CN102827259A - Porcine circovirus type 2 (PCV2) Cap protein gene and application thereof - Google Patents
Porcine circovirus type 2 (PCV2) Cap protein gene and application thereof Download PDFInfo
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- CN102827259A CN102827259A CN2012103157763A CN201210315776A CN102827259A CN 102827259 A CN102827259 A CN 102827259A CN 2012103157763 A CN2012103157763 A CN 2012103157763A CN 201210315776 A CN201210315776 A CN 201210315776A CN 102827259 A CN102827259 A CN 102827259A
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Abstract
The invention relates to a porcine circovirus type 2 (PCV2) Cap protein gene and an application thereof. The amino acid sequence of the protein is SEQ ID NO:1, and the nucleotide sequence of the protein is SEQ ID NO:2. The PCV2 Cap protein gene provided by the invention can be applied to preparation of a PCV2 Cap protein gene engineering subunit vaccine. The PCV2 Cap protein gene engineering subunit vaccine obtained in the invention is inoculated to five PCV2 negative weaned pigs of 21 days old through muscles, and another five weaned pigs are taken as a control group. Blood is sampled for detecting PCV2 antibodies 28 days after inoculation, which indicate that all PCV2 antibodies in an immune group are positive, the effective rate is up to 100 percent, and antibodies are not detected in the control group. As proved by a result, a prepared recombinant protein has high immunogenicity, and a further prepared PCV2 Cap protein gene engineering subunit vaccine can cause the immune response of piglets.
Description
Technical field
The invention belongs to domestic animal recombinant vaccine technical field, be specifically related to a kind of porcine circovirus 2 type Cap protein gene and application thereof.
Background technology:
Porcine circovirus 2 type (Porcine Circovirus type 2; PCV2) be pmws (Postweaning multsystemic wasting syndrome; PMWS) cause of disease, this virus can cause symptoms such as weanling pig generation progressive emaciation, cough, expiratory dyspnea, diarrhoea, palor or xanthochromia, lymphadenectasis, the greyish white oedema of kidney.Simultaneously should virus also can cause to a certain extent immunosuppression, cause the secondary or the accompanying infection of other disease easily.Since Canada in 1991 found clinical pig circular ring virus first, this disease had caused enormous economic loss for whole world pig industry.Therefore, the research to porcine circovirus 2 type is one of the emphasis in poultry farming field always.
The Cap albumen of porcine circovirus 2 type is the primary structure albumen of PCV2; Can the oneself be assembled into viral particle; And contain the viral epitope that can neutralize, and can induce the stronger antibody response of body generation and obtain immunoprotection, be the preferred object gene of design PCV2 new generation vaccine.At present, abroad the genetic engineering subunit vaccine of the Cap protein Preparation of existing baculovirus expression porcine circovirus 2 type is realized commercial applications.But along with the porcine circovirus 2 type strain constantly makes a variation, the immune protective efficiency of existing vaccine constantly descends.Therefore, the Cap albumen of screening current popular strain carries out that the vaccine research and development have important practical significance undoubtedly and the market development is worth.
Summary of the invention:
The purpose of this invention is to provide a kind of porcine circovirus 2 type Cap protein gene and application thereof, promptly a kind of new porcine circovirus 2 type Cap protein gene, and as the application of vaccine.
One aspect of the invention provides a kind of circovurus type 2 Cap albumen, comprises that aminoacid sequence is the albumen of SEQ ID NO:1.
Another aspect of the present invention relates to the above-mentioned proteic Nucleotide of coding, and its sequence is SEQ ID NO:2.
Another aspect of the present invention relates to the application of circovurus type 2 Cap albumen in preparation porcine circovirus 2 type genetic engineering subunit vaccine.
The porcine circovirus 2 type genetic engineering subunit vaccine that the present invention obtains is through the negative weanling pig of intramuscular inoculation 21 age in days PCV2; Inoculate and get blood survey PCV2 antibody after 28 days; Immune group PCV2 antibody is all positive, efficiently reaches 100%, and control group does not detect antibody.The result shows that the recombinant protein immunogenicity of preparation is good, and its porcine circovirus 2 type genetic engineering subunit vaccine that further prepares can cause the immunne response of piglet.
Figure of description
Fig. 1: the vector construction collection of illustrative plates that the present invention is used.
Embodiment:
Further describe the present invention below in conjunction with embodiment; But what those skilled in the art should understand that is; Under the situation that does not depart from technical scheme of the present invention, can make amendment or replace, but these modifications and replacement all fall in the protection domain of the present invention the details of technical scheme of the present invention and form.
One, the amplification of porcine circovirus 2 type Cap protein gene
After the pathological material of disease of the doubtful PMWS of clinical separation being handled the PK-15 cell, blind passage three generations of the no PCV1 pollution of back inoculation, pcr amplification porcine circovirus 2 type full length gene, primer sequence is following:
P1: 5′--GCTGGCTGAACTTTTGAAAG--3′
P2： 5?′--AAATTTCTGACAAACGTTAC--3′
The condition of amplification is following: 94 ℃ of 5min sex change, and 94 ℃ of 30S, 65 ℃ of 30S, 72 ℃ of 1.5min, 30 circulations, 72 ℃ are extended 10min.
With the Blast comparison on NCBI of Cap protein gene sequence part, homology was up to 97% after amplified production checked order, and the sample that shows amplification is the virus strain that morphs.The aminoacid sequence of its translation is SEQ ID NO:1.The Cap protein gene is carried out the transformation of intestinal bacteria rare codon, and improved nucleotides sequence is classified SEQ ID NO:2 as.Nucleotide (SEQ ID NO:2) 5 ' end and the 3 ' end of improved Cap protein gene is added BamH I and Hind III restriction enzyme site respectively, send genome company to carry out full gene and synthesize.
Two, the acquisition of the structure of engineered protein expression vector and engineering bacteria
1, is connected into the corresponding restriction enzyme site of pET30a carrier behind the Cap protein gene double digestion that above-mentioned amplification obtains, makes up pET30a/mCap expression vector (Fig. 1).
2, use CaCl
2Method is transformed into e. coli bl21 (DE3) with the pET30a/mCap expression vector, coats the agar plate that contains 50 μ g/ml kantlex, 37 ℃ of incubated overnight.Choose 10 single bacterium colonies and extract plasmids, BamH I and Hind III double digestion are verified the evaluation of further checking order of male bacterium colony.Fermentation culture to 0.6～0.8 an o'clock adding 0.3mM IPTG in the LB substratum induced 4～5 hours with the positive colony after the sequence verification, and centrifugal collection thalline runs the SDS-PAGE electrophoresis, sets up simultaneously and does not induce thalline as contrast.The result induces back positive colony comparison to have more a protein band at the 30KD place according to bacterium, and consistent with the recombinant protein theoretical molecular, expression amount is about more than 35%.Through the calibrating of PCV2 antibody mediated immunity trace, show positive reaction.The positive colony that proof obtains is for efficiently expressing the engineering bacteria of engineered protein (SEQ ID NO:1).
The preparation of three, fermentation, purifying and porcine circovirus 2 type genetic engineering subunit vaccine
1, zymotechnique
1) the LB substratum contains 5g/L glucose and 5g/L MgSO as seed culture medium
47H
2The LB substratum of O is as fermention medium, and feed supplement is glucose 400g/L, peptone 24g/L, yeast extract 10 g/L, NaCl 5 g/L, MgSO
47H
2O 5 g/L.
2) fermenting process
The engineering bacteria that picking was identified is inoculated in the LB substratum that the concentration that contains kantlex is 50 μ g/ml, and 37 ℃ of shaking culture 8 hours are as seed liquor.Seed liquor is inoculated in the fermentor tank by 2% inoculum size, regulates each parameter, 37 ℃, 200 change, and dissolved oxygen is controlled at more than 20%.Fermenting began flow feeding after 4 hours, fermented after 6 hours to add 0.3mmol/L IPTG and carry out abduction delivering, expressed back 6 hours fermentation ends.
3) ni-sepharose purification, desalination
4) affinity chromatography
Adopt nickel ion metal chelate chromatography post, recombinant protein can wash with the elutriant that contains the 300mmol/L imidazoles, and purity reaches more than 90%
5) desalination
The recombinant protein elutriant of collecting is put into dialysis tubing, and PBS liquid is as extracellular fluid dialysis, and dialysis desalting promptly gets recombinant protein liquid.
2, the preparation of porcine circovirus 2 type genetic engineering subunit vaccine
With the sequence of preparation is that 96 parts of the recombinant proteins of SEQ ID NO:1 fully mix as water with 4 parts of tween-80s of sterilizing.Simultaneously the injection white oil is 94 parts, add 6 parts department this 80,2 parts of StAls, mix, behind the autoclaving as oil phase.Can prepare the porcine circovirus 2 type genetic engineering subunit vaccine by water and the emulsification of oil phase 1:3 mixed.It is all qualified to carry out proterties, steriling test, safety verification by the method for inspection of veterinary biologics rules.
Four, the animal experiment of the porcine circovirus 2 type genetic engineering subunit vaccine of recombinant protein preparation
Through 5 of the negative weanling pigs of intramuscular inoculation 21 age in days PCV2, other gets 5 as contrast with the porcine circovirus 2 type genetic engineering subunit vaccine for preparing.Inoculate and get blood after 28 days and survey PCV2 antibody, immune group PCV2 antibody is all positive, efficiently reaches 100%, and control group does not detect antibody.The result shows that the recombinant protein immunogenicity of preparation is good, and its porcine circovirus 2 type genetic engineering subunit vaccine that further prepares can cause the immunne response of piglet.And,, can more effectively carry out immunoprotection to piglet because porcine circovirus 2 type gene of the present invention is new allelotrope.
Claims (4)
1. a circovurus type 2 Cap albumen is characterized in that, described proteic aminoacid sequence is SEQ ID NO:1.
2. Nucleotide, the described Nucleotide described circovurus type 2 Cap of claim 1 albumen that is used to encode.
3. Nucleotide as claimed in claim 2 is characterized in that, the sequence of described Nucleotide is SEQ ID NO:2.
4. the application of the described circovurus type 2 Cap of claim 1 albumen in preparation porcine circovirus 2 type genetic engineering subunit vaccine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827289A (en) * | 2012-08-30 | 2012-12-19 | 青岛康地恩药业股份有限公司 | Porcine circovirus type 2 Cap protein and thymosin alpha1 fusion protein and application |
| CN105833262A (en) * | 2016-04-15 | 2016-08-10 | 山东畜牧兽医职业学院 | Porcine circovirus II type inactivated vaccine composition and preparation method |
| CN106399138A (en) * | 2016-09-07 | 2017-02-15 | 复旦大学 | Oral porcine circovirus II-like particle vaccine, and preparation method and application thereof |
| CN113845575A (en) * | 2020-06-28 | 2021-12-28 | 山西农业大学 | Porcine circovirus type 2 Cap protein and coding gene and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296089A (en) * | 2011-04-20 | 2011-12-28 | 中国兽医药品监察所 | Method for efficiently preparing porcine circovirus 2 type empty capsid |
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- 2012-08-30 CN CN2012103157763A patent/CN102827259A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296089A (en) * | 2011-04-20 | 2011-12-28 | 中国兽医药品监察所 | Method for efficiently preparing porcine circovirus 2 type empty capsid |
Non-Patent Citations (2)
| Title |
|---|
| 宋月等: "猪圆环病毒2型Cap蛋白在杆状病毒系统表达及鉴定", 《东北农业大学学报》 * |
| 黄立平等: "抗猪圆环病毒2型Cap蛋白中和性单克隆抗体的制备及鉴定", 《中国预防兽医学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827289A (en) * | 2012-08-30 | 2012-12-19 | 青岛康地恩药业股份有限公司 | Porcine circovirus type 2 Cap protein and thymosin alpha1 fusion protein and application |
| CN102827289B (en) * | 2012-08-30 | 2014-04-09 | 青岛蔚蓝生物股份有限公司 | Porcine circovirus type 2 Cap protein and thymosin alpha1 fusion protein and application |
| CN105833262A (en) * | 2016-04-15 | 2016-08-10 | 山东畜牧兽医职业学院 | Porcine circovirus II type inactivated vaccine composition and preparation method |
| CN106399138A (en) * | 2016-09-07 | 2017-02-15 | 复旦大学 | Oral porcine circovirus II-like particle vaccine, and preparation method and application thereof |
| CN106399138B (en) * | 2016-09-07 | 2019-11-12 | 复旦大学 | Porcine Circovirus takes orally virus sample particle vaccines and its preparation method and application |
| CN113845575A (en) * | 2020-06-28 | 2021-12-28 | 山西农业大学 | Porcine circovirus type 2 Cap protein and coding gene and application thereof |
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