CN107007832A - A kind of preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position - Google Patents
A kind of preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position Download PDFInfo
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- CN107007832A CN107007832A CN201610054517.8A CN201610054517A CN107007832A CN 107007832 A CN107007832 A CN 107007832A CN 201610054517 A CN201610054517 A CN 201610054517A CN 107007832 A CN107007832 A CN 107007832A
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- staphylococcus aureus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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Abstract
The invention discloses a kind of preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA A target position, extract L-form staphylococcus aureus, according to the ClfA gene orders being had been filed on GenBank, separately design sense primer and anti-sense primer, and upstream, with introducing Bam H I and Hind III digestions site respectively in anti-sense primer, recombinant plasmid pET ClfA A are made in primer；Enter performing PCR amplification, recombinant plasmid pET ClfA A are transferred in competent cell BL21 (DE3), induced with IPTG, great expression and purifying are carried out to destination protein；Rabbit is taken, blood sampling obtains serum after being immunized, and is detected；Blood sampling obtains serum after immune programme for children, determines after antibody titer, and staphylococcus aureus mastitis for milk cows vaccine is made.The preparation for the effective staphylococcus aureus agglutination factor albumen (ClfA A) that the present invention is set up, is the vaccine research using ClfA A as target position, and the research to staphylococcus aureus property mastitis for milk cows vaccine provides test basis.
Description
Technical field
The present invention relates to animal gene technical field, specifically a kind of staphylococcus aureus cow breast of ClfA-A target position
The preparation method of scorching vaccine.
Background technology
Mastitis for milk cows (Bovine Mastitis) is the common disease of milk cow, is the inflammation of cow mammary gland tissue, mainly
It is common disease, the frequently-occurring disease of milk cow caused by breast tissue pathogenic infection microorganism.In decades, both at home and abroad for milk cow
Many effort have been made in the preventing and treating of mammitis, but the disease be always perplex China's dairy cultivation development the most serious disease it
One.It makes Lactation of Dairy Cow and Reproductive Performance sustain damage, and not only reduces the output of milk of milk cow, eliminates milk cow, influence breast
Quality causes serious economic loss, and even human health is had influence on when serious.
The first step of S.a μ reus infection is exactly to be colonized in host, and realizes that the premise being colonized is tissue of the bacterium in host
With sticking for cell surface, bacterium has the component that can be attached to tissue surface to turn into adhesin, and S.aureus, which sticks, to be have
Fibrin primary agglutinin A, B (Fibronectin-binding proteins clumping factor A and B, ClfA,
ClfB), fibrin A, B (Fibronectin-binding protein A and B, FnBPA, FnBPB), collagen
Associated proteins (collagen-binding protein Can) and A albumen (protein A), ClfA is Staphylococcus aureus
One of main adhesin of bacterium, can mediate staphylococcus aureus to be incorporated into the cellulose connection proteinogen of cell surface, make
Bacterial adhesion promotes invasion of the cell to host tissue in host cell surface.ClfA be by bacterial adhesion to fibrin and
Decision sex factor on solvable and insoluble fibrin is former.
The content of the invention
It is an object of the invention to provide a kind of preparation side of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position
Method, to solve the problems mentioned in the above background technology.
To achieve the above object, the present invention provides following technical scheme：
A kind of preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position, is comprised the steps of：
1) recombinant plasmid pET-ClfA-A structure
L-form staphylococcus aureus is extracted, according to the ClfA gene orders being had been filed on GenBank, upstream is separately designed and draws
Thing and anti-sense primer, and upstream primer, with introducing Bam HI and Hind III digestions site respectively in anti-sense primer, is made
Recombinant plasmid pET-ClfA-A；
Sense primer P1 sequences such as SEQ ID NO：Shown in 1,
Anti-sense primer P2 sequences such as SEQ ID NO：Shown in 2,
Then performing PCR amplification is entered to recombinant plasmid pET-ClfA-A, pcr amplification product is detected through agarose gel electrophoresis；Through
The positive colony that blue hickie screening and double digestion identification are obtained carries out Sequence analysis；
2) expression product of induction
Above-mentioned recombinant plasmid pET-ClfA-A is transferred in competent cell BL21 (DE3), with final concentration of 0.8-1.2mM's
IPTG (isopropylthiogalactoside) is induced, and takes the expression product of different time sections to carry out SDS-PAGE (polypropylene
Acrylamide gel electrophoresis) detection；
3) great expression of destination protein and purifying
According to SDS-PAGE detect determine optimal induction time and IPTG concentration, to destination protein carry out great expression and
Purifying；
4) screening of animal is immunized
Body weight 2~2.5kg rabbit are chosen, blood sampling obtains serum after being immunized, and is detected；
5) immune programme for children：
First immunisation：On destination protein and immunizing rabbit after Freund's complete adjuvant emulsification, every rabbit injection 650-750 μ g
State destination protein, dorsal sc multi-point injection；
After first immunisation every 15d booster immunizations once, using incomplete Freund's adjuvant as adjuvant, every rabbit 250-350 μ g
Destination protein, the 3rd immune rear 10d blood sampling obtains serum, determines after antibody titer, staphylococcus aureus milk is made
Garget vaccine.
It is used as further scheme of the invention：PCR amplification conditions：94℃5min；94℃30s、62℃45s、72℃1min
30s, 35 circulations；72℃10min.
It is used as further scheme of the invention：Step 2) in IPTG final concentration of 1.0mM.
It is used as further scheme of the invention：First immunisation：Destination protein and immunizing rabbit after Freund's complete adjuvant emulsification, often
Rabbit injects 700 μ g destination proteins.
It is used as further scheme of the invention：After first immunisation every 15d booster immunizations once, using incomplete Freund's adjuvant as
Adjuvant, every μ g destination protein of rabbit 300.
Staphylococcus aureus mastitis for milk cows vaccine is made according to the above method.
Compared with prior art, the beneficial effects of the invention are as follows：
The preparation for the effective staphylococcus aureus agglutination factor albumen (ClfA-A) that the present invention is set up, is with ClfA-A
For the vaccine research of target position, the research to staphylococcus aureus property mastitis for milk cows vaccine provides test basis.
Brief description of the drawings
Fig. 1 is the amplification figure of ClfA-A areas gene, M：DNA molecular quality standard, 1：PCR primer；
Fig. 2 is the SDS-PAGE analysis charts of destination protein, M：Protein standard, 1：Under IPTG inductions
Expression of the pET-32a-ClfA-A in BL21,2：Expression of the pET-32a-ClfA-A in BL21 when being induced without IPTG,
3：Empty carrier under IPTG inductions.
Embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, shown
So, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, all
Belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, a kind of preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position,
Concrete operation step：
1st, recombinant plasmid pET-ClfA-A structure
According to bibliography extraction L-form staphylococcus aureus, the ClfA gene orders (Z18852) that GenBank is logged in,
Upstream and downstream primer is designed with Primer5.0,
Sense primer P1 sequences：5’-CGCGGATCCGTAGCTGCAGATGCACC-3’；
Anti-sense primer P2 sequences：5’-CCCAAGCTTCTCATCAGGTTGTTCAGG-3’。
Its directed cloning, Bam HI and Hind III digestions site (underscore part) is introduced in upstream and downstream respectively for convenience,
PCR amplification conditions：94℃5min；94 DEG C of 30s, 62 DEG C of 45s, 72 DEG C of 1min30s, 35 circulations；72 DEG C of 10min,
Pcr amplification product is detected through agarose gel electrophoresis.Screened through blue hickie and double digestion identifies that obtained positive colony carries out sequence
Row determine analysis.
Expressed after sequencing correctly.(see Fig. 1)
2nd, the expression product of induction
The recombinant plasmid of above-mentioned structure is transferred in competent cell BL21 (DE3), carried out with final concentration of 1.0mM IPTG
Induction, takes the expression product of different time sections to carry out SDS-PAGE detections.
3rd, the great expression of albumen and purifying
Identified, destination protein exists with soluble form, expands after culture, the optimal induction time determined according to preliminary experiment
With IPTG concentration, great expression and purifying are carried out.(see Fig. 2)
4th, the screening of animal is immunized
Body weight 2~2.5kg rabbit are chosen, blood sampling obtains serum after being immunized, and is detected.
5th, immune programme for children：
First immunisation：Destination protein and immunizing rabbit after Freund's complete adjuvant emulsification, every 700 above-mentioned recombinant proteins of μ g,
Dorsal sc multi-point injection.
After first immunisation every 15d booster immunizations once, using incomplete Freund's adjuvant as adjuvant, every μ g egg of rabbit 300
In vain, the 3rd immune rear 10d blood sampling obtains serum, determines antibody titer,
6th, expression product ELISA is detected
100 μ L BFGs (4 μ g/mL) are coated with 96 hole elisa Plates, and 4 DEG C overnight；200 μ L gelatin (1g/L)
Closing, 37 DEG C of incubation 2h；100 μ L destination protein (5 μ g/mL) is added, to be not added with the hole of destination protein as control,
37 DEG C of incubation 2h；Add homemade primary antibody (rabbit anteserum of 1: 4000 dilution) 100 μ L, 37 DEG C of incubation 2h；Add sheep
ELIAS secondary antibody (1: 1000 dilution) 100 the μ L, 37 DEG C of incubation 2h of anti-rabbit；Add the μ L of nitrite ion 100,37 DEG C of incubations
10~30min；Add the μ L of terminate liquid 100；Determine OD values at 490nm.
7th, antibody anti-adhesion ability is detected
The μ L of 4 μ g/mL BFGs 100 are coated with 96 hole elisa Plates, and 4 DEG C are overnight, after TBST buffer solutions are washed 2 times,
Softly pat dry；Add the closing of 100 μ L gelatin (1g/L), 37 DEG C of incubation 2h；The staphylococcus aureus of exponential phase,
Add isometric self-control primary antibody, 37 DEG C of incubation 30min；The 1/2 of hole count is selected to add through making serum by oneself in the enzyme mark hole being coated with
The μ L of staphylococcus aureus 100 of incubation, remaining 1/2 adds the golden yellow grape of the exponential phase without sera incubation
Coccus, 37 DEG C of incubation 2h；After TBST buffer solutions are washed 3 times, softly pat dry；250mL/L formaldehyde is added, room temperature is placed
10~30min；After TBST buffer solutions are washed 3 times, softly pat dry；Add the washing of violet staining 30min, TBST buffer solution
After 3 times, softly pat dry；Ethanol decolorization 20min is added, crystal violet all abjections are made as far as possible；Determine OD values at 583nm.
8th, result
The detection of antibody Adhering capacity shows, adds the OD583 in the enzyme mark hole of the staphylococcus aureus without sera incubation
The OD583 of (1.031 ± 0.23, n=13) apparently higher than the enzyme mark hole for adding the staphylococcus aureus through sera incubation
(0.693 ± 0.23, n=13), the i.e. staphylococcus aureus without sera incubation are adhered to the energy on BFG
Power is stronger, and the ability that the staphylococcus aureus after sera incubation is adhered to BFG is decreased obviously.
Therefore, by the above method, the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position can be prepared, is
The preventing and treating of mastitis for milk cows provides the foundation from now on.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, and do not carrying on the back
In the case of spirit or essential attributes from the present invention, the present invention can be realized in other specific forms.Therefore, no matter from
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit is required rather than described above is limited, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each embodiment is only included
One independent technical scheme, this narrating mode of specification is only that for clarity, those skilled in the art should be by
Specification is as an entirety, and the technical solutions in the various embodiments may also be suitably combined, and forming those skilled in the art can
With the other embodiment of understanding.
Claims (6)
1. a kind of preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position, it is characterised in that by
Following steps are constituted：
1) recombinant plasmid pET-ClfA-A structure
L-form staphylococcus aureus is extracted, according to the ClfA gene orders being had been filed on GenBank, upstream is separately designed and draws
Thing and anti-sense primer, and upstream primer, with introducing Bam H I and Hind III digestions site respectively in anti-sense primer, is made
Recombinant plasmid pET-ClfA-A；
Sense primer P1 sequences such as SEQ ID NO：Shown in 1,
Anti-sense primer P2 sequences such as SEQ ID NO：Shown in 2,
Then performing PCR amplification is entered to recombinant plasmid pET-ClfA-A, pcr amplification product is detected through agarose gel electrophoresis；Through
The positive colony that blue hickie screening and double digestion identification are obtained carries out Sequence analysis；
2) expression product of induction
Above-mentioned recombinant plasmid pET-ClfA-A is transferred in competent cell BL21 (DE3), with final concentration of 0.8-1.2mM's
IPTG is induced, and takes the expression product of different time sections to carry out SDS-PAGE detections；
3) great expression of destination protein and purifying
According to SDS-PAGE detect determine optimal induction time and IPTG concentration, to destination protein carry out great expression and
Purifying；
4) screening of animal is immunized
Body weight 2~2.5kg rabbit are chosen, blood sampling obtains serum after being immunized, and is detected；
5) immune programme for children：
First immunisation：On destination protein and immunizing rabbit after Freund's complete adjuvant emulsification, every rabbit injection 650-750 μ g
State destination protein, dorsal sc multi-point injection；
After first immunisation every 15d booster immunizations once, using incomplete Freund's adjuvant as adjuvant, every rabbit 250-350 μ g
Destination protein, the 3rd immune rear 10d blood sampling obtains serum, determines after antibody titer, staphylococcus aureus milk is made
Garget vaccine.
2. the preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position according to claim 1,
Characterized in that, PCR amplification conditions：94℃5min；94 DEG C of 30s, 62 DEG C of 45s, 72 DEG C of 1min 30s, 35 are followed
Ring；72℃10min.
3. the preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position according to claim 1,
Characterized in that, step 2) in IPT6 final concentration of 1.0mM.
4. the preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position according to claim 1,
Characterized in that, first immunisation：Destination protein injects 700 μ g with immunizing rabbit after Freund's complete adjuvant emulsification, every rabbit
Destination protein.
5. the preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position according to claim 1,
Characterized in that, after first immunisation every 15d booster immunizations once, using incomplete Freund's adjuvant as adjuvant, every rabbit 300
μ g destination proteins.
6. staphylococcus aureus mastitis for milk cows vaccine is made according to any described preparation methods of claim 1-5.
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| CN201610054517.8A CN107007832A (en) | 2016-01-28 | 2016-01-28 | A kind of preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position |
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| CN201610054517.8A CN107007832A (en) | 2016-01-28 | 2016-01-28 | A kind of preparation method of the staphylococcus aureus mastitis for milk cows vaccine of ClfA-A target position |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1109577A4 (en) * | 1998-08-31 | 2002-03-13 | Inhibitex Inc | Multicomponent vaccines |
| CN102847172A (en) * | 2012-10-17 | 2013-01-02 | 西北农林科技大学 | Preparation method of genetic engineering vaccine for preventing staphylococcus caprae mastitis application |
-
2016
- 2016-01-28 CN CN201610054517.8A patent/CN107007832A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1109577A4 (en) * | 1998-08-31 | 2002-03-13 | Inhibitex Inc | Multicomponent vaccines |
| CN102847172A (en) * | 2012-10-17 | 2013-01-02 | 西北农林科技大学 | Preparation method of genetic engineering vaccine for preventing staphylococcus caprae mastitis application |
Non-Patent Citations (2)
| Title |
|---|
| 姜晓娟: "奶牛乳腺炎金黄色葡萄球菌凝聚因子A A区基因的克隆表达", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 姜晓娟等: "金黄色葡萄球菌凝聚因子 A区基因的克隆表达及表达产物的免疫学特性", 《中国兽医科学》 * |
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Application publication date: 20170804 |