation, can be obtained into
Product.
25 rabbit of selection same condition are randomly divided into 5 groups, every group 5.It is respectively as follows: 1 group using Alfalfa, 2 groups are adopted
With RHDV oil emulsion vaccine, 3 groups use Agrobacterium empty plasmid, and 4 groups of PBS control groups, 5 groups use Alfalfa vaccine.In 1 group of feeding
Feed Alfalfa 500g/ only, in 2 groups and 4 groups of every injection RHEV oil emulsion vaccines and 1000 μ of PBS L/, 3 groups of every rabbit are adopted
Agrobacterium empty plasmid is injected with quadriceps muscle of thigh, every time 1000 μ g/.5th group of feeding Alfalfa plant vaccine, 500g/ is only.Often
Group animal interval is immunized 2 times for 3 weeks.The Culling heart blood after 0d, 7d, 14d, 21d, 28d, 35d carries out amynologic index detection.Inspection
Survey the result shows that: 3 groups and 4 groups do not generate antibody, do not generate immunization, and the Alfalfa plant vaccine of building generates antibody
Slightly below 2 groups of level, but it is above the level of 3 groups and 4 groups, there is preferable immune effect.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (7)
1. a kind of preparation method of Alfalfa plant vaccine, which is characterized in that specific step is as follows:
Step 1 constructs intermediate transfer carrier: by the downstream of the structural proteins VP60 insertion combined promoter of RHDV, by IL-18
Gene is inserted into the downstream of Gene expression, constructs the chicken pox containing RHDV structural proteins VP60 and rabbit interleukins IL-18
Viral intermediate transfer carrier pUTAL-VP60-IL18；
Step 2, the connection of exogenous dna fragment and intermediate transfer carrier: by the bird pox virus intermediate transfer carrier prepared and outside
Source DNA segment is added in 2 μ L10 × connection buffer, adds water to 20 μ L thereto and is uniformly mixed, then T4 is added thereto
It DNA ligase and is uniformly mixed, then moment is centrifuged, and water bath processing obtains connection product, 3-4 μ L connection product is taken to be converted into
Competent cell；
Step 3, the identification of recombinant plasmid: competent cell is cultivated, and obtains single bacterium colony, and single bacterium colony is being contained
It is inoculated on the solid medium of antibiotic, and is gently dipped on the tube wall of the tubule of sterilizing, bacterium colony is made to be stained with the tube wall of tubule
Upper culture, obtains recombinant plasmid, recombinant plasmid is carried out PCR detection, test passes enter in next step；
Step 4 prepares Alfalfa plant vaccine: preparing fresh Agrobacterium EH105 competence using Calcium Chloride Method, will recombinate
Plasmid transfection is into the positive Agrobacterium competent cell prepared, and ice bath 30 minutes, liquid nitrogen frozen 5 minutes, 37 water-baths 2 minutes
Afterwards, it after 28 DEG C of 600 μ L LB liquid medium cultures being added 6 hours, is applied to added on corresponding antibiotic culture dish, is turned
Change spot to carry out being accredited as positive plasmid by PCR, obtain positive Agrobacterium recombinant plasmid, by clover grass seed using at thimerosal
Reason, is inoculated on solid medium, the illumination cultivation in plant incubator, is then inoculated in positive Agrobacterium recombinant plasmid and contains
Have in the fluid nutrient medium of antibiotic, be centrifuged, collects thallus, remove supernatant, thallus is resuspended with fluid nutrient medium, is diluted to original
Volume is cultivated, and after waiting the growth of Alfalfa seedling, Alfalfa seedling is moved into soil plantation, finished product can be obtained.
2. the preparation method of Alfalfa plant vaccine according to claim 1, which is characterized in that external source in the step 2
The amount of the substance of DNA fragmentation is 2-10 times of the amount of the substance of bird pox virus intermediate transfer carrier.
3. the preparation method of Alfalfa plant vaccine according to claim 1, which is characterized in that compound in the step 1
Promoter is ATI-P7.5 × 20, and Gene expression is P7.5 × 16.
4. the preparation method of Alfalfa plant vaccine according to claim 1 or 2, which is characterized in that in the step 2
Water bath processing be 15-18 DEG C of holding 15-24 hours or 24-27 DEG C holding 0.8-2 hours.
5. the preparation method of Alfalfa plant vaccine according to claim 1, which is characterized in that chicken pox in the step 2
The preceding digestion filling-in using 100-200pmol and dephosphorization in connection buffer is added in viral intermediate transfer carrier.
6. the preparation method of Alfalfa plant vaccine according to claim 1 or 5, which is characterized in that in the step 2
The revolving speed of centrifugation is 1000rpm.
7. the preparation method of Alfalfa plant vaccine according to claim 1, which is characterized in that sterilized in the step 4
The temperature of liquid is 28-33 DEG C, and solid medium uses MS solid medium.
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Citations (3)
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| CN102008720A (en) * | 2010-07-20 | 2011-04-13 | 东北农业大学 | Clostridium welchii disease resistant transgenic plant vaccine and preparation method thereof |
| CN104436187A (en) * | 2014-11-10 | 2015-03-25 | 张文波 | Vaccine for expressing rabbit hemorrhagic disease virus VP60 protein |
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2019
- 2019-07-04 CN CN201910599970.0A patent/CN110302370A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002018426A2 (en) * | 2000-09-01 | 2002-03-07 | Consejo Superior De Investigaciones Cientificas | Rabbit hemorrhagic disease vaccine and antigens |
| CN102008720A (en) * | 2010-07-20 | 2011-04-13 | 东北农业大学 | Clostridium welchii disease resistant transgenic plant vaccine and preparation method thereof |
| CN104436187A (en) * | 2014-11-10 | 2015-03-25 | 张文波 | Vaccine for expressing rabbit hemorrhagic disease virus VP60 protein |
Non-Patent Citations (5)
| Title |
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| YURI Y. GLEBA 等: "Plant Viral Vectors for Delivery by Agrobacterium", 《CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY》 * |
| 张大鹏: "VP60蛋白的原核表达、抗体制备及VP60基因转化的研究", 《中国博士学位论文全文数据库（电子期刊）农业科技辑》 * |
| 江文正 等: "共表达HIV-1中国流行株gp120与IL-18重组鸡痘病毒的构建及其免疫原性观察", 《生物工程学报》 * |
| 金宁一 等: "含中国流行株 HIV-1Gag-gp120 融合蛋白的重组鸡痘病毒的构建", 《中国免疫学杂志》 * |
| 霍晓伟: "亚洲1型***核酸疫苗与重组鸡痘疫苗的构建及实验免疫研究", 《中国博士学位论文全文数据库（电子期刊）农业科技辑》 * |
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