CN103305534A - Reverse genetics operating system of mink enteritis virus and application thereof - Google Patents
Reverse genetics operating system of mink enteritis virus and application thereof Download PDFInfo
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- CN103305534A CN103305534A CN2013101925556A CN201310192555A CN103305534A CN 103305534 A CN103305534 A CN 103305534A CN 2013101925556 A CN2013101925556 A CN 2013101925556A CN 201310192555 A CN201310192555 A CN 201310192555A CN 103305534 A CN103305534 A CN 103305534A
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Abstract
The invention relates to a reverse genetics operating system of a mink enteritis virus and an application thereof. The nucleotide sequence of the mink enteritis virus gene provided by the invention is shown as 1) or 2): 1) the nucleotide sequence shown as SEQ ID No.1; and 2) the nucleotide sequence shown as SEQ ID No.1 from the 2323rd-4577th sites at 3' end. The reverse genetics operating system of the mink enteritis virus established by the invention can be used for toxicity analysis of mink enteritis virus proteins, and a novel inactivated vaccine or attenuated vaccine can be prepared according to the system. The operating system further can use mutation sites as a genetic mark to detect whether virus in mink is wild toxicity or toxicity caused by vaccine, so that the system is of significance in production practice.
Description
Technical field
The present invention relates to the reverse genetics field operation of virus, relate to foundation and the application thereof of mink enteritis virus reverse genetics operating system particularly.
Background technology
(Mink enteritis virus MEV) is the cause of disease that causes mink viral enteritis to mink enteritis virus.Schofield F. in 1949 in Canadian reported first mink viral enteritis.Jiang Tingxiu equal reported first in 1981 China's mink farming this disease takes place.Clinically, mink viral enteritis significantly is reduced to feature to contain come off pipe, the blood leucocyte of intestinal mucosa, scleroproein and intestines mucus of canescence in serious diarrhoea, the ight soil, and morbidity is anxious, mortality ratio is high.Often can cause enormous economic loss.At present, this disease almost all has in all foster ermine countries and takes place and popular report.
According to the report of ICTV the 9th subseries, MEV belongs to Parvoviridae, parvovirus subfamily, parvovirus genus.MEV is the same with other parvoviruss, and physics and chemistry factor has powerful resistibility to external world, can tolerate the processing of fatsolvent and comparatively high temps and does not lose its infectivity.This chemical constitution and constructional feature with virus is relevant.MEV does not have cyst membrane, does not contain lipid and carbohydrate, and sturdy construction is tight, can tolerate 65 ℃ of 30min heating and does not lose its infectivity.At pH3-9, keep infectivity 60min at least in 56 ℃.Ether, chloroform, alcohols and deoxycholate are had resistibility, and it is infectious and blood clotting is all unaffected.Sterilizing agent can deactivation MEV, effectively formalin, beta-propiolactone, ammoniacal liquor and fluorizating agent etc.The red corpuscle of animals such as MEV energy aggegation pig, rhesus monkey.
Parvovirus is all in the nucleus internal breeding.Except dependovirus virus, other parvoviruss all can self-replicating, but need depend on some function in the host cell mitotic division process.So go down to posterity when cultivating in the external virus of carrying out, must be when passage be cultivated or virus inoculation in 24h at the latest, namely connect poison synchronously, just can reach the purpose of the good propagation of virus.MEV grows in the F81 cat kidney cell line, inoculates after 4～6 days, and virus titer can reach the highest.
MEV and other parvoviruss have common morphological structure feature.Virus particle does not have cyst membrane, and the about 18～26nm of diameter is the icosahedron symmetry, and capsid is made of the capsomere of 32 long 3～4nm approximately.The buoyant density of virus particle in cesium chloride solution is 1.39～1.42g/cm
3The capsid of three-dimensional symmetry surrounds the negative adopted DNA of wire, sub-thread of a molecule.Zhao Xintai (1991) has cloned and has measured the MEV genome sequence of nearly total length, obtains the fragment of 5064nt size altogether.According to the sequence of GenBank report, the full genome of MEV is 5094nt(sequence number D00765.1).
The genomic structure of MEV is very special, at its 3 '-end and 5 '-end a hairpin structure is arranged respectively, and the centre is the strand part.The long 205nt of hairpin structure of 3 '-end becomes y-type structure, sees Fig. 1.And 5 '-end hairpin structure length only is 62nt, becomes U-shaped structure.
The part of the terminal reverse complemental of genome is the primer of parvovirus dna replication dna.3 '-OH with 3 ' end hairpin structure is the complementary strand of primer synthetic gene group earlier, forms rf (RF) intermediate, produces the genome of progeny virus again by RF.The intermediate sequence of two hairpin structures is about 4700nt, two open reading frame (ORF) are arranged in the sequence hereinto, 3 '-end ORF coding Nonstructural Protein (NS), 5 '-end ORF coding constitutes the structural protein (VP) of nucleocapsid, and 3 kinds of capsid protein: VP1, VP2 and VP3 altogether encode.Through the formed mRNA of different modes montage coding, VP1 is Duoed 154 amino-acid residues at the N end than VP2 by same transcription product for VP1 and VP2, and VP3 falls 15-20 the product behind the amino-acid residue in the N of VP2 end-grain cutting, as Fig. 2.
Each nucleocapsid is made of the protein subunit that 60 VP1, VP2 and VP3 form, and wherein the copy number of VP1 albumen is 6-10, and the 4-13 residue of its N end has nuclear target ability, replaces Lys6, Arg7, Arg9 then to make it lose this nuclear target ability with Gly.So Lys6, Arg7 and Arg9 are important for nuclear target ability.VP2 is major capsid protein and protective antigen, and VP2 albumen can self-assembling assembly virus-like particle; In addition, also has a spot of VP3 albumen.There is not VP3 in the hollow capsid.
Birth and the development of reverse genetics (reverse genetics) and associated molecule biology techniques for we address the above problem, provide strong theoretical foundation and technique means.Reverse genetics is on the basis of known sequence, utilizes molecular biological theory and technology, and means such as the sudden change by nucleotide sequence, disappearance, insertion are created the phenotypic effect that mutant and research sudden change cause.Namely directly start with to study the biological function of gene from the genetic material of biology, set forth essential phenomenon and rule that life takes place.The various technology relevant with reverse genetics operation are referred to as reverse genetic learn a skill (reverse genetics techniques).
Set up dna virus reverse genetic manipulation technical system and mainly comprise contents such as the evaluation of structure, cotransfection rescue virus particle and newborn viral related characteristics of the clone of genome total length and transformation, sequencing, helper plasmid and application.It sets up process is exactly by external artificial genetic manipulation, makes the system of foundation satisfy virus replication and the needed various prerequisites of packing.At present a lot of dna virus reverse genetics service platforms are all set up, as PCV-II, simplexvirus and adenovirus etc.But some viral genome is too big or genome structure is too complicated, is difficult to make up full-length gene group clone.
At present, to the research of whole self-replicating parvovirus also seldom, particularly to the research of mink enteritis parvovirus molecular biology aspect.In the nineties in last century, the mink enteritis viral genome overwhelming majority has been cloned in the Zhao Xintai segmentation, and has carried out sequencing, but does not carry out the research work such as determine in the rescue of virus and molecule pathogenesis thereof, relevant virulence site.Because the genome structure of self-replicating parvovirus is special, is difficult to clone its full-length gene group, has had a strong impact on the foundation of its reverse genetics service platform.Do not see the research report that relevant parvovirus reverse genetics service platform is set up so far, greatly influenced its molecule pathogenesis, with the further investigation of the interaction relationship of host cell.
Find through patent retrieval, the patent relevant with the reverse genetics service platform has 8, comprise CN200310115457.9, CN201210314154.9, CN200910094223.8, CN200910094354.6, CN201010267524.9, CN200980136612.0, CN201010620151.9, CN201210346779.3,2 of the patents relevant with mink enteritis virus, comprise CN201010566499.4, CN201110254418.1, but all irrelevant with the foundation of MEV reverse genetics service platform.United States Patent (USP), European patent, Japanese Patent etc. are not found relevant therewith patent yet by retrieval.
At present, research and the application about mink enteritis virus has several problems to need to solve: the virulence of the pathogenesis of the one, MEV and viral major antigen albumen still imperfectly understands; The 2nd, clinically, vaccine virus and wild poison are often inseparable, and what do not know viral enteritis is owing to the infection of wild poison or owing to vaccine inactivation does not thoroughly cause.
Summary of the invention
At above-mentioned technical problem, the present invention has set up the reverse genetics service platform of mink enteritis virus, and introduced can be for the genetics mark of identifying, for the development of carrying out this virus virulence research and new generation vaccine later on provides technical guarantee.
One aspect of the present invention provides a kind of mink enteritis virogene, and its nucleotide sequence is as following 1) or 2) shown in:
1) nucleotide sequence shown in the SEQ ID No.1;
2) hold the nucleotide sequence shown in the 2323-4577 position by SEQ ID No.1 from 3 '.
Wherein, hold the nucleotide sequence shown in the 2323-4577 position through shearing coding VP1 albumen behind the nucleotide sequence shown in 3 ' the end 2356-2441 position by SEQ ID No.1 from 3 '.
The present invention provides the carrier that contains said gene on the other hand.In an embodiment of the present invention, the carrier that sets out is pBluescript ‖ SK (+).
Further aspect of the present invention provides the host cell that contains said gene or above-mentioned carrier.
Another aspect of the invention provides a kind of genetic marker, and it is G for 5 ' end 3530bp place base from sequence shown in the SEQ ID No.1.
The present invention provides a kind of recombinant virus on the other hand, and its genome sequence is classified the nucleotide sequence shown in the SEQ ID No.1 as.
The present invention provides a kind of antiviral vaccine on the other hand, and its activeconstituents is any of said gene, above-mentioned carrier, above-mentioned recombinant virus.
The present invention provides described gene, described carrier, the application of described recombinant virus in the preparation antiviral vaccine on the other hand.
Wherein, described antiviral vaccine is anti-mink enteritis virus vaccines.
The present invention has following beneficial effect: the mink enteritis virus reverse genetics operating system that the present invention sets up can be used for the virulence analysis of mink enteritis viral protein, and can prepare novel deactivation vaccine or attenuated vaccine accordingly.Also can be according to the mutational site as genetic marker, detect virus in the disease ermine body and be wild poison or since vaccine cause, significant in production practice, for research mink enteritis parvovirus gene structure and aspects such as function and Study on Pathogenicity provide platform, also lay the foundation for studying at the vaccine of mink enteritis etc.
Description of drawings
Fig. 1 is the Y type hairpin structure that MEV genome 3 '-end is made up of 205nt;
Fig. 2 is parvovirus genome structure and phraseology synoptic diagram thereof; TR is reverse terminal repeat, and D is donor splicing site, and A is acceptor;
Fig. 3 A-D is experimental road line chart of the present invention;
Fig. 4 is the growth curve (connect poison synchronously and survey virus titer) of wild poison and rescue virus.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The structure of embodiment 1 sudden change mink enteritis virus vector
Transform carrier pBluescript ‖ SK (+) (available from precious biotechnology company limited): Kpn I Afe I Stu I Sma I will be transform as between Kpn I and the Sma I; Synthetic MEV genome 3 ' end 109bp(fragment I) by Kpn I, Afe I is cloned on the transformed carrier; Synthetic MEV genome 3 ' end 82bp(fragment II) is connected with the fragment I by the Infusion clone technology; The carrier that contains MEV genome 3 ' end with Stu I linearizing; According to three pairs of primers of the relative conserved regions design of mink enteritis virogene (seeing Table 1), non-hairpin in the middle of the amplification mink enteritis parvovirus genome couples together three fragments (fragment III) by Over-lapPCR.The fragment III is cloned on the pEASY-Blunt carrier (available from sky, Beijing root biochemical technology company limited).The base A of 3530bp place is mutated into bases G introduces BamH I site as genetic marker evaluation rescue virus.Design primer I nfusion III SF, Infusion III SR(sees Table 1) the amplification fragment III of suddenling change.Synthetic MEV genome 5 ' end 222bp is cloned into pMD-19T carrier (available from precious biotechnology company limited).Design primer I nfusion IV SF, Infusion IV SR(sees Table 1) amplification MEV genome 5 ' end (the fragment IV contains pMD-19T part sequence).With the Infusion clone technology with the linearizing carrier that contains MEV genome 3 ' end, the fragment III of suddenly change and contain the fragment IV seamless link that MEV genome 5 ' is held, this plasmid called after pMEV (specifically construction strategy is seen Fig. 3 A-D).Final synthetic nucleotide sequence is shown in SEQ ID No.1.
Table 1 primer sequence
| Title | Sequence |
| SF198-218 | AGGTGGCGGGCTAATTGTGGG |
| SR2326-2346 | GCTCTCTTTGCCGGAGGTGCC |
| SF2062-2081 | ACAAGCGGCAAGCAATCCTC |
| SR3669-3688 | TGGTAAGCCCAATGCTCTAT |
| SF3292-3311 | TGCTACTCAGCCACCAACTA |
| SR4864-4884 | AACATATTCTAAGGGCAAACC |
| InfusionⅢSF | TTGGGCGGGAAAAGGTGGCGGGCTAATTGTGGGGCGT |
| InfusionⅢSR | CTTAACATATTCTAAGGGCAAACCAACCAACCACCCACAC |
| InfusionⅣSF | TTAGAATATGTTAAGGACCAAAAAA |
| InfusionⅣSR | TCCCCGGGAGTCAGGGAGCGAGGAAGCGGAAGA |
The preparation of embodiment 2 rescue viruses
Inoculation F81 cell (available from China Veterinary Drugs Supervisory Inst.) is put in six orifice plates, and cell density carries out transfection during for 80%-90%.Add 5 μ l(5 μ g in every hole) plasmid pMEV and 10 μ l Lipofectamine2000 (available from Invitrogen company) the transfection reagents mixing that vibrates in the Opti-MEM of 500 μ l, carry out transfection according to the transfection reagent specification sheets.And observation of cell pathology day by day.Occurred cytopathy (CPE) on the 3rd day, collected the cell transfecting thing on the 5th day and freeze repeatedly molten three times and go down to posterity.After collecting the virocyte viral cultures multigelation three times in the 7th generation, it is standby to put-70 ℃ of preservations.
The evaluation of embodiment 3 rescue viruses
The rescue virus of embodiment 2 preparations is carried out titration with parental virus, the result shows: parental virus MEV and the viral rMEV TCID50 of rescue and hemagglutinative titer do not have significant difference, carrying out the multistep growth curve simultaneously measures, after 10h virus began to copy after the result was presented at and infects, reach at 36h and to copy the peak period, the difference between wild poison and the rescue virus is remarkable (Fig. 4) not.
The result shows that we utilize reverse genetic operating system to obtain to have with wild poison the full-length infectious clone of similar growth characteristics, illustrate that simultaneously the genetic marker of introducing in viral genome does not influence the growth of whole virus.The genetic marker BamH I site of introducing among the full-length infectious clone of mink enteritis parvovirus still can stable existence after going down to posterity, and can be used as to identify wild poison and rescue virus genetic marker reliably.
The preparation of embodiment 4 mink parvovirus inactivated vaccines
(1) the mink parvovirus liquid after will suddenling change is put into microfiltration systems and is filtered from head tank, obtains penetrating fluid and concentrated solution, and described concentrated solution returns head tank, after the parvovirus fluid solid content is more than or equal to 1g/L, parvovirus liquid is discharged; (2) penetrating fluid is carried out uf processing, further concentrate venom, reach to 2 up to the viral hemoagglutination valency
8～2
13In time, stop to concentrate, and obtains concentrated solution; (3) concentrated solution is refiltered with microfiltration systems, obtaining the blood clotting valency after handling is 2
8～2
13The parvovirus venom; (4) with the deactivation of parvovirus venom and will concentrate and deactivation after viral liquid be prepared into vaccine product.
Be prepared into vaccine product after testing and have good immunogenicity and stability.
Though 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 all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. mink enteritis virogene, its nucleotide sequence is as following 1) or 2) shown in:
1) nucleotide sequence shown in the SEQ ID No.1;
2) hold the nucleotide sequence shown in the 2323-4577 position by SEQ ID No.1 from 3 '.
2. the carrier that contains the described gene of claim 1.
3. carrier according to claim 2 is characterized in that, the carrier that sets out is pBluescript ‖ SK (+).
4. the host cell that contains the described gene of claim 1 or claim 2 or 3 described carriers.
5. a genetic marker is characterized in that, holding 3530bp place base from 3 ' of sequence shown in the SEQ ID No.1 is G.
6. recombinant virus, its genome sequence is classified the nucleotide sequence shown in the SEQ ID No.1 as.
7. antiviral vaccine, its activeconstituents be following any: the described gene of claim 1, claim 2 or 3 described carriers, the described recombinant virus of claim 6.
8. the described gene of claim 1, claim 2 or 3 described carriers or the described recombinant virus of claim 6 application in the preparation antiviral vaccine.
9. application according to claim 8 is characterized in that, described antiviral vaccine is anti-mink enteritis virus vaccines.
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Cited By (3)
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| CN103952378A (en) * | 2014-04-29 | 2014-07-30 | 中国农业科学院特产研究所 | Natural non-hemagglutinating mink enteritis virus strain MEV-NH and preparation method and application thereof |
| CN108410885A (en) * | 2018-03-29 | 2018-08-17 | 中国农业科学院北京畜牧兽医研究所 | The full-length infectious DNA clone and its construction method of a kind of pig parvoviral and application |
| CN111334528A (en) * | 2020-03-20 | 2020-06-26 | 山东农业大学 | Mink enteritis parvovirus whole genome infectious clone and construction method and application thereof |
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Cited By (4)
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| CN103952378A (en) * | 2014-04-29 | 2014-07-30 | 中国农业科学院特产研究所 | Natural non-hemagglutinating mink enteritis virus strain MEV-NH and preparation method and application thereof |
| CN108410885A (en) * | 2018-03-29 | 2018-08-17 | 中国农业科学院北京畜牧兽医研究所 | The full-length infectious DNA clone and its construction method of a kind of pig parvoviral and application |
| CN111334528A (en) * | 2020-03-20 | 2020-06-26 | 山东农业大学 | Mink enteritis parvovirus whole genome infectious clone and construction method and application thereof |
| CN111334528B (en) * | 2020-03-20 | 2022-02-18 | 山东农业大学 | Mink enteritis parvovirus whole genome infectious clone and construction method and application thereof |
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