CN104195154B - Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain reverse genetic operating system and application thereof - Google Patents
Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain reverse genetic operating system and application thereof Download PDFInfo
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Abstract
The present invention relates to the reverse genetic operating system of a kind of Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain and application thereof, this system comprises: transcribe plasmid, this transcribe plasmid include described in be controlled by the full-length genome cDNA sequence of Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of eukaryotic promoter；Three be controlled by eukaryotic promoter transcribe helper plasmid, this helper plasmid includes encoding the cDNA sequence of the nucleoprotein (NP) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain respectively, encodes the cDNA sequence of the Phospoprotein (P) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain and encode the cDNA sequence of big polymerase protein (L) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain.Utilize the reverse genetic operating system that the present invention sets up, successfully rescue and obtained wild type recombinant Newcastle disease virus, for carry out further with this Avian pneumo-encephalitis virus be carrier the relevant basic research of vaccine development, tumor oncolytic therapy virus and NDV virus lay a good foundation.
Description
Technical field
The present invention relates to viral genetic field operation, more particularly it relates to an Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain reverse genetic operating system and application thereof.
Background technology
nullAngelaRomer-Oberdorfer etc. and BENP.H.PEETERS is equal within 1999, publishing an article " GenerationofrecombinantlentogenicNewcastlediseasevirusfr omcDNA " (JournalofGeneralVirology (1999) respectively first,80,2987 2995) and " RescueofNewcastleDiseaseVirusfromClonedcDNA:Evidencethat CleavabilityoftheFusionProteinIsaMajorDeterminantforViru lence " (JOURNALOFVIROLOGY,June1999,p.5001–5009)，Disclosing one utilizes T7 promoter to control re-recording system，Save Avian pneumo-encephalitis virus low virulent strain，Some are had again to save the Avian pneumo-encephalitis virus of different strain successively afterwards，The rescue system of all these Avian pneumo-encephalitis virus is all controllable by T7 promoter，Such Avian pneumo-encephalitis virus reverse genetic operating system is all the special cells system utilizing and expressing t7 rna polymerase，And need the recombinant poxvirus expressing t7 rna polymerase to be additionally provided t7 rna polymerase and effectively save recombinant Newcastle disease virus.In this class system, the full-length genome cDNA clone of Avian pneumo-encephalitis virus is under T7 promoter, and the indispensable protein NP needed for its Revive virus, the gene of P and L also more to be implemented on the plasmid by the control of T7 promoter.In the process of Revive virus, the cell line (such as BHK-T7) that can express t7 rna polymerase is infected firstly the need of with the recombinant poxvirus expressing t7 rna polymerase, afterwards again by helper plasmid and this cell line of full-length genome plasmid co-transfection, the t7 rna polymerase generated in cell is relied on to transcribe out the geneome RNA of Avian pneumo-encephalitis virus, and with helper plasmid express NP, P and L be assembled into nucleocapsid, finally generate recombinant Newcastle disease virus in the mode of sprouting.This kind of reverse genetic operating system there is problems of the cell line needing to prepare stably express t7 rna polymerase, the process of preparation cell line is complicated, technology requires height, cycle is long, and the incubation of cell line needs constantly add the antibiotic maintaining pressure to keep its stably express t7 rna polymerase, but being as the increase of passage number of times, its ability expressing t7 rna polymerase can constantly reduce, until disappearing；The recombinant poxvirus expressing t7 rna polymerase additionally infected in transfection process can always present in cell, not only need to add galactoside to suppress the duplication of this poxvirus in rescue recombinant Newcastle disease virus process, and also need to by filtering or the mode of repeatedly diluted passage removes poxvirus after saving out recombinant virus, this not only can reduce the rescue efficiency of Avian pneumo-encephalitis virus, there is also the risk that poxvirus pollutes, in the research of this method, if not filtering the cell culture of transfection, the rescue efficiency of Avian pneumo-encephalitis virus is higher, but it is very complicated to remove poxvirus, if filtering the cell culture of transfection, then the rescue efficiency of Avian pneumo-encephalitis virus is very low, even failed.
This good carrier of Avian pneumo-encephalitis virus is utilized in order to more effective, for the shortcoming in prior art described above, the Avian pneumo-encephalitis virus mesogenic Mukteswar vaccine strain reverse genetic operating system that we set up has abandoned the t7 rna polymerase system under the T7 promoter control of original use completely, but utilize the eukaryotic cell promoter that all cultivation cell line has, and the expression of helper plasmid is also controllable by eukaryotic promoter, this system can fasten the process of Revive virus in various kinds of cell, efficiency is unaffected (such as Vero, BHK21, the cell lines such as DFEI), the cell line that our rescue system uses is widely used in the Vero cell of production of vaccine, this has established foundation for security for the vaccine that this strain of Future Development is carrier.Current studies in China person has saved out new castle disease LaSota attenuated vaccine strain (Ge Jinying etc., NewcastleDiseaseVirus-BasedLiveAttenuatedVaccineComplete lyProtectsChickensandMicefromLethalChallengeofHomologous andHeterologousH5N1AvianInfluenzaViruses) and High Virulent Newcastle Disease Virus, these reverse genetic operating systems are also all built upon under the control of T7 promoter, but low virulent strain is low due to virulence, utilizing status is limited, and virulent strain exists bio-safety problem.There is presently no others and 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, powerful will be provided for the Genetic evolution between the research low virulent strain of Avian pneumo-encephalitis virus and high virulence, also lay a good foundation with the vaccine that this strain is carrier for exploitation.
Instant invention overcomes in prior art the shortcoming that must be provided t7 rna polymerase by specific cells system and recombinant poxvirus, it is provided that a kind of can use the various kinds of cell system method to save recombinant Newcastle disease virus.
Summary of the invention
It is an object of the present invention to provide the reverse genetic operating system of a kind of Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain, this system comprises:
(1) transcribing plasmid, this transcribes the full-length genome cDNA sequence that plasmid includes being controlled by the Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of eukaryotic promoter；
Transcribe helper plasmid for (2) three, this helper plasmid includes encoding the cDNA sequence of the nucleoprotein (NP) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain respectively, encodes the cDNA sequence of the Phospoprotein (P) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain and encode the cDNA sequence of big polymerase protein (L) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain, and three helper plasmids are controlled by eukaryotic promoter.
In reverse genetic operating system of the present invention; preferably; described plasmid of transcribing also includes encoding the sequence of the nuclease with self splicing function, encodes the sequence of hepatitis D ribozyme (Rbz) and be positioned at the genetic tags within full-length genome cDNA sequence; after described full-length genome cDNA sequence is positioned at eukaryotic promoter and encodes the sequence of nuclease with self splicing function; before the sequence of coding hepatitis D ribozyme (Rbz), collectively form transcription templates.
In reverse genetic operating system of the present invention, preferably, the wherein said nuclease with self splicing function is fist shape ribozyme (HamRz), described genetic tags is in the NotI restriction enzyme site within full-length genome cDNA sequence and BglII restriction enzyme site, and described eukaryotic promoter is CMV promoter.
In reverse genetic operating system of the present invention, it is preferred that the described nucleotide sequence transcribing plasmid is such as shown in SEQIDNO.1, called after pCI-NDV.
In reverse genetic operating system of the present invention, preferably, transcribe helper plasmid and be the cDNA sequence by the nucleoprotein (NP) by encoding described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain, the cDNA sequence encoding the Phospoprotein (P) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain and the cDNA sequence of big polymerase protein (L) encoding described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain is cloned into respectively in carrier pCI and obtains for described three.
nullIn 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 such as shown in SEQIDNO.2，Encode the cDNA sequence of Phospoprotein (P) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain such as shown in SEQIDNO.3，Encode the cDNA sequence of big polymerase protein (L) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain such as shown in SEQIDNO.4，Wherein，The cDNA sequence of the cDNA sequence of encoding nuclear proteins (NP) and coding phosphor acid albumin (P) is utilized respectively NheI and XbaI enzyme cutting site is implemented on pCI carrier，The cDNA sequence encoding big polymerase protein (L) is to utilize NheI and NotI restriction enzyme site to be implemented on pCI carrier，Transcribe helper plasmid and be respectively designated as pCI-NP for three obtained，PCI-P and pCI-L.
In reverse genetic operating system of the present invention, it is preferred that also comprise the host cell for virus rescue, described host cell is Vero cell, BHK21 cell or DFEI cell, more preferably Vero cell.
The method that it is a further object to provide a kind of reverse genetic operating system utilized described in any of the above item rescue recombinant Newcastle disease virus, comprises the following steps:
(1) use transcribing plasmid and transcribing the helper plasmid cotransfection host cell for virus rescue in the reverse genetic operating system described in any of the above item, cultivate the host cell after transfection；
(2) from the supernatant of transfectional cell, recombinant Newcastle disease virus is saved.
In method of the present invention, it is preferred that transcribe plasmid pCI-NDV and transfect according to the ratio that mass ratio is 4:2:2:1 with helper plasmid pCI-NP, pCI-P and pCI-L.
It is also another object of the present invention to provide the application in rescue recombinant Newcastle disease virus Mukteswar strain of the described reverse genetic operating system.And the application that described reverse genetic operating system is in preparation is with newcastle disease virus strain for the live vector vaccine of skeleton.
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 the used t7 rna polymerase system of prior art completely, there is no loaded down with trivial details cell line preparation and maintenance process, the complex technology of the recombinant poxvirus of t7 rna polymerase is expressed also without preparation, therefore need not use recombinant poxvirus in the rescue process of recombinant virus, after also just not suppressing poxvirus over-replicate and virus rescue, remove the complicated processes of poxvirus.The present invention can utilize all of cultivation cell line to save recombinant Newcastle disease virus, and this lays the foundation for setting up from now on and saving such paramyxovirus.In the recombinant Newcastle disease virus rescue system of the present invention, the cell line that we use is preferably the Vero cell line widely used in production of vaccine field, this cell line is the safe cell line of the production of vaccine generally acknowledged at present, and this is that Future Development establishes foundation for security with the vaccine that this strain Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain strain is carrier.
Accompanying drawing explanation
Fig. 1 is the structure schematic diagram of Avian pneumo-encephalitis virus Mukteswar strain full-length genome cDNA；
Full-length genome is divided into 4 sections to clone, and in full-length genome cDNA clone, add genetic tags, it is in the NotI restriction enzyme site of genome 4953 respectively and is positioned at the BglII restriction enzyme site of 8597, genome head is fist shape ribozyme (HamRz), it is restriction enzyme site-the NheI being connected with pCI carrier before ribozyme, it is connected by restriction enzyme site SacII between first paragraph with second segment, connected by restriction enzyme site MluI between second segment and the 3rd section, it is connected by restriction enzyme site MluI between 3rd section with the 4th section, 4th segment trailer is hepatitis D ribozyme (Rbz), NotI is the restriction enzyme site that full-length genome cDNA clone is connected with pCI carrier；
The plasmid that Fig. 2 is Avian pneumo-encephalitis virus Mukteswar strain reverse genetic operating system constitutes schematic diagram；
Three helper plasmids are all implemented on pCI carrier, are controlled by CMV promoter；
Fig. 3 is Avian pneumo-encephalitis virus Mukteswar strain reverse genetic operating system transfected Vero cells, saves out the schematic diagram of recombinant virus；
Representing three kinds of albumen that the multiple small circles in the circle of cell represent that helper plasmid pCI-NP, pCI-P and pCI-L express respectively, in figure, three circles with multiple projections in the lower right corner represent the recombinant virus saved out；
Fig. 4 is for after transfection supernatant inoculating cell, and the recombinant Newcastle disease virus Mukteswar strain infection cell saved out forms picture under plasmodial microscope and Normocellular picture；
Fig. 4 A and Fig. 4 B are that Fig. 4 C figure is the result of 200x thing Microscopic observation, and the round speckle in figure is the syncytium of formation by result at 100x thing Microscopic observation after transfection supernatant inoculation Vero cell 40h；Fig. 4 D figure is normal Vero cell controls；
Fig. 5 is the result utilizing the recombinant virus that the primer pair rescue of this viral gene group-specific obtains to carry out pcr amplification.
Detailed description of the invention
Further describe the present invention by the following examples and in conjunction with accompanying drawing, it should be understood that these embodiments only for illustration purposes, do not 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, pBluescriptIIks (+/-) Phagemids is purchased from Shanghai JaRa Bioisystech Co., Ltd；Archaeal dna polymerase purchased from TAKARA company, T4DNA ligase and other restricted enzyme all purchased from NEB company；Viral RNA extracts test kit and middle amount prepares test kit purchased from QIAGEN company, M-MLV Reverse Transcription box and liposome 2000 test kit purchased from IVENTROGEN company.
2, method
2.1 structures transcribing plasmid containing Avian pneumo-encephalitis virus Mukteswar strain full-length genome cDNA sequence
For setting up the reverse genetic operating system of Avian pneumo-encephalitis virus Mukteswar strain, must first build corresponding full-length genome cDNA clone, head at Avian pneumo-encephalitis virus full-length genome cDNA is the fist dress ribozyme with self splicing function, it is the full-length genome cDNA of virus afterwards, nucleic acid-protein gene (NP), Phospoprotein gene (P), matrix gene (M), antigen-4 fusion protein gene (F), hemagglutinin gene (HA) and big polymerase protein gene (L) successively in order, is connected to hepatitis D ribozyme at genomic afterbody.In the clone of full-length genome cDNA, we will according to the restriction enzyme site of virus Mukteswar vaccine strain gene order, it is divided into 4 sections of clones completing full-length genome cDNA, and in full-length genome cDNA clone, add genetic tags, being in the NotI restriction enzyme site of genome 4953 and be positioned at the BglII restriction enzyme site of 8597 respectively, whole genome cDNA is positioned between NheI and the NotI multiple clone site of PCI carrier (Fig. 1).Specifically, 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 Embryo Gallus domesticus, after inoculating 3 days, gathers in the crops allantoic fluid, and encapsulation is placed in cryogenic refrigerator and preserves.The viral RNA utilizing QIAGEN extracts test kit and extracts viral RNA, operate to specifications, the M-MLV Reverse Transcription box utilizing IVENTROGEN afterwards carries out reverse transcription, the specific primer of recycling synthesis carries out PCR, and glue reclaims PCR primer and is cloned into the EcoRV site of carrier pBluescriptIIks (+/-) Phagemids.The full-length 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, being linked together each section by the mode of enzyme action, connection according to Strategies For The Cloning is cloned on pCI carrier, build and obtain transcribing plasmid containing Avian pneumo-encephalitis virus Mukteswar strain full-length genome cDNA sequence, the whole nucleotide sequence of this plasmid is such as shown in SEQIDNO.1, called after pCI-NDV.
Three structures transcribing helper plasmid in 2.2 Avian pneumo-encephalitis virus Mukteswar strains
nullEncode the cDNA sequence of nucleoprotein (NP) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain such as shown in SEQIDNO.2，Encode the cDNA sequence of Phospoprotein (P) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain such as shown in SEQIDNO.3，Encode the cDNA sequence of big polymerase protein (L) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain such as shown in SEQIDNO.4，Wherein，The cDNA sequence of the cDNA sequence of encoding nuclear proteins (NP) and coding phosphor acid albumin (P) is utilized respectively NheI and XbaI enzyme cutting site is implemented on pCI carrier，The cDNA sequence encoding big polymerase protein (L) is to utilize NheI and NotI restriction enzyme site to be implemented on pCI carrier，Transcribe helper plasmid and be respectively designated as pCI-NP for three obtained，PCI-P and pCI-L (Fig. 2).
2.3 transcribe plasmid and three helper plasmids transformed competence colibacillus cell Top10 respectively by above-mentioned containing Avian pneumo-encephalitis virus Mukteswar strain full-length genome cDNA sequence, large volume shakes bacterium afterwards, utilize amount in the plasmid of QIAGEN to prepare the plasmid of test kit preparation transfection, and measure the concentration of each plasmid.
Embodiment 2 utilizes the reverse genetic operating system that embodiment 1 structure obtains to carry out the rescue of recombinant virus
1, method:
Vreo cell is inoculated in 6 porocyte culture plates, when cell monolayer length to about 60%～80%, starts transfection.With plasma-free DMEM medium, washed cell 2 times, add Opti-MEM, 2ml/ hole afterwards；Transfection reagent is liposome 2000 test kit of Invitrogen company, transfection carries out according to test kit description, transcribe plasmid pCI-NDV and helper plasmid pCI-NP, pCI-P, pCI-L is the ratio of 4:2:2:1 according to mass ratio, transfect with the amount of every hole 4ug, after transfection 6h～8h, discard transfection supernatant, add the DMEM containing 5%FBS, when the substantially all death of transfectional cell, draw the Vero cell in the culture bottle that supernatant inoculation is new, day by day observation of cell pathological changes afterwards, when there being significant syncytium to generate, show to save out recombinant virus (Fig. 3), the recombinant virus saved out can be inoculated afterwards in Vero cell with amplicon virus, and detect HA-HI test with the chicken erythrocyte of 1%.
2, result
In order to identify the virus being rescued, by the Vero cell of the supernatant inoculated and cultured culture in glassware in transfection process, with amplicon virus, wild type strains produces significant cytopathy, namely has syncytium to generate, it is possible to carry out blood clotting or RT-PCR tests to detect recombinant virus.Microscopy results finds: the recombinant Newcastle disease virus Mukteswar strain vero cells infection saved out all has syncytium to produce (Fig. 4 A, Fig. 4 B and Fig. 4 C), and normal Vero cell does not have syncytium to produce (Fig. 4 D).Utilizing Trizol to extract total serum IgE by rescuing the virus obtained, utilize random primer to carry out reverse transcription, utilize the primer of this viral gene group-specific to carry out pcr amplification afterwards, result amplifies specificity purpose band (Fig. 5).Above experimental result shows, by reverse genetics manipulation technology, utilization transcribes plasmid and the clone of three ancillary transcription plasmids containing Avian pneumo-encephalitis virus Mukteswar strain full-length genome cDNA's, has 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 the recombinant Newcastle disease virus rescue system of the present invention, the cell line that we use is the Vero cell line widely used in production of vaccine field, this cell line is the safe cell line of the production of vaccine generally acknowledged at present, and this is that Future Development establishes foundation for security with the vaccine that this strain Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain strain is carrier.
The foregoing is only the preferred embodiments of the present invention, be merely illustrative for the purpose of the present invention, and nonrestrictive；Those of ordinary skill in the art understand, and it can be carried out many changes in the spirit and scope that the claims in the present invention limit, amendment, and even equivalence is changed, but falls within protection scope of the present invention.
Claims (12)
1. a reverse genetic operating system for Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain, this system comprises:
(1) transcribing plasmid, this transcribes the full-length genome cDNA sequence that plasmid includes being controlled by the Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain of eukaryotic promoter；
Transcribe helper plasmid for (2) three, this helper plasmid includes encoding the cDNA sequence of the nucleoprotein (NP) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain respectively, encodes the cDNA sequence of the Phospoprotein (P) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain and encode the cDNA sequence of big polymerase protein (L) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain, and three helper plasmids are controlled by eukaryotic promoter.
2. reverse genetic operating system according to claim 1; wherein said plasmid of transcribing also includes encoding the sequence of the nuclease with self splicing function, encodes the sequence of hepatitis D ribozyme (Rbz) and be positioned at the genetic tags within full-length genome cDNA sequence; after described full-length genome cDNA sequence is positioned at eukaryotic promoter and encodes the sequence of nuclease with self splicing function; before the sequence of coding hepatitis D ribozyme (Rbz), collectively form transcription templates.
3. reverse genetic operating system according to claim 2, the wherein said nuclease with self splicing function is fist shape ribozyme (HamRz), described genetic tags is in the NotI restriction enzyme site within full-length genome cDNA sequence and BglII restriction enzyme site, and described eukaryotic promoter is CMV promoter.
4. the reverse genetic operating system as described in any one of claim 1-3, the wherein said nucleotide sequence transcribing plasmid is such as shown in SEQIDNO.1, called after pCI-NDV.
5. reverse genetic operating system according to claim 1, transcribes helper plasmid and is the cDNA sequence by the nucleoprotein (NP) by encoding described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain, the cDNA sequence encoding the Phospoprotein (P) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain and the cDNA sequence of big polymerase protein (L) encoding described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain is cloned into respectively in carrier pCI and obtains for wherein said three.
null6. 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 such as shown in SEQIDNO.2，Encode the cDNA sequence of Phospoprotein (P) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain such as shown in SEQIDNO.3，Encode the cDNA sequence of big polymerase protein (L) of described Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain such as shown in SEQIDNO.4，Wherein，The cDNA sequence of the cDNA sequence of encoding nuclear proteins (NP) and coding phosphor acid albumin (P) is utilized respectively NheI and XbaI enzyme cutting site is implemented on pCI carrier，The cDNA sequence encoding big polymerase protein (L) is to utilize NheI and NotI restriction enzyme site to be implemented on pCI carrier，Transcribe helper plasmid and be respectively designated as pCI-NP for three obtained，PCI-P and pCI-L.
7. reverse genetic operating system according to claim 1, wherein also comprises the host cell for virus rescue, and described host cell is Vero cell, BHK21 cell or DFEI cell.
8. reverse genetic operating system according to claim 7, wherein said host cell is Vero cell.
9. the method utilizing the reverse genetic operating system described in any one of claim 1-8 to save recombinant Newcastle disease virus, comprises the following steps:
(1) use transcribing plasmid and transcribing the helper plasmid cotransfection host cell for virus rescue in the reverse genetic operating system described in any one of claim 1-8, cultivate the host cell after transfection；
(2) from the supernatant of transfectional cell, recombinant Newcastle disease virus is saved.
10. method according to claim 9, its transcription plasmid pCI-NDV and helper plasmid pCI-NP, pCI-P and pCI-L transfect according to the ratio that mass ratio is 4:2:2:1.
11. the application that the reverse genetic operating system described in any one of claim 1-8 is in rescue recombinant Newcastle disease virus Mukteswar strain.
12. the application that the reverse genetic operating system described in any one of claim 1-8 is in preparation is with newcastle disease virus strain for the live vector vaccine of skeleton.
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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 |
| CN110592108B (en) * | 2019-09-20 | 2021-03-26 | 华农（肇庆）生物产业技术研究院有限公司 | Infectious recombinant cloning method for II-type VII-type epidemic NDV strain DHN3 |
| 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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