KR102332652B1 - Recombinant vector using Newcatsle Disease Virus isolated from wild bird in Korea and method for preparing thereof - Google Patents

Abstract

The present invention relates to a recombinant vector prepared by inserting the full-length gene of the novel Newcastle disease virus strain KU001 (Accession No.: KCTC18794P) isolated from wild domestic ducks, and a method for producing the same.

Description

Newcastle disease virus recombinant vector isolated from domestic wild duck and its manufacturing method {Recombinant vector using Newcatsle Disease Virus isolated from wild bird in Korea and method for preparing thereof}

The present invention relates to a recombinant vector prepared by inserting the full-length gene of a novel Newcastle disease virus isolated from domestic wild ducks, and a method for preparing the same.

Newcastle disease virus (NDV) is a negative sense single-stranded RNA virus that belongs to the Paramyxoviridae family, the Mononegavirales order, and the Avulavirus genus. am. This virus is a highly contagious virus that occurs frequently in poultry, especially chickens and ducks. The length of the genome is 15,186 nucleotides. NDV has six transcription units in its genome that encode different viral proteins: nucleocapsid protein (NP), phosphoprotein (P), matrix (M), fusion protein (F), and hemagglutinin neuraminidase ( HN) and polymerase (L). The synthesis of mRNA encoded by these transcription units is catalyzed by viral RNA-dependent RNA polymerase in the cytoplasm of infected cells. All transcription units are flanked by conserved gene initiation and gene end sequences. In the course of viral replication, mRNA synthesis causes a gradient of mRNA transcription, with genes proximal to the 3' end, such as NP, being transcribed more than genes located far from the 3' end, such as L. Thus, the expression level of a new transcriptional unit will depend on its position relative to the 3' end.

Newcastle disease virus has an envelope, which contains the HN protein, which allows the virus to bind to the host cell, and the F protein, which causes the fusion of the envelope with the host cell. F protein and HN protein are glycoproteins and are distributed on the surface of the envelope.

F protein forms a trimeric structure as a °-type membrane glycoprotein. The F protein is made in the inactive precursor form (F0) and cleaved into the active forms F1 and F2 during transit through the Golgi membranes. This process exposes the hydrophobic domain at the amino terminus of the F1 subunit, which plays an important role in the biological activity of the mature protein. A hydrophobic domain called a fusion peptide is highly conserved in the paramyxovirus F protein and is thought to be directly involved in mediating membrane fusion. Paramyxovirus F protein has several common structural conformations, including seven heptad repeats and two regions likely to form an alpha helix structure. Of the two repeats, the longest 7 repeat A (heptad repeat A) is adjacent to the hydrophobic fusion peptide at the amino terminus of F1, and the 7 repeat B is in close contact with the upper part of the transmembrane region. Seven repeats B consist of a highly conserved sequence of leucine or isoleucine every seven residues.

NDV infects a variety of poultry species. It causes severe respiratory and nervous system diseases in many poultry species. Strains of NDV have been classified according to virulence: lentogenic strains that cause asymptomatic infection in birds; mesogenic strains causing respiratory diseases in birds; A velogenic strain that causes fulminant infection in birds, often with neurological symptoms. Several pharmacologically pathogenic strains of NDV have recently been used as live attenuated vaccines in chickens.

An ideal vaccine for the protection of the general public or poultry from infectious diseases is one that is easily manufactured and rapidly elicits a strong protective immune response. Live viral vaccines have been demonstrated to be effective against a variety of viral diseases and induced strong and persistent humoral and cellular immune responses. Recombinant NDV is a good candidate for use as a live viral vaccine vector because it is not pathogenic to humans, humans do not have pre-existing immunity to it, and because it is readily grown in embryonated eggs for large-scale production.

Republic of Korea Patent No. 10-1099629

An object of the present invention is to provide a recombinant vector into which the full-length gene of Newcastle disease virus is inserted and a method for producing the same, compared to the B1 strain or Lasota strain used as the existing Newcastle disease virus vaccine strain.

However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

According to an embodiment of the present invention, there is provided a method for preparing a recombinant vector into which the full-length gene sequence of Newcastle disease virus strain KU001 (Accession No.: KCTC18794P) is inserted, comprising the following steps, wherein cDNA is prepared using RNA extracted from Newcastle disease virus. synthesizing; The nucleotide sequence encoding the NP protein, the nucleotide sequence encoding the P protein, the nucleotide sequence encoding the M protein, the nucleotide sequence encoding the F protein, the nucleotide sequence encoding the HN protein, and the nucleotide sequence encoding the L protein are sequentially sequenced inserting into a plasmid; Including, wherein the recombinant vector is composed of the nucleotide sequence of SEQ ID NO: 1, a method for producing a recombinant vector is provided.

According to one side, the nucleotide sequence encoding the NP protein consists of the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence encoding the P protein consists of the nucleotide sequence of SEQ ID NO: 3, and the base encoding the M protein The sequence consists of the nucleotide sequence of SEQ ID NO: 4, the nucleotide sequence encoding the F protein consists of the nucleotide sequence of SEQ ID NO: 5, and the nucleotide sequence encoding the HN protein consists of the nucleotide sequence of SEQ ID NO: 6, The nucleotide sequence encoding the L protein may be composed of the nucleotide sequence of SEQ ID NO: 7.

According to one side, the Newcastle disease virus KU001 strain may be isolated from domestic wild ducks.

According to another embodiment of the present invention, the full-length gene sequence of the Newcastle disease virus strain KU001 (accession number: KCTC18794P) is inserted, and a recombinant vector is provided, comprising the nucleotide sequence of SEQ ID NO: 1.

According to one aspect, the recombinant vector may be a plasmid vector.

The vector prepared by the method for preparing the recombinant vector of the present invention contains the full-length gene of the Newcastle disease virus, thereby preventing diseases such as avian influenza as well as Newcastle disease as a backbone vector for the preparation of a multivalent vaccine. can be used

In addition, since the recombinant virus prepared from the recombinant vector of the present invention has superior productivity compared to the B1 strain or Lasota strain used as the existing Newcastle disease virus vaccine strain, the production cost is reduced during mass production of the recombinant virus using the recombinant vector of the present invention. This is possible.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a schematic diagram of a recombinant vector of the present invention.
Figure 2 is a schematic diagram of the recombinant vector construction process of the present invention.
3 shows a phylogenetic tree analysis table using the fusion protein (F) sequence of Newcastle disease virus.
Figure 4 confirms whether the recombinant vector of the present invention is constructed using the PCR technique.
Figure 5 confirms whether a recombinant virus is prepared from the recombinant vector of the present invention using the RT-PCR technique.
6 shows whether a recombinant virus is prepared from the recombinant vector of the present invention using a Genetic tag (NotI).
7 is a conventional Newcastle disease virus (rNDV/B1/wt (rB1 wt), rNDV/Lasota/wt (rLasota wt))
and the growth characteristics of the virus (rKU001) prepared with the recombinant vector of the present invention.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. Like reference numerals in each figure indicate like elements.

Various modifications may be made to the embodiments described below. It should be understood that the embodiments described below are not intended to limit the embodiments, and include all modifications, equivalents or substitutes thereto.

Terms used in the examples are only used to describe specific examples, and are not intended to limit the examples. The singular expression includes the plural expression unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the possibility of addition or existence of numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

In addition, in the description with reference to the accompanying drawings, the same components are assigned the same reference numerals regardless of the reference numerals, and the overlapping description thereof will be omitted. In the description of the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

In the present invention, the term "cDNA" refers to DNA complementary to mRNA, and preferably refers to DNA having a nucleotide sequence complementary to mRNA of Newcastle disease virus.

In the present invention, the term "vector" refers to a means for introducing a foreign gene so that the foreign gene can be expressed, and a plasmid vector, cosmid vector, bacteriophage vector and adenovirus capable of self-replicating itself. vectors, retroviral vectors, adeno-associated viral vectors, and the like.

In the present invention, the term "restriction enzyme" is an endonuclease, which is one of nucleolytic enzymes that recognizes a specific nucleotide sequence of DNA or RNA and cuts the nucleotide chain. In genetic engineering, recombinant DNA or RNA It means a special enzyme used to make it.

In the present invention, the term "primer" refers to DNA or RNA having a short gene sequence complementary to a specific gene sequence to be synthesized, and means synthesized for the purpose of use in PCR, DNA sequencing, etc.

In the present invention, the term "promoter" refers to a region on DNA where RNA polymerase specifically binds and starts transcription. It may further include enhancers that can be used for facilitation and regulation.

According to an embodiment of the present invention, there is provided a method for preparing a recombinant vector into which the full-length gene sequence of Newcastle disease virus strain KU001 (Accession No.: KCTC18794P) is inserted, comprising the following steps, wherein cDNA is prepared using RNA extracted from Newcastle disease virus. synthesizing; The nucleotide sequence encoding the NP protein, the nucleotide sequence encoding the P protein, the nucleotide sequence encoding the M protein, the nucleotide sequence encoding the F protein, the nucleotide sequence encoding the HN protein, and the nucleotide sequence encoding the L protein are sequentially sequenced inserting into a plasmid; Including, wherein the recombinant vector is composed of the nucleotide sequence of SEQ ID NO: 1, a method for producing a recombinant vector is provided.

The nucleotide sequence encoding the NP protein consists of the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence encoding the P protein consists of the nucleotide sequence of SEQ ID NO: 3, and the nucleotide sequence encoding the M protein is SEQ ID NO: 4 consists of the nucleotide sequence of, the nucleotide sequence encoding the F protein consists of the nucleotide sequence of SEQ ID NO: 5, the nucleotide sequence encoding the HN protein consists of the nucleotide sequence of SEQ ID NO: 6, and the L protein The nucleotide sequence may be one consisting of the nucleotide sequence of SEQ ID NO: 7.

The Newcastle disease virus strain KU001 is isolated from domestic wild ducks, and refers to a strain that has been granted accession number KCTC18794P by the Korea Research Institute of Bioscience and Biotechnology as of October 31, 2019. The genotype corresponds to Genotype I (FIG. 3). Since the recombinant vector of the present invention contains the full-length gene of the NDV/KU001 strain isolated from domestic wild ducks, when it is used as a backbone vector, a recombinant virus vaccine effective for preventing the Newcastle disease virus prevalent in Korea can be prepared.

According to another embodiment of the present invention, the full-length gene of Newcastle disease virus strain KU001 (accession number: KCTC18794P) is inserted, and a recombinant vector comprising the nucleotide sequence of SEQ ID NO: 1 is provided. Here, the recombinant vector is a plasmid vector, and pSL1180 vector may be used, but is not limited thereto.

The recombinant vector of the present invention can be used to prepare a recombinant virus by reverse genetics. A specific method for preparing the recombinant virus is as follows. First, in order to provide T7 RNA polymerase, a virus (MVA/T7 virus) expressing T7 RNA polymerase in Modified Vaccinia Ankara strain is infected with Hep-2 or A549 cells. Three plasmids (NP, P. L) expressing the NDV RNA polymerase protein and the recombinant vector pNDV/KU001 of the present invention are simultaneously transfected into the Hep-2 or A549 cells. 24 hours after transfection, co-culture with chicken embryo fibroblast (CEF) cells is performed. After culturing for more than 48 hours, the cell supernatant is injected into 10-day-old fertilized eggs and cultured for 72 hours, then urine fluid is obtained.

Hereinafter, the experimental materials and methods used in the examples of the present invention will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited by the examples.

Example 1: Construction of the recombinant vector

To construct the recombinant vector of the present invention, RNA was extracted from Newcastle disease virus strain KU001 using TRIzol (Invitrogen, USA), and then ^{reverse transcription PCR was performed with Superscript TM} II Reverse Transcriptase (Invitrogen, USA) to synthesize cDNA. . Then, 7 gene segments (segment 1, T7 promoter to nucleotide 1,755 nucleotide sequence; fragment 2, nucleotide 1 nucleotide sequence to 3,321 nucleotide sequence; fragment 3, nucleotide 1,755 nucleotide sequence to 6,580 nucleotide sequence; fragment 4 , nucleotide sequence 6,151 to nucleotide 10,210; segment 5, nucleotide 7,381 to nucleotide 11,351; segment 6, nucleotide 11,351 to nucleotide 14,995; segment 7, nucleotide 14,701 to nucleotide 15,186 up to the second sequence) was obtained by PCR, the hepatitis delta virus ribozyme and T7 terminator gene sequences were inserted following the segment 7 gene, and the entire gene sequence was sequentially inserted into the pSL1180 (Amersham Parmacia Biotech, UK) plasmid to pNDV/KU001. The vector was completed. Here, p in pNDV means plasmid.

Example 2: Verification of whether the recombinant vector was constructed

In order to verify the pNDV/KU001 vector constructed in Example 1, a PCR technique was performed as described in (A) of FIG. 4 . The NP-P-M-F section and the HN-L section were used as controls. The primers used were P1/P2 (FIG. 4B), P4/P6 (FIG. 4C), and P10/P12 (FIG. 4D). The primer P1 used (forward direction; 5'-ACCAAACAGAGAATCCGTGAG-3') is the nucleotide sequence information in front of the NP gene of NDV, P2 (reverse direction; 5'-AGATGAGGGGGTTCGTTTGTAG-3') is the nucleotide sequence information before the 1701th of NDV, P4 (forward direction; 5'-CGCGCCCGTCGCAGTGACC-3') is the 4292th, P6 (reverse direction; 5'-CGTTTCCCACCGAACCGC-3') is the 7514th, and P10 (forward direction; 5'-ACCGACCCCAACACAGTTCC-3') is the 13825th , P12 (reverse direction; 5'-ACCAAACAAAGATTTGGTG-3') is each designed based on the last nucleotide sequence information of NDV. As a result, as shown in B to D of FIG. 4 , it was confirmed that the pNDV/KU001 plasmid in which the F protein-coding nucleotide sequence and the HN protein-coding nucleotide sequence were linked was constructed.

Example 3: Verification of production of recombinant virus

It was confirmed whether a recombinant virus was actually produced using the recombinant vector constructed in Example 1 above. Hereinafter, r in rNDV means recombinant, and rNDV means recombinant virus.

3-1. RT- PCR Recombination using techniques rNDV Verification of production of /KU001 virus

The rNDV/KU001 virus was prepared by the reverse genetics method described below. First, in order to provide T7 RNA polymerase, a virus expressing T7 RNA polymerase (MVA/T7 virus) in Modified Vaccinia Ankara strain was infected with Hep-2 or A549 cells. The pNDV/KU001 vector constructed in Example 1 and three plasmids expressing the NP, P, and L proteins were transfected into Hep-2 or A549 cells infected with the transfection. After incubation for more than 48 hours. Chicken embryo fibroblast (CEF) cells were added and co-cultured for 2-3 days. Next, the cell supernatant is obtained and inoculated into the allantoic cavity of 9-10-day-old chicken fertilized eggs. After 2-3 days, the allantoic fluid is obtained and the presence of the virus is confirmed by performing hemagglutination assay and hemagglutination-inhibition assay.

In order to verify that the rNDV/KU001 virus was actually prepared from the recombinant vector of the present invention, total RNA was extracted from the virus and cDNA was prepared using a random hexamer. Using the prepared cDNA as a template, PCR was performed to amplify the HN gene (FIG. 5A), and comparative analysis was performed with the control rNDV/Lasota/wt using restriction enzyme XbaI present only in the KU001 strain (FIG. 5B) )

As a result, as shown in FIG. 5, only the HN gene of the KU001 strain was cut, and it was confirmed that the virus prepared through this was derived from the recombinant vector of the present invention.

3-2. Genetic tag( NotI ) using recombination rNDV Verification of production of /KU001 virus

Since the rNDV/KU001 virus produced by reverse genetics has a NotI restriction enzyme recognition site that is not found in the wild type NDV KU001 strain, NotI was used as a genetic marker to distinguish the wild-type virus from the recombinant virus prepared by the reverse genetics method. .

After RNA of wild-type virus and rNDV/KU001 virus was extracted, RT-PCR was performed to obtain a PCR gene product containing NotI restriction enzyme (FIG. 6A), and then the gene product was treated with restriction enzyme NotI (FIG. 6). B).

As a result, as shown in FIG. 6 , in the case of rNDV/KU001 virus, it was confirmed that the PCR product was cleaved when the restriction enzyme NotI was treated. Through this, it was confirmed that the rNDV/KU001 virus prepared by the reverse genetics method was not contaminated with the wild type NDV KU001 strain and was a novel virus derived from the recombinant vector of the present invention.

Example 4: Evaluation of the productivity of recombinant virus

The recombinant NDV KU001 virus prepared in Example 3 and the Newcastle disease virus strain B1 and Lasota strain used as existing vaccine strains were prepared, and each virus _{was infected with a titer of 100 TCID 50} into 10-day-old fertilized eggs. Urine was obtained from fertilized eggs 12, 24, 48 and 72 hours after virus infection, and virus titers were measured by hemagglutinin assay (HA), and are shown in FIG. 7 .

As a result, as shown in FIG. 7 , it was confirmed that the growth titer of the recombinant virus of the present invention was about 10 ² times higher than that of the Lasota strain, and about 10 ⁵ times higher than that of the B1 strain. These results suggest that production costs can be reduced when a vaccine is mass-produced using the recombinant vector of the present invention.

As described above, although the embodiments have been described with reference to the limited embodiments and drawings, various modifications and variations are possible from the above description by those skilled in the art. For example, even if the described techniques are performed in an order different from the described method, and/or the described components are combined or combined in a different form from the described method, or replaced or substituted by other components or equivalents Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Korea Institute of Bioscience and Biotechnology KCTC18794P 20191031

<110> KOREA UNIVERSITY RESEARCH & BUSINESS FOUNDATION <120> Newcastle disease virus backbone vector using NDV KU001 strain and method for preparing thereof <130> APC-2018-0568 <150> KR 10-2018-0134494 <151> 2018-11-05 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 15186 <212> DNA <213> Newcastle disease virus <400> 1 accaaacaga gaatccgtaa ggtacgatag aaggcgaagg agcaatcgaa gtcgtacggg 60 tagaaggtgt gaatctcgag tgcgagcccg aagctcaaac tcgagagagc cttctgccaa 120 aatgtcttct gtattcgatg agtacgagca gctcctcgcg gctcagactc gccccaatgg 180 agctcatggc ggaggagaga aggggagcac cttaaaggta gaagtcccgg tattcactct 240 caacagtgat gacccagaag atagatggaa ctttgcagtg ttttgtcttc ggattgctgt 300 tagcgaggat gccaacaaac cacttaggca aggtgctctc atatctctct tatgttccca 360 ctctcaagtg atgaggaacc atgttgccct tgcggggaaa cagaatgagg ccacactggc 420 tgttcttgag atcgatggtt ttaccaacgg cgtgccccag ttcaacaaca ggagtggagt 480 gtctgaagag agagcacaga gattatgat gatagcaggg tctctccctc gggcatgcag 540 caacggtacc ccgttcgtca cagctggggt tgaagatgat gcaccagaag acattactga 600 taccctggag aggatcctct ctatccaggc tcaagtatgg gtcacggtgg caaaggccat 660 gactgcatat gagacagcag atgagtcaga aacaagaaga atcaataagt acatgcagca 720 aggcagggtc cagaagaagt acatcctcca ccccgtatgc aggagcgcaa tccaactcac 780 aatcagacag tctctggcgg tccgcatctt tttggttagc gagcttaaga gaggccgcaa 840 cacggcaggt gggacctcca cctattacaa cttggtgggg gatgtagact catacatcag 900 gaacactggg ctaactgcat tcttcctgac acttaaatat ggaattaaca ccaagacatc 960 agcccttgca cttagcagcc tctcaggcga tatccagaaa atgaagcagc tcatgcgctt 1020 gtatcggatg aaaggagata atgcgccgta catgacattg ctcggtgaca gtgaccagat 1080 gagctttgca cctgccgagt atgcacaact ttactccttt gccatgggta tggcatcagt 1140 cctagataaa ggaactagca aataccaatt tgccagggac tttatgagca catcattctg 1200 gagacttgga gtagagtacg ctcaggctca aggaagtagc atcaatgagg atatggccgc 1260 cgagctaaag ctaaccccag cagcaaggag aggcctggca gctgctgccc aaagagtgtc 1320 tgaggagacc agcagcatgg acatgcccac ccaacaagcc ggggtcctca ctggactcag 1380 cgacggaggc tcccaagccc cccaaggtgc actgaacaga tcacaagggc aaccggacac 1440 cggggatggg gagacccaat ttctggatct gatgagagcg gtggcaaata gcatgagaga 1500 agcgccaaac tctgcgcagg gcacccctca accggggcct cccccaaccc ctgggccctc 1560 tcaagacaat gacaccgact gggggtactg accgacagca cccagtttgc ttctatgagg 1620 tcatcccaat tcctctgccc acaccccacc cctcaatccg caatcccgca tggtcaaacc 1680 cacaaacgaa cccccctgtc tccctcctct cccccagccc cacaacccca cctgcccagg 1740 gcaacatagg tacaatgcga cccactaata atcaatacag ggccaaagaa attagaaaaa 1800 agtacgggta gaagggagac attcagagat cagggcgagt cacccgggtc tctgctctcc 1860 cttctaccta gtggattagg atggagatgg ccacctttac agatgcggag atcgacgagc 1920 tatttgagac cagtggaact gtcattgaca gcataattac ggcccaggga aaaccagtag 1980 agactgttgg aaggagtgca atcccacaag gcaaaactaa ggctttgagc gcagcatggg 2040 agaagcatgg gagcatccag tcaccagcca gccaagacac ccctgatcga caggacagat 2100 cagataaaca actgtccaca cccgagcaag cgagtccaaa cgacagcccc ccagccacat 2160 ccactgacca gcctcccact caggctgcag atgaggccgg cgatacacag ctcaagaccg 2220 gagcaagcaa ctctctgctg tcgatgcttg ataaactcag caataagtca tctaatgcta 2280 aaaagggccc agggtcgagc cctcaagaaa ggcatcatca acgtctgact caacaacagg 2340 ggagtcaaca aagccgcgga aacagccaag agagaccgca gaaccaggcc aaggccatcc 2400 ctggaaacca ggtcacagac gcgaacacag catatcatgg acaatgggag gagtcacaac 2460 tatcagctgg tgcaacccat catgctctcc gatcagagca gagccaagac aatactcctg 2520 cacctgtgga tcatgtccag ctacctgtcg actttgtgca ggcgatgatg tctatgatgg 2580 aggcgatatc acagagggta agtaaagttg actatcagct ggaccttgtc ttgaaacaga 2640 catcttctat ccccatgatg cggtctgaaa tccagcagct gaaaacgtct gttgcggtca 2700 tggaagccaa tttgggcatg atgaagatcc tggaccctgg ttgtgccaac gtttcatctc 2760 taagtgatct acgggcagtt gcccgatccc acccggtttt aatttctggc cccggagacc 2820 catctcctta tgtgacccaa gggggcgaaa tggcactcaa taaactttcg caaccggtgc 2880 aacacccctc tgaattgatt aaacccgcca cggcaagcgg gcctgatata ggagtggaga 2940 aagacactgt ccgtgcattg atcatgtcac gccctatgca tccgagctct tcagctaggc 3000 tcttgagcaa actggacgca gccggatcga ttgaggaaat cagaaaaatc aagcgccttg 3060 cactgaatgg ctaatcacca ccgcaacccg cagcagatcc ctgtccaccc agcaccacac 3120 ggtatctgca ccaagctcct ctctgcaaac ccaaggtcca acaccccgag cgacaaccct 3180 gtcctgcttc ctctgcccca ctaaatgatc gcgcagctgc aatcaattca gctatattaa 3240 ggattaagaa aaaatacggg tagaatcgga gtgccccgat tgtgccaaga tggactcatc 3300 taggacaatc gggctgtact ttgattctac ccttccttct agcaacctgc tagcattccc 3360 gatagtccta caagacacag gggacgggaa gaagcaaatc gccccgcaat acaggatcca 3420 gcgtcttgac tcgtggacag acagcaaaga agactcggta ttcatcacca cctatggatt 3480 catctttcag gttgggaatg aagaagccac tgtcggcatg atcaatgata atcccaagcg 3540 cgagttactt tccactgcca tgctatgcct agggagtgta ccaaatgtcg gagatcttgt 3600 tgagctggca agggcctgcc tcactatggt ggtaacatgc aagaagagtg caactaacac 3660 cgagagaatg gtcttctcag tagtgcaggc accccaggtg ctgcaaagct gtagggttgt 3720 ggcaaacaaa tactcgtcgg tgaatgcagt caagcacgtg aaagcaccag agaagattcc 3780 tgggagcgga accctagagt acaaagtgaa ctttgtctct ctgaccgtgg tgccaagaaa 3840 ggacgtctac aagataccaa ctgcagcact taaggtctct ggctcaagtc tgtacaatct 3900 tgcgctcaat gtcactattg atgtggaggt agacccgaag agcccgttgg tcaaatccct 3960 ttccaagtcc gacagtgggt actatgctaa tctcttctta catattgggc ttatgtccac 4020 tgtagataag aaggggaaga aagtgacatt tgacaagctg gaaaggaaga taaggagact 4080 tgatctatct gtagggctta gtgacgtgct cggaccttcc gtgcttgtaa aggcgagagg 4140 tgcacggact aagctgctgg cacctttctt ctctagcagt gggacagcct gctatcccat 4200 agcaaatgcc tctcctcagg tggccaagat actctggagc caaaccgcgt acctgcggag 4260 tgtaaaagtc attatccaag cgggcaccca gcgtgctgtc gcagtgaccg ccgaccacga 4320 ggttacctct actaagctgg agaaggggca taccattgcc aaatacaatc ccttcaagaa 4380 ataggctgca tctctgagat tgcactccgc ccatcttccc ggatcaccat gacactaaat 4440 aatgatctgt cttgattact tatagttagt tcgcctgtct atcaaattag aaaaaacacg 4500 ggtagaagat tctggatccc ggttggcgcc ttcaaggtgc aagatgggct ccagatcttc 4560 taccaggatc ccagtacctc tcatgctgac cgtccgagtc atgttggcac tgagttgcgt 4620 ctgtccgacc agcgcccttg atggcaggcc tcttgcagct gcagggattg tggtaacggg 4680 agacaaagca gtcaacatat acacctcatc tcagacaggg tcaatcataa tcaagttact 4740 cccaaatatg cccaaggata aagaggcgtg tgcaaaagcc ccgttggagg catacaacag 4800 gacattgact actttgctca ccccccttgg tgattctatc cgtaggatac aagagtctgt 4860 gaccacgtcc ggaggaggga aacagggacg tcttataggc gccattatcg gtggtgtagc 4920 tctcggggtt gcaaccgctg cacagataac agcagcctcg gctctgatac aagccaatca 4980 aaatgctgcc aacatactcc ggctaaaaga gagcattgct gcaaccaatg aggctgtgca 5040 cgaggtcact aatggattat cacaactagc agtggcagtt gggaagatgc agcaatttgt 5100 taatgaccag tttaataaaa cagctcagga attggactgt ataaaaatta cacagcaggt 5160 tggtgtagaa ctcaacctgt acctaactga attgactaca gtattcgggc cacaaatcac 5220 ttcccctgcc ttaactcagc tgactatcca ggcgctttac aatctagctg gtgggaatat 5280 ggattacttg ttgactaagt taggtgtggg gaacaaccaa ctcagctcat taattagtag 5340 tggcctgatc accggcaacc ctattctgta cgactcacag actcaactct tgggtataca 5400 ggtaacccta ccctcagtcg ggaacctaaa taatatgcgt gccacctacc tggaaacctt 5460 gtctgtaagt acaaccaaag gatttgcctc agcacttgtc ccaaaagtag tgacacaggt 5520 cggttccgtg atagaagagc ttgacacctc gtactgtata gagaccgatt tggatctata 5580 ttgtacaaga atagtgacat tccctatgtc tcctggtatt tattcctgtt tgagtggcaa 5640 tacatctgct tgcatgtact caaagactga aggcgcactc actacgccgt atatgaccct 5700 caaaggctca gttattgcta actgtaagat gacaacatgt agatgtgcag accccccggg 5760 tatcatatcg caaaattatg gagaagctgt gtctctaata gataggcaat catgcaatat 5820 cttatcctta gacgggataa ctttgaggct cagtggggaa tttgatgcaa cttatcaaaa 5880 gaatatctca atacaagatt ctcaagtaat agtgacaggc aatcttgata tctcgactga 5940 gcttgggaat gtcaacaact cgataagtaa tgctttggat aagttagagg aaagcaacag 6000 caaactagat aaggtcaatg tcaaactgac cagcacatcc gctcttatta cctatatcgt 6060 tttaactgtc atatctcttg tatgtggtat acttagcctg gttctagcat gctacctgat 6120 gtacaagcaa aaggcgcaac agaagacctt gttgtggctt gggaataata ccctagacca 6180 gatgagggcc actacaaaaa tgtgaatgcg gatgagaggc agaaacatcc ccaatagcag 6240 tttgtgtgta aagtctgaca gcctgttaat tagaagaatt aagaaaaaac taccggatgt 6300 agatgaccaa agggcgatat acgggtagaa cggtcgggga ggccgtccct caatcgggag 6360 ccgggcctca caacatccgt tctaccgcat caccaatagc agttttcagt catggaccgc 6420 gcagttagcc aagttgcgct ggagaatgat gaaagagagg caaagaatac atggcgcttg 6480 gtattccgga tcgcaatcct actctcaacg gtggtgacct tagccatctc tgcagccgcc 6540 cttgcatata gcatggaggc cagcacacct agcgatcttg taggcatacc gactgcgatc 6600 tctagagcag aggaaaagat tacatctgca ctcggttcca atcaagatgt agtagatagg 6660 atatataagc aggtggccct cgaatctcca ctggcattgc taaacaccga atctacaatt 6720 atgaacgcaa taacgtctct ctcttatcga atcaatgggg ccgcaaatag cagcggatgt 6780 ggagcaccca ttcatgatcc agattatatt ggaggaatag gtaaagaact tattgtagat 6840 gatgctagcg acgtcacatc atactatccc tctgcgttcc aagaacacct gaactttatc 6900 ccggcgccta ctacaggatc aggttgcact cggataccct catttgacat gagcgctacc 6960 cactactgtt atactcacaa tgtgatatta tctggctgca gagatcactc gcactcacat 7020 caatatttag cacttggtgt gcttcggaca tctgcaacag ggagggtatt cttttccact 7080 ctgcgttcca tcaatctgga tgacacccaa aatcggaagt cttgcagtgt gagtgcaacc 7140 cccttgggtt gtgatatgct gtgctctaaa gtcacagaga ctgaagaaga ggattataac 7200 tcagctatcc ccacgtcgat ggtacatgga aggttagggt tcgacggcca ataccacgag 7260 aaggacctag atgtcacaac actattcgag gactgggtgg caaactaccc aggagtaggg 7320 ggcgggtctt ttattgacaa ccgcgtatgg ttcccagttt acggagggct aaaacccaat 7380 tcgcccagtg acaccgcaca agaagggaaa tatgtaatat acaagcgata caatgacaca 7440 tgtccagatg agcaagatta tcagattcaa atggctaagt cttcatataa gcctgggcgg 7500 tttggaggga aacgcgtaca gcaggccatc ttatctatca aagtgtcaac atccttgggc 7560 gaggacccgg tactgactgt accgcccaac acagtaacac tcatgggggc cgaaggcaga 7620 gttctcacag tagggacatc tcatttcctt tatcagcgag ggtcatcata cttctcccct 7680 gccctactat atcctatgat agtcagcaac aaaacagcca ctcttcatag tccttataca 7740 ttcaatgcct tcactcgacc aggtagtgtc ccttgccagg cttcagcaag atgccctaac 7800 tcatgtgtta ccggagtcta tactgatcca tatcccttgg tcttctatag gaaccacacc 7860 ttgcgagggg tattcgggac gatgcttgat gataaacaag caagactcaa ccctgtatct 7920 gcagtatttg acagcatatc ccgcagtcgc ataacccggg tgagttcaag cagcaccaag 7980 gcagcataca caacatcaac atgttttaaa gttgtaaaga ccaataaaac ctattgtctc 8040 agcattgccg aaatatccaa taccctcttc ggggaattca gaatcgtccc tttactagtt 8100 gagattctca aggatgatgg ggttagagaa gccaggtcta gccggttgag tcaactgcga 8160 gagggttgga aagatgacat tgtatcacct atcttttgcg acgccaagaa tcaaactgaa 8220 taccggcgcg agctcgagtc ctacgctgcc agttggccat aatcagctag tgctaatgtg 8280 attagattaa gtcttgtcgg tagtcacttg attaagaaaa aatgtgggtg gtagcgggat 8340 ataaggcaaa acaactcaag gaggatagca cgggtaggac atggcgagct ccggtcccga 8400 gagggcggag catcagatta tcctaccaga gtcacacctg tcttcaccat tagtcaagca 8460 caaactactc tattactgga aattaactgg gctaccactc cctgacgagt gtgacttcga 8520 ccacctcatt ctcagccgac aatggaagaa aatacttgaa tcggcctccc ctgacactga 8580 gagaatgata aaacttggaa gggcagtgca ccagactctc aaccacaatt ccaagataac 8640 cggagtactc catcccaggt gtttagaaga attggctagt attgaggttc ctgactcaac 8700 caacaagttt cggaagatcg agaagaaaat ccaaattcac aacacaaggt atggagaact 8760 gttcacaaga ctgtgcacgc atgtagagaa gaaattgttg ggatcatctt ggtctaataa 8820 tgtccccccgg tcagaagagt tcaacagcat ccgtacagat ccggcattct ggtttcactc 8880 aaaatggtcc acaactaagt ttgcatggct ccatataaaa cagattcaaa ggcatctgat 8940 tgtggcagca agaacaaggt ccgcagccaa caaattggtg acgctgaccc ataaggtagg 9000 ccaagtcttt gttactcctg agcttgtcat tgtgacacat acagatgaga acaagttcac 9060 gtgtcttacc caggaacttg tgttgatgta tgcagatatg atggagggca gagatatggt 9120 caacataata tcatccacgg cggcacatct caggagccta tcagagaaaa ttgatgacat 9180 tctgcggtta gtagatgccc tggcaaaaga tctgggtaat caagtctacg atgttgtagc 9240 actcatggag ggatttgcat acggcgccgt ccagctgctt gagccgtcag gtacattcgc 9300 aggggatttc ttcgcattca acctgcagga gctcaaagac actttgatcg gcctccttcc 9360 taaggatata gcagaatctg tgactcacgc aatagccact gtattctctg gcttagaaca 9420 aaatcaagcg gctgagatgc tgtgcctgtt gcgtctatgg ggccacccat tacttgagtc 9480 ccgtattgcg gcaaaagcag taaggagcca aatgtgcgca ccaaaaatgg tagactttga 9540 tatgatcctc caggtattgt ctttctttaa aggaacaatc atcaacggat acagaaagaa 9600 gaatgcaggt gtttggccac gtgtcaaagt agaaacgata tacgggaagg tcattgggca 9660 gctacacgct gattcagcgg agatttcaca cgatatcatg ttgagagagt acaagagttt 9720 atctgcgctt gaattcgagc catgtataga atacgaccct atcaccaatc tgagcatgtt 9780 tctaaaagac aaggcgatcg cacacccgaa agacaactgg ctcgccgcgt ttaggcgaaa 9840 ccttctctct gaggaccaga agaaacatgt aaaggaggca acctctacta accgtctctt 9900 gatagagttc ttagagtcaa atgattttga tccatataag gagatggaat atctgacgac 9960 ccttgagtac ctaagagatg acaatgtggc agtatcatac tcgctcaagg agaaggaagt 10020 gaaggttaat gggcggattt ttgctaagct aacaaagaaa ttaaggaact gtcaagtgat 10080 ggcggaaggg atcttagctg accagattgc acctttcttt caagggaatg gggtcattca 10140 ggatagcata tctttaacca agagtatgct agcgatgagt caattgtctt tcaacagcaa 10200 taagaaacgt atcactgact gcaaagaaag agtagcctca aaccgcaatc acgatcaaaa 10260 gagcaagaat cgtcggagag ttgccacttt tataacgact gacctgcaaa agtactgtct 10320 taattggaga tatcagacaa tcaaactgtt cgctcatgcc atcaatcagc tgatgggctt 10380 acctcacttc ttcgaatgga ttcatctaag actaatggat actacgatgt ttgtaggaga 10440 ccctttcaat cccccaagtg acccaactga ctgtgatctc tcaagagtcc caaatgatga 10500 catatatatt gtcagtgcta gagggggtat tgagggatta tgtcagaagc tatggacaat 10560 gatctcaatt gctgcaatcc aacttgctgc agcaagatca cattgtcgcg tcgcctgtat 10620 ggtacagggt gacaatcaag taatagctgt aacgagagag gtaaggtcag atgactcccc 10680 ggaaatggtg ttaacacaat tgcatcaagc cagtgataat ttcttcaagg aattgattca 10740 tgttaatcat ttgattggcc ataatttgaa ggatcgtgaa acaatcagat cagacacatt 10800 cttcatatac agcaaacgaa tattcaaaga tggagcaata ctcagtcaag tcctcaaaaa 10860 ttcatctaaa ttagtgctaa tatcaggcga ccttagtgaa aacaccgtaa tgtcctgtgc 10920 caacattgca tctactatag cacggctgtg cgagaacggg cttccaaagg atttctgtta 10980 tacttaaac tacctgatga gttgcgtgca gacatacttt gattctgagt tttccatcac 11040 taacagctcg caccccgatt ctaaccagtc gtggattgaa gacatctctt ttgtgcactc 11100 atatgtcctg acccctgccc agctaggggg actgagcaac ctccaatact caaggctcta 11160 cacgaggaac atcggtgacc cgggaactac tgcttttgca gagatcaagc gattagaagc 11220 agtggggtta ctaagttcta gtattatgac taacatctta actaggccgc ctggaaatgg 11280 agattgggcc agtctgtgta acgaccctta ctctttcaat tttgagactg tcgcgagtcc 11340 aaatattgtc cttaagaaac atacacaaag agtcctattt gaaacttgtt caaatccctt 11400 attatctggc gtgcatacag aggataatga ggcagaagag aaggcgttgg ctgaattttt 11460 actcaatcaa gaagtaattc atccacgtgt cgcacatgct atcatggaag caagctctat 11520 aggtaggagg aagcagattc aagggcttgt tgacacaaca aacaccgtaa tcaagattgc 11580 attgactagg aggccacttg gcatcaagag gctgatgcgg atagttaact actcgagcat 11640 gcatgcaatg ctgtttagag acgatgtttt ctcatctaac aggtctaacc accccttagt 11700 ttcctctaat atgtgttctc tgacgctagc agactatgca cggaatagaa gctggtcacc 11760 attgacgggg ggtagaaaga tactgggtgt atctaatcct gatactatag aacttgtaga 11820 gggtgagatc cttagcgtca gcggaggatg cacaagatgt gacagcggag atgaacaatt 11880 cacttggttc catcttccga gcaatataga actgaccgat gacaccagca agaatcctcc 11940 gatgagagtg ccgtacctcg ggtcaaagac tcaagagagg agggccgcct cgcttgcgaa 12000 aatagctcat atgtcaccac atgtgaaagc tgctctaagg gcatcatccg tgttgatctg 12060 ggcttatgga gacaacgaag taaattggac tgctgctctt aaaattgcaa gatctcggtg 12120 caatataaac tcagagtatc ttcgactatt gtccccctta cccacagctg ggaatctcca 12180 acatagactg gatgacggca taactcagat gacattcacc cctgcatctc tctacagggt 12240 gtcaccttat attcacatat ccaatgattc tcaaaggtta ttcacggaag aaggagtcaa 12300 agagggaaat gtagtttatc agcaaatcat gctcttgggt ttatctctaa tcgaatcact 12360 cttcccgatg acgacaacca ggacatacga tgagatcaca ttgcacctcc acagtaaatt 12420 tagctgctgt atcagggaag caccggttgc agttcctttc gagttactcg ggatggcacc 12480 agaactaagg acagtgacct caaataagtt tatgtatgat cctagtcctg tatcggaggg 12540 tgactttgcg agacttgact tagctatctt taagagttat gagcttaatc tagaatcata 12600 tcccacaata gagctaatga acattctttc aatatccagc gggaagttaa tcggccagtc 12660 tgtggtttct tatgatgaag atacctccat aaagaatgac gccataatag tgtatgacaa 12720 cacccggaat tggatcagcg aagctcagaa ttcagatgtg gtccgcctat tcgagtatgc 12780 agcacttgaa gtgcttctcg actgttctta tcagctctac tatctgagag taagaggcct 12840 agacaatatc gtgttgtata tgagtgactt atataagaat atgccaggaa ttctactttc 12900 caacattgca gctacaatat ctcatcccat cattcattca agattgcatg cagtaggcct 12960 ggtcaatcac gacgggtcac accaacttgc agacacagat ttcatcgaaa tgtctgcaaa 13020 actattagtc tcttgcactc gacgcgtggt ctcaggttta tatgcaggga ataagtatga 13080 tctgctgttc ccgtctgtct tagatgataa cctgagtgag aagatgcttc agctgatatc 13140 tcggttatgc tgcctgtata cggtgctctt tgctacaaca agagagatcc cgaaaataag 13200 aggcttatct gcagaagaga agtgttcagt acttactgag tacctactgt cagatgctgt 13260 gaaaccata cttagttctg agcaagtgag ctctatcatg tctcctaaca tagttacgtt 13320 cccagctaat ctatattaca tgtctcggaa gagccttaat ttgattaggg aaagagagga 13380 cagggacact atcttggcat tgttgttccc ccaagagcca ctacttgagt tccccttagt 13440 acaagatatt ggcgctcgag tgaaagatcc attcacccga caacctgcgg cgtttttaca 13500 agaattagat ttgagcgctc cagcaaggta tgacgcattt acacttagtc aggttcattc 13560 tgaacacaca tcaccaaatc cggaggacga ctacttagta cgatacctgt tcagaggaat 13620 agggaccgcg tcctcctctt ggtataaggc atctcacctt ctttctgtac ctgaggtcag 13680 atgtgcaagg cacgggaatt ccttatactt ggcagaagga agcggagcca ttatgagtct 13740 tctcgaactg catgtgccgc atgagactat ctattacaat acgctcttct caaacgagat 13800 gaacccccca cagcggcatt tcggaccgac cccaacacag tttctgaatt cagttgttta 13860 taggaatcta caggcggagg taccatgtaa ggatggattt gtccaggagt tccgtccatt 13920 atggagagag aatacagaag aaagcgatct gacctcagat aaagcagtgg gttacatcac 13980 atctgcagtg ccctaccggt ctgtatcatt gctgcactgt gcattgaga ttcctccagg 14040 atccaatcaa agcttactgg atcaactggc taccaatcta tctctgattg ccatgcattc 14100 tgtaagggag ggcggggtcg tgatcatcaa agtgttgtat gcaatgggat attacttcca 14160 tctactcatg aacttgttca ctccgtgttc tacgaaagga tatattctct ctaatggcta 14220 tgcatgtaga ggggatatgg agtgttacct ggtatttgtc atgggctatc gaggtgggcc 14280 tacatttgta catgaggtag tgaggatggc aaaaactcta gtgcagcggc acggtacact 14340 tttgtccaaa tcagatgaga tcacactgac taggttattt acctcacagc ggcagcgtgt 14400 aacagacatc ctatccagtc ctttaccgag actaataaag ttcttgagaa agaatatcga 14460 tactgcgcta attgaagccg ggggacaacc cgtccgtcca ttctgtgcag agagcttggt 14520 gaggacacta gcggacacaa ctcagatgac ccagatcatc gctagtcaca ttgacacagt 14580 cattcgatct gtgatctaca tggaggctga gggtgatctc gccgacacag tgttcttatt 14640 taccccctac aatctctcta cagacggtaa aaagagaaca tcacttaaac agtgcacaag 14700 gcagatctta gaggtcacaa tattgggtct tagagttgaa aatctcaata aagtaggtga 14760 tgtagtcagt ctagtactta aaggtatgat ttctctggag gacctgatcc ctctaagaac 14820 atacttgaag cgtagtacct gccctaagta tttgaagtct gttctaggta tactaaact 14880 caaagaaatg tttacagaca cctctttatt atacttgact cgtgctcaac aaaaattcta 14940 catgaaaact ataggcaacg cagtcaaggg atactacagt aactgtgact cttaaaaata 15000 atcacatatt aataggctcc ttttctagtt aactgagccc ttgttgattt aatgatacta 15060 tattagaaaa aagttgcact ccgatccttt aggactcgtg ttcgaattca aataattgtc 15120 ttagaaaaaa gttgcgcgta attgttcttg aatgtagtcc tgtcattcac caaatctttg 15180 tttggt 15186 <210> 2 <211> 1470 <212> DNA <213> Newcastle disease virus <400> 2 atgtcttctg tattcgatga gtacgagcag ctcctcgcgg ctcagactcg ccccaatgga 60 gctcatggcg gaggagagaa ggggagcacc ttaaaggtag aagtcccggt attcactctc 120 aacagtgatg acccagaaga tagatggaac tttgcagtgt tttgtcttcg gattgctgtt 180 agcgaggatg ccaacaaacc acttaggcaa ggtgctctca tatctctctt atgttcccac 240 tctcaagtga tgaggaacca tgttgccctt gcggggaaac agaatgaggc cacactggct 300 gttcttgaga tcgatggttt taccaacggc gtgccccagt tcaacaacag gagtggagtg 360 tctgaagaga gagcacagag atttatgatg atagcagggt ctctccctcg ggcatgcagc 420 aacggtaccc cgttcgtcac agctggggtt gaagatgatg caccagaaga cattactgat 480 accctggaga ggatcctctc tatccaggct caagtatggg tcacggtggc aaaggccatg 540 actgcatatg agacagcaga tgagtcagaa acaagaagaa tcaataagta catgcagcaa 600 ggcagggtcc agaagaagta catcctccac cccgtatgca ggagcgcaat ccaactcaca 660 atcagacagt ctctggcggt ccgcatcttt ttggttagcg agcttaagag aggccgcaac 720 acggcaggtg ggacctccac ctattacaac ttggtggggg atgtagactc atacatcagg 780 aacactgggc taactgcatt cttcctgaca cttaaatatg gaattaacac caagacatca 840 gcccttgcac ttagcagcct ctcaggcgat atccagaaaa tgaagcagct catgcgcttg 900 tatcggatga aaggagataa tgcgccgtac atgacattgc tcggtgacag tgaccagatg 960 agctttgcac ctgccgagta tgcacaactt tactcctttg ccatgggtat ggcatcagtc 1020 ctagataaag gaactagcaa ataccaattt gccagggact ttatgagcac atcattctgg 1080 agacttggag tagagtacgc tcaggctcaa ggaagtagca tcaatgagga tatggccgcc 1140 gagctaaagc taaccccagc agcaaggaga ggcctggcag ctgctgccca aagagtgtct 1200 gaggagacca gcagcatgga catgcccacc caacaagccg gggtcctcac tggactcagc 1260 gacggaggct cccaagcccc ccaaggtgca ctgaacagat cacaagggca accggacacc 1320 ggggatgggg agacccaatt tctggatctg atgagagcgg tggcaaatag catgagagaa 1380 gcgccaaact ctgcgcaggg cacccctcaa ccggggcctc ccccaacccc tgggccctct 1440 caagacaatg acaccgactg ggggtactga 1470 <210> 3 <211> 1194 <212> DNA <213> Newcastle disease virus <400> 3 atggagatgg ccacctttac agatgcggag atcgacgagc tatttgagac cagtggaact 60 gtcattgaca gcataattac ggcccaggga aaaccagtag agactgttgg aaggagtgca 120 atcccacaag gcaaaactaa ggctttgagc gcagcatggg agaagcatgg gagcatccag 180 tcaccagcca gccaagacac ccctgatcga caggacagat cagataaaca actgtccaca 240 cccgagcaag cgagtccaaa cgacagcccc ccagccacat ccactgacca gcctcccact 300 caggctgcag atgaggccgg cgatacacag ctcaagaccg gagcaagcaa ctctctgctg 360 tcgatgcttg ataaactcag caataagtca tctaatgcta aaaagggccc agggtcgagc 420 cctcaagaaa ggcatcatca acgtctgact caacaacagg ggagtcaaca aagccgcgga 480 aacagccaag agagaccgca gaaccaggcc aaggccatcc ctggaaacca ggtcacagac 540 gcgaacacag catatcatgg acaatgggag gagtcacaac tatcagctgg tgcaacccat 600 catgctctcc gatcagagca gagccaagac aatactcctg cacctgtgga tcatgtccag 660 ctacctgtcg actttgtgca ggcgatgatg tctatgatgg aggcgatatc acagagggta 720 agtaaagttg actatcagct ggaccttgtc ttgaaacaga catcttctat ccccatgatg 780 cggtctgaaa tccagcagct gaaaacgtct gttgcggtca tggaagccaa tttgggcatg 840 atgaagatcc tggaccctgg ttgtgccaac gtttcatctc taagtgatct aggggcagtt 900 gcccgatccc acccggtttt aatttctggc cccggagacc catctcctta tgtgacccaa 960 gggggcgaaa tggcactcaa taaactttcg caaccggtgc aacacccctc tgaattgatt 1020 aaacccgcca cggcaagcgg gcctgatata ggagtggaga aagacactgt ccgtgcattg 1080 atcatgtcac gccctatgca tccgagctct tcagctaggc tcttgagcaa actggacgca 1140 gccggatcga ttgaggaaat cagaaaaatc aagcgccttg cactgaatgg ctaa 1194 <210> 4 <211> 1095 <212> DNA <213> Newcastle disease virus <400> 4 atggactcat ctaggacaat cgggctgtac tttgattcta cccttccttc tagcaacctg 60 ctagcattcc cgatagtcct acaagacaca ggggacggga agaagcaaat cgccccgcaa 120 tacaggatcc agcgtcttga ctcgtggaca gacagcaaag aagactcggt attcatcacc 180 acctatggat tcatctttca ggttgggaat gaagaagcca ctgtcggcat gatcaatgat 240 aatcccaagc gcgagttact ttccactgcc atgctatgcc tagggagtgt accaaatgtc 300 ggagatcttg ttgagctggc aagggcctgc ctcactatgg tggtaacatg caagaagagt 360 gcaactaaca ccgagagaat ggtcttctca gtagtgcagg caccccaggt gctgcaaagc 420 tgtagggttg tggcaaacaa atactcgtcg gtgaatgcag tcaagcacgt gaaagcacca 480 gagaagattc ctgggagcgg aaccctagag tacaaagtga actttgtctc tctgaccgtg 540 gtgccaagaa aggacgtcta caagatacca actgcagcac ttaaggtctc tggctcaagt 600 ctgtacaatc ttgcgctcaa tgtcactatt gatgtggagg tagacccgaa gagcccgttg 660 gtcaaatccc tttccaagtc cgacagtggg tactatgcta atctcttctt acatattggg 720 cttatgtcca ctgtagataa gaaggggaag aaagtgacat ttgacaagct ggaaaggaag 780 ataaggagac ttgatctatc tgtagggctt agtgacgtgc tcggaccttc cgtgcttgta 840 aaggcgagag gtgcacggac taagctgctg gcacctttct tctctagcag tgggacagcc 900 tgctatccca tagcaaatgc ctctcctcag gtggccaaga tactctggag ccaaaccgcg 960 tacctgcgga gtgtaaaagt cattatccaa gcgggcaccc agcgtgctgt cgcagtgacc 1020 gccgaccacg aggttacctc tactaagctg gagaaggggc ataccattgc caaatacaat 1080 cccttcaaga aatag 1095 <210> 5 <211> 1662 <212> DNA <213> Newcastle disease virus <400> 5 atgggctcca gatcttctac caggatccca gtacctctca tgctgaccgt ccgagtcatg 60 ttggcactga gttgcgtctg tccgaccagc gcccttgatg gcaggcctct tgcagctgca 120 gggattgtgg taacgggaga caaagcagtc aacatataca cctcatctca gacagggtca 180 atcataatca agttactccc aaatatgccc aaggataaag aggcgtgtgc aaaagccccg 240 ttggaggcat acaacaggac attgactact ttgctcaccc cccttggtga ttctatccgt 300 aggatacaag agtctgtgac cacgtccgga ggagggaaac agggacgtct tataggcgcc 360 attatcggtg gtgtagctct cggggttgca accgctgcac agataacagc agcctcggct 420 ctgatacaag ccaatcaaaa tgctgccaac atactccggc taaaagagag cattgctgca 480 accaatgagg ctgtgcacga ggtcactaat ggattatcac aactagcagt ggcagttggg 540 aagatgcagc aatttgttaa tgaccagttt aataaaacag ctcaggaatt ggactgtata 600 aaaattacac agcaggttgg tgtagaactc aacctgtacc taactgaatt gactacagta 660 ttcgggccac aaatcacttc ccctgcctta actcagctga ctatccaggc gctttacaat 720 ctagctggtg ggaatatgga ttacttgttg actaagttag gtgtggggaa caaccaactc 780 agctcattaa ttagtagtgg cctgatcacc ggcaacccta ttctgtacga ctcacagact 840 caactcttgg gtatacaggt aaccctaccc tcagtcggga acctaaataa tatgcgtgcc 900 acctacctgg aaaccttgtc tgtaagtaca accaaaggat ttgcctcagc acttgtccca 960 aaagtagtga cacaggtcgg ttccgtgata gaagagcttg acacctcgta ctgtatagag 1020 accgatttgg atctatattg tacaagaata gtgacattcc ctatgtctcc tggtatttat 1080 tcctgtttga gtggcaatac atctgcttgc atgtactcaa agactgaagg cgcactcact 1140 acgccgtata tgaccctcaa aggctcagtt attgctaact gtaagatgac aacatgtaga 1200 tgtgcagacc ccccgggtat catatcgcaa aattatggag aagctgtgtc tctaatagat 1260 aggcaatcat gcaatatctt atccttagac gggataactt tgaggctcag tggggaattt 1320 gatgcaactt atcaaaagaa tatctcaata caagattctc aagtaatagt gacaggcaat 1380 cttgatatct cgactgagct tgggaatgtc aacaactcga taagtaatgc tttggataag 1440 ttagaggaaa gcaacagcaa actagataag gtcaatgtca aactgaccag cacatccgct 1500 cttattacct atatcgtttt aactgtcata tctcttgtat gtggtatact tagcctggtt 1560 ctagcatgct acctgatgta caagcaaaag gcgcaacaga agaccttgtt gtggcttggg 1620 aataataccc tagaccagat gagggccact acaaaaatgt ga 1662 <210> 6 <211> 1851 <212> DNA <213> Newcastle disease virus <400> 6 atggaccgcg cagttagcca agttgcgctg gagaatgatg aaagagaggc aaagaataca 60 tggcgcttgg tattccggat cgcaatccta ctctcaacgg tggtgacctt agccatctct 120 gcagccgccc ttgcatatag catggaggcc agcacaccta gcgatcttgt aggcataccg 180 actgcgatct ctagagcaga ggaaaagatt acatctgcac tcggttccaa tcaagatgta 240 gtagatagga tatataagca ggtggccctc gaatctccac tggcattgct aaacaccgaa 300 tctacaatta tgaacgcaat aacgtctctc tcttatcgaa tcaatggggc cgcaaatagc 360 agcggatgtg gagcacccat tcatgatcca gattatattg gaggaatagg taaagaactt 420 attgtagatg atgctagcga cgtcacatca tactatccct ctgcgttcca agaacacctg 480 aactttatcc cggcgcctac tacaggatca ggttgcactc ggataccctc atttgacatg 540 agcgctaccc actactgtta tactcacaat gtgatattat ctggctgcag agatcactcg 600 cactcacatc aatatttagc acttggtgtg cttcggacat ctgcaacagg gagggtattc 660 ttttccactc tgcgttccat caatctggat gacacccaaa atcggaagtc ttgcagtgtg 720 agtgcaaccc ccttgggttg tgatatgctg tgctctaaag tcacagagac tgaagaagag 780 gattataact cagctatccc cacgtcgatg gtacatggaa ggttagggtt cgacggccaa 840 taccacgaga aggacctaga tgtcacaaca ctattcgagg actgggtggc aaactaccca 900 ggagtagggg gcgggtcttt tattgacaac cgcgtatggt tcccagttta cggagggcta 960 aaacccaatt cgcccagtga caccgcacaa gaagggaaat atgtaatata caagcgatac 1020 aatgacacat gtccagatga gcaagattat cagatcaaa tggctaagtc ttcatataag 1080 cctgggcggt ttggagggaa acgcgtacag caggccatct tatctatcaa agtgtcaaca 1140 tccttgggcg aggacccggt actgactgta ccgcccaaca cagtaacact catgggggcc 1200 gaaggcagag ttctcacagt agggacatct catttccttt atcagcgagg gtcatcatac 1260 ttctcccctg ccctactata tcctatgata gtcagcaaca aaacagccac tcttcatagt 1320 ccttatacat tcaatgcctt cactcgacca ggtagtgtcc cttgccaggc ttcagcaaga 1380 tgccctaact catgtgttac cggagtctat actgatccat atcccttggt cttctatagg 1440 aaccacacct tgcgaggggt attcgggacg atgcttgatg ataaacaagc aagactcaac 1500 cctgtatctg cagtatttga cagcatatcc cgcagtcgca taacccgggt gagttcaagc 1560 agcaccaagg cagcatacac aacatcaaca tgttttaaag ttgtaaagac caataaaacc 1620 tattgtctca gcattgccga aatatccaat accctcttcg gggaattcag aatcgtccct 1680 ttactagttg agattctcaa ggatgatggg gttagagaag ccaggtctag ccggttgagt 1740 caactgcgag agggttggaa agatgacatt gtatcaccta tcttttgcga cgccaagaat 1800 caaactgaat accggcgcga gctcgagtcc tacgctgcca gttggccata a 1851 <210> 7 <211> 6615 <212> DNA <213> Newcastle disease virus <400> 7 atggcgagct ccggtcccga gagggcggag catcagatta tcctaccaga gtcacacctg 60 tcttcaccat tagtcaagca caaactactc tattactgga aattaactgg gctaccactc 120 cctgacgagt gtgacttcga ccacctcatt ctcagccgac aatggaagaa aatacttgaa 180 tcggcctccc ctgacactga gagaatgata aaacttggaa gggcagtgca ccagactctc 240 aaccacaatt ccaagataac cggagtactc catcccaggt gtttagaaga attggctagt 300 attgaggttc ctgactcaac caacaagttt cggaagatcg agaagaaaat ccaaattcac 360 aacacaaggt atggagaact gttcacaaga ctgtgcacgc atgtagagaa gaaattgttg 420 ggatcatctt ggtctaataa tgtcccccgg tcaagaagagt tcaacagcat ccgtacagat 480 ccggcattct ggtttcactc aaaatggtcc acaactaagt ttgcatggct ccatataaaa 540 cagatcaaa ggcatctgat tgtggcagca agaacaaggt ccgcagccaa caaattggtg 600 acgctgaccc ataaggtagg ccaagtcttt gttactcctg agcttgtcat tgtgacacat 660 acagatgaga acaagttcac gtgtcttacc caggaacttg tgttgatgta tgcagatatg 720 atggagggca gagatatggt caacataata tcatccacgg cggcacatct caggagccta 780 tcagagaaaa ttgatgacat tctgcggtta gtagatgccc tggcaaaaga tctgggtaat 840 caagtctacg atgttgtagc actcatggag ggatttgcat acggcgccgt ccagctgctt 900 gagccgtcag gtacattcgc aggggatttc ttcgcattca acctgcagga gctcaaagac 960 actttgatcg gcctccttcc taaggatata gcagaatctg tgactcacgc aatagccact 1020 gtattctctg gcttagaaca aaatcaagcg gctgagatgc tgtgcctgtt gcgtctatgg 1080 ggccacccat tacttgagtc ccgtattgcg gcaaaagcag taaggagcca aatgtgcgca 1140 ccaaaaatgg tagactttga tatgatcctc caggtattgt ctttctttaa aggaacaatc 1200 atcaacggat acagaaagaa gaatgcaggt gtttggccac gtgtcaaagt agaaacgata 1260 tacgggaagg tcattgggca gctacacgct gattcagcgg agatttcaca cgatatcatg 1320 ttgagagagt acaagagttt atctgcgctt gaattcgagc catgtataga atacgaccct 1380 atcaccaatc tgagcatgtt tctaaaagac aaggcgatcg cacacccgaa agacaactgg 1440 ctcgccgcgt ttaggcgaaa ccttctctct gaggaccaga agaaacatgt aaaggaggca 1500 acctctacta accgtctctt gatagagttc ttagagtcaa atgattttga tccatataag 1560 gagatggaat atctgacgac ccttgagtac ctaagagatg acaatgtggc agtatcatac 1620 tcgctcaagg agaaggaagt gaaggttaat gggcggattt ttgctaagct aacaaagaaa 1680 ttaaggaact gtcaagtgat ggcggaaggg atcttagctg accagattgc acctttcttt 1740 caagggaatg gggtcattca ggatagcata tctttaacca agagtatgct agcgatgagt 1800 caattgtctt tcaacagcaa taagaaacgt atcactgact gcaaagaaag agtagcctca 1860 aaccgcaatc acgatcaaaa gagcaagaat cgtcggagag ttgccacttt tataacgact 1920 gacctgcaaa agtactgtct taattggaga tatcagacaa tcaaactgtt cgctcatgcc 1980 atcaatcagc tgatgggctt acctcacttc ttcgaatgga ttcatctaag actaatggat 2040 actacgatgt ttgtaggaga ccctttcaat cccccaagtg acccaactga ctgtgatctc 2100 tcaagagtcc caaatgatga catatatatt gtcagtgcta gagggggtat tgagggatta 2160 tgtcagaagc tatggacaat gatctcaatt gctgcaatcc aacttgctgc agcaagatca 2220 cattgtcgcg tcgcctgtat ggtacagggt gacaatcaag taatagctgt aacgagagag 2280 gtaaggtcag atgactcccc ggaaatggtg ttaacacaat tgcatcaagc cagtgataat 2340 ttcttcaagg aattgattca tgttaatcat ttgattggcc ataatttgaa ggatcgtgaa 2400 acaatcagat cagacacatt cttcatatac agcaaacgaa tattcaaaga tggagcaata 2460 ctcagtcaag tcctcaaaaa ttcatctaaa ttagtgctaa tatcaggcga ccttagtgaa 2520 aacaccgtaa tgtcctgtgc caacattgca tctactatag cacggctgtg cgagaacggg 2580 cttccaaagg atttctgtta tacttaaac tacctgatga gttgcgtgca gacatacttt 2640 gattctgagt tttccatcac taacagctcg caccccgatt ctaaccagtc gtggattgaa 2700 gacatctctt ttgtgcactc atatgtcctg acccctgccc agctaggggg actgagcaac 2760 ctccaatact caaggctcta cacgaggaac atcggtgacc cgggaactac tgcttttgca 2820 gagatcaagc gattagaagc agtggggtta ctaagttcta gtattatgac taacatctta 2880 actaggccgc ctggaaatgg agattgggcc agtctgtgta acgaccctta ctctttcaat 2940 tttgagactg tcgcgagtcc aaatattgtc cttaagaaac atacacaaag agtcctattt 3000 gaaacttgtt caaatccctt attatctggc gtgcatacag aggataatga ggcagaagag 3060 aaggcgttgg ctgaattttt actcaatcaa gaagtaattc atccacgtgt cgcacatgct 3120 atcatggaag caagctctat aggtaggagg aagcagattc aagggcttgt tgacacaaca 3180 aacaccgtaa tcaagattgc attgactagg aggccacttg gcatcaagag gctgatgcgg 3240 atagttaact actcgagcat gcatgcaatg ctgtttagag acgatgtttt ctcatctaac 3300 aggtctaacc accccttagt ttcctctaat atgtgttctc tgacgctagc agactatgca 3360 cggaatagaa gctggtcacc attgacgggg ggtagaaaga tactgggtgt atctaatcct 3420 gatactatag aacttgtaga gggtgagatc cttagcgtca gcggaggatg cacaagatgt 3480 gacagcggag atgaacaatt cacttggttc catcttccga gcaatataga actgaccgat 3540 gacaccagca agaatcctcc gatgagagtg ccgtacctcg ggtcaaagac tcaagagagg 3600 agggccgcct cgcttgcgaa aatagctcat atgtcaccac atgtgaaagc tgctctaagg 3660 gcatcatccg tgttgatctg ggcttatgga gacaacgaag taaattggac tgctgctctt 3720 aaaattgcaa gatctcggtg caatataaac tcagagtatc ttcgactatt gtcccccctta 3780 cccacagctg ggaatctcca acatagactg gatgacggca taactcagat gacattcacc 3840 cctgcatctc tctacagggt gtcaccttat attcacatat ccaatgattc tcaaaggtta 3900 ttcacggaag aaggagtcaa agagggaaat gtagtttatc agcaaatcat gctcttgggt 3960 ttatctctaa tcgaatcact cttcccgatg acgacaacca ggacatacga tgagatcaca 4020 ttgcacctcc acagtaaatt tagctgctgt atcagggaag caccggttgc agttcctttc 4080 gagttactcg ggatggcacc agaactaagg acagtgacct caaataagtt tatgtatgat 4140 cctagtcctg tatcggaggg tgactttgcg agacttgact tagctatctt taagagttat 4200 gagcttaatc tagaatcata tcccacaata gagctaatga acattctttc aatatccagc 4260 gggaagttaa tcggccagtc tgtggtttct tatgatgaag atacctccat aaagaatgac 4320 gccataatag tgtatgacaa cacccggaat tggatcagcg aagctcagaa ttcagatgtg 4380 gtccgcctat tcgagtatgc agcacttgaa gtgcttctcg actgttctta tcagctctac 4440 tatctgagag taagaggcct agacaatatc gtgttgtata tgagtgactt atataagaat 4500 atgccaggaa ttctactttc caacattgca gctacaatat ctcatcccat cattcattca 4560 agattgcatg cagtaggcct ggtcaatcac gacgggtcac accaacttgc agacacagat 4620 ttcatcgaaa tgtctgcaaa actattagtc tcttgcactc gacgcgtggt ctcaggttta 4680 tatgcaggga ataagtatga tctgctgttc ccgtctgtct tagatgataa cctgagtgag 4740 aagatgcttc agctgatatc tcggttatgc tgcctgtata cggtgctctt tgctacaaca 4800 agagagatcc cgaaaataag aggcttatct gcagaagaga agtgttcagt acttactgag 4860 tacctactgt cagatgctgt gaaaccatta cttagttctg agcaagtgag ctctatcatg 4920 tctcctaaca tagttacgtt cccagctaat ctatattaca tgtctcggaa gagccttaat 4980 ttgattaggg aaagagagga cagggacact atcttggcat tgttgttccc ccaagagcca 5040 ctacttgagt tccccttagt acaagatatt ggcgctcgag tgaaagatcc attcacccga 5100 caacctgcgg cgtttttaca agaattagat ttgagcgctc cagcaaggta tgacgcattt 5160 acacttagtc aggttcattc tgaacacaca tcaccaaatc cggaggacga ctacttagta 5220 cgatacctgt tcagaggaat agggaccgcg tcctcctctt ggtataaggc atctcacctt 5280 ctttctgtac ctgaggtcag atgtgcaagg cacgggaatt ccttatactt ggcagaagga 5340 agcggagcca ttatgagtct tctcgaactg catgtgccgc atgagactat ctattacaat 5400 acgctcttct caaacgagat gaacccccca cagcggcatt tcggaccgac cccaacacag 5460 tttctgaatt cagttgttta taggaatcta caggcggagg taccatgtaa ggatggattt 5520 gtccaggagt tccgtccatt atggagagag aatacagaag aaagcgatct gacctcagat 5580 aaagcagtgg gttacatcac atctgcagtg ccctaccggt ctgtatcatt gctgcactgt 5640 gacattgaga ttcctccagg atccaatcaa agcttactgg atcaactggc taccaatcta 5700 tctctgattg ccatgcattc tgtaagggag ggcggggtcg tgatcatcaa agtgttgtat 5760 gcaatgggat attacttcca tctactcatg aacttgttca ctccgtgttc tacgaaagga 5820 tatattctct ctaatggcta tgcatgtaga ggggatatgg agtgttacct ggtatttgtc 5880 atgggctatc gaggtgggcc tacatttgta catgaggtag tgaggatggc aaaaactcta 5940 gtgcagcggc acggtacact tttgtccaaa tcagatgaga tcacactgac taggttattt 6000 acctcacagc ggcagcgtgt aacagacatc ctatccagtc ctttaccgag actaataaag 6060 ttcttgagaa agaatatcga tactgcgcta attgaagccg ggggacaacc cgtccgtcca 6120 ttctgtgcag agagcttggt gaggacacta gcggacacaa ctcagatgac ccagatcatc 6180 gctagtcaca ttgacacagt cattcgatct gtgatctaca tggaggctga gggtgatctc 6240 gccgacacag tgttcttatt taccccctac aatctctcta cagacggtaa aaagagaaca 6300 tcacttaaac agtgcacaag gcagatctta gaggtcacaa tattgggtct tagagttgaa 6360 aatctcaata aagtaggtga tgtagtcagt ctagtactta aaggtatgat ttctctggag 6420 gacctgatcc ctctaagaac atacttgaag cgtagtacct gccctaagta tttgaagtct 6480 gttctaggta tactaaact caaagaaatg tttacagaca cctctttatt atacttgact 6540 cgtgctcaac aaaaattcta catgaaaact ataggcaacg cagtcaaggg atactacagt 6600 aactgtgact cttaa 6615 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> P1 primer <400> 8 accaaacaga gaatccgtga g 21 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> P2 primer <400> 9 agatgagggg gttcgtttgt ag 22 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> P4 primer <400> 10 cgcgccgtcg cagtgacc 18 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> P6 primer <400> 11 cgtttcccac cgaaccgc 18 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P10 primer <400> 12 accgacccca acacagttcc 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> P12 primer <400> 13 accaaacaaa gatttggtg 19

Claims (5)

A method for producing a recombinant vector into which the full-length gene sequence of the Newcastle disease virus strain KU001 (Accession No.: KCTC18794P) is inserted, comprising the steps of:
synthesizing cDNA using RNA extracted from the Newcastle disease virus strain KU001;
The nucleotide sequence encoding the NP protein, the nucleotide sequence encoding the P protein, the nucleotide sequence encoding the M protein, the nucleotide sequence encoding the F protein, the nucleotide sequence encoding the HN protein and the nucleotide sequence encoding the L protein are sequentially sequenced inserting into a plasmid;
including,
The recombinant vector is composed of the nucleotide sequence of SEQ ID NO: 1,
The Newcastle disease virus KU001 strain is isolated from domestic wild ducks,
The plasmid is a pSL1180 plasmid,
The recombinant vector prepared by the above production method has a ^{virus production titer that is improved 10 2 times compared to the Lasota strain or 10 5} times compared to the B1 strain, the method for producing a recombinant vector.

delete According to claim 1,
The nucleotide sequence encoding the NP protein consists of the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence encoding the P protein consists of the nucleotide sequence of SEQ ID NO: 3, and the nucleotide sequence encoding the M protein is SEQ ID NO: 4 consists of the nucleotide sequence of, the nucleotide sequence encoding the F protein consists of the nucleotide sequence of SEQ ID NO: 5, the nucleotide sequence encoding the HN protein consists of the nucleotide sequence of SEQ ID NO: 6, and the L protein A method for producing a recombinant vector, wherein the nucleotide sequence consists of the nucleotide sequence of SEQ ID NO: 7.
The full-length gene sequence of the Newcastle disease virus strain KU001 (Accession No.: KCTC18794P) is inserted into the pSL1180 plasmid, and consists of the nucleotide sequence of SEQ ID NO: 1, wherein the Newcastle disease virus KU001 strain is isolated from domestic wild ducks, and compared to Lasota strain 10 ^{2 Recombinant vector having a 10 5-} fold improved virus production titer compared to the fold or B1 strain.

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