CN103451198B - The full-length infectious CDNA of oncolytic type newcastle disease virus D 90 strain and construction process thereof and application - Google Patents
The full-length infectious CDNA of oncolytic type newcastle disease virus D 90 strain and construction process thereof and application Download PDFInfo
- Publication number
- CN103451198B CN103451198B CN201310364591.6A CN201310364591A CN103451198B CN 103451198 B CN103451198 B CN 103451198B CN 201310364591 A CN201310364591 A CN 201310364591A CN 103451198 B CN103451198 B CN 103451198B
- Authority
- CN
- China
- Prior art keywords
- strain
- newcastle disease
- pbr
- full
- virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The invention discloses the full-length infectious CDNA of oncolytic type newcastle disease virus D 90 strain and construction process thereof and application.The full-length infectious CDNA of described oncolytic type newcastle disease virus D 90 strain can form reverse genetic operating system with helper plasmid.This infectious CDNA and reverse genetic operating system thereof can be used for saving the Avian pneumo-encephalitis virus with oncolytic function, and the present invention is that experiment basis has been established in the research oncolytic function of Avian pneumo-encephalitis virus and molecular mechanism, the treatment of exploitation antineoplastic genetic engineering etc.
Description
Technical field
The present invention relates to the infections clone of sub-thread minus-stranded rna virus, particularly relate to the new castle disease virus infected cDNA and construction process thereof and application with oncolytic function, the Avian pneumo-encephalitis virus reverse genetic operating system that the present invention is made up of this new castle disease virus infected cDNA, minigenome and helper plasmid thereof.
Background technology
Avian pneumo-encephalitis virus (Newcastlediseasevirus, NDV) belongs to the member of Paramyxoviridae fowl Rubulavirus, and be sub-thread minus-stranded rna virus, its geneome RNA total length is about 15kb.Rna gene group is the leader sequence of 55 Nucleotide at 3' end, and 5 ' end is the tailer sequence of 114 Nucleotide, and transcribing and copying of these two sequence pair viruses is all most important.The order of six gene RNAs of virus is followed successively by 3'-NP-P-M-F-HN-L-5'.Six kinds of structural protein that these six genes encodings of Avian pneumo-encephalitis virus are main: nucleocapsid protein (Nucleocapsidprotein, NP), phosphorprotein (Phosphateprotein, P), stromatin (Matrixprotein, M), fusion rotein (Fusionprotein, F), hemagglutinin neuraminidase (HemagglutininNeuraminidaseprotein, and RNA polymerase (LargeRNA-dependentRNApolymeraseprotein, L) HN).The rna editing of P gene produces two other Nonstructural Protein V and W (Mebatsionetal., 2001; Stewardetal., 1993).Virus genome RNA and NP, P, L form ribonucleoprotein complex (RNP) jointly, and RNP can, as rna transcription and the template copied, be the minimum infectious unit (Hamaguchietal., 1983) that virus has functionally active.
The final purpose of oncotherapy removes cancer cell under condition healthy cell not being produced to obviously damage.Multiple oncolytic C-type virus C identified and and its potential anti-tumor function, specificity, effect and security are detected.In numerous oncolytic virus, Avian pneumo-encephalitis virus has been considered to oncolytic preparation likely and has been applied to clinical more than 40 years as experimental therapy.Some clinical trials show that NDV treats preparation safely and effectively.Avian pneumo-encephalitis virus has now become the five kind viruses one of of clinical assessment as oncolytic, gene and immunostimulatory treatment carrier, has Development volue.
Reverse genetic operating system is one of the important tool in Molecular level study virus.By carrying out various external manual operation to rna virus cdna group on DNA level, as carried out the transformations such as transgenation, genetically deficient, gene insertion, gene replacement and gene complementation (structure embedded virus), be difficult to operate this difficult problem to rna virus cdna group to solve, be very easy to the molecular mechanism of the function that people study virogene on a molecular scale and the biological characteristics had thereof.
Research shows, Avian pneumo-encephalitis virus oncolytic strain 73-T can kill various human cancer cell.In nude mice model, intra-tumoral injection 73-T obviously can suppress multiple heteroplastic tumour, comprises fibrosarcoma and neurospongioma.Oncolytic type MTH-68 strain can obvious part or all of anticancer transfevent tumour.One clinical trial phase shows, oncolytic newcastle disease PV701 strain has the anti-tumor function of wide spectrum.
Summary of the invention
An object of the present invention is to provide a kind of full-length infectious CDNA with the newcastle disease virus D 90 strain of oncolysis.
The full-length infectious CDNA clones of described oncolytic type Avian pneumo-encephalitis virus is compared with parent's newcastle disease D90 strain native sequences (shown in SEQIDNO:1), disappearance falls a KpnI restriction enzyme site, only has the molecular label that single KpnI restriction enzyme site is full-length infectious CDNA clones.
The full-length infectious CDNA of oncolytic type newcastle disease virus D 90 of the present invention strain, is characterized in that: described full-length infectious CDNA sequence is for shown in SEQIDNO:2.
Two of object of the present invention is to provide a kind of method building the full-length infectious CDNA of described oncolytic type newcastle disease virus D 90 strain, comprising:
1) with the RNA of newcastle disease virus D 90 strain for template, points of 11 sections amplification D90 pnca gene groups, carry out whole genome sequence mensuration, the whole genome sequence of the newcastle disease D90 strain virus obtained is as shown in SEQIDNO:1;
2) 4 fragments of newcastle disease virus D 90 pnca gene group each several part are obtained by RT-PCR method, increased PCR fragment adopted the assembling of the method for homologous recombination to be cloned in plasmid vector, build the plasmid vector of the full-length infectious CDNA with oncolytic type Avian pneumo-encephalitis virus.
(newcastle disease virus D 90 strain is to the restraining effect of lung cancer A549 cell in wherein said newcastle disease virus D 90 strain, Fu Fang etc., " Chinese Preventive Veterinary Medicine report ", 02 phase in 2007) be separated by Scientia Agricultura Sinica research Harbin veterinary institute pig infectious ward swine disease molecular diagnosis group and preserved.
In the present invention, preferably, the sequence of described full-length infectious CDNA is as shown in SEQIDNO:2, and compared with the parent's newcastle disease D90 strain native sequences shown in SEQIDNO:1, its disappearance falls a KpnI restriction enzyme site.
In the present invention, preferably, described plasmid vector is the pBR322 carrier adding T7 promotor, ribozyme and T7 terminator.
Three of object of the present invention is to provide a kind of oncolytic Avian pneumo-encephalitis virus reverse genetic operating system, it is characterized in that: this oncolytic type Avian pneumo-encephalitis virus reverse genetic operating system comprises plasmid vector and three helper plasmids of the full-length infectious CDNA comprising oncolytic type newcastle disease virus D 90 of the present invention strain, three wherein said helper plasmids are by oncolytic type newcastle disease virus D 90 strain NP, and multiple clone site that the open reading frame ORFcDNA of phosphorprotein P and polymerase protein L gene is cloned in pCI-neo carrier T7 promotor downstream respectively builds and obtains.
In the present invention, preferably, the open reading frame ORFcDNA of described oncolytic type newcastle disease virus D 90 strain nucleoprotein NP gene is as shown in SEQIDNO:3, the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain phosphorprotein P gene is as shown in SEQIDNO:4, and the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain polymerase protein L gene is as shown in SEQIDNO:5.
Four of object of the present invention is to provide the application of described full-length infectious CDNA in rescue Avian pneumo-encephalitis virus, it comprises described full-length infectious CDNA and three helper plasmid cotransfection mammalian cells, rescue obtains Avian pneumo-encephalitis virus, three wherein said helper plasmids are by oncolytic type newcastle disease virus D 90 strain NP, and multiple clone site that the open reading frame ORFcDNA of phosphorprotein P and polymerase protein L gene is cloned in pCI-neo carrier T7 promotor downstream respectively builds and obtains.
In the present invention, preferably, the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain nucleoprotein NP gene is as shown in SEQIDNO:3, the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain phosphorprotein P gene is as shown in SEQIDNO:4, and the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain polymerase protein L gene is as shown in SEQIDNO:5.
In the present invention, preferably, described mammalian cell is BHK-21 cell.
Four of object of the present invention is to provide the described application of reverse genetic operating system in rescue Avian pneumo-encephalitis virus.
Experiment shows, utilizes the full-length infectious CDNA of the oncolytic type Avian pneumo-encephalitis virus constructed by the present invention successfully can save out oncolytic type Avian pneumo-encephalitis virus.
The successful structure of infectious CDNA of the present invention is by providing a strong gene manipulation techniques platform for research oncolytic type Avian pneumo-encephalitis virus, convenient to the genomic operation of Newcastle Disease toxicity.
Accompanying drawing explanation
Fig. 1 is D90 full-length cDNA plasmid KpnI single endonuclease digestion result;
M:MarkerDL15,000; 1:D90 total length plasmid; 2:D90 total length plasmid KpnI single endonuclease digestion product;
Fig. 2 is minigenome and helper plasmid functional verification result;
A:D90minigenome and helper plasmid pCI-NP, pCI-P, pCI-L; B:D90minigenome
Fig. 3: Revive virus F1, F5 and F10 are for molecular label sequencing result;
Fig. 4 Revive virus and the parental virus growth kinetics curve on chick embryo fibroblast CEF;
Fig. 5 mtt assay detects the oncolytic properties of Revive virus;
Fig. 6 is pBR322-THT Vector map.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1:D90 strain whole genome sequence measures
1, biomaterial prepares:
(newcastle disease virus D 90 strain is to the restraining effect of lung cancer A549 cell in newcastle disease virus D 90 strain, Fu Fang etc., " Chinese Preventive Veterinary Medicine report ", 02 phase in 2007) be separated by Scientia Agricultura Sinica research Harbin veterinary institute pig infectious ward swine disease molecular diagnosis group and preserved.
Increase with the primer 4501-6484U/4501-6484L comprising NDVF cracking site, through sequence alignment, result shows, D90 strain F cracking site has strong malicious R-R-Q-K-R-F-I sequence signature, therefore, according to the strong malicious sequences Design of NDV primer as shown in table 1, amplification D90 strain whole genome sequence.
2, the purifying of virus
Adopt the method purifying NDVD90 virus of SPF chicken embryo limiting dilution purifying.First the HA of D90 is measured, diluent inoculation SPF chicken embryo (100ul/ piece) of most highly diluted multiple will be got after the dilution of viral continuous gradient, discard chicken embryo dead in 24 hours, HA is measured every 3 days results chick embryo allantoic liquids, use same method serial dilution viral purification 3 generation again, the 3rd generation viral allantoic fluid of results is used for the mensuration of whole genome sequence as kind of poison.
3, the extraction of viral RNA, reverse transcription and RT-PCR
Get D90 virus allantoic fluid, extract test kit specification sheets according to QiagenRNA and extract geneome RNA, cDNA is obtained afterwards with after the reverse transcription of PromegaM-MLV ThermoScript II, by PrimerStar high-fidelity DNA polymerase pcr amplification PCR primer, each fragment does three reactions, and test kit purifying to specifications received by PCR primer purification kit or glue afterwards.PCR reaction product after purifying send Beijing Hua Da genome company to check order.Pcr amplification primer used is in table 1:
Table 1:D90 genome sequencing primer
| Primer | Primer sequence |
| 1-1495U | ACCAAACAGAGAATCTGTGAGGTACG |
| 1-1495L | CATGCTGTTCGCCACTGCTCT |
| 348L | CACCTGAGAATGGGAGCATAAGA |
| 1258-3042U | TGCTGAGCTAAAACTAACCCTG |
| 1258-3042L | GGCGTTTGATCTTCCTGATCTC |
| 2817-4706U | CCCGGAGACCCATCTCCTTAC |
| 2817-4706L | GACCCTGTCTGAGACGAGGTGTA |
| 4501-6484U | AACACGGGTAGAAGAGTCTGG |
| 4501-6484L | GCGCCATGTGTTCTTTGCTTC |
| 9859-11885U | GGACCAGAAGAAACAGATAAAAGAG |
| 9859-11885L | CTGCTCATCTCCGCTGTCACATT |
| 11675-13649U | TTCTTATCCAATAGATCCAACCACCC |
| 11675-13649L | CCAAGAAGATGAAGCAGTCCCTAT |
| 13528-15155U | AGCAAGGTACGACGCATTTACAC |
| 13528-15155L | GGGACGACTTTGCGCACTCTTGCTT |
| 14947-15193U | CATGAAAACCATAGGTAATGCTG |
| 14947-15193L | ACCAAACAGAGATTTGGTGAATG |
| 6299-8150U | CTGGGAACAAGCAACCAAAGAG |
| 6299-8150L | CGGCCAAGTCTAGCTTCTTAAACTC |
| 8000-9978U | CATCGACATGTTTTAAAGTTGTCAAG |
| 8000-9978L | GGTACTCGAGGGTTGTCAGGTATT |
| Annex 15193L | ACCAWACAYAGATTTGGTGYATG |
| Annex 1U | ACCAAACAGAGAATCYGTGAGRTACG |
4, result:
D90 full-length gene group sequence is 15192bp, and concrete nucleotide sequence is as shown in SEQIDNO.1.
The structure of the infectious full-length cDNA of embodiment 2:D90 strain
According to above-mentioned oncolytic type newcastle disease virus D 90 strain whole genome sequence measurement result and Cleavage Map, D90 strain total length is divided into 4 sections, designs 4 pairs of primers, in table 2.
Table 2
Respectively with table 2 primer PCR amplification F1-F4 fragment and linker, wherein, utilize overlapPCR technology (Primer is: MF4-F-L and MF4-R-U) in F4 section, introduce D90 strain full-length infectious CDNA molecular label (the 13996th A-T), thus the sudden change of KpnI restriction enzyme site fallen, thus to make in full-length cDNA only existence anduniquess KpnI restriction enzyme site.
Upstream and downstream linker primer, annealing forms double-stranded DNA.Linker comprises StuI, PsiI, SpeI, MluI and SmaI restriction enzyme site.There is StuI and SmaI restriction enzyme site between T7 promotor and ribozyme HdvRz, with StuI and SmaI double digestion pBR322-THT carrier, Vector map as shown in Figure 6, is connected with linker, called after pBR-linker carrier.
According to the order of F4-F1-F2-F3, utilize the method for homologous recombination, be connected into the pBR322 carrier (pBR322-THT) comprising T7 promotor, ribozyme and T7 terminator successively, concrete operations are: cut carrier pBR-linker with MluI, SmaI enzyme, the F4 fragment homologous recombination obtained with PCR, the positive colony pBR-F4 of acquisition; PBR-F4 carrier after cutting with StuI, PsiI enzyme and F1 fragment homologous recombination, obtain pBR-F4F1; Cut pBR-F4F1 and F2 fragment homologous recombination with Psi, SpeI enzyme, obtain pBR-F4F1F2; Cut pBR-F4F1F2 and F3 fragment homologous recombination with Spe, MluI enzyme, obtain the pBR-D90 finally comprising D90 full-length cDNA.Wherein, the nucleotide sequence of D90 strain full-length infectious CDNA is as shown in SEQIDNO.2.
Genome sequencing is carried out to the infections clone built, ensures the fidelity of base sequence.And introduce successfully with KpnI single endonuclease digestion qualification checking molecular label.Result as shown in Figure 1.
The structure of embodiment 3:D90 minigenome plasmid, helper plasmid and functional verification.
1, the structure of helper plasmid
Get D90 virus allantoic fluid, extract test kit specification sheets according to QiagenRNA and extract geneome RNA, cDNA is obtained afterwards with after the reverse transcription of PromegaM-MLV ThermoScript II, design the cDNA that three pairs of primers are respectively used to amplification coding NP, phosphorprotein P and polymerase protein L gene ORF, helper plasmid builds the primer, in table 3:
Table 3
The cDNA of increase the encoding nuclear proteins NP, the phosphorprotein P that obtain and polymerase protein L gene ORF is carried out respectively enzyme cut rear clone adopt same enzyme carry out enzyme cut after the multiple clone site in pCI-neo carrier T7 promotor downstream, the helper plasmid be built into is called after pCI-NP, pCI-P and pCI-L respectively.Wherein, the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain nucleoprotein NP gene is as shown in SEQIDNO:3, the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain phosphorprotein P gene is as shown in SEQIDNO:4, and the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain polymerase protein L gene is as shown in SEQIDNO:5
2, the structure of D90 minigenome plasmid
By the D90 strain Leader district (ACCAAACAGAGAATCTGTGAGGTACGATAAAAGGCGAAGAAGCAATCGAGATCGTA CGGGTAGAAGGTGTGAACCCCGAGCGCGAGGCCGAAGCTTGAACTTGAGGGAACCT TCTACCGAT) that pcr amplification obtains, DNA fragmentation LGT reverse cloning is obtained in pBR322-THT carrier, the minigenome plasmid called after minigenome be built into after trailer district (GGGCAATCGTACGCCAATCAGTTATCTTCTTAACTGATGACTCCCTCACTGACTTA gTTATACCGGgTTAGAAAAAAGTTAAATTCCGACTCTTTGGAACTCGTATTCGGAT TCAGTTAGTTAACTTTAAGCAAAAGTGCGCAAAGTCGTCCCTAATTATAGTTATGT CATTCACCAAATCTCTGTTTGGT) and eGFP sequence overlap.By the minigenome plasmid minigenome of 2ug altogether and helper plasmid pCI-NP, pCI-P and pCI-L according to transfection reagent Lipofectamine
tM2000 operation instructions cotransfection BHK-21 cells, using the combination of untransfected minigenome plasmid as negative control.Transfection, after 24 hours, observes the expression of green fluorescent protein under inverted fluorescence microscope.The results are shown in Figure 2, result shows to prove that the leader sequence of D90 and tailer sequence can have the activity of packaging virus under T7 promotor, and also demonstrate the helper plasmid pCI-NP of structure, pCI-P and pCI-L has functionally active.
Embodiment 4: the rescue of oncolytic type newcastle disease virus D 90 strain virus
1, the rescue of oncolytic type newcastle disease virus D 90 strain virus
BHK-21 cell is laid in six orifice plates and grows to 80-90% individual layer, DMEM is changed to opti-MEM substratum, incite somebody to action 10ug total length pBR322 plasmid-D90, pCI-NP, pCI-P and pCI-L transfection reagent Lipofectamine altogether
tM2000 cotransfection BHK-21 cells, changed liquid after 6 hours, continued to hatch 3 days.Results nutrient solution and cell, inoculate 9 age in days SPF chicken embryos after multigelation 3 times, postvaccinal SPF chicken embryo continues Avian pneumo-encephalitis virus is carried out in cultivation HA test after 3 days, the allantoic fluid of the results HA positive, after packing-70 DEG C frozen for subsequent use.Revive virus passed for 15 generations continuously in SPF chicken embryo, and withhold with F1, F5, F10 the chick embryo allantoic liquid obtained respectively and extract total serum IgE, after reverse transcription, the DNA fragmentation of amplification containing label segment checks order, and confirms the existence of label.As shown in Figure 3, result shows F1, F5, F10 molecular label stable existence for Revive virus to result.
2, the growth kinetics curve of Revive virus rD90
For whether the growth characteristics measuring Revive virus are consistent with parent's poison, 6 orifice plates having covered with individual layer CEF cell with MOI=0.01 inoculation of Revive virus and parental virus.12 hours after inoculation, 24 hours, 36 hours, 48 hours, 60 hours and sampling in 72 hours, after multigelation three times, in 96 orifice plates covering with individual layer CEF cell, measure the TCID50 of each time point harvested cell culture, draw the growth kinetics curve of Revive virus and parent's poison.The results are shown in Figure 4, result shows that Revive virus and the parental virus growth kinetics on chick embryo fibroblast CEF is substantially identical, and Revive virus maintains the growth characteristics of parental virus.
3, the checking of Revive virus rD90 oncolytic properties
Respectively parent's poison D90 and Revive virus rD90 is covered with the A549 lung carcinoma cell of individual layer with MOI=1 inoculation, after inoculation 40h, measure cell survival rate with mtt assay, the results are shown in Figure 5, result shows that Revive virus maintains the oncolytic properties of parental virus.
Claims (8)
1. the full-length infectious CDNA of oncolytic type newcastle disease virus D 90 strain, is characterized in that: described full-length infectious CDNA sequence is for shown in SEQIDNO:2.
2. build a method for the full-length infectious CDNA of oncolytic type newcastle disease virus D 90 according to claim 1 strain, comprising:
1) with the RNA of newcastle disease virus D 90 strain for template, points of 11 sections amplification D90 pnca gene groups, carry out whole genome sequence mensuration, the whole genome sequence of the newcastle disease D90 strain virus obtained is as shown in SEQIDNO:1;
2) according to above-mentioned oncolytic type newcastle disease virus D 90 strain whole genome sequence measurement result and Cleavage Map, D90 strain total length is divided into 4 sections, designs following primer:
Respectively with above-mentioned primer PCR amplification F1-F4 fragment and linker, wherein, utilize overlapPCR technology with MF4-F-L and MF4-R-U for primer introduces D90 strain full-length infectious CDNA molecular label in F4 section, thus the sudden change of KpnI restriction enzyme site fallen, thus to make in full-length cDNA only existence anduniquess KpnI restriction enzyme site;
Upstream and downstream linker primer, annealing forms double-stranded DNA, and Linker comprises StuI, PsiI, SpeI, MluI and SmaI restriction enzyme site; There is StuI and SmaI restriction enzyme site between T7 promotor and ribozyme HdvRz, comprise the pBR322 carrier of T7 promotor, ribozyme and T7 terminator with StuI and SmaI double digestion, be connected with linker, called after pBR-linker carrier;
According to the order of F4-F1-F2-F3, utilize the method for homologous recombination, be connected into the pBR322 carrier comprising T7 promotor, ribozyme and T7 terminator successively, concrete operations are: cut carrier pBR-linker with MluI, SmaI enzyme, the F4 fragment homologous recombination obtained with PCR, the positive colony pBR-F4 of acquisition; PBR-F4 carrier after cutting with StuI, PsiI enzyme and F1 fragment homologous recombination, obtain pBR-F4F1; Cut pBR-F4F1 and F2 fragment homologous recombination with Psi, SpeI enzyme, obtain pBR-F4F1F2; Cut pBR-F4F1F2 and F3 fragment homologous recombination with Spe, MluI enzyme, obtain the pBR-D90 finally comprising D90 full-length infectious CDNA;
Wherein, the sequence of described full-length infectious CDNA is as shown in SEQIDNO:2, and compared with the parent's newcastle disease D90 strain native sequences shown in SEQIDNO:1, its disappearance falls a KpnI restriction enzyme site.
3. an oncolytic type Avian pneumo-encephalitis virus reverse genetic operating system, it is characterized in that: this oncolytic type Avian pneumo-encephalitis virus reverse genetic operating system comprises plasmid vector and three helper plasmids of the full-length infectious CDNA comprising oncolytic type newcastle disease virus D 90 according to claim 1 strain, wherein, the plasmid vector comprising the full-length infectious CDNA of oncolytic type newcastle disease virus D 90 according to claim 1 strain builds by the following method and obtains:
1) with the RNA of newcastle disease virus D 90 strain for template, points of 11 sections amplification D90 pnca gene groups, carry out whole genome sequence mensuration, the whole genome sequence of the newcastle disease D90 strain virus obtained is as shown in SEQIDNO:1;
2)) according to above-mentioned oncolytic type newcastle disease virus D 90 strain whole genome sequence measurement result and Cleavage Map, D90 strain total length is divided into 4 sections, designs following primer:
3) respectively with above-mentioned primer PCR amplification F1-F4 fragment and linker, wherein, utilize overlapPCR technology with MF4-F-L and MF4-R-U for primer introduces D90 strain full-length infectious CDNA molecular label in F4 section, thus the sudden change of KpnI restriction enzyme site fallen, thus to make in full-length cDNA only existence anduniquess KpnI restriction enzyme site;
4) upstream and downstream linker primer, annealing forms double-stranded DNA, and Linker comprises StuI, PsiI, SpeI, MluI and SmaI restriction enzyme site; There is StuI and SmaI restriction enzyme site between T7 promotor and ribozyme HdvRz, comprise the pBR322 carrier of T7 promotor, ribozyme and T7 terminator with StuI and SmaI double digestion, be connected with linker, called after pBR-linker carrier;
According to the order of F4-F1-F2-F3, utilize the method for homologous recombination, be connected into the pBR322 carrier comprising T7 promotor, ribozyme and T7 terminator successively, concrete operations are: cut carrier pBR-linker with MluI, SmaI enzyme, the F4 fragment homologous recombination obtained with PCR, the positive colony pBR-F4 of acquisition; PBR-F4 carrier after cutting with StuI, PsiI enzyme and F1 fragment homologous recombination, obtain pBR-F4F1; Cut pBR-F4F1 and F2 fragment homologous recombination with Psi, SpeI enzyme, obtain pBR-F4F1F2; Cut pBR-F4F1F2 and F3 fragment homologous recombination with Spe, MluI enzyme, obtain the pBR-D90 finally comprising D90 full-length infectious CDNA;
Wherein, the sequence of described full-length infectious CDNA is as shown in SEQIDNO:2, and compared with the parent's newcastle disease D90 strain native sequences shown in SEQIDNO:1, its disappearance falls a KpnI restriction enzyme site; Wherein, described plasmid vector is the pBR322 carrier adding T7 promotor, ribozyme and T7 terminator;
Three wherein said helper plasmids are by oncolytic type newcastle disease virus D 90 strain NP, and multiple clone site that the open reading frame ORFcDNA of phosphorprotein P and polymerase protein L gene is cloned in pCI-neo carrier T7 promotor downstream respectively builds and obtains.
4. reverse genetic operating system as claimed in claim 3, it is characterized in that: the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain nucleoprotein NP gene is as shown in SEQIDNO:3, the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain phosphorprotein P gene is as shown in SEQIDNO:4, and the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain polymerase protein L gene is as shown in SEQIDNO:5.
5. the application of full-length infectious CDNA according to claim 1 in rescue Avian pneumo-encephalitis virus, it is characterized in that comprising by described full-length infectious CDNA and three helper plasmid cotransfection mammalian cells, rescue obtains Avian pneumo-encephalitis virus, three wherein said helper plasmids are by oncolytic type newcastle disease virus D 90 strain NP, and multiple clone site that the open reading frame ORFcDNA of phosphorprotein P and polymerase protein L gene is cloned in pCI-neo carrier T7 promotor downstream respectively builds and obtains.
6. apply as claimed in claim 5, it is characterized in that: the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain nucleoprotein NP gene is as shown in SEQIDNO:3, the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain phosphorprotein P gene is as shown in SEQIDNO:4, and the open reading frame ORFcDNA of oncolytic type newcastle disease virus D 90 strain polymerase protein L gene is as shown in SEQIDNO:5.
7. apply as claimed in claim 5, it is characterized in that: described mammalian cell is BHK-21 cell.
8. the application of the reverse genetic operating system described in claim 3 or 4 in rescue Avian pneumo-encephalitis virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310364591.6A CN103451198B (en) | 2013-08-20 | 2013-08-20 | The full-length infectious CDNA of oncolytic type newcastle disease virus D 90 strain and construction process thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310364591.6A CN103451198B (en) | 2013-08-20 | 2013-08-20 | The full-length infectious CDNA of oncolytic type newcastle disease virus D 90 strain and construction process thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103451198A CN103451198A (en) | 2013-12-18 |
| CN103451198B true CN103451198B (en) | 2015-11-25 |
Family
ID=49734035
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310364591.6A Expired - Fee Related CN103451198B (en) | 2013-08-20 | 2013-08-20 | The full-length infectious CDNA of oncolytic type newcastle disease virus D 90 strain and construction process thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103451198B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676117A (en) * | 2016-11-09 | 2017-05-17 | 山东省农业科学院家禽研究所 | Construction and authentication of newcastle disease virus overall-length cDNA clone |
| CN110484517A (en) * | 2019-09-02 | 2019-11-22 | 中国科学院武汉病毒研究所 | A kind of composition and preparation method of the Rift Valley fever virus being used to prepare weak poison, RVFV attenuated vaccine |
| 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 |
| CN110564766A (en) * | 2019-09-20 | 2019-12-13 | 华农（肇庆）生物产业技术研究院有限公司 | Preparation method of whole genome expression vector pBR322-DHN3 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1772909A (en) * | 2005-09-02 | 2006-05-17 | 中国农业科学院哈尔滨兽医研究所 | Reverse genetic operation system of New castle disease LaSota vaccine strain and its applciation |
| CN101056646A (en) * | 2004-11-12 | 2007-10-17 | 拜耳先灵医药股份有限公司 | Recombinant newcastle disease virus |
| CN101235364A (en) * | 2007-02-01 | 2008-08-06 | 中国农业科学院哈尔滨兽医研究所 | Newcastle disease virus D90 strain and application thereof |
-
2013
- 2013-08-20 CN CN201310364591.6A patent/CN103451198B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101056646A (en) * | 2004-11-12 | 2007-10-17 | 拜耳先灵医药股份有限公司 | Recombinant newcastle disease virus |
| CN1772909A (en) * | 2005-09-02 | 2006-05-17 | 中国农业科学院哈尔滨兽医研究所 | Reverse genetic operation system of New castle disease LaSota vaccine strain and its applciation |
| CN101235364A (en) * | 2007-02-01 | 2008-08-06 | 中国农业科学院哈尔滨兽医研究所 | Newcastle disease virus D90 strain and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 应用新城疫病毒治疗肿瘤的研究进展;吴云舟等;《生物工程学报》;20100825;第26卷(第8期);第2、3节 * |
| 新城疫病毒D90 株对肺癌A549 细胞的抑制作用;符芳等;《中国预防兽医学报》;20070228;第29卷(第2期);摘要 * |
| 鹅源新城疫病毒拯救体系的建立;胡顺林等;《微生物学通报》;20071213;第34卷(第3期);426-429 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103451198A (en) | 2013-12-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rout et al. | The large polymerase protein is associated with the virulence of Newcastle disease virus | |
| EP3153577B1 (en) | Method for modification of heat resistance of newcastle disease virus and use thereof | |
| CN103451198B (en) | The full-length infectious CDNA of oncolytic type newcastle disease virus D 90 strain and construction process thereof and application | |
| CN101532030A (en) | Recombinant influenza virus vector carrying foreign genes in NA segment and preparation method and application thereof | |
| CN105734023B (en) | A kind of recombinant Newcastle disease virus is preparing the application in medicines resistant to liver cancer | |
| CN104195154B (en) | Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain reverse genetic operating system and application thereof | |
| Zhang et al. | Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism | |
| Liu et al. | Two-plasmid system to increase the rescue efficiency of paramyxoviruses by reverse genetics: the example of rescuing Newcastle disease virus | |
| CN105396143A (en) | MiRNA of specificity of Ebola virus and method for inhibiting Ebola virus through miRNA | |
| CN115725657A (en) | Segmented vesicular stomatitis virus vector and preparation method and application thereof | |
| Kim et al. | Reverse genetics for Newcastle disease virus as a vaccine vector | |
| Neumann | Influenza reverse genetics—historical perspective | |
| CN102807989B (en) | Preparation method of recombination live vector vaccines for diseases of canid and/or feline | |
| CN103484462B (en) | The recombinant adenoviral vector of Survivin promoter regulation CD gene builds and application | |
| CN103981202B (en) | A kind of method building canine distemper virus reverse genetics system | |
| CN111100845B (en) | Recombinant measles virus and preparation method and application thereof | |
| CN104013954B (en) | The application in resisiting influenza virus of ISG20 albumen and encoding gene thereof | |
| US11512117B1 (en) | Whole avian-origin reverse genetic system and recombinant H5N2 subtype avian influenza virus, vaccine and uses thereof | |
| CN107735493A (en) | Poxvirus derives promoter and the carrier comprising promoter | |
| Javaheri et al. | Avian paramyxovirus 4 antitumor activity leads to complete remissions and long-term protective memory in preclinical melanoma and colon carcinoma models | |
| CN108103097B (en) | Attenuated measles virus mRNA methyltransferase deficient vaccine strain and application thereof | |
| CN106399591A (en) | One-step process PCR detection method for I-type duck hepatitis viruses and duck plague viruses | |
| CN101397572B (en) | Method for rescuing influenza virus and bidirectional transcription vector special therefor | |
| CN114480303B (en) | Group A influenza virus attenuated strain and preparation method and application thereof | |
| RU2787724C1 (en) | Set of recombinant plasmid dna for obtaining recombinant viruses sendai strain moscow (options) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151125 Termination date: 20170820 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |