KR20150026817A - New castle disease virus bp acnd with improved thermostable property - Google Patents

Abstract

The present invention relates to chimera new castle disease virus BP ACND with improved heat resistance. The chimera new castle disease virus of the present invention has similar antigenicity to strong pathogenicity new castle disease virus spreading in South Korea and Asia, but the pathogenicity is similar or significantly lower than vaccine strains recently used, and the heat insulation is extremely high for inducing high immunogenicity even in a real situation of inoculating vaccine into birds.

Description

NEW CASTLE DISEASE VIRUS BP ACND WITH IMPROVED THERMOSTABLE PROPERTY

The present invention relates to a Newcastle disease virus BP ACND having improved heat resistance, and more particularly to a method for producing a chimeric Newcastle disease virus having improved heat resistance, a method for producing the chimeric Newcastle disease virus, an immunogenic composition or vaccine comprising the chimeric Newcastle disease virus, The present invention relates to a method of inoculating a virus into a bird subject by spray administration.

Newcastle Disease (ND) is one of the 15 most important animal diseases in the world, acute febrile respiratory disease, a 100% deadly infectious disease of the first type when infected with unimmunized poultry.

Korea is a Newcastle disease virus (NDV) overland area, and there are considerable difficulties in eradicating this disease. In addition, as there are various Newcastle disease viruses in Southeast Asia, China, and Taiwan, which are actively trading with Korea, they are a potential risk factor and it is time to develop an Asian type Newcastle vaccine.

Newcastle disease virus is a single stranded RNA virus that belongs to the genus Avulavirus genus. The Newcastle Disease virus has an envelope and the envelope contains the Haemagglutinin-Neuraminidase (HN) protein, which allows the virus to bind to the host cell, and the F (fusion) protein, which causes the envelope and host cell fusion. The F and HN proteins are glycoproteins distributed on the surface of the envelope.

Newcastle disease virus is classified as a genotype from I to IX by the phylogenetic analysis of the base sequence of the F gene. Genotype VII is reported to be a problem in Indonesia, China, Taiwan and Japan, including Asian countries.

Newcastle disease viruses are classified as phathotypes according to disease severity in chickens: 1) velogenic NDV with intestinal lesions and a high mortality rate; And respiratory and neurological manifestations and high mortality; 2) methogenic NDV with low mortality, acute respiratory disease and neurotic symptoms in some birds; And 3) lentogenic and apathogenic NDV causing mild or asymptomatic respiratory infections.

For the Newcastle virus to become infectious to cells, a precursor glycoprotein Fo that is cleaved with F1 and F2 is needed. This post-translational cleavage is mediated by host cell proteases. Strong pathogenic and mild disease Fo proteins of NDV can be cleaved by a wide range of proteases leading to fatal systemic infections whereas Fo proteins of pharmacogenetic NDVs are cleaved only in regions with trypsin-like enzymes such as the respiratory or intestinal tract Only local infection is possible.

Amino acid degradation of the Fo precursor has a single arginine (R) at the cleavage recognition site linking the F2 and F1 chains with the pathogenic viruses, while the isolates with intermediate toxicity have cleavage recognition sites of (112) RK / RXK / RRF (R) or lysine (K) are present as a pair. In addition, the F2 chain of NDV with pathogenicity above the severity of the disease is generally initiated as a phenylalanine (F ₁₁₇ ) residue whereas the F2 chain of NDV, which is less pathogenic, is generally referred to as leucine (L ₁₁₇ ).

HN protein is a type II membrane glycoprotein that forms a tetramer on the surface of the virus envelope and penetrates the cell membrane (Gorman et al., 1988; Ng et al., 1989). The HN protein functions to position the virion on the host cell surface by binding to sialic acids of glycoconjugates.

The HN protein is divided into three regions: a transmembrane domain, a stalk domain, and a globular domain. Both the antigenic receptor binding and the neuraminidase active site are located in the spherical domain. Fusion induction activity is located in the stem domain, which interacts with the F protein (Sergei et al., 1993).

The currently commercialized Newcastle disease inactivated vaccine is produced by using the pathogenic NDV clone 30 or lasoquant, and the F and HN protein coding sequences of the pathogenic Newcastle virus developed by the present inventors by the reverse genetics technology, Inactivated vaccine (KBNP-C4152, Korean Patent Registration No. 10-0454870) substituted with F and HN protein coding sequences of Newcastle disease virus. Reverse Genetics technology of negative strand RNA viruses has been proposed as a technique for recovering infectious viruses from viral genomes (see US Patent No. 5,166,057, etc.). Although these techniques were originally proposed to manipulate the influenza virus genome, it has been proposed that the RNA viruses other than the influenza virus, such as Rabies Virus, Respiratory Syncytial Virus, Senda virus, Have been successfully applied to segmented negative strand RNA viruses.

However, in the case of the conventional inactivated vaccine (KBNP-C4152), La Sota strain, which is not heat-resistant, is used. However, since it is an environment in which an actual vaccine is used due to its low heat resistance, The function as a vaccine is weakened.

The present invention provides a chimeric Newcastle disease virus (BP ACND) having improved heat resistance. The present invention provides a method for producing the chimeric Newcastle disease virus having improved heat resistance. The present invention is intended to provide an immunogenic composition or vaccine comprising said thermostable chimeric Newcastle disease virus and at least one veterinarily acceptable carrier or excipient. The present invention also provides a method for inoculating a bird, comprising spraying and administering the chimeric Newcastle disease virus having improved heat resistance to a bird.

The chimeric Newcastle disease virus with improved heat resistance according to the present invention is a gene consisting of a sequence encoding a 3'-NP-P-M-F-HN-L-5 'protein; A leader gene linked to the 3 'terminus; And a trailer region gene linked to the 5 ' end,

The leader, NP, P, M, L and trailer region genes are derived from the pathogenic thermostable Newcastle disease virus,

The F and HN genes are derived from a strongly pathogenic Newcastle disease virus,

The 112 th amino acid sequence of the purine cleavage site 112-RRQKRF-117 of the F gene is substituted with glycine (G), and the 115 th amino acid sequence is replaced with an alanine (A) coding group selected from the group consisting of GCU, GCC, GCA, , And the 117th amino acid sequence is substituted with a leucine (L) coding group.

The chimeric Newcastle disease virus having improved heat resistance according to the present invention has antigenicity similar to that of a river pathogenic Newcastle disease virus which is prevalent in both domestic and Asian countries. However, the virulence is similar or significantly lower than that of currently used vaccines, There is an advantage of inducing immunogenicity.

Figure 1 shows the amino acid sequence of the HN linear antigen site of Newcastle disease virus.
Fig. 2 shows a process for producing a non-pathogenic thermostable chimeric NDV expression vector according to Example 2.1.
3 shows the entire nucleotide sequence of the non-pathogenic thermostable chimeric NDV expression vector prepared according to Example 2.2.
Fig. 4 shows the results of comparative analysis of the L gene nucleotide sequences in the pharmacogenetic NDV expression vector prepared according to Example 2.1 and the non-pathogenic thermostable chimeric NDV expression vector.
FIG. 5 shows a non-pathogenic NDV production process in which a heat resistant NDV L gene according to Example 3 is introduced.
Figure 6 shows RT-PCR results of non-pathogenic NDV production confirmation with heat resistant L gene introduced according to Example 3.2.1.
7 shows the hemagglutination reaction results of the non-pathogenic NDV virus in which the heat resistant L gene was introduced according to Example 3.2.2.
Figure 8 shows the stability test results for heat of a conventional vaccine strain and non-pathogenic thermostability according to Example 4.2.1.
FIG. 9 is a graph showing the cytopathic effect of virus after infecting the fetal kidney cells with each virus heat-treated at 56 ° C. according to Example 4.2.2. FIG.

Unless otherwise defined, can be performed by molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing and routine techniques commonly used in the art of recombinant DNA within the skill of those skilled in the art. These techniques are known to those skilled in the art and are described in many standardized textbooks and references.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Various scientific dictionaries, including the terms contained herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing of the present application, some methods and materials have been described. Should not be construed as limiting the invention to the particular methodology, protocols, and reagents, as they may be used in various ways in accordance with the context in which those skilled in the art use them.

As used herein, the singular forms include plural objects unless the context clearly dictates otherwise. Also, unless otherwise indicated, nucleic acids are written from left to right, amino acid sequences from left to right, amino to carboxyl.

The numerical range includes numerical values defined in the above range. All numerical limitations of all the maximum numerical values given throughout this specification include all lower numerical limitations as the lower numerical limitations are explicitly stated. All the minimum numerical limitations given throughout this specification include all higher numerical limitations as the higher numerical limitations are explicitly stated. All numerical limitations given throughout this specification will include all better resin ranges within a broader numerical range, as narrower numerical limitations are explicitly stated.

The subject matter provided herein should not be construed as limiting the following embodiments in various aspects or as a reference throughout the specification.

Expression vector

According to one embodiment, a gene consisting of a sequence encoding a 3'-NP-PMF-HN-L-5 'protein, a leader gene linked to the 3' end, and a trailer region linked to the 5 ' a chimeric Newcastle disease expression vector comprising the trailer region gene is provided.

The term " chimeric " or " recombinant " as used herein in reference to a nucleic acid, protein or vector means that a nucleic acid, protein or vector is modified by introduction of a heterologous nucleic acid or protein or by alteration of a native nucleic acid or protein . Thus, chimeras and recombinant vectors can include nucleic acid sequences that are not found in the native (non-chimeric or non-recombinant) form of the vector. The chimeric Newcastle disease expression vector refers to a Newcastle disease expression vector comprising a nucleic acid sequence encoding a heterologous polypeptide.

The term " expression vector " as used herein refers to a nucleic acid construct recombinantly or synthetically produced through a series of specified nucleic acid elements capable of transcribing a particular nucleic acid in a host cell. The expression vector may be a plasmid, a virus or a fragment. Typically, the expression vector comprises a nucleic acid to be transcribed that is operably linked to a promoter. The expression vector may also include other forms of vectors having functions equivalent to those known or known in the art, for example, replication vectors, shuttle vectors, plasmids, phage or viral particles, DNA constructs, and cassettes .

As is well known in the art, in order to increase the level of expression of a gene introduced in a host cell, the gene must be operably linked to transcriptional and detoxification control sequences that function in a selected expression host. For example, an expression control sequence and the gene are contained within an expression vector containing a selectable marker and a replication origin. If the expression host is a eukaryotic cell, the expression vector should further include a useful expression marker in the eukaryotic expression host.

As used herein, the term "gene" refers to a 5 'untranslated (5' UTR) or leader sequence as well as the preceding and succeeding coding regions, such as the intervening sequence between the respective coding fragments (exons) Quot; means a polynucleotide sequence that may or may not include a traunt sequence (3 'UTR) or trailer sequence. "Heterologous gene" means that it does not occur naturally in the host cell. The heterologous gene may be a naturally occurring gene, a mutant gene, or a synthetic gene. "Homologous gene" means native or naturally occurring in host cells.

As used herein, the term " promoter " means a nucleic acid control sequence array that directs the transcription of a nucleic acid. The promoter may optionally include a distal enhancer or repressor element that may be located at a distance of a few thousand base pairs from the transcription start site. Promoters include constitutive and inducible promoters. A "constitutive" promoter is a promoter that is active in most environmental and developmental conditions, and an "inducible" promoter is a promoter that is active under environmental or developmental conditions.

As used herein, the term " terminator " refers to a nucleic acid sequence that serves to bring about the end of transcription or to effect it.

As used herein, the term " operably linked " means that, when affecting transcription of a gene, the promoter sequence affects the transcription of the sequence encoding the gene.

The terms " nucleic acid " and " polynucleotide ", as used herein, are used interchangeably and refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses known nucleotide analogs or modified backbone residues or linkages, including synthetic, naturally occurring, and non-covalently occurring, having similar binding properties as the reference nucleic acid, similar to the reference nucleotide Lt; RTI ID = 0.0 > metabolism. ≪ / RTI > Unless otherwise indicated, a particular nucleic acid sequence also includes conservatively modified variants thereof (e. G., Degenerate codon substitutions) and complementarity sequences as well as well-defined sequences. Specifically, it is possible to achieve degenerative codon substitution by generating a sequence in which the third position of one or more selected or all codons is substituted with a mixed-base and / or deoxyinosine residue. &Quot; Nucleic acid " is used interchangeably with genes, cDANs, mRNAs, oligonucleotides and polynucleotides.

As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably, meaning a polymer of amino acid residues. The term includes both naturally occurring and non-naturally occurring amino acid polymers as well as amino acid polymers that are artificial chemical mimics of the corresponding naturally occurring amino acids with which one or more amino acid residues are corresponding.

According to this embodiment, the purine cleavage site of the F gene may have an amino acid sequence represented by 112-GRQARL-117.

When the purine cleavage site of the F gene has the amino acid sequence as described above, recognition and cleavage by purine hardly occur and cleavage occurs only by locally existing intracellular or extracellular proteolytic enzymes. Thus, fatal systemic infection The pathogenicity can be lowered.

(G) of the purine cleavage site 112-RRQKRF-117 of the F gene, and the 115th amino acid sequence is substituted with an alanine (A) coding group selected from the group consisting of GCU, GCC, GCA and GCG And the 117th amino acid is substituted with a leucine (L) coding group.

The F gene has a molecularly attenuated genotype VII virus, which represents a virulent Newcastle disease virus in Korea, and 112-RQQR-117 as 112-GRQARL-117 so that the protease recognition site of the F gene is non-pathogenic. Designed artificial synthetic genes were used.

According to this embodiment, the sequence encoding the HN protein is derived from a strongly pathogenic Newcastle disease virus, and an artificial synthetic gene designed to have the characteristics of the gene of the VII type Newcastle disease virus was used.

According to this embodiment, the sequences encoding the NP, P, M and L proteins are derived from the pathogenic thermostable Newcastle disease virus. In one embodiment, NP, P, M and L proteins derived from the BP- Respectively. The pharmacologically resistant thermostable Newcastle disease virus may be selected from the group consisting of Type I or Type II Newcastle disease virus strains.

According to this embodiment, the leader gene linked to the 3 'terminal and the trailer region gene linked to the 5' terminal may each include a leader and trailer region gene derived from the pathogenic thermostable Newcastle disease virus. The pharmacologically resistant thermostable Newcastle disease virus may be selected from the group consisting of Type I or Type II Newcastle disease virus strains. In one embodiment, the BP-AT attention leader gene and the trailer region gene were used.

According to this embodiment, the chimeric Newcastle disease expression vector may be BP-ACND (Accession No: KCTC12444BP), which has the same antigenicity as an outdoor river pathogenic strain but has no pathogenicity and heat resistance.

According to another embodiment, there is provided a chimeric Newcastle disease virus comprising an expression vector according to this embodiment. Specifically, the chimeric Newcastle disease virus according to the present invention comprises a gene consisting of a sequence encoding a 3'-NP-P-M-F-HN-L-5 'protein; A leader gene linked to the 3 'terminus; And a trailer region gene linked to the 5 ' end,

The leader, NP, P, M, L and trailer region genes are derived from the pathogenic thermostable Newcastle disease virus,

The F and HN genes are derived from a strongly pathogenic Newcastle disease virus,

The 112 th amino acid sequence of the purine cleavage site 112-RRQKRF-117 of the F gene is substituted with glycine (G), and the 115 th amino acid sequence is replaced with an alanine (A) coding group selected from the group consisting of GCU, GCC, GCA, , And the 117th amino acid sequence may be substituted with a leucine (L) coding group.

The chimeric Newcastle Disease virus according to the above embodiment is a virus which has a complete loss of immunogenicity within 25 minutes after heat treatment at a temperature of 50 to 60 ° C, but more than 30 minutes and more than 40 minutes after heat treatment at a temperature of 50 to 60 ° C , 50 minutes, or even 60 minutes or more. Therefore, the chimeric Newcastle disease virus according to the present invention has a remarkably improved heat resistance.

The immunogenic composition or vaccine

According to another embodiment, there is provided an immunogenic composition or vaccine comprising a chimeric Newcastle virus according to the invention and at least one veterinarily acceptable carrier or excipient.

The term " immunogenic composition " as used herein refers to any composition that induces an immune response to an antigen or immunogen of interest expressed from a Newcastle disease expression vector of the invention, for example, A composition capable of eliciting an immune response against a target immunogen or antigen of interest. The term " vaccine " refers to a composition that induces or defends a protective immune response against an antigen of interest, such as, for example, a protective immune response against a target antigen or immunogen Or provide effective protection against an antigen or immunogen expressed from a Newcastle disease expression vector of the invention.

In such an embodiment, the veterinarily acceptable carrier or diluent is well known in the art including, but not limited to, sterile water, saline solution or phosphate buffer.

In this embodiment, the vaccine may include, but is not limited to, a subunit vaccine, a vector vaccine, a chimeric vaccine, and a DNA vaccine produced using a gene of a virulence vaccine, a sadox vaccine, a Newcastle disease virus strain.

Inoculation method

There is provided a method of inoculating an algae subject, comprising spraying an immunogenic composition or vaccine comprising a chimeric Newcastle disease virus according to the present invention to a bird subject.

As used herein, " algae " means any and all domestic and wild birds belonging to the Aves River, including, but not limited to, newborn and alveoli. The newest nomenclature is composed of geese, goose neck, buxalifomes, rhubarb, thyme, hornbill, chickpea, pigeon neck, bluebird neck, cuckoo neck, Pine tree neck, pine tree neck, shrimp neck, silkworm's neck, hoopoe's neck and so on, sofas, bamboo shoots, office tocopomes, Neck and the like. The alpine tops include kiwifruit, vetch, air conditioning, lea neck, ostrich neck, and tinia mimetic. The milking object may include mature algae, bird algae and avian embryos / eggs.

In such embodiments, the immunogenic compositions may be administered by in the ovarian, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, conjunctival, and subcutaneous routes, It is not. In particular, the immunogenic compositions according to the present invention have good heat resistance and can therefore be adapted to the route of collective administration for agricultural purposes, such as through drinking water or spraying.

Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited by the following examples.

≪ Example 1 > Newcastle disease virus gene selection

1.1. Selection of F, HN gene from a pathogenic antigenic mutant

SNU4152 strain isolated from the Bird Disease Department of Veterinary Medicine, Seoul National University was selected as a virus that can represent the Newcastle disease virus in Korea.

We analyzed molecular epidemiology and antigenic determinants of Korean Newcastle disease virus in 65 domestic isolates. The results of the systematic analysis of the nucleotide sequence using the Neighbor-Joining method for the isolated F gene diversity region (384 bp) are as follows.

The amino acid sequence of the HN linear antigen site of Newcastle disease virus is shown in FIG.

1.2. Reduced F, HF Gene Synthesis

The cleavage site of the F gene of the river pathogenic SNU 4152 Newcastle disease virus selected in Example 1.1 was designed to have a non-pathogenic amino acid codon as 112-GRQARL-117, and the genotype VII type NDV F, HN gene was synthesized.

1.2.1 Domestic Separation Heat Resistance The main BP-AT nucleotide sequence analysis

In order to clone NP, P, M and L genes of non-pathogenic thermostable Newcastle disease virus, the nucleotide sequence of the corresponding gene part of BP-AT virus was analyzed. The corresponding nucleotide and amino acid sequences of the BP-AT strain are shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4

1.2.2. Cloning NP, P, M and L genes of BP-AT

NP, P, and ML gene, leader, and trailer regions of the BP-AT gene for the production of the non-pathogenic heat-resistant Newcastle disease virus expression vector were PCR-cloned. At this time, the AvrII restriction enzyme recognition site used for L gene introduction of heat resistant NDV was removed from the M gene of BP-AT using Overlap extension PCR method, and the recognition site of PacI restriction enzyme used for introducing BP-AT trailer sequence was designated as T7 promoter sequence and the trailer sequence of the vector at the 5 'end of the pathogenic pTMH-Lasota expression vector was replaced with the BP-AT sequence using the Counter-Selection method of the Red / ET Recombination Kit (Gene bridge).

<Example 2> Preparation of non-pathogenic thermostable Newcastle disease virus expression vector

2.1. A non-pathogenic thermostable vector as a basic back bone

Replacing the pTMH-LaSota eukaryotic cell expression vector prepared in the same manner as the 10-0862049 patent registered by the same inventor with previously cloned genes to make a non-pathogenic heat-resistant chimeric NDV expression vector having outdoor antagonistic antigenicity . The restriction enzyme used in the replacement and the replacement sequence are shown in Fig.

2.2. Sequence analysis of chimeric Newcastle disease virus expression vector

The entire nucleotide sequence of the constructed chimeric NDV expression vector was analyzed using the following primer sets and the results are shown in FIG. The nucleotide sequences before and after the introduction of the L gene are shown in FIG. As can be seen from FIG. 4, it was confirmed that the recombinant non-pathogenic NDV vector L gene sequence (below) with heat resistant NDV L gene was different from the nucleotide sequence of the pathogenic NDV vector before heat resistant L gene introduction (above).

Primer Primer sequence (5- > 3) Uls8661F GTT TAG AAG AAC TGG CTA GT (SEQ ID NO: 5) Uls9381-F TGA CTC ACG CAA TAG CCA CT (SEQ ID NO: 6) Uls10092 TCT TAG CTG ACC AGA TTG CA (SEQ ID NO: 7) Uls11952 CGT ATC TCG GGT CAA AGA CT (SEQ ID NO: 8) Uls12674 GAT GAA GAT ACC TCC ATA AAG (SEQ ID NO: 9) Uls13462 GAA AGA TCC ATT TAC CCG ACA (SEQ ID NO: 10) Uls10691 ATG GTG TTA ACA CAA TTG CAT C (SEQ ID NO: 11) Uls11290-F GCC AGT CTG TGT AAC GAC C (SEQ ID NO: 12) LaUls14357-F ATC AGA TGA GAT CAC ACT GAC (SEQ ID NO: 13) La4941-F ACA GCG GCA CAG ATA ACA GCA (SEQ ID NO: 14) La5545-F AGC TCG ACA CCT CAT ACT GCA (SEQ ID NO: 15) LaUls-6183-F GAT CAG ATG AGA GCC ACT ACA (SEQ ID NO: 16) La6844-F AGT GGA CGA CAT CAG TGA TGT (SEQ ID NO: 17) Uls8264-R CAA CTG GCA GCG TAG GAC TCG (SEQ ID NO: 18) La60-F GTA GAA GGT GTG AAT CTC GAG (SEQ ID NO: 19) La755-F GTA TGC AGG AGC ACA ATC CAA (SEQ ID NO: 20) La2026-R AGC ACC TTG GTC TTC CCT TGT (SEQ ID NO: 21) La15396-F ATG CGG TGT GAA ATA CCG CAC (SEQ ID NO: 22) La16846-R CTT CTG ACA ACG ATC GGA GGA (SEQ ID NO: 23) La16712-F ATC GTG GTG TCA CGC TCG TCG (SEQ ID NO: 24) La17372-F AGT GCC ACC TGA CGT CTA AGA (SEQ ID NO: 25) La7989-F AGC ATA CAC GAC ATC GAC ATG (SEQ ID NO: 26) La264-R TAT CTT CTG GGT CAT CAC TGT (SEQ ID NO: 27) La4010 CTT ATG ACC ACC GTA GAT AGG (SEQ ID NO: 28) La1901 GAT GCA GAG ATC GAC GAG C (SEQ ID NO: 29) La2581 AGG CGA TAT CAC AGA GAG TA (SEQ ID NO: 30) La3271 GTG CCC CAA TTG TGC CAA G (SEQ ID NO: 31)

Example 3 Production of non-pathogenic thermostable NDV

From the recombinant full-length NDV cDNA vector, which replicates the entire NDV gene, ND virus having heat resistance representing the pathogenic antigenicity of the attenuated strain was constructed as shown in FIG.

3.1. Generation of Chimera NDV

Hep-2 cell lines were grown in a 6-well plate at 80% and infected with the vaccinia T7 virus. It is then initiated by the T7 promoter in the cell line and initiated by the pCR-TM-NP, pCR-TM-P, pCR-TM-L plasmid vectors and T7 promoter, And pTMH-HT-CNDr, which is a plasmid capable of producing a complete chimeric heat resistant NDV genome, was prepared. Were mixed at a ratio of 1: 1: 0.1: 1 and mixed with Lipofectamine ^TM (Invitrogen. Then, 1 ug / ml of acetylated trypsin was added to induce heat-resistant non-pathogenic recombinant virus to become infectious. After culturing for 2-3 days at 37 ° C, 6 wells of cells were harvested and infectious NDV was obtained by inoculation on SPF developmental bones at 11 days of age.

3.2. Virus verification experiment

After 11 hours of SPF development, inoculation was carried out every 24 hours to confirm the insult. After 72 hours of inoculation, the inoculated cells were refrigerated at 4 ° C, and vaginal fluid was sampled to confirm the virus.

3.2.1. RT-PCT

To 150 μl of urethral lavage, 150 μl of Virus genome Ex Kit (BIOPOA) lysis buffer was added, homogeneously mixed and allowed to stand at room temperature for 10 minutes. To the column, 300 μl of binding buffer was added and homogenously mixed. The column was washed with washing buffer A and B, and the RNA attached to the column was eluted with elution buffer.

2 ㎕ of RNA of the extracted heat-resistant main virus was amplified by the ONE-STEP RT-PCR Kit (iNtRON) using the following NDVcomF / NDVcomR primer at 45 캜 / 30 min-94 캜 / 5 min- (94 캜 / 20 sec-50 캜 / The RNA was extracted with MG-taq polymerase (macrogen) using NDVcomF / NDVcomR primer to confirm the false positive reaction by the DNA used for transfection. , And the results are shown in Fig. 6: &lt; RTI ID = 0.0 &gt;

Virus detection RT-PCR: 45 ° C / 30min-94 ° C / 5min- (94 ° C / 20sec-50 ° C / 15sec-72 ° C / 30sec) 30cycles

False positive confirmation PCR: 94 ° C / 5min- (94 ° C / 20sec-50 ° C / 15sec-72 ° C / 30sec) 30cycles

primer primer sequence (5- > 3) NDVcomF ATA CAC CTC RTC YCA GAC AG (SEQ ID NO: 32) NDVcomR TGC CAC TGM TAG TTG YGA TA (SEQ ID NO: 33)

3.2.2. Hemagglutination reaction

The extracted urogenital fluid was reacted with chicken blood cells to identify recombinant NDV virus produced by hemagglutination. The hemagglutination reaction of the non-pathogenic NDV virus into which the produced heat-resistant L gene was introduced After the first production confirmation, the virus was cultured through the hemagglutination reaction two times, three times in the subculture, and the result was shown in FIG. 7 .

Example 4 Characterization of Chimeric Newcastle Disease Virus

4.1. Pathogenicity analysis

Measurement of Intracerebral Pathogenicity Index (ICPI)

Each 10 day old chicks is used, Isolator is made of hard paper sealed box and the back side is glass-wool filter to prevent cross-contamination of virus by air filtration. . The NDV used for the test was inoculated into the fetal membranes of the fetal kidney, and 50 μl of the viral solution obtained by diluting 1 μl of sterile saline into the retinas of the dead fetus was inoculated into the brain at a rate of 5 mm on the back of the head using a 1 cc syringe. Were placed in an isolator, and the number of pathogenesis and mortality was recorded every day for 8 days. The pathogenicity index in the brain was determined according to Alexander 's method. That is, the normal chick was scored as 0, the chick with the disease as 1, and the dead chick as 2, and the total score was divided by 80 for a total of 8 days. The pathogenicity was classified as 0.0 ~ 0.2, the pathogenicity was 0.2 ~ 0.5, the severity was 1.0 ~ 1.5, and the disease severity was 1.5 ~ 2.0. In order to utilize the virus as a vaccine in Korea, the virus should have a virulence index of 0.5 or less. Therefore, chimeric viruses ranging from 0.0 to 0.5 were selected. The results are shown in Table 3 below.

No. Strain Clinical Days Score (live) view One 2 3 4 5 6 7 8 One BP-ACND Sign 0 0 0 0 0 0 0 0 Dead 0 0 0 0 0 0 0 0 Normal 10 10 10 10 10 10 10 10 Total 0 0 0 0 0 0 0 0 0 2 Avinew Sign 0 0 One 2 One One 0 0 Dead 0 0 0 One 3 4 5 5 Normal 10 10 9 7 6 5 5 5 Total 0 0 One 4 7 9 10 10 0.26 3 KBNP-C4152 Sign 0 0 0 0 0 0 0 0 Dead 0 0 0 0 0 0 0 0 Normal 10 10 10 10 10 10 10 10 Total 0 0 0 0 0 0 0 0 0 Scoring: normal = 0, sick = 1, dead = 2 ICPI = score number / Asymptomatic: 0.0-0.2, lentogenic: 0.2-0.5, mesogenic: 1.0-1.5, velogenic: 1.5-2.0

As can be seen from the above Table 3, the ICPI of the produced BP-ACND ND virus is lower than the vaccine strain Avinew's 0.26 ICPI, confirming that it is a safe virulent vaccine candidate.

4.2. Heat resistance test

4.2.1. Stability test of non-pathogenic thermostable virus against heat

The change of each hemocyte cohesion ability after heat treatment at 56 캜 was measured, and the result is shown in Fig. As can be seen from FIG. 8, the heat-resistant BP-AT isolated from the field showed hemagglutination ability up to 20 minutes, and all of the existing vaccine strains KBNP-C4152 and LaSota lost their hemagglutination ability at 56 ° C., , The blood coagulation ability of BP-ACND was observed for up to 10 minutes, indicating that heat resistance was obtained.

4.2.2. Cytotoxic effect test of heat-treated non-pathogenic thermostable virus

In order to confirm whether BP-ACND exhibits heat resistance, a test was performed to infect a fetal kidney cell with a virus to confirm the cytopathic effect of the virus, and the results are shown in FIG.

As can be seen from FIG. 9, in the case of KBNP-C4152, the infectivity completely disappeared after 25 minutes of heat treatment, but in case of BP-ACND, 25% of infectivity remained to 60 minutes, indicating that the heat resistance was remarkably improved.

Korea Biotechnology Research Institute KCTC12444BP 20130715

<110> BioPoA, Inc. <120> NEW CASTLE DISEASE VIRUS BP ACND WITH IMPROVED THERMOSTABLE          PROPERTY <130> DPP-2013-0283 <150> KR 13 / 102,060 <151> 2013-08-28 <160> 33 <170> Kopatentin 2.0 <210> 1 <211> 1470 <212> DNA <213> BP-AT NP gene <400> 1 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> 2 <211> 1188 <212> DNA <213> BP-AT P gene <400> 2 atggccacct ttacagatgc ggagatcgac gagctatttg agaccagtgg aactgtcatt 60 gacagcataa ttacggccca gggaaaacca gtagagactg ttggaaggag tgcaatccca 120 caaggcaaaa ctaaggcttt gagcgcagca tgggagaagc atgggagcat ccagtcacca 180 gccagccaag acacccctga tcgacaggac agatcagata aacaactgtc cacacccgag 240 caagcgagtc caaacgacag ccccccagcc acatccactg accagcctcc cactcaggct 300 gcagatgagg ccggcgatac acagctcaag accggagcaa gcaactctct gctgtcgatg 360 cttgataaac tcagcaataa gtcatctaat gctaaaaagg gcccagggtc gagccctcaa 420 gaaaggcatc atcaacgtct gactcaacaa caggggagtc aacaaagccg cggaaacagc 480 caagagagac cgcagaacca ggccaaggcc atccctggaa accaggtcac agacgcgaac 540 acagcatatc atggacaatg ggaggagtca caactatcag ctggtgcaac ccatcatgct 600 ctccgatcag agcagagcca agacaatact cctgcacctg tggatcatgt ccagctacct 660 gtcgactttg tgcaggcgat gatgtctatg atggaggcga tatcacagag ggtaagtaaa 720 gttgactatc agctggacct tgtcttgaaa cagacatctt ctatccccat gatgcggtct 780 gaaatccagc agctgaaaac gtctgttgcg gtcatggaag ccaatttggg catgatgaag 840 atcctggacc ctggttgtgc caacgtttca tctctaagtg atctacgggc agttgcccga 900 tcccacccgg ttttaatttc tggccccgga gacccatctc cttatgtgac ccaagggggc 960 gaaatggcac tcaataaact ttcgcaaccg gtgcaacacc cctctgaatt gattaaaccc 1020 gccacggcaa gcgggcctga tataggagtg gagaaagaca ctgtccgtgc attgatcatg 1080 tcacgcccta tgcatccgag ctcttcagct aggctcttga gcaaactgga cgcagccgga 1140 tcgattgagg aaatcagaaa aatcaagcgc cttgcactga atggctaa 1188 <210> 3 <211> 1095 <212> DNA <213> BP-AT M gene <400> 3 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> 4 <211> 6615 <212> DNA <213> BP-AT L gene <400> 4 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 tcagaagagt tcaacagcat ccgtacagat 480 ccggcattct ggtttcactc aaaatggtcc acaactaagt ttgcatggct ccatataaaa 540 cagattcaaa 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 agatacgata 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 ttacttaaac 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 ctaagtccta 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 gcatcgtccg tgttgatctg ggcttatgga gacaacgaag taaattggac tgctgctctt 3720 aaaattgcaa gatctcggtg caatataaac tcagagtatc ttcgactatt gtccccctta 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 cggtaggcct ggtcaatcac gacgggtcac accaacttgc agacacagat 4620 ttcatcgaaa tgtctgcaaa actattagtc tcttgcactc gacgtgtggt 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 taccaatctg 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 ttactaaact caaagaaatg tttacagaca cctctttatt atacttgact 6540 cgtgctcaac aaaaattcta catgaaaact ataggcaacg cagtcaaggg atactacagt 6600 aactgtgact cttaa 6615 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 5 gtttagaaga actggctagt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Uls9381-F <400> 6 tgactcacgc aatagccact 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Uls10092 <400> 7 tcttagctga ccagattgca 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 8 cgtatctcgg gtcaaagact 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Uls13462 <400> 9 gatgaagata cctccataaa g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 10 gaaagatcca tttacccgac a 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 11 atggtgttaa cacaattgca tc 22 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 12 gccagtctgt gtaacgacc 19 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 13 atcagatgag atcacactga c 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 14 acagcggcac agataacagc a 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 15 agctcgacac ctcatactgc a 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 16 gatcagatga gagccactac a 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 17 agtggacgac atcagtgatg t 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 18 caactggcag cgtaggactc g 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 19 gtagaaggtg tgaatctcga g 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 20 gtatgcagga gcacaatcca a 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 21 agcaccttgg tcttcccttg t 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 22 atgcggtgtg aaataccgca c 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 23 cttctgacaa cgatcggagg a 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 24 atcgtggtgt cacgctcgtc g 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 25 agtgccacct gacgtctaag a 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 26 agcatacacg acatcgacat g 21 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 27 tatcttctgg gtcatcactg t 21 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 28 cttatgacca ccgtagatag g 21 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 29 gatgcagaga tcgacgagc 19 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 30 aggcgatatc acagagagta 20 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 31 gtgccccaat tgtgccaag 19 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 32 atacacctcr tcycagacag 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer sequence <400> 33 tgccactgmt agttgygata 20

Claims (10)

A gene consisting of a sequence encoding a 3'-NP-PMF-HN-L-5 'protein; A leader gene linked to the 3 'terminus; And a trailer region gene linked to the 5 ' end,
The leader, NP, P, M, L and trailer region genes are derived from the pathogenic thermostable Newcastle disease virus,
The F and HN genes are derived from a strongly pathogenic Newcastle disease virus,
The 112 th amino acid sequence of the purine cleavage site 112-RRQKRF-117 of the F gene is substituted with glycine (G), and the 115 th amino acid sequence is replaced with an alanine (A) coding group selected from the group consisting of GCU, GCC, GCA, , And the 117th amino acid sequence is substituted with a leucine (L) coding group.
The method according to claim 1,
Wherein the pharmacologically resistant thermostable Newcastle disease virus is a Type I or II Newcastle disease virus strain.
3. The method of claim 2,
Wherein the pharmacologically resistant thermostable Newcastle disease virus is a BP-AT strain, the thermostable chimeric Newcastle disease virus.
The method according to claim 1,
Wherein the Strong Pathogenic Newcastle Disease Virus is selected from the group consisting of Type VII Newcastle Disease virus strains.
The method according to claim 1,
Wherein the chimeric Newcastle disease virus is BP-ACND (Accession No .: KCTC12444BP).
The method according to claim 1,
Wherein the chimeric Newcastle disease virus maintains the infectivity for at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes after the heat treatment at a temperature of 50 to 60 占 폚.
7. An immunogenic composition or vaccine comprising a thermostable chimeric Newcastle virus according to any one of claims 1 to 6 and at least one veterinarily acceptable carrier or excipient.
8. The method of claim 7,
Wherein the vaccine is selected from the group consisting of a subunit vaccine, a vector vaccine, a chimeric vaccine, and a DNA vaccine produced using a gene of a virulence vaccine, a sadox vaccine, a Newcastle disease virus strain.
A method of vaccinating a bird, comprising spraying an immunogenic composition or vaccine comprising a chimeric Newcastle disease virus having improved heat resistance according to any one of claims 1 to 6 to a bird.
10. The method of claim 9,
Wherein the algae comprises mature algae, algae, avian embryos or avian eggs.

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