CN104195154A - Reverse genetic operation system of Newcastle disease virus Mukteswar medium-toxicity vaccine strain and application of reverse genetic operation system - Google Patents
Reverse genetic operation system of Newcastle disease virus Mukteswar medium-toxicity vaccine strain and application of reverse genetic operation system Download PDFInfo
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- CN104195154A CN104195154A CN201410373399.8A CN201410373399A CN104195154A CN 104195154 A CN104195154 A CN 104195154A CN 201410373399 A CN201410373399 A CN 201410373399A CN 104195154 A CN104195154 A CN 104195154A
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Abstract
The invention relates to a reverse genetic operation system of a Newcastle disease virus Mukteswar medium-toxicity vaccine strain and an application of the reverse genetic operation system. The system comprises a transcription plasmid and three transcription auxiliary plasmids, wherein the transcription plasmid comprises a whole genome cDNA sequence of the Newcastle disease virus Mukteswar medium-toxicity vaccine strain which is controlled by an eukaryotic promoter; the three transcription auxiliary plasmids are controlled by the eukaryotic promoter; and each transcription auxiliary plasmid comprises a cDNA sequence which is used for encoding the nucleoprotein (NP) of the Newcastle disease virus Mukteswar medium-toxicity vaccine strain, a cDNA sequence which is used for encoding the phosphoprotein (P) of the Newcastle disease virus Mukteswar medium-toxicity vaccine strain and a cDNA sequence which is used for encoding the large polymerase (L) of the Newcastle disease virus Mukteswar medium-toxicity vaccine strain. According to the reverse genetic operation system, the wild type recombined Newcastle disease virus is successfully rescued, and a foundation is laid for the further development of the vaccine development by taking the Newcastle disease virus as a carrier and the fundamental researches related to the tumor oncolytic treating virus and the NDV (Newcastle Disease Virus).
Description
Technical field
The present invention relates to viral genetic manipulation field, more specifically, the present invention relates to a kind of Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain reverse genetic operating system and application thereof.
Background technology
Angela Romer-Oberdo rfer etc. and BEN P.H.PEETERS < < Generation of recombinant lentogenic Newcastle disease virus from cDNA > > (the Journal of General Virology (1999) that equals to publish an article first respectively for 1999,80,2987 – 2995) and < < Rescue of Newcastle Disease Virus from Cloned cDNA:Evidence that Cleavability of the Fusion Protein Is a Major Determinant for Virulence > > (JOURNAL OF VIROLOGY, June1999, p.5001 – 5009), disclose a kind of T7 of utilization promotor and controlled re-reading system, save Avian pneumo-encephalitis virus low virulent strain, there are again afterwards some to save successively the Avian pneumo-encephalitis virus of different strains, the rescue system of all these Avian pneumo-encephalitis virus is to be all controlled by T7 promotor, such Avian pneumo-encephalitis virus reverse genetic operating system is all to utilize the special cells system of expressing t7 rna polymerase, and the recombinant poxvirus that need to express t7 rna polymerase additionally provides t7 rna polymerase effectively to save recombinant Newcastle disease virus.In this type systematic, the full Genomic cDNA clone of Avian pneumo-encephalitis virus is under T7 promotor, and indispensable protein NP, the P that its rescue virus is required and the gene of L be implemented on the plasmid that is subject to the control of T7 promotor also more.In the viral process of rescue, first need to infect with the recombinant poxvirus of expressing t7 rna polymerase the clone (as BHK-T7) that can express t7 rna polymerase, afterwards again by helper plasmid and complete this clone of geneome plasmid cotransfection, rely on the t7 rna polymerase generating in cell to transcribe out the geneome RNA of Avian pneumo-encephalitis virus, and be assembled into nucleocapsid with NP, P and L that helper plasmid is expressed, finally in the mode of sprouting, generate recombinant Newcastle disease virus.The problem of this kind of reverse genetic operating system existence is to prepare the clone of stably express t7 rna polymerase, the process of preparation clone is complicated, technical requirements is high, cycle is long, and in the culturing process of clone, need constantly to add maintain pressure microbiotic to keep its stably express t7 rna polymerase, but along with the increase of passage number of times, its ability of expressing t7 rna polymerase can constantly reduce, until disappear, the recombinant poxvirus of the expression t7 rna polymerase infecting in transfection process in addition can be present in cell always, not only in rescue recombinant Newcastle disease virus process, need to add Arabinoside to suppress copying of this poxvirus, and after saving out recombinant virus, also need by filter or repeatedly the mode of diluted passage remove poxvirus, this not only can reduce the rescue efficiency of Avian pneumo-encephalitis virus, also have the risk that poxvirus pollutes, in the research of this method, if do not filter the cell culture of transfection, the rescue efficiency of Avian pneumo-encephalitis virus is higher, but it is very complicated to remove poxvirus, if filter the cell culture of transfection, the rescue efficiency of Avian pneumo-encephalitis virus is very low, even failed.
In order more effectively to utilize this good carrier of Avian pneumo-encephalitis virus, for the above shortcoming of the prior art, the t7 rna polymerase system of the T7 promotor that the Avian pneumo-encephalitis virus mesogenic Mukteswar vaccine strain reverse genetic operating system that we set up has been abandoned original use completely under controlling, but the eukaryotic cell promotor of utilizing all culturing cell cordings to have, and the expression of helper plasmid is to be also controlled by eukaryotic promoter, this system can have been fastened the viral process of rescue in various kinds of cell, efficiency is unaffected (as Vero, BHK21, the clones such as DFEI), the clone that our rescue system is used is the Vero cell that is widely used in production of vaccine, this vaccine that is carrier for this strain of Future Development has been established foundation for security.Current domestic investigator saves out new castle disease LaSota attenuated vaccine strain (Ge Jinying etc., Newcastle Disease Virus-Based Live Attenuated Vaccine Completely Protects Chickens and Mice from Lethal Challenge of Homologous and Heterologous H5N1Avian Influenza Viruses) and High Virulent Newcastle Disease Virus, these reverse genetic operating systems are also all based upon T7 promotor and control lower, but low virulent strain is because virulence is low, utilize effect limited, and there is Biosafety problem in virulent strain.Also do not have at present others to save out Avian pneumo-encephalitis virus Mukteswar strain, this strain virus is Avian pneumo-encephalitis virus mesogenic strains, the foundation of this strain reverse genetic operating system, by the Genetic evolution between the research low virulent strain of Avian pneumo-encephalitis virus and high virulence, provide powerful, also for exploitation, take the vaccine that this strain is carrier and lay a good foundation.
The present invention has overcome in prior art must be provided the shortcoming of t7 rna polymerase by specific cells system and recombinant poxvirus, providing a kind of can be the method for saving recombinant Newcastle disease virus by various kinds of cell.
Summary of the invention
The reverse genetic operating system that an object of the present invention is to provide a kind of Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain, this system comprises:
(1) transcribe plasmid, this transcribes the full genome cDNA sequence that plasmid comprises the Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain that is controlled by eukaryotic promoter;
Transcribe helper plasmid for (2) three, this helper plasmid comprises respectively the cDNA sequence of the nucleoprotein (NP) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of encoding, the cDNA sequence of the cDNA sequence of the phosphoric acid albumen (P) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of encoding and the large polymerase protein (L) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of encoding, and three helper plasmids are all controlled by eukaryotic promoter.
In reverse genetic operating system of the present invention; preferably; the described plasmid of transcribing also comprises that coding has the sequence of the nuclease of self splicing function, the sequence of coding fourth liver ribozyme (Rbz) and the hereditary label that is positioned at full genome cDNA sequence inside; described full genome cDNA sequence is positioned at after eukaryotic promoter and coding have the sequence of nuclease of self splicing function; before the sequence of coding fourth liver ribozyme (Rbz), common formation is transcribed template.
In reverse genetic operating system of the present invention, preferably, the wherein said nuclease with self splicing function is fist shape ribozyme (HamRz), described hereditary label is NotI restriction enzyme site and the Bgl II restriction enzyme site that is positioned at full genome cDNA sequence inside, and described eukaryotic promoter is CMV promotor.
In reverse genetic operating system of the present invention, preferred, the described nucleotide sequence of transcribing plasmid as shown in SEQ ID NO.1, called after pCI-NDV.
In reverse genetic operating system of the present invention, preferably, described three transcribe helper plasmid be by by the cDNA sequence of nucleoprotein (NP) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of coding, the cDNA sequence of the cDNA sequence of the phosphoric acid albumen (P) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of encoding and the large polymerase protein (L) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of encoding is cloned into respectively in carrier pCI obtains.
In reverse genetic operating system of the present invention, preferably, encode the cDNA sequence of nucleoprotein (NP) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain as shown in SEQ ID NO.2, encode the cDNA sequence of phosphoric acid albumen (P) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain as shown in SEQ ID NO.3, encode the cDNA sequence of large polymerase protein (L) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain as shown in SEQ ID NO.4, wherein, the cDNA sequence of coding nucleoprotein (NP) and the cDNA sequence of coding phosphor acid albumin (P) utilize respectively NheI and XbaI enzyme cutting site to be implemented on pCI carrier, the cDNA sequence of large polymerase protein (L) of encoding is to utilize NheI and NotI restriction enzyme site to be implemented on pCI carrier, transcribe helper plasmid called after pCI-NP respectively for three that obtain, pCI-P and pCI-L.
In reverse genetic operating system of the present invention, preferred, also comprise the host cell for virus rescue, described host cell is Vero cell, BHK21 cell or DFEI cell, more preferably Vero cell.
Another object of the present invention has been to provide a kind of method of utilizing the reverse genetic operating system rescue recombinant Newcastle disease virus described in above any one, comprises the following steps:
(1) use transcribing plasmid and transcribing helper plasmid cotransfection for the host cell of virus rescue in the reverse genetic operating system described in above any one, cultivate the host cell after transfection;
(2) from the supernatant liquor of transfectional cell, save recombinant Newcastle disease virus.
In method of the present invention, preferred, transcribe plasmid pCI-NDV and helper plasmid pCI-NP, the ratio that pCI-P and pCI-L are 4:2:2:1 according to mass ratio is carried out transfection.
A further object of the present invention is to provide the application of described reverse genetic operating system in rescue recombinant Newcastle disease virus Mukteswar strain.And the described reverse genetic operating system application of take in the live vector vaccine that newcastle disease virus strain is skeleton in preparation.
Compared to prior art, the invention has the beneficial effects as follows:
The foundation of Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain reverse genetic operating system in the present invention, abandon prior art completely and used t7 rna polymerase system, there is no loaded down with trivial details clone preparation and maintenance process, do not need preparation to express the complex technology of the recombinant poxvirus of t7 rna polymerase yet, therefore in the rescue process of recombinant virus, do not need to use recombinant poxvirus, just do not suppress to remove after poxvirus over-replicate and virus rescue the complicated processes of poxvirus yet.It is to save recombinant Newcastle disease virus that the present invention can utilize all culturing cells, and this lays the foundation for setting up from now on and save such paramyxovirus.In recombinant Newcastle disease virus rescue system of the present invention, the clone that we use is preferably the Vero clone of widely applying in production of vaccine field, this clone is the safe clone of at present generally acknowledged production of vaccine, and this take for Future Development the vaccine that the strain of this strain Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain is carrier and establishes foundation for security.
Accompanying drawing explanation
Fig. 1 is the structure schematic diagram of the full genome cDNA of Avian pneumo-encephalitis virus Mukteswar strain;
Full gene element is 4 sections and clones, and add hereditary label in full Genomic cDNA clone, respectively be positioned at the NotI restriction enzyme site of genome 4953 and be positioned at 8597 Bgl II restriction enzyme site, genome head is fist shape ribozyme (HamRz), before ribozyme, be the restriction enzyme site-NheI being connected with pCI carrier, between first paragraph and second segment, by restriction enzyme site SacII, be connected, second segment with the 3rd section between by restriction enzyme site MluI, be connected, the 3rd section with the 4th section between by restriction enzyme site MluI, be connected, the 4th segment trailer is fourth liver ribozyme (Rbz), NotI is the restriction enzyme site that full Genomic cDNA clone is connected with pCI carrier,
Fig. 2 is that the plasmid of Avian pneumo-encephalitis virus Mukteswar strain reverse genetic operating system forms schematic diagram;
Three helper plasmids are all implemented on pCI carrier, controlled by CMV promotor;
Fig. 3 is Avian pneumo-encephalitis virus Mukteswar strain reverse genetic operating system transfection Vero cell, saves out the schematic diagram of recombinant virus;
Represent that a plurality of small circles in the circle of cell represent respectively three kinds of albumen that helper plasmid pCI-NP, pCI-P and pCI-L express, in figure, three of the lower right corner circles with a plurality of projections represent the recombinant virus of saving out;
After Fig. 4 is transfection supernatant inoculating cell, the recombinant Newcastle disease virus Mukteswar strain cells infected of saving out forms picture under plasmodial microscope, and Normocellular picture;
Fig. 4 A and Fig. 4 B be by after transfection supernatant inoculation Vero cell 40h in the result of 100x thing Microscopic observation, Fig. 4 C figure is the result of 200x thing Microscopic observation, the round spot in figure is the synplasm of formation; Fig. 4 D figure is normal Vero cell contrast;
Fig. 5 utilizes the specific primer pair of this viral genome to save the result that the recombinant virus obtaining carries out pcr amplification.
Embodiment
Further describe the present invention by the following examples and by reference to the accompanying drawings, it should be understood that these embodiment, only for the object of illustration, never limit the scope of the invention.
The structure of embodiment 1 Avian pneumo-encephalitis virus Mukteswar strain reverse genetic operating system
1, material source
Avian pneumo-encephalitis virus Mukteswar strain is purchased from Harbin Wei Ke biotechnology development company; Plasmid pCI purchased from
PROMEGA company, pBluescript II ks (+/-) Phagemids is purchased from Shanghai JaRa Bioisystech Co., Ltd; Archaeal dna polymerase is purchased from TAKARA company, and T4DNA ligase enzyme and other restriction enzyme are all purchased from NEB company; Viral RNA extracts test kit and middle amount is prepared test kit purchased from QIAGEN company, and M-MLV reverse transcription test kit and liposome 2000 test kits are purchased from IVENTROGEN company.
2, method
2.1 structures of transcribing plasmid that contain the full genome cDNA sequence of Avian pneumo-encephalitis virus Mukteswar strain
For setting up the reverse genetic operating system of Avian pneumo-encephalitis virus Mukteswar strain, must first build corresponding full Genomic cDNA clone, head at the full genome cDNA of Avian pneumo-encephalitis virus is the fist dress ribozyme with self splicing function, the full genome cDNA of virus afterwards, nucleic acid-protein gene (NP), phosphoric acid protein gene (P), matrix gene (M), antigen-4 fusion protein gene (F), hemagglutinin gene (HA) and large polymerase protein gene (L) successively, be connected with fourth liver ribozyme at genomic afterbody in order.In the clone of full genome cDNA, we will according to the restriction enzyme site of viral Mukteswar vaccine strain gene order, be divided into 4 sections of clones that complete full genome cDNA, and add hereditary label in full Genomic cDNA clone, be be respectively positioned at the NotI restriction enzyme site of genome 4953 and be positioned at 8597 Bgl II restriction enzyme site, whole genome cDNA is positioned between the NheI of PCI carrier and NotI multiple clone site (Fig. 1).Particularly, experimental procedure is as follows:
The allantoic cavity amplicon virus of Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain inoculation 9～11 age in days SPF chicken embryos, inoculated after 3 days, results allantoic fluid, and encapsulation is placed in cryogenic refrigerator and preserves.Utilize the viral RNA of QIAGEN to extract test kit extraction viral RNA, operation to specifications, utilize afterwards the M-MLV reverse transcription test kit of IVENTROGEN to carry out reverse transcription, recycle synthetic Auele Specific Primer and carry out PCR, glue reclaims PCR product and is cloned into the EcoR V site of carrier pBluescript II ks (+/-) Phagemids.The full genome cDNA sequence of Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain is divided into 4 sections according to restriction enzyme site, each section of cloned plasmids is after order-checking confirms that sequence is errorless, the mode of being cut, being connected by enzyme according to Strategies For The Cloning links together each section to be cloned on pCI carrier, structure obtains the plasmid of transcribing that contains the full genome cDNA sequence of Avian pneumo-encephalitis virus Mukteswar strain, the complete nucleotide sequence of this plasmid as shown in SEQ ID NO.1, called after pCI-NDV.
Three structures of transcribing helper plasmid in 2.2 Avian pneumo-encephalitis virus Mukteswar strains
Encode the cDNA sequence of nucleoprotein (NP) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain as shown in SEQ ID NO.2, encode the cDNA sequence of phosphoric acid albumen (P) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain as shown in SEQ ID NO.3, encode the cDNA sequence of large polymerase protein (L) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain as shown in SEQ ID NO.4, wherein, the cDNA sequence of coding nucleoprotein (NP) and the cDNA sequence of coding phosphor acid albumin (P) utilize respectively NheI and XbaI enzyme cutting site to be implemented on pCI carrier, the cDNA sequence of large polymerase protein (L) of encoding is to utilize NheI and NotI restriction enzyme site to be implemented on pCI carrier, transcribe helper plasmid called after pCI-NP respectively for three that obtain, pCI-P and pCI-L (Fig. 2).
2.3 by above-mentioned contain the full genome cDNA sequence of Avian pneumo-encephalitis virus Mukteswar strain transcribe plasmid and three helper plasmids transformed competence colibacillus cell Top10 respectively, large volume is shaken bacterium afterwards, utilize in the plasmid of QIAGEN amount to prepare test kit and prepare the plasmid that transfection is used, and measure the concentration of each plasmid.
The reverse genetic operating system that embodiment 2 utilizes embodiment 1 structure to obtain carries out the rescue of recombinant virus
1, method:
Vreo cell is inoculated in to 6 porocyte culture plates, when cell monolayer grows to 60%～80% left and right, starts transfection.With serum-free DMEM substratum, washed cell 2 times, adds Opti-MEM afterwards, 2ml/ hole, transfection reagent is liposome 2000 test kits of Invitrogen company, transfection is carried out according to test kit specification sheets, transcribe plasmid pCI-NDV and helper plasmid pCI-NP, pCI-P, the ratio that pCI-L is 4:2:2:1 according to mass ratio, amount with every hole 4ug is carried out transfection, after transfection 6h～8h, discard transfection supernatant, add the DMEM containing 5%FBS, when the basic all death of transfectional cell, draw supernatant and inoculate the Vero cell in new culturing bottle, day by day observation of cell pathology afterwards, when having significant synplasm to generate, show to save out recombinant virus (Fig. 3), can inoculate afterwards the recombinant virus saved out in Vero cell with amplicon virus, and detect blood clotting valency with 1% chicken red blood corpuscle.
2, result
In order to identify the virus of being rescued, by the Vero cell of cultivating in the supernatant liquor inoculation culture bottle in transfection process, with amplicon virus, wild-type strain produces significant cytopathy, there is synplasm to generate, can carry out blood clotting or RT-PCR tests to detect recombinant virus.Microscopic examination found that: the recombinant Newcastle disease virus Mukteswar strain vero cells infection of saving out all has synplasm to produce (Fig. 4 A, Fig. 4 B and Fig. 4 C), and normal Vero cell does not have synplasm to produce (Fig. 4 D).By rescuing the virus obtaining, utilize Trizol to extract total RNA, utilize random primer to carry out reverse transcription, utilize afterwards the specific primer of this viral genome to carry out pcr amplification, result amplifies specificity object band (Fig. 5).Above experimental result shows, by reverse genetic manipulation technology, utilize contain the full genome cDNA of Avian pneumo-encephalitis virus Mukteswar strain transcribe plasmid and three auxiliary clones that transcribe plasmid, successfully saved recombinant Newcastle disease virus.
The present invention utilizes the reverse genetic operating system of Avian pneumo-encephalitis virus Mukteswar strain successfully to save recombinant Newcastle disease virus, in recombinant Newcastle disease virus rescue system of the present invention, the clone that we use is the Vero clone of widely applying in production of vaccine field, this clone is the safe clone of at present generally acknowledged production of vaccine, and this take for Future Development the vaccine that the strain of this strain Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain is carrier and establishes foundation for security.
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, revise, and even equivalence change, but all will fall within the scope of protection of the present invention.
Claims (10)
1. a reverse genetic operating system for Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain, this system comprises:
(1) transcribe plasmid, this transcribes the full genome cDNA sequence that plasmid comprises the Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain that is controlled by eukaryotic promoter;
Transcribe helper plasmid for (2) three, this helper plasmid comprises respectively the cDNA sequence of the nucleoprotein (NP) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of encoding, the cDNA sequence of the cDNA sequence of the phosphoric acid albumen (P) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of encoding and the large polymerase protein (L) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of encoding, and three helper plasmids are all controlled by eukaryotic promoter.
2. reverse genetic operating system according to claim 1; the wherein said plasmid of transcribing also comprises that coding has the sequence of the nuclease of self splicing function, the sequence of coding fourth liver ribozyme (Rbz) and the hereditary label that is positioned at full genome cDNA sequence inside; described full genome cDNA sequence is positioned at after eukaryotic promoter and coding have the sequence of nuclease of self splicing function; before the sequence of coding fourth liver ribozyme (Rbz), common formation is transcribed template.
3. reverse genetic operating system according to claim 2, the wherein said nuclease with self splicing function is fist shape ribozyme (HamRz), described hereditary label is NotI restriction enzyme site and the Bgl II restriction enzyme site that is positioned at full genome cDNA sequence inside, and described eukaryotic promoter is CMV promotor.
4. the reverse genetic operating system as described in claim 1-3 any one, wherein said nucleotide sequence of transcribing plasmid as shown in SEQ ID NO.1, called after pCI-NDV.
5. reverse genetic operating system according to claim 1, wherein said three transcribe helper plasmid be by by the cDNA sequence of nucleoprotein (NP) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of coding, the cDNA sequence of the cDNA sequence of the phosphoric acid albumen (P) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of encoding and the large polymerase protein (L) of the described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of encoding is cloned into respectively in carrier pCI obtains.
6. reverse genetic operating system according to claim 5, wherein encode the cDNA sequence of nucleoprotein (NP) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain as shown in SEQ ID NO.2, encode the cDNA sequence of phosphoric acid albumen (P) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain as shown in SEQ ID NO.3, encode the cDNA sequence of large polymerase protein (L) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain as shown in SEQ ID NO.4, wherein, the cDNA sequence of coding nucleoprotein (NP) and the cDNA sequence of coding phosphor acid albumin (P) utilize respectively NheI and XbaI enzyme cutting site to be implemented on pCI carrier, the cDNA sequence of large polymerase protein (L) of encoding is to utilize NheI and NotI restriction enzyme site to be implemented on pCI carrier, transcribe helper plasmid called after pCI-NP respectively for three that obtain, pCI-P and pCI-L.
7. according to any one reverse genetic operating system in claim 1-4, wherein also comprise the host cell for virus rescue, described host cell is Vero cell, and BHK21 cell or DFEI cell, be preferably Vero cell.
8. a method of utilizing the reverse genetic operating system rescue recombinant Newcastle disease virus described in claim 1-7 any one, comprises the following steps:
(1) transcribing plasmid and transcribing helper plasmid cotransfection for the host cell of virus rescue in the reverse genetic operating system described in right to use requirement 1-7 any one, cultivate the host cell after transfection, preferably, transcribe plasmid pCI-NDV and helper plasmid pCI-NP, the ratio that pCI-P and pCI-L are 4:2:2:1 according to mass ratio is carried out transfection;
(2) from the supernatant liquor of transfectional cell, save recombinant Newcastle disease virus.
9. the application in rescue recombinant Newcastle disease virus Mukteswar strain according to the reverse genetic operating system described in claim 1-7 any one.
10. the application of take in the live vector vaccine that newcastle disease virus strain is skeleton in preparation according to the reverse genetic operating system described in claim 1-7 any one.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109321598A (en) * | 2018-09-27 | 2019-02-12 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Method and its application are saved based on helper plasmid and the blue tongue virus reverse genetic of PCR product |
| CN110684081A (en) * | 2019-09-02 | 2020-01-14 | 天益健康科学研究院（镇江）有限公司 | Recombinant oncolytic newcastle disease virus for coding secretory polypeptide amino acid and preparation method thereof |
| CN111218475A (en) * | 2018-11-23 | 2020-06-02 | 成都生物制品研究所有限责任公司 | System and method for rescuing measles Schwarz/Moraten vaccine strain |
| CN111218474A (en) * | 2018-11-23 | 2020-06-02 | 成都生物制品研究所有限责任公司 | Paramyxovirus rescue system and method |
| CN111926025A (en) * | 2020-03-31 | 2020-11-13 | 华南农业大学 | Rescue method of gene VII type Newcastle disease virus through codon replacement |
| WO2021051906A1 (en) * | 2019-09-20 | 2021-03-25 | 华农（肇庆）生物产业技术研究院有限公司 | Infectious recombinant cloning method for class ii type vii epidemic ndv strain dhn3 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010115133A2 (en) * | 2009-04-03 | 2010-10-07 | Merial Limited | Newcastle disease virus vectored avian vaccines |
| CN102559607A (en) * | 2012-01-04 | 2012-07-11 | 扬州大学 | Newcastle disease virus high mucosal immunity level lentogen vaccine strain NDV/LX and reverse genetic construction system |
| CN102776156A (en) * | 2012-07-12 | 2012-11-14 | 扬州大学 | Gene VIb subtype Rubulavirus Newcastle disease virus attenuated strain VIbI4 and construction method thereof |
| CN103146571A (en) * | 2013-04-01 | 2013-06-12 | 南京工业大学 | Column type immobilized reactor and process for producing rhamnose gum by fermentation |
| CN103468651A (en) * | 2013-10-08 | 2013-12-25 | 扬州大学 | Recombination Newcastle vaccine strain rAI4-S1 for expressing infectious bronchitis virus S1 protein and generating method thereof |
-
2014
- 2014-07-31 CN CN201410373399.8A patent/CN104195154B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010115133A2 (en) * | 2009-04-03 | 2010-10-07 | Merial Limited | Newcastle disease virus vectored avian vaccines |
| CN102559607A (en) * | 2012-01-04 | 2012-07-11 | 扬州大学 | Newcastle disease virus high mucosal immunity level lentogen vaccine strain NDV/LX and reverse genetic construction system |
| CN102776156A (en) * | 2012-07-12 | 2012-11-14 | 扬州大学 | Gene VIb subtype Rubulavirus Newcastle disease virus attenuated strain VIbI4 and construction method thereof |
| CN103146571A (en) * | 2013-04-01 | 2013-06-12 | 南京工业大学 | Column type immobilized reactor and process for producing rhamnose gum by fermentation |
| CN103468651A (en) * | 2013-10-08 | 2013-12-25 | 扬州大学 | Recombination Newcastle vaccine strain rAI4-S1 for expressing infectious bronchitis virus S1 protein and generating method thereof |
Cited By (8)
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|---|---|---|---|---|
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| CN109321598B (en) * | 2018-09-27 | 2021-08-13 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Helper plasmid and PCR product-based reverse genetic rescue method for bluetongue virus and application thereof |
| CN111218475A (en) * | 2018-11-23 | 2020-06-02 | 成都生物制品研究所有限责任公司 | System and method for rescuing measles Schwarz/Moraten vaccine strain |
| CN111218474A (en) * | 2018-11-23 | 2020-06-02 | 成都生物制品研究所有限责任公司 | Paramyxovirus rescue system and method |
| CN110684081A (en) * | 2019-09-02 | 2020-01-14 | 天益健康科学研究院（镇江）有限公司 | Recombinant oncolytic newcastle disease virus for coding secretory polypeptide amino acid and preparation method thereof |
| WO2021051906A1 (en) * | 2019-09-20 | 2021-03-25 | 华农（肇庆）生物产业技术研究院有限公司 | Infectious recombinant cloning method for class ii type vii epidemic ndv strain dhn3 |
| CN111926025A (en) * | 2020-03-31 | 2020-11-13 | 华南农业大学 | Rescue method of gene VII type Newcastle disease virus through codon replacement |
| CN111926025B (en) * | 2020-03-31 | 2023-05-05 | 华南农业大学 | Rescue method of codon-replaced gene VII type newcastle disease virus |
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