Pigeon-derived Newcastle disease virus recombinant vaccine strain and construction method and application thereof
阅读说明：本技术 一种鸽源新城疫病毒重组疫苗株及其构建方法和应用 (Pigeon-derived Newcastle disease virus recombinant vaccine strain and construction method and application thereof ) 是由 于晓慧 王静静 刘华雷 舒波 李峥 李阳 蒋文明 于 2021-10-08 设计创作，主要内容包括：本发明提供一种鸽源新城疫病毒重组疫苗株,其中用于构建鸽源新城疫病毒重组疫苗株的过程中使用的基因片段包含了毒性致弱的鸽源新城疫病毒的囊膜糖蛋白F蛋白基因和HN蛋白基因。其中一种具体的鸽源新城疫病毒重组弱毒疫苗株的保藏编号为CGMCC No.21900。本发明提供的鸽源新城疫病毒重组疫苗株在鸡胚中具有高生长滴度和低致病力的生物学特性,遗传稳定,对鸽源新城病毒具有良好的免疫保护效果,并能有效抑制排毒,能够用于防控目前流行的鸽源新城疫病毒,填补了我国缺少鸽新城疫防控专用疫苗产品的空白。(The invention provides a pigeon-derived newcastle disease virus recombinant vaccine strain, wherein a gene fragment used in the process of constructing the pigeon-derived newcastle disease virus recombinant vaccine strain comprises an envelope glycoprotein F protein gene and a HN protein gene of a pigeon-derived newcastle disease virus with attenuated toxicity. The preservation number of one specific pigeon source newcastle disease virus recombinant attenuated vaccine strain is CGMCC No. 21900. The recombinant vaccine strain of the pigeon-derived newcastle disease virus provided by the invention has the biological characteristics of high growth titer and low pathogenicity in chicken embryos, is stable in heredity, has a good immune protection effect on the pigeon-derived newcastle disease virus, can effectively inhibit toxin expelling, can be used for preventing and controlling the existing popular pigeon-derived newcastle disease virus, and fills the gap that China lacks a special vaccine product for preventing and controlling the pigeon newcastle disease.)
1. A gene segment for constructing a recombinant attenuated vaccine strain of a pigeon-derived newcastle disease virus is characterized by comprising an envelope glycoprotein F protein gene and a HN protein gene of the attenuated pigeon-derived newcastle disease virus; the envelope glycoprotein F protein gene of the pigeon source newcastle disease virus with weakened toxicity is obtained by mutating a site 112R-R-Q-K-R-F117 of an F gene with an amino acid sequence of SEQ ID NO. 1 into 112G-R-Q-G-R-L117.
2. The gene fragment of claim 1, wherein the HN protein gene has the amino acid sequence of SEQ ID NO 2.
3. The gene fragment of claim 1, wherein the HN protein gene has the amino acid sequence of SEQ ID NO 3.
4. The gene fragment of claim 1, wherein the nucleotide sequence of the gene fragment is SEQ ID NO. 4.
5. The use of the gene fragment of any one of claims 1-4 in the construction of recombinant attenuated pigeon-derived newcastle disease virus vaccine strains.
6. A recombinant attenuated vaccine strain of pigeon-derived Newcastle disease virus, which is constructed by replacing the gene fragment of the corresponding part of the genome of the gene type II LaSota strain with the gene fragment according to any one of claims 1 to 4.
7. The recombinant attenuated pigeon-originated newcastle disease virus vaccine strain according to claim 6, wherein the nucleotide sequence of the genome of the genotype II LaSota strain is SEQ ID NO. 5.
8. The recombinant attenuated pigeon-originated newcastle disease virus vaccine strain according to claim 6 or 7, which is constructed by the method comprising the following steps:
1) constructing a recombinant plasmid containing the genome full-length cDNA of the gene II type Newcastle disease virus LaSota strain;
2) on the recombinant plasmid constructed in 1), replacing a corresponding partial fragment of the LaSota strain genome with the gene fragment of any one of claims 1-4 to obtain a full-length recombinant plasmid;
3) co-transfecting the full-length recombinant plasmid constructed in the step 2) and eukaryotic expression plasmids expressing genes NP, P and L of the Newcastle disease virus LaSota strain to cells capable of expressing T7 polymerase to obtain the pigeon-sourced Newcastle disease virus recombinant attenuated vaccine strain.
9. The recombinant pigeon derived newcastle disease virus attenuated vaccine strain according to claim 6, wherein the preservation number of the recombinant pigeon derived newcastle disease virus attenuated vaccine strain is CGMCC No. 21900.
10. A vaccine, wherein the antigen of said vaccine comprises the recombinant attenuated vaccine strain of pigeon-derived newcastle disease virus according to any of claims 6-9.
Technical Field
The invention belongs to the field of biological products for livestock, and particularly relates to a pigeon-derived Newcastle disease virus recombinant vaccine strain and a construction method and application thereof.
Background
Newcastle Disease (ND) is an important infectious disease caused by infection of birds with a virulent strain of Newcastle Disease Virus (NDV). The disease is a highly-contact acute septic infectious disease which is characterized by respiratory tract and digestive tract injuries as typical characteristics, has high transmission speed and high disease death rate, and causes huge economic loss to the poultry industry in the world. The world animal health Organization (OIE) ranks the Newcastle disease as an animal epidemic disease which is legally reported, and the national agricultural rural area ranks the Newcastle disease as a type of animal epidemic disease.
The NDV is a single-strand, negative-strand and nonsegmented RNA virus belonging to the family Paramyxoviridae and has a genome structure of 3 '-NP-P-M-F-HN-L-5'. The virus can infect more than 240 kinds of birds, and the domestic chicken and the guinea fowl are most susceptible to infection, but in recent years, the host range of Newcastle disease virus infection and pathogenesis tends to be expanded.
Pigeon Newcastle disease, commonly known as Pigeon plague, is a highly contagious disease caused by Pigeon paramyxovirus (1, PPMV-1, Gene type VI NDV) and has a clinical manifestation very similar to that of chicken Newcastle disease. The new castle disease of the pigeons is found in Germany in the last 60 th century, and is introduced into China in the 80 th century, and along with the vigorous development of the pigeon breeding industry in China, the new castle disease of the pigeons becomes a main disease threatening the pigeon breeding industry, and the healthy development of the pigeon breeding industry in China is seriously hindered.
Vaccine immunization is an important means for preventing newcastle disease, and currently widely used newcastle disease vaccines mainly comprise Mukuswwar I vaccine, B1 II vaccine, LaSota IV vaccine and V4 live vaccines, A-VII oil emulsion inactivated vaccine, LaSota oil emulsion inactivated vaccine and the like. The vaccines are developed for preventing and controlling the newcastle disease, but no vaccine product aiming at the newcastle disease exists in China at present, and the pigeon farm can only use the newcastle disease vaccines such as LaSota to immunize pigeon groups. However, the pigeon I-type paramyxovirus PPMV-1 belongs to gene VI type NDV, has a longer genetic distance with the vaccine strain, has obvious antigenic difference with the LaSota strain, and still can outbreak pigeon Newcastle disease in immune pigeon groups during production, which indicates that the risk of immune failure exists in the prevention and control of the pigeon Newcastle disease by using the chicken Newcastle disease vaccine. Therefore, in order to promote the healthy development of the pigeon industry in China, the development of the special vaccine for preventing and controlling the new castle disease of the pigeons has important significance.
Disclosure of Invention
The invention aims to construct and obtain a pigeon-derived Newcastle disease virus recombinant vaccine strain and a construction method and application thereof, thereby solving the problem that a pigeon Newcastle disease vaccine product is lacked in the prior art.
The invention firstly provides a gene segment for constructing a recombinant attenuated vaccine strain of the pigeon-derived newcastle disease virus, wherein the gene segment comprises an envelope glycoprotein F protein gene and a HN protein gene of the attenuated pigeon-derived newcastle disease virus;
the envelope glycoprotein F protein gene of the pigeon source newcastle disease virus with attenuated toxicity is obtained by mutating a site 112R-R-Q-K-R-F117 of an F gene with an amino acid sequence of SEQ ID NO. 1 into 112G-R-Q-G-R-L117;
the HN protein gene has an amino acid sequence of SEQ ID NO. 2;
furthermore, the length of HN protein is extended from 571aa to 577aa, and the amino acid sequence is SEQ ID NO. 3;
specifically, the sequence of the gene fragment containing the envelope glycoprotein F protein gene and HN protein gene of the attenuated pigeon-derived Newcastle disease virus is SEQ ID NO. 4;
furthermore, the LaSota strain genome is obtained by dividing the full-length genome into 5 fragments according to the characteristics of the full-length cDNA sequence of the LaSota strain, amplifying the 5 fragments, carrying out corresponding mutation modification on partial fragments of a primer pair so as to ensure the unicity of enzyme cutting sites used for cloning, introducing a T7 promoter sequence into the 5 'upstream of the first amplified fragment, introducing three continuous G bases behind a T7 promoter to enhance the transcription activity of the T7 promoter sequence, introducing a hepatitis D ribozyme (HdvRz) sequence and a T7 terminator sequence into the 3' end of the last amplified fragment, and sequentially connecting the fragments, wherein one specific nucleotide sequence is SEQ ID NO. 5;
the invention also provides a pigeon source Newcastle disease virus recombinant attenuated vaccine strain, and the construction method comprises the following steps:
1) constructing a recombinant plasmid containing the genome full-length cDNA of the gene II type Newcastle disease virus LaSota strain;
2) on the recombinant plasmid constructed in the step 1), replacing a corresponding partial fragment of a LaSota strain genome with the gene fragment for constructing the pigeon-derived Newcastle disease virus recombinant attenuated vaccine strain to obtain a full-length recombinant plasmid;
3) co-transfecting the full-length recombinant plasmid constructed in the step 2) and eukaryotic expression plasmids expressing genes NP, P and L of the LaSota strain of the newcastle disease virus with cells capable of expressing T7 polymerase to obtain a recombinant newcastle disease virus attenuated vaccine strain expressing genes VI type F and HN proteins;
the cells are BSR-T7/5 cells;
preferably, the recombinant attenuated vaccine strain of the pigeon-derived newcastle disease virus is a newcastle disease virus rLa-VI-QH17 strain, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and has a preservation address of the institute of microorganisms of China academy of sciences No. 3, North Chen West Lu No. 1 of the Chao-Yangxi, Beijing, and a preservation date of: 2021, 7/9, with a deposit number: CGMCC No. 21900.
The invention also aims to provide the application of the recombinant vaccine strain of the pigeon-derived newcastle disease virus in the preparation of vaccines;
the vaccine is an inactivated vaccine, and is obtained by inactivating a low virulent strain through formaldehyde.
The recombinant pigeon-derived newcastle disease virus vaccine strain provided by the invention is obtained by replacing envelope glycoprotein F protein gene and HN protein gene fragments of attenuated pigeon-derived newcastle disease virus with corresponding partial genes of a LaSota strain genome on the basis of an established reverse genetic operation platform of a gene II type newcastle disease virus LaSota strain, and rescuing the attenuated pigeon-derived newcastle disease virus recombinant attenuated vaccine strain. The recombinant vaccine strain has the biological characteristics of high growth titer and low pathogenicity in chick embryos, is stable in heredity, has a good immune protection effect on the pigeon-derived newcastle disease virus, can effectively inhibit toxin expelling, can be used for preventing and controlling the existing popular pigeon-derived newcastle disease virus, and fills the gap that China lacks special vaccine products for preventing and controlling the pigeon newcastle disease.
Drawings
FIG. 1 is a schematic diagram of the construction of pOK-rLaSota full-length plasmid, in which the inverted triangle is the molecular marker (mutated single enzyme cutting site) introduced by the sequence different from the wild strain;
FIG. 2 is a diagram showing the sequencing result of the mutation position of the full-length cDNA of the rescued virus rLaSota strain;
FIG. 3 is a schematic diagram of the construction of full-length genomic plasmids pOK-rLa-VI-QH17 and pOK-rLa-VI-571-QH 17;
FIG. 4 shows the sequencing comparison of the F gene cleavage sites of candidate strains rLa-VI-QH17 and rLa-VI-571-QH17 of the first, second, third and fourth generations of recombinant vaccine of Pigeon Newcastle disease virus and Pigeon Newcastle disease virus QH 1344/2017;
FIG. 5 is a graph showing the elimination curve of antibodies against Pigeon Newcastle disease after pigeon is immunized by recombinant vaccine strain rLa-VI-QH17 of Pigeon Newcastle disease virus and LaSota strain; indicates that the antibody level of the group is significantly different from the rLa-VI-QH17 immune group; indicates that the antibody levels in this group are very significantly different from those in the rLa-VI-QH17 immune group; indicates that the antibody levels in this group are very significantly different from those in the rLa-VI-QH17 immune group;
FIG. 6 shows the HI antibody detection results 28 days and 35 days after pigeon immunization by the recombinant vaccine strain rLa-VI-QH17 and LaSota; indicates that the antibody level of the group is significantly different from the rLa-VI-QH17 immune group; indicates that the antibody levels in this group are very significantly different from those in the rLa-VI-QH17 immune group; indicates that the antibody levels in this group were very significantly different from those in the rLa-VI-QH17 immune group.
Detailed Description
The invention designs a construction method of a recombinant attenuated vaccine rLa-VI-QH17 for expressing pigeon-derived newcastle disease virus F and HN genes, which is characterized in that on the basis of establishing a reverse genetic operation platform of a gene II-type newcastle disease virus LaSota strain, the envelope glycoprotein F protein gene and HN protein gene fragments of a pigeon-derived newcastle disease virus gene VI.2.1.2.2 subtype isolate QH1344/2017 which is mainly popular in China are replaced with corresponding partial genes of a LaSota strain genome, meanwhile, a virulent cracking site (112R-R-Q-K-R-F117) of an F protein is mutated into an F protein cracking site (112G-R-Q-G-R-L117) of an attenuated strain LaSota, the length of the HN protein is prolonged from 571aa to 577aa, and a full-length cDNA pOK-rH-QH-17 plasmid for expressing the pigeon-derived newcastle disease virus gene F.2.1.2.2.2 subtype F and the HN genes is constructed, after co-transfecting BSR-T7/5 cells with helper plasmids, the recombinant pigeon-derived Newcastle disease virus attenuated vaccine strain rLa-VI-QH17 is rescued.
The invention also provides a construction method of the pigeon source newcastle disease virus recombinant attenuated vaccine strain rLa-VI-QH17, which comprises the following steps:
(1) constructing genome full-length cDNA plasmid pOK-rLaSota of gene II type Newcastle disease virus LaSota strain;
(2) constructing a recombinant plasmid pOK-rLa-VI-QH17, cutting a B fragment and a C fragment by Bgl II and Hind III restriction endonucleases on the basis of pOK-rLaSota, replacing an F gene and an HN gene fragment of a pigeon-derived Newcastle disease virus QH1344/2017 strain with a corresponding part of a gene of a LaSota strain genome by combining a homologous recombination method, mutating a virulent cleavage site (112R-R-Q-K-R-F117) of an F protein into an F protein cleavage site (112G-R-Q-G-R-L117) of a low virulent strain LaSota, and prolonging the length of the HN protein from 571aa to 577aa to obtain a recombinant plasmid pOK-rLa-VI-QH 17;
(3) rescuing the recombinant attenuated strain rLa-VI-QH17, co-transfecting BSR-T7/5 cells expressing T7 polymerase with full-length plasmids pOK-rLa-VI-QH17 and three eukaryotic expression plasmids including pCI-NP, pCI-P and pCI-L expressing NDV LaSota strain NP, P and L genes, adding TPCK pancreatin after 6h of transfection, incubating for 72h, repeatedly freezing and thawing the transfected samples, and inoculating 9-11-day-old SPF chick embryos to obtain the recombinant Newcastle disease virus attenuated vaccine strain rLa-VI-QH17 expressing gene VI type F and HN proteins.
In the method, pOK-rLaSota in the step (1) is constructed by the following method: according to the characteristics of the full-length cDNA sequence of LaSota strain, the full-length genome is divided into 5 segments of a, b, c, d and e. Designing a primer according to cloning requirements to perform corresponding mutation modification on a part of fragments so as to ensure the uniqueness of enzyme cutting sites used for cloning, wherein a mutated base sequence can be used as a molecular label for rescuing viruses, introducing a T7 promoter sequence at the 5 'upstream of a fragment, introducing three continuous G bases behind a T7 promoter to enhance the transcriptional activity of the fragment, and introducing a hepatitis B ribozyme (HdvRz) sequence and a T7 terminator sequence at the 3' end of an e fragment. The fragments were then cloned sequentially into pOK12 vector, and the full-length genome-constructing plasmid was designated pOK-rLaSota.
The invention further aims to provide application of the recombinant pigeon derived Newcastle disease virus vaccine strain rLa-VI-QH 17.
The biological product containing the expression pigeon source newcastle disease virus F and the prolonged HN protein gene recombinant attenuated vaccine strain rLa-VI-QH17 belongs to the protection scope of the invention.
The genetic stability of the recombinant vaccine strain rLa-VI-QH17 is analyzed, and the result shows that the rLa-VI-QH17 strain is continuously passaged to the 15 th generation in 9-11 day old SPF chick embryos, and the 1 st (F1), the 5 th (F5), the 10 th (F10) and the 15 th (F15) viruses all contain F (mutant cleavage site) and HN sequences of the pigeon-derived Newcastle disease virus through sequence determination, contain molecular tags introduced during construction, and have good virus genetic stability.
The recombinant vaccine strain rLa-VI-QH17 of the pigeon source newcastle disease virus is characterized in that a virulent cleavage site 112R-R-Q-K-R-F117 of a F protein of the pigeon source newcastle disease virus is mutated into an F protein cleavage site 112G-R-Q-G-R-L117 of a low virulent strain LaSota, the length of HN protein is prolonged from 571aa to 577aa, the sequence after mutation can reduce the virulence of the recombinant vaccine strain, and the safety of the virulent strain is better.
The pigeon source newcastle disease virus recombinant vaccine strain rLa-VI-QH17 designed by the invention uses NP, P, M and L genes of a chicken newcastle disease gene II type classical vaccine strain LaSota strain as a framework, the characteristic of high propagation titer of the LaSota virus strain is kept, and the recombinant vaccine strain shows excellent growth characteristic on a chicken embryo.
The vaccine is an inactivated vaccine, and is obtained by inactivating a low virulent strain through formaldehyde.
The immune protection experiment result shows that the inactivated vaccine prepared by the recombinant vaccine strain rLa-VI-QH17 is safe to pigeons and has no side reaction; high levels of protective antibodies were induced after immunization of 4-week-old squabs for 5 weeks. After immunization for 5 weeks, the pigeon Newcastle disease virus QH1344/2017 virulent strain is used for virus challenge, the immunized rLa-VI-QH17 pigeon only has no any disease symptoms or death, and the immune protection rate reaches 100%. On 3, 5, 7 and 10 days after immune challenge, the oropharynx and cloaca swab of the pigeon in the immune rLa-VI-QH17 group did not detect the detoxification, while the immune LaSota group and the challenge control group showed different degrees of morbidity, mortality and swab virus detoxification detection rate.
The present invention will be further described with reference to the following specific examples.
The specific experimental procedures described in the examples, unless otherwise specified, are all conventional procedures well known to those skilled in the art, and all starting materials are commercially available. The test materials used in the examples were purchased from conventional biochemicals, unless otherwise specified.
Example 1: rescue of LaSota Strain
1. Virus purification
The purification of the virus was carried out by limiting dilution method to purify the LaSota strain in order to obtain a single virus clone, and the detailed procedure was as follows: diluting the virus solution by 10 times, and respectively taking each titer of virus solution to inoculate SPF (specific pathogen free) chick embryos (100 mu L/embryo) of 9-11 days old after dilution, and inoculating 5 virus embryos at each dilution. Chick embryo allantoic fluid was harvested after 4d for HA activity. Allantoic fluid with the highest dilution of HA activity was selected as the next generation of purified virus fluid. The virus is continuously purified for 5 generations by the same method for multiple dilution and chick embryo inoculation, and the virus liquid of the 5 th generation is subpackaged and stored as the original seed virus for the next test.
2. Viral sequence determination
The purified cDNA of the viral reverse transcription genome was used as a template, and the LaSota genome was amplified by PCR using 13 pairs of primers (Table 1) designed to amplify the full-length sequence of the LaSota strain, and the two terminal sequences of the viral genome were determined using 3 '-RACE and 5' -RACE kits. The determined sequences were spliced by DNA Star biology software. The result shows that the total genome length of the NDV LaSota strain is 15186 bp.
Table 1: primer table for LaSota strain NDV whole genome sequencing
3. Construction of LaSota full-Length cDNA cloning plasmid
According to the sequence characteristics of the LaSota genome, the full-length genome is divided into 5 segments (segments a-e) by enzyme cutting site screening. Primers are designed to amplify the 5 segments (table 2), and three enzyme cutting sites on the P gene, the M gene and the L gene are mutated so as to ensure the unicity of the enzyme cutting sites of Bgl II and Hind III, and the mutated base sequence can be used as a molecular tag for rescuing viruses. The T7 promoter sequence is introduced into the 5 'upstream of the a fragment, three continuous G bases are introduced behind the T7 promoter to enhance the transcriptional activity, and the hepatitis D ribozyme (Hdvrz) sequence and the T7 terminator sequence are introduced into the 3' end of the e fragment. The individual fragments were then ligated sequentially into the pOK12 cloning vector to construct the strategy as shown in FIG. 1. After the cloning of the full-length genome is finished, carrying out sequence determination, wherein the nucleotide sequence is SEQ ID NO. 5; the constructed and sequenced correct viral genome plasmid was named pOK-rLaSota.
Table 2: primer table for construction of LaSota strain genome full-length cDNA clone
4. Construction of helper plasmids
According to the determined LaSota strain genome sequence, primers are designed to respectively amplify the open reading frames (table 3) of the encoded nucleocapsid protein NP, phosphoprotein P and polymerase protein L, 3 ORFs are respectively cloned to the downstream of a CMA promoter of a eukaryotic expression vector pCI-neo, 3 auxiliary plasmids pCI-NP, pCI-P and pCI-L are constructed, and no gene mutation is generated through sequencing verification.
Table 3: primer table for LaSota helper plasmid construction
5. Rescue of virus
The constructed full-length plasmid (pOK-rLaSota) and 3 helper plasmids (pCI-NP, pCI-P, and pCI-L) were co-transfected into BSR-T7/5 cells. After 6h of transfection, the medium was changed to DMEM medium containing 2% serum, 1% antibiotic, 5. mu.g/mL TPCK-Trypsin. And (3) after transfection for 72h, harvesting culture solution and cells, inoculating 9-11 day-old SPF (specific pathogen free) chick embryos (200 mu L/embryo), incubating at 37 ℃, harvesting chick embryo allantoic fluid after 3-4 days, and performing Hemagglutination (HA) test. The result shows that HA is positive, which preliminarily indicates that the virus rescue is successful.
6. Identification and biological characteristic analysis of rescued virus rLaSota
And extracting HA positive virus liquid RNA, and performing whole genome sequence determination on the rescued virus. The sequence comparison result shows that the rescued virus genome contains a molecular tag (mutated enzyme cutting site, figure 2) introduced during construction, which indicates that the LaSota strain genome cDNA clone is successfully rescued, and the rescued virus is named as rLaSota. Continuously conveying the eggs in SPF (specific pathogen free) chick embryos of 9-11 days oldTo generation 5. The hemagglutination titer of each generation can be detected to be 210～211In the meantime. Sequence determination is carried out on F5-generation virus liquid, and the result shows that the molecular label has no back mutation, which indicates that the rescued rLaSota has better stability. Determination of the amount of infection (EID) in half of the embryos of chickens that rescued the virus from the parental virus50) Average chick embryo Mortality (MDT) and intracerebral inoculation pathogenicity index (ICPI) for 1 day old chicks, table 4 shows that the rescued viruses have the same biological properties and pathogenicity as the parental viruses.
Table 4: biological characterization of rescued virus and parental strains
In conclusion, the reverse genetic operation platform of the Newcastle disease virus LaSota strain is successfully constructed, and can be used as a good vaccine vector.
EXAMPLE 2 rescue of recombinant Pigeon vaccine candidate seed Virus
1. Screening and purification of viruses
Selecting a gene VI.2.1.2.2 subtype Pigeon-derived Newcastle disease virus which is separated and stored in a laboratory and mainly prevails in the current Chinese pigeon group for reproduction and rejuvenation, and further purifying 5 strains by adopting a limiting dilution method, wherein the hemagglutination titer of each strain in different generations is shown in Table 5. Finally, the strain QH1344/2017 purified after 5 generations is selected as an original seed virus for construction of full-length cDNA clone.
Table 5: respective generation hemagglutination titer tables of different strains of pigeon-derived newcastle disease virus
2. Determination and analysis of full-length genome sequence of strain QH1344/2017
The purified QH1344/2017 virus reverse transcription genome cDNA is used as a template, 13 pairs of primers (shown in Table 6) for amplifying a VI type NDV full-length sequence are utilized to perform PCR amplification on the QH1344/2017 genome, and simultaneously, 3 'RACE and 5' RACE kits are used for determining two terminal sequences of a virus genome. The determined sequences were spliced by DNA Star biology software. The result shows that the total genome length of the strain QH1344/2017 is 15192 bp.
Table 6: primer table for sequencing type VI NDV whole genome
3. Construction of full-length cDNA of vaccine candidate strain genome
The method is characterized in that pOK-LaSota full-length plasmid is used as a platform to design and synthesize a new gene sequence QH17-571HN-bc in the middle of Bgl II enzyme cutting site to Hind III enzyme cutting site in the full-length sequence, wherein the 6539bp total comprises that envelope glycoprotein F protein gene and HN protein gene fragments of a pigeon source Newcastle disease virus QH1344/2017 strain are replaced with corresponding partial genes of LaSota strain genome, meanwhile, a virulent cleavage site (112R-R-Q-K-R-F117) of an F protein (SEQ ID NO:1) is mutated into an F protein cleavage site (112G-R-Q-G-R-L117) of a weak strain LaSota, other sequences are kept unchanged, and a recombinant virus rLa-VI-571-QH17 strain is designed.
Based on the above mutation, a recombinant virus rLa-VI-QH17 strain was designed by synthesizing a new gene sequence QH17-577HN-bc (SEQ ID NO:4) comprising the HN protein with a length extending from 571aa (SEQ ID NO:2) to 577aa (SEQ ID NO:3) for a total of 6539 bp.
The pOK-rLaSota full-length plasmid was double digested with Bgl II and Hind III restriction enzymes, and the original b and c fragments were cut off. Primers were designed to amplify the synthesized target genes QH17-571HN-bc and QH17-577HN-bc fragments (Table 7), and the target fragments QH17-571HN-bc and QH17-577HN-bc fragments recovered from the gel were ligated to the digested vector, respectively, to construct the strategy shown in FIG. 3. After the full-length genome cloning was completed, sequencing was performed, and the constructed and sequenced correct viral genome plasmids were named pOK-rLa-VI-571-QH17 and pOK-rLa-VI-QH17, respectively.
TABLE 7 primer set for amplification of QH17-571HN-bc and QH17-577HN-bc fragments
4. Rescue of vaccine candidate strains
Two full-length recombinant plasmids (pOK-rLa-VI-571-QH17 and pOK-rLa-VI-QH17) which have been constructed were co-transfected with 3 helper plasmids (pCI-NP, pCI-P and pCI-L) of LaSota virus to BSR-T7/5 cells, respectively. After 6h of transfection, the medium was changed to DMEM medium containing 2% serum, 1% antibiotic, 5. mu.g/mL TPCK-Trypsin. And (3) harvesting culture solution and cells 72h after transfection, inoculating 9-11 day-old SPF (specific pathogen free) chick embryos (200 mu L/embryo), incubating at 37 ℃, harvesting chick embryo allantoic fluid after 3-4 days, and performing Hemagglutination (HA) test. The results are all shown to be HA positive, which preliminarily shows that the virus rescue is successful.
5. Sequence identification and genetic stability of vaccine candidate strains
And (3) selecting HA positive virus liquid to extract RNA, carrying out reverse transcription, and then carrying out whole genome sequence determination. The sequence alignment result shows that the genome of the rescued virus rLa-VI-571-QH17 takes a LaSota strain as a framework, F and HN genes of a QH1344/2017 strain are embedded, and the F gene cleavage site is mutated; the genome of the rescued virus rLa-VI-QH17 takes a LaSota strain as a framework, F and HN genes of a QH1344/2017 strain are embedded, the F gene cleavage site is mutated, and the length of HN protein is prolonged from 571aa to 577aa, which indicates that two strains of viruses are successfully rescued.
Continuously carrying out continuous passage on the rescued virus in SPF chick embryos of 9-11 days old for 15 times. The hemagglutination titer of each generation of virus is 210～211In the meantime. F and HN genes of the 1 st, 5 th, 10 th and 15 th generation recombinant viruses were selected for sequence determination. Sequencing results show that the F and HN genes of the 1 st, 5 th, 10 th and 15 th generation recombinant viruses are allConsistent with the inserted sequence, the F protein cleavage site also did not undergo back mutation (fig. 4), indicating that the two recombinant viruses are genetically stable during passage.
6. Biological characterization of vaccine candidate strains
Selecting rLa-VI-QH17 and rLa-VI-571-QH17 vaccine candidate strains of the 15 th generation to measure EID thereof50MDT and ICPI, and comparing the virulence changes of the viruses by taking the Lasota virus strain and the QH1344/2017 virus strain as controls. The test data in Table 8 show that the rescued rLa-VI-QH17 and rLa-VI-571-QH17 vaccine candidate strains have obviously reduced virulence, and have the biological characteristics that the rLa-VI-QH17 vaccine candidate strain with the HN protein length prolonged has higher growth titer in chick embryos and lower pathogenicity in 1-day-old chicks compared with the rLa-VI-571-QH17 vaccine candidate strain. Therefore, the rLa-VI-QH17 vaccine candidate strain constructed by the invention can be used as a preferred vaccine strain for vaccine production.
Table 8: biological characterization of rescued virus and parental strains
Example 3 safety test and immune Effect test of rLa-VI-QH17 vaccine Strain on non-immune Pigeon
1. Preparation of inactivated vaccine
Diluting the rLa-VI-QH17 vaccine strain by 10000 times by using sterilized normal saline, inoculating SPF (specific pathogen free) chick embryos of 9-11 days old, inoculating 0.1mL of each embryo, and placing at 37 ℃ for continuous incubation. Discarding the dead embryos within 24h after inoculation, immediately placing the dead embryos for 24 h-120 h at 4 ℃, collecting the mixed sample, and determining the HA of the vaccine preparation poison to be 210.5Viral titer of 109.48EID500.1 mL. Introducing the newcastle disease virus solution with the determined titer into an inactivation tank, adding a formaldehyde solution with the final concentration of 0.1% to fully mix the newcastle disease virus solution, inactivating the newcastle disease virus solution at 4 ℃ for 48 hours, and shaking the newcastle disease virus solution once every 2 hours. Preparing the inactivated oil adjuvant inactivated vaccine by the inactivated virus stock solution according to a conventional method. The LaSota inactivated vaccine with the same dose is prepared simultaneously according to the same method.
2. Safety test of inactivated vaccine
In order to determine the safety of the rLa-VI-QH17 vaccine strain rescued in the embodiment 2 of the invention to non-immune pigeons, 10 non-immune pigeons with the age of 4 weeks are immunized by the prepared rLa-VI-QH17 inactivated vaccine, 0.2mL of vaccine is injected into each muscle, 5 control animals are set at the same time, the pigeons are raised under the same condition, the pigeons are continuously observed for 14 days, and the feeding, drinking and clinical conditions of the test pigeons are recorded. The result shows that the vaccine absorption effect is good after the pigeon is immunized, no local and systemic adverse reaction occurs in the immunized pigeon, and the state of the pigeon is completely normal.
3. Antibody monitoring of inactivated vaccines
In order to determine the law of antibody growth in the non-immune pigeons generated by the rLa-VI-QH17 vaccine strain rescued in the example 2, the non-immune pigeons raised in an isolator are randomly divided into two groups, wherein each group comprises 5 animals, one group is the rLa-VI-QH17 inactivated vaccine immune group prepared in the step 1 of the example 3, and the other group is the LaSota inactivated vaccine immune group prepared in the step 1 of the example 3. 0.2mL of vaccine is injected into pigeon intramuscular, and pigeon is subjected to wing vein blood collection to separate serum and determine antibody respectively at 1, 3, 5, 7, 9, 11 and 13 weeks after immunization. The results show that both groups of the immune groups can generate certain titer of antibodies from the end of the monitoring period of 13 weeks after the immunization, and the titer of the antibodies of the rLa-VI-QH17 inactivated vaccine immune group is obviously higher than that of the LaSota inactivated vaccine immune group, and the difference is obvious (figure 5). The rLa-VI-QH17 inactivated vaccine immunization group has the highest antibody level of the immunized pigeons at the 9 th week after immunization, and the average value of HI antibody titer is 8.5log2And it produces higher antibody levels from weeks 3 to 13 after immunization, indicating that the immune antibody protection period can be at least 3 months.
4. Immunoprotection assay for inactivated vaccines
To determine the immune protection effect of the rLa-VI-QH17 vaccine strain rescued in example 2 of the invention on non-immune pigeons, the non-immune pigeons raised in an isolator were randomly divided into three groups of 10 animals, one group was the rLa-VI-QH17 inactivated vaccine immune group prepared in step 1 of example 3, one group was the LaSota inactivated vaccine immune group prepared in step 1 of example 3, and the other group was a control group. Each pigeon in the immunized group was injected intramuscularly with 0.2mL of vaccine, and the control group was injected with the same dose of PBS. Respectively 28 days and 3 days after immunizationBlood was collected from the wing vein for 5 days to separate serum and measure antibodies. After 35 days of blood collection, the virulent strain QH1344/2017 of the clinically isolated gene VI wild strain is used for counteracting the virulent strain, and the quantity of the virulent strain is 10 for each pigeon6EID50The dose is subjected to virus counteracting through intramuscular injection, the pigeon is fed in an isolator, the morbidity and the mortality of the pigeon are recorded every day after 14 days of observation, the oropharynx swab and the cloaca swab of the pigeon are collected on 3 rd, 5 th, 7 th and 10 th days after virus counteracting, the chick embryo is inoculated for virus separation, and the detoxification condition is detected.
The results show that the immune group can generate antibodies with certain titer after being immunized for 28 days, the antibody titer is obviously improved after being immunized for 35 days, the antibody titer of the rLa-VI-QH17 inactivated vaccine immune group is obviously higher than that of the LaSota inactivated vaccine immune group, and the difference is obvious (figure 6).
After 35 days of immunization for virus challenge, all pigeons in the rLa-VI-QH17 inactivated vaccine immunization group have no morbidity or mortality, and the immune protection rate reaches 100%; two pigeons in the LaSota inactivated vaccine immunity group show Newcastle disease infection symptoms such as askew neck, vertical wings and the like, and no pigeons die; all pigeons in the PBS group were ill, and one pigeons died (table 9).
Table 9: toxicity attacking protective test result table of vaccine strain
The detoxification condition shows that the pigeons immunized with the rLa-VI-QH17 inactivated vaccine group have no detoxification phenomenon in 3, 5, 7 and 10 days after challenge, while the pigeons immunized with the LaSota inactivated vaccine group still have partial detoxification phenomenon, and the detoxification rate of the blank challenge control group is 100% (Table 10).
Table 10: toxin expelling detection result table for test pigeons in each group after toxin attack
In conclusion, the inactivated pigeon source newcastle disease virus recombinant vaccine strain rLa-VI-QH17 prepared by the invention has good safety and protection effect on immune pigeons and has wide application prospect.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention should not be limited thereby, that is, all the simple changes, equivalents and modifications made to the above embodiment according to the claims and the description of the present invention are within the scope of the present invention.
Sequence listing
<110> China center for animal health and epidemiology
<120> pigeon-derived Newcastle disease virus recombinant vaccine strain and construction method and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 553
<212> PRT
<213> Pigeon paramyxovirus type I (Pigeon paramyxovirus 1)
<400> 1
Met Asp Ser Lys Pro His Ile Arg Ile Pro Ala Ser Pro Thr Leu Ile
1 5 10 15
Thr Arg Ile Thr Leu Val Leu Ser Tyr Ile Cys Ser Thr Ser Ser Leu
20 25 30
Asp Gly Arg Pro Leu Ala Ala Ala Gly Ile Val Val Thr Gly Asp Lys
35 40 45
Ala Ile Asn Ile Tyr Thr Ser Ser Gln Thr Gly Ser Ile Ile Val Lys
50 55 60
Leu Leu Pro Asn Met Pro Lys Asp Lys Glu Ala Cys Ala Lys Ala Pro
65 70 75 80
Leu Glu Ala Tyr Asn Arg Thr Leu Thr Thr Leu Leu Thr Pro Leu Gly
85 90 95
Asp Ser Ile Arg Arg Ile Gln Gly Ser Val Ser Thr Ser Gly Gly Arg
100 105 110
Arg Gln Lys Arg Phe Ile Gly Ala Ile Ile Gly Ser Val Ala Leu Gly
115 120 125
Val Ala Thr Ser Ala Gln Ile Thr Ala Ala Ala Ala Leu Ile Gln Ala
130 135 140
Asn Gln Asn Ala Ala Asn Ile Leu Arg Leu Lys Glu Ser Ile Ala Ala
145 150 155 160
Thr Asn Glu Ala Val His Glu Val Thr Asp Gly Leu Ser Gln Leu Ala
165 170 175
Val Ala Ile Gly Lys Met Gln Gln Phe Val Asn Asp Gln Phe Asn Asn
180 185 190
Thr Ala Arg Glu Leu Asp Cys Ile Lys Ile Ser Gln Gln Val Gly Ile
195 200 205
Glu Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gly Pro Gln
210 215 220
Ile Thr Ser Pro Ala Leu Thr Gln Leu Thr Ile Gln Ala Leu Tyr Asn
225 230 235 240
Leu Ala Gly Gly Asn Met Asp Tyr Leu Leu Thr Lys Leu Gly Ile Gly
245 250 255
Asn Asn His Leu Ser Ser Leu Ile Gly Ser Gly Leu Ile Thr Gly Asn
260 265 270
Pro Ile Leu Tyr Asp Ser Gln Thr Gln Leu Leu Gly Ile Gln Val Asn
275 280 285
Leu Pro Ser Val Gly Asn Leu Asn Asn Met Arg Ala Thr Tyr Leu Glu
290 295 300
Thr Leu Ser Val Ser Thr Thr Lys Gly Phe Ala Ser Ala Leu Val Pro
305 310 315 320
Lys Val Val Thr Gln Val Gly Ser Val Ile Glu Glu Leu Asp Thr Ser
325 330 335
Tyr Cys Ile Glu Ser Asp Leu Asp Leu Tyr Cys Thr Arg Ile Val Thr
340 345 350
Phe Pro Met Ser Pro Gly Ile Tyr Ser Cys Leu Ser Gly Asn Thr Ser
355 360 365
Ala Cys Met Tyr Ser Lys Thr Glu Gly Ala Leu Asn Thr Pro Tyr Met
370 375 380
Ala Leu Lys Gly Ser Val Ile Ala Asn Cys Lys Ile Thr Thr Cys Arg
385 390 395 400
Cys Ala Asp Pro Pro Gly Ile Ile Ser Gln Asn Tyr Gly Glu Ala Val
405 410 415
Ser Leu Ile Asp Arg His Ser Cys Asn Val Leu Ser Leu Asp Gly Ile
420 425 430
Thr Leu Arg Leu Ser Gly Glu Phe Asp Ala Thr Tyr Gln Lys Asn Ile
435 440 445
Ser Ile Leu Asp Ser Gln Val Ile Val Thr Gly Asn Leu Asp Ile Ser
450 455 460
Thr Glu Leu Gly Asn Val Asn Asn Ser Ile Ser Asn Ala Leu Asp Arg
465 470 475 480
Leu Ala Glu Ser Asn Ser Lys Leu Asp Lys Val Asn Val Lys Leu Thr
485 490 495
Ser Thr Ser Ala Leu Ile Thr Tyr Ile Ile Leu Thr Ile Ile Ser Leu
500 505 510
Val Phe Gly Ala Leu Ser Leu Val Leu Ala Cys Tyr Leu Met Tyr Lys
515 520 525
Gln Arg Ala Gln Gln Lys Thr Leu Leu Trp Leu Gly Asn Asn Thr Leu
530 535 540
Asp Gln Met Arg Ala Thr Thr Arg Thr
545 550
<210> 2
<211> 571
<212> PRT
<213> Pigeon paramyxovirus type I (Pigeon paramyxovirus 1)
<400> 2
Met Asp His Ala Val Ser Arg Val Ala Leu Glu Asn Glu Glu Arg Glu
1 5 10 15
Ala Lys Ser Thr Trp Arg Leu Val Phe Arg Ile Ala Val Leu Leu Leu
20 25 30
Thr Val Val Thr Leu Ala Ile Ser Val Ala Ala Leu Val Tyr Ser Met
35 40 45
Gly Ala Ser Thr Pro Arg Asp Leu Val Gly Val Ser Thr Met Val Ser
50 55 60
Lys Val Glu Asp Lys Ile Thr Ser Ser Ile Ser Ser Ser Gln Asp Val
65 70 75 80
Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro Leu Ala Leu
85 90 95
Leu Asn Thr Glu Ser Ile Ile Met Asn Ala Ile Thr Ser Leu Ser His
100 105 110
Gln Ile Asn Gly Ala Ala Asn Asn Ser Arg Cys Gly Ala Pro Ile His
115 120 125
Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile Val Asp Asp
130 135 140
Ile Ser Gly Val Thr Ser Phe Tyr Pro Ser Ala Tyr Gln Glu His Leu
145 150 155 160
Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro
165 170 175
Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn Val Ile
180 185 190
Leu Ser Gly Cys Ile Asp His Ser His Ser His Gln Tyr Leu Ala Leu
195 200 205
Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe Phe Ser Thr Leu
210 215 220
Arg Ser Ile Asn Leu Asp Asp Asn Gln Asn Arg Lys Ser Cys Ser Val
225 230 235 240
Ser Ala Thr Pro Leu Gly Cys Asp Ile Leu Cys Ser Lys Val Thr Glu
245 250 255
Ala Glu Glu Glu Asp Tyr Lys Ser Val Ala Pro Thr Ser Met Val His
260 265 270
Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp Leu Asp Thr
275 280 285
Thr Val Leu Phe Lys Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Ser
290 295 300
Gly Ser Phe Ile Asp Asp Arg Val Trp Phe Pro Val Tyr Gly Gly Leu
305 310 315 320
Lys Pro Asn Ser Pro Ser Asp Thr Ala Gln Glu Gly Lys Tyr Ile Ile
325 330 335
Tyr Lys Arg Tyr Asn Asn Thr Cys Pro Asp Gly Gln Glu Tyr Gln Ile
340 345 350
Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Ser Gly Lys Arg
355 360 365
Val Gln Gln Ala Ile Leu Ser Ile Lys Val Ser Thr Ser Leu Gly Glu
370 375 380
Asp Pro Val Leu Thr Val Pro Pro Asn Thr Ile Thr Leu Met Gly Ala
385 390 395 400
Glu Gly Arg Val Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gln Arg
405 410 415
Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val His
420 425 430
Asn Lys Thr Ala Thr Leu His Asn Pro Tyr Thr Phe Asn Ala Phe Thr
435 440 445
Arg Pro Gly Ser Val Pro Cys Gln Ala Ser Ala Arg Cys Pro Asn Pro
450 455 460
Cys Ile Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Val Phe Tyr Arg
465 470 475 480
Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Asp Glu Gln
485 490 495
Thr Arg Leu Asn Pro Val Ser Ala Val Phe Asp Asn Ile Ser Arg Ser
500 505 510
Arg Val Thr Arg Val Ser Ser Ser Ser Thr Lys Ala Ala Tyr Thr Thr
515 520 525
Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Val Tyr Cys Leu Ser
530 535 540
Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro
545 550 555 560
Leu Leu Val Glu Ile Leu Lys Asp Glu Arg Ala
565 570
<210> 3
<211> 577
<212> PRT
<213> Pigeon paramyxovirus type I (Pigeon paramyxovirus 1)
<400> 3
Met Asp His Ala Val Ser Arg Val Ala Leu Glu Asn Glu Glu Arg Glu
1 5 10 15
Ala Lys Ser Thr Trp Arg Leu Val Phe Arg Ile Ala Val Leu Leu Leu
20 25 30
Thr Val Val Thr Leu Ala Ile Ser Val Ala Ala Leu Val Tyr Ser Met
35 40 45
Gly Ala Ser Thr Pro Arg Asp Leu Val Gly Val Ser Thr Met Val Ser
50 55 60
Lys Val Glu Asp Lys Ile Thr Ser Ser Ile Ser Ser Ser Gln Asp Val
65 70 75 80
Val Asp Arg Ile Tyr Lys Gln Val Ala Leu Glu Ser Pro Leu Ala Leu
85 90 95
Leu Asn Thr Glu Ser Ile Ile Met Asn Ala Ile Thr Ser Leu Ser His
100 105 110
Gln Ile Asn Gly Ala Ala Asn Asn Ser Arg Cys Gly Ala Pro Ile His
115 120 125
Asp Pro Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile Val Asp Asp
130 135 140
Ile Ser Gly Val Thr Ser Phe Tyr Pro Ser Ala Tyr Gln Glu His Leu
145 150 155 160
Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro
165 170 175
Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn Val Ile
180 185 190
Leu Ser Gly Cys Ile Asp His Ser His Ser His Gln Tyr Leu Ala Leu
195 200 205
Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe Phe Ser Thr Leu
210 215 220
Arg Ser Ile Asn Leu Asp Asp Asn Gln Asn Arg Lys Ser Cys Ser Val
225 230 235 240
Ser Ala Thr Pro Leu Gly Cys Asp Ile Leu Cys Ser Lys Val Thr Glu
245 250 255
Ala Glu Glu Glu Asp Tyr Lys Ser Val Ala Pro Thr Ser Met Val His
260 265 270
Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu Lys Asp Leu Asp Thr
275 280 285
Thr Val Leu Phe Lys Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Ser
290 295 300
Gly Ser Phe Ile Asp Asp Arg Val Trp Phe Pro Val Tyr Gly Gly Leu
305 310 315 320
Lys Pro Asn Ser Pro Ser Asp Thr Ala Gln Glu Gly Lys Tyr Ile Ile
325 330 335
Tyr Lys Arg Tyr Asn Asn Thr Cys Pro Asp Gly Gln Glu Tyr Gln Ile
340 345 350
Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Ser Gly Lys Arg
355 360 365
Val Gln Gln Ala Ile Leu Ser Ile Lys Val Ser Thr Ser Leu Gly Glu
370 375 380
Asp Pro Val Leu Thr Val Pro Pro Asn Thr Ile Thr Leu Met Gly Ala
385 390 395 400
Glu Gly Arg Val Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gln Arg
405 410 415
Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val His
420 425 430
Asn Lys Thr Ala Thr Leu His Asn Pro Tyr Thr Phe Asn Ala Phe Thr
435 440 445
Arg Pro Gly Ser Val Pro Cys Gln Ala Ser Ala Arg Cys Pro Asn Pro
450 455 460
Cys Ile Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Val Phe Tyr Arg
465 470 475 480
Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Asp Glu Gln
485 490 495
Thr Arg Leu Asn Pro Val Ser Ala Val Phe Asp Asn Ile Ser Arg Ser
500 505 510
Arg Val Thr Arg Val Ser Ser Ser Ser Thr Lys Ala Ala Tyr Thr Thr
515 520 525
Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Val Tyr Cys Leu Ser
530 535 540
Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro
545 550 555 560
Leu Leu Val Glu Ile Leu Lys Asp Glu Arg Ala Arg Glu Ala Arg Ser
565 570 575
Gly
<210> 4
<211> 6539
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
agatcttgtc ttgaaacaga catcctccat ccctatgatg cggaccgaaa tccaacagct 60
gaaaacatct gttgcagtca tggaagccaa cttgggaatg atgaagattc tggatcccgg 120
ttgtgccaac atttcatctc tgagtgatct acgggcagtt gcccgatctc acccggtttt 180
agtttcaggc cctggagacc cctctcccta tgtgacacaa ggaggcgaaa tggcacttaa 240
taaactttcg caaccagtgc cacatccatc tgaattgatt aaacccgcca ctgcatgcgg 300
gcctgatata ggagtggaaa aggacactgt ccgtgcattg atcatgtcac gcccaatgca 360
cccgagttct tcagccaagc tcctaagcaa gttagatgca gccgggtcga tcgaggaaat 420
caggaaaatc aagcgccttg ctctaaatgg ttaattacta ctgccacacg tagcgggtcc 480
ctgtccactc ggcatcacac ggaatctgca ccgagttccc ccccgcagac ccaaggtcca 540
actctccaag cggcaatcct ctctcgcttc ctcagcccca ctgaatgatc gcgtaaccgt 600
aattaatcta gctacattta agattaagaa aaaatacggg tagaattgga gtgccccaat 660
tgtgccaaga tggactcatc taggacaatt gggctgtact ttgattctgc ccattcttct 720
agcaacctgt tagcatttcc gatcgtccta caagacacag gagatgggaa gaagcaaatc 780
gccccgcaat ataggatcca gcgccttgac ttgtggactg atagtaagga ggactcagta 840
ttcatcacca cctatggatt catctttcaa gctgggaatg aagaagccac tgtcggcatg 900
atcgatgata aacccaagcg cgagttactt tccgctgcga tgctctgcct aggaagcgtc 960
ccaaataccg gagaccttat tgagctggca agggcctgtc tcactatgat agtcacatgc 1020
aagaagagtg caactaatac tgagagaatg gttttctcag tagtgcaggc accccaagtg 1080
ctgcaaagct gtagggttgt ggcaaacaaa tactcatcag tgaatgcagt caagcacgtg 1140
aaagcgccag agaagattcc cgggagtgga accctagaat acaaggtgaa ctttgtctcc 1200
ttgactgtgg taccgaagaa ggatgtctac aagatcccag ctgcagtatt gaaggtttct 1260
ggctcgagtc tgtacaatct tgcgctcaat gtcactatta atgtggaggt agacccgagg 1320
agtcctttgg ttaaatctct gtctaagtct gacagcggat actatgctaa cctcttcttg 1380
catattggac ttatgaccac cgtagatagg aaggggaaga aagtgacatt tgacaagctg 1440
gaaaagaaaa taaggagcct tgatctatct gtcgggctca gtgatgtgct cgggccttcc 1500
gtgttggtaa aagcaagagg tgcacggact aagctcttgg cacctttctt ctctagcagt 1560
gggacagcct gctatcccat agcaaatgct tctcctcagg tggccaagat actctggagt 1620
caaaccgcgt gcctgcggag cgttaaaatc attatccaag caggtaccca acgcgctatc 1680
gcagtgaccg ccgaccacga ggttacctct actaagctgg agaaggggca cacccttgcc 1740
aaatacaatc cttttaagaa ataagctgcg tctctgagat tgcgctccgc ccactcaccc 1800
agatcatcat gacacaaaaa actaatctgt cttgattatt tacagttagt ttacctgtct 1860
atcaagttag aaaaaacacg ggtagaagat tctggatccc ggttggcgcc ctccaggtgc 1920
aagatggact ccaaacccca catcaggatc ccggcatctc cgacgctgat cactcgaatc 1980
actctggtac tgagctacat ctgctcgacg agctctcttg atggcaggcc acttgcagct 2040
gcggggattg tggtaacagg agataaagca atcaatatat acacctcatc ccagacaggg 2100
tcaatcatag tcaagttgct cccgaatatg cccaaggaca aagaggcatg tgcgaaagcc 2160
ccactagagg catacaacag aacactgacc actttactca cccccctcgg tgattccatc 2220
cgcaggatac aaggatctgt gtccacatca ggaggaggga gacaggggcg ccttataggt 2280
gccattatag gcagtgtagc tcttggggtt gcgacatcgg cacagataac agcagctgcg 2340
gccctaatac aagctaacca gaatgccgcc aacatcctcc ggcttaagga gagcatcgct 2400
gcgaccaatg aagctgtgca tgaggtcact gacggattat cgcaactagc agtggcaatt 2460
gggaagatgc aacagtttgt aaacgaccaa tttaataata cagcgcggga attggactgt 2520
ataaaaattt cacaacaagt cggtatagaa ctcaacttat acctaactga actgactaca 2580
gtgttcgggc cacaaatcac ttcccctgcc ctaactcagc tgaccatcca agcgctttat 2640
aatttagctg gcggtaatat ggattaccta ttgactaaat taggtatagg gaacaatcat 2700
ctcagctcat taataggcag tggcttgatc acaggcaacc ctatactgta tgactcacag 2760
actcaactct tgggcatcca ggtaaattta ccctccgtcg ggaaccttaa taatatgcgt 2820
gccacctacc tggaaacttt atctgtaagc acaaccaaag ggtttgcctc agcacttgtc 2880
ccgaaggtag tgacacaagt cggctccgtg atagaagaac ttgacacctc atactgtata 2940
gaatctgatc tggatttata ctgtacaagg atagtgacat tccctatgtc tccaggaatt 3000
tattcttgtc tgagcggtaa tacatcagct tgcatgtact caaagactga aggcgcactc 3060
aatacgccat acatggccct caaaggctca gtcattgcca attgcaaaat aacaacctgc 3120
agatgtgcag accccccagg tatcatatcg caaaattatg gagaagctgt atctcttata 3180
gataggcatt catgcaatgt cttatcatta gatgggataa ccctgaggct cagtggggag 3240
tttgatgcaa cttatcaaaa gaatatctca atactagatt ctcaagtcat cgtgacaggc 3300
aaccttgata tatcaaccga gcttggaaat gtcaacaatt caataagcaa tgctctggac 3360
aggttagcag agagtaacag caaactagac aaagtcaatg tcaaattaac cagcacatct 3420
gctctcatta cctatatcat cctaaccatc atatctcttg tttttggtgc acttagcttg 3480
gttttagcat gctatctaat gtacaaacaa agggcacaac aaaagaccct actgtggctt 3540
gggaacaaca ccctcgatca gatgagagcc actacaagaa catgaacaca gatgaggaac 3600
gaaggtttcc ctaatagtaa tttatgtgaa agttctggta gtctgtcagt tcagagagtt 3660
aagaaaaaac taccggttgt agatgaccaa aggacgatat acgggtagaa cggtaagaga 3720
ggccgcccct caattgcgag ccaggcttca caacctccgt tctaccgctt caccgacaac 3780
agtcctcaat catggaccat gcagtcagca gagttgcgtt agagaatgag gaaagagaag 3840
cgaagagcac atggcgcttg gttttccgga tcgcggtttt acttttaaca gtggtgacct 3900
tagctatctc tgtagctgcc ttggtataca gcatgggggc cagtacgccc cgcgacctcg 3960
taggcgtatc gactatggtc tctaaggtgg aagacaagat tacgtcctca atcagttcga 4020
gtcaagatgt agtagacagg atatacaaac aagtagctct tgaatctccg ctggcactgc 4080
taaatactga atctataatt atgaatgcaa taacgtctct ctctcatcag attaatgggg 4140
ctgcaaacaa tagcaggtgt ggggcgccta ttcatgaccc agattatatc ggggggatag 4200
gtaaagaact catagtagac gacatcagtg gtgttacatc attctaccca tctgcatacc 4260
aagaacacct gaattttatc ccagcgccca ccacaggatc aggctgcact cggataccct 4320
catttgatat gagtgctacc cactactgtt atactcataa cgtgatattg tctggctgca 4380
tagaccactc acattcacat caatacttag cacttggtgt gcttcggaca tccgcaacag 4440
ggagggtatt cttttctact ctacgttcca tcaacttaga tgacaaccaa aatcggaagt 4500
cctgcagtgt gagtgcaact cctctaggtt gtgatatcct gtgctctaaa gtcacggagg 4560
ctgaagaaga ggattataag tcagttgccc ctacatcaat ggtgcacgga agattaggtt 4620
ttgacggtca gtaccatgag aaggacttag acaccacagt cttatttaag gattgggtag 4680
caaattaccc gggggttggg agcggatctt ttattgatga ccgtgtctgg ttcccagttt 4740
acggagggtt gaaacctaat tcgcccagcg acactgcaca agaagggaaa tatataatat 4800
acaagcgcta taataacaca tgccctgatg gacaggagta tcaaatccgg atggctaagt 4860
cttcatacaa gcctgggcgt ttcagtggaa agcgtgtaca gcaagccatc ttatctatca 4920
aagtgtcgac atctttgggt gaggacccgg tgctgactgt tccacccaat acaattacac 4980
tcatgggggc cgaaggaaga gtcctcacgg tagggacgtc tcacttcttg tatcaacgag 5040
ggtcttcata tttctcccct gccttattat atcctatgac agtccataac aaaacagcta 5100
ctcttcataa tccttataca tttaatgcct tcacccggcc aggtagtgtc ccttgtcagg 5160
catcagcaag atgccccaac ccgtgtatca ctggagtcta tactgatccg taccctctgg 5220
tcttctacag gaaccacact ttgcgagggg tattcgggac aatgcttgat gatgaacaaa 5280
caagactcaa ccccgtatct gcagtattcg acaatatatc tcgcagtcgt gtaaccagag 5340
taagttcaag cagcaccaag gcagcataca cgacatcaac atgcttcaaa gttgtcaaga 5400
ccaataaagt ttattgcctc agcattgccg aaatatccaa caccctattt ggggaattca 5460
gaatcgttcc tttgttagtt gagatcctaa aggatgaaag ggctagagaa gctagatctg 5520
gttagttgag tcaattataa aggagttgga aagatggcat tgtatcacct atcctctgcg 5580
acatcaagaa tcaaaccgaa tgccggcgcg tgctcgaatt ccatgttgcc agttgaccac 5640
aatcagccag tgctcatgcg atcagattaa gccttgtcaa tagtctcttg atttagaaaa 5700
aatgtaagtg gcaatgagat acaaggcaaa acagctcatg gtaaataata cgggtaggac 5760
atggcgagct ccggtcctga aagggcagag catcagatta tcctaccaga gtcacacctg 5820
tcttcaccat tggtcaagca caaactactc tattactgga aattaactgg gctaccgctt 5880
cctgatgaat gtgacttcga ccacctcatt ctcagccgac aatggaaaaa aatacttgaa 5940
tcggcctctc ctgatactga gagaatgata aaactcggaa gggcagtaca ccaaactctt 6000
aaccacaatt ccagaataac cggagtgctc caccccaggt gtttagaaga actggctaat 6060
attgaggtcc cagattcaac caacaaattt cggaagattg agaagaagat ccaaattcac 6120
aacacgagat atggagaact gttcacaagg ctgtgtacgc atatagagaa gaaactgctg 6180
gggtcatctt ggtctaacaa tgtcccccgg tcagaggagt tcagcagcat tcgtacggat 6240
ccggcattct ggtttcactc aaaatggtcc acagccaagt ttgcatggct ccatataaaa 6300
cagatccaga ggcatctgat ggtggcagct aggacaaggt ctgcggccaa caaattggtg 6360
atgctaaccc ataaggtagg ccaagtcttt gtcactcctg aacttgtcgt tgtgacgcat 6420
acgaatgaga acaagttcac atgtcttacc caggaacttg tattgatgta tgcagatatg 6480
atggagggca gagatatggt caacataata tcaaccacgg cggtgcatct cagaagctt 6539
<210> 5
<211> 15342
<212> DNA
<213> Newcastle disease virus (Newcastle disease virus)
<400> 5
taatacgact cactataggg accaaacaga gaatccgtga gttacgttaa aaggcgaagg 60
agcaattgaa gtcgcacggg tagaaggtgt gactctcgag tgcgagcccg aagcacaaac 120
tcgagaaagc cttctgccaa catgtcttcc gtatttgatg agtacgaaca gctcctcgcg 180
gctcagactc gccccaatgg agctcatgga gggggagaaa aagggagtac cttaaaagta 240
gacgtcccgg tattcactct taacagtgat gacccagaag atagatggag ctttgtggta 300
ttctgcctcc ggattgctgt tagcgaagat gccaacaaac cactcaggca aggtgctctc 360
atatctcttt tatgctccca ctcacaggta atgaggaacc atgttgccct tgcagggaaa 420
cagaatgaag ccacattggc cgtgcttgag attgatggct ttgccaacgg cacgccccag 480
ttcaacaata ggagtggagt gtctgaagag agagcacaga gatttgcgat gatagcagga 540
tctctccctc gggcatgcag caacggaacc ccgttcgtca cagccggggc cgaagatgat 600
gcaccagaag acatcaccga taccctggag aggatcctct ctatccaggc tcaagtatgg 660
gtcacagtag caaaagccat gactgcgtat gagactgcag atgagtcgga aacaaggcga 720
atcaataagt atatgcagca aggcagggtc caaaagaaat acatcctcta ccccgtatgc 780
aggagcacaa tccaactcac gatcagacag tctcttgcag tccgcatctt tttggttagc 840
gagctcaaga gaggccgcaa cacggcaggt ggtacctcta cttattataa cctggtaggg 900
gacgtagact catacatcag gaataccggg cttactgcat tcttcttgac actcaagtac 960
ggaatcaaca ccaagacatc agcccttgca cttagtagcc tctcaggcga catccagaag 1020
atgaagcagc tcatgcgttt gtatcggatg aaaggagata atgcgccgta catgacatta 1080
cttggtgata gtgaccagat gagctttgcg cctgccgaat atgcacaact ttactccttt 1140
gccatgggta tggcatcagt cctagataaa ggtactggga aataccaatt tgccagggac 1200
tttatgagca catcattctg gagacttgga gtagagtacg ctcaggctca gggaagtagc 1260
attaacgagg atatggctgc cgagctaaag ctaaccccag cagcaaggag gggcctggca 1320
gctgctgccc aacgggtctc cgaggagacc agcagcatag acatgcctac tcaacaagtc 1380
ggagtcctca ctgggcttag cgaggggggg tcccaagctc tacaaggcgg atcgaataga 1440
tcgcaagggc aaccagaagc cggggatggg gagacccaat tcctggatct gatgagagcg 1500
gtagcaaata gcatgaggga ggcgccaaac tctgcacagg gcactcccca atcggggcct 1560
cccccaactc ctgggccatc ccaagataac gacaccgact gggggtattg atggacaaaa 1620
cccagcctgc ttccacaaaa acatcccaat gccctcaccc gtagtcgacc cctcgatttg 1680
cggctctata tgaccacacc ctcaaacaaa catccccctc tttcctccct ccccctgctg 1740
tacaactccg cacgccctag ataccacagg cacaatgcgg ctcactaaca atcaaaacag 1800
agccgaggga attagaaaaa agtacgggta gaagagggat attcagagat cagggcaagt 1860
ctcccgagtc tctgctctct cctctacctg atagaccagg acaaacatgg ccacctttac 1920
agatgcagag atcgacgagc tatttgagac aagtggaact gtcattgaca acataattac 1980
agcccagggt aaaccagcag agactgttgg aaggagtgca atcccacaag gcaagaccaa 2040
ggtgctgagc gcagcatggg agaagcatgg gagcatccag ccaccggcca gtcaagacaa 2100
ccccgatcga caggacaggt ctgacaaaca accatccaca cccgagcaaa cgaccccgca 2160
tgacagcccg ccggccacat ccgccgacca gccccccacc caggccacag acgaagccgt 2220
cgacacacag ctcaggaccg gagcaagcaa ctctctgctg ttgatgcttg acaagctcag 2280
caataaatcg tccaatgcta aaaagggccc atggtcgagc ccccaagagg ggaatcacca 2340
acgtccgact caacagcagg ggagtcaacc cagtcgcgga aacagtcagg aaagaccgca 2400
gaaccaagtc aaggccgccc ctggaaacca gggcacagac gtgaacacag catatcatgg 2460
acaatgggag gagtcacaac tatcagctgg tgcaacccct catgctctcc gatcaaggca 2520
gagccaagac aatacccttg tatctgcgga tcatgtccag ccacctgtag actttgtgca 2580
agcgatgatg tctatgatgg aggcgatatc acagagagta agtaaggttg actatcagct 2640
agatcttgtc ttgaaacaga catcctccat ccctatgatg cggaccgaaa tccaacagct 2700
gaaaacatct gttgcagtca tggaagccaa cttgggaatg atgaagattc tggatcccgg 2760
ttgtgccaac atttcatctc tgagtgatct acgggcagtt gcccgatctc acccggtttt 2820
agtttcaggc cctggagacc cctctcccta tgtgacacaa ggaggcgaaa tggcacttaa 2880
taaactttcg caaccagtgc cacatccatc tgaattgatt aaacccgcca ctgcatgcgg 2940
gcctgatata ggagtggaaa aggacactgt ccgtgcattg atcatgtcac gcccaatgca 3000
cccgagttct tcagccaagc tcctaagcaa gttagatgca gccgggtcga tcgaggaaat 3060
caggaaaatc aagcgccttg ctctaaatgg ttaattacta ctgccacacg tagcgggtcc 3120
ctgtccactc ggcatcacac ggaatctgca ccgagttccc ccccgcagac ccaaggtcca 3180
actctccaag cggcaatcct ctctcgcttc ctcagcccca ctgaatgatc gcgtaaccgt 3240
aattaatcta gctacattta agattaagaa aaaatacggg tagaattgga gtgccccaat 3300
tgtgccaaga tggactcatc taggacaatt gggctgtact ttgattctgc ccattcttct 3360
agcaacctgt tagcatttcc gatcgtccta caagacacag gagatgggaa gaagcaaatc 3420
gccccgcaat ataggatcca gcgccttgac ttgtggactg atagtaagga ggactcagta 3480
ttcatcacca cctatggatt catctttcaa gctgggaatg aagaagccac tgtcggcatg 3540
atcgatgata aacccaagcg cgagttactt tccgctgcga tgctctgcct aggaagcgtc 3600
ccaaataccg gagaccttat tgagctggca agggcctgtc tcactatgat agtcacatgc 3660
aagaagagtg caactaatac tgagagaatg gttttctcag tagtgcaggc accccaagtg 3720
ctgcaaagct gtagggttgt ggcaaacaaa tactcatcag tgaatgcagt caagcacgtg 3780
aaagcgccag agaagattcc cgggagtgga accctagaat acaaggtgaa ctttgtctcc 3840
ttgactgtgg taccgaagaa ggatgtctac aagatcccag ctgcagtatt gaaggtttct 3900
ggctcgagtc tgtacaatct tgcgctcaat gtcactatta atgtggaggt agacccgagg 3960
agtcctttgg ttaaatctct gtctaagtct gacagcggat actatgctaa cctcttcttg 4020
catattggac ttatgaccac cgtagatagg aaggggaaga aagtgacatt tgacaagctg 4080
gaaaagaaaa taaggagcct tgatctatct gtcgggctca gtgatgtgct cgggccttcc 4140
gtgttggtaa aagcaagagg tgcacggact aagctcttgg cacctttctt ctctagcagt 4200
gggacagcct gctatcccat agcaaatgct tctcctcagg tggccaagat actctggagt 4260
caaaccgcgt gcctgcggag cgttaaaatc attatccaag caggtaccca acgcgctatc 4320
gcagtgaccg ccgaccacga ggttacctct actaagctgg agaaggggca cacccttgcc 4380
aaatacaatc cttttaagaa ataagctgcg tctctgagat tgcgctccgc ccactcaccc 4440
agatcatcat gacacaaaaa actaatctgt cttgattatt tacagttagt ttacctgtct 4500
atcaagttag aaaaaacacg ggtagaagat tctggatccc ggttggcgcc ctccaggtgc 4560
aagatgggct ccagaccttc taccaagaac ccagcaccta tgatgctgac tatccgggtt 4620
gcgctggtac tgagttgcat ctgtccggca aactccattg atggcaggcc tcttgcagct 4680
gcaggaattg tggttacagg agacaaagcc gtcaacatat acacctcatc ccagacagga 4740
tcaatcatag ttaagctcct cccgaatctg cccaaggata aggaggcatg tgcgaaagcc 4800
cccttggatg catacaacag gacattgacc actttgctca ccccccttgg tgactctatc 4860
cgtaggatac aagagtctgt gactacatct ggagggggga gacaggggcg ccttataggc 4920
gccattattg gcggtgtggc tcttggggtt gcaactgccg cacaaataac agcggccgca 4980
gctctgatac aagccaaaca aaatgctgcc aacatcctcc gacttaaaga gagcattgcc 5040
gcaaccaatg aggctgtgca tgaggtcact gacggattat cgcaactagc agtggcagtt 5100
gggaagatgc agcagtttgt taatgaccaa tttaataaaa cagctcagga attagactgc 5160
atcaaaattg cacagcaagt tggtgtagag ctcaacctgt acctaaccga attgactaca 5220
gtattcggac cacaaatcac ttcacctgct ttaaacaagc tgactattca ggcactttac 5280
aatctagctg gtggaaatat ggattactta ttgactaagt taggtgtagg gaacaatcaa 5340
ctcagctcat taatcggtag cggcttaatc accggtaacc ctattctata cgactcacag 5400
actcaactct tgggtataca ggtaactcta ccttcagtcg ggaacctaaa taatatgcgt 5460
gccacctact tggaaacctt atccgtaagc acaaccaggg gatttgcctc ggcacttgtc 5520
ccaaaagtgg tgacacaggt cggttctgtg atagaagaac ttgacacctc atactgtata 5580
gaaactgact tagatttata ttgtacaaga atagtaacgt tccctatgtc ccctggtatt 5640
tattcctgct tgagcggcaa tacgtcggcc tgtatgtact caaagaccga aggcgcactt 5700
actacaccat acatgactat caaaggttca gtcatcgcca actgcaagat gacaacatgt 5760
agatgtgtaa accccccggg tatcatatcg caaaactatg gagaagccgt gtctctaata 5820
gataaacaat catgcaatgt tttatcctta ggcgggataa ctttaaggct cagtggggaa 5880
ttcgatgtaa cttatcagaa gaatatctca atacaagatt ctcaagtaat aataacaggc 5940
aatcttgata tctcaactga gcttgggaat gtcaacaact cgatcagtaa tgctttgaat 6000
aagttagagg aaagcaacag aaaactagac aaagtcaatg tcaaactgac tagcacatct 6060
gctctcatta cctatatcgt tttgactatc atatctcttg tttttggtat acttagcctg 6120
attctagcat gctacctaat gtacaagcaa aaggcgcaac aaaagacctt attatggctt 6180
gggaataata ctctagatca gatgagagcc actacaaaaa tgtgaacaca gatgaggaac 6240
gaaggtttcc ctaatagtaa tttatgtgaa agttctggta gtctgtcagt tcagagagtt 6300
aagaaaaaac taccggttgt agatgaccaa aggacgatat acgggtagaa cggtaagaga 6360
ggccgcccct caattgcgag ccaggcttca caacctccgt tctaccgctt caccgacaac 6420
agtcctcaat catggaccgc gccgttagcc aagttgcgtt agagaatgat gaaagagagg 6480
caaaaaatac atggcgcttg atattccgga ttgcaatctt attcttaaca gtagtgacct 6540
tggctatatc tgtagcctcc cttttatata gcatgggggc tagcacacct agcgatcttg 6600
taggcatacc gactaggatt tccagggcag aagaaaagat tacatctaca cttggttcca 6660
atcaagatgt agtagatagg atatataagc aagtggccct tgagtctccg ttggcattgt 6720
taaatactga gaccacaatt atgaacgcaa taacatctct ctcttatcag attaatggag 6780
ctgcaaacaa cagtgggtgg ggggcaccta tccatgaccc agattatata ggggggatag 6840
gcaaagaact cattgtagat gatgctagtg atgtcacatc attctatccc tctgcatttc 6900
aagaacatct gaattttatc ccggcgccta ctacaggatc aggttgcact cgaataccct 6960
catttgacat gagtgctacc cattactgct acacccataa tgtaatattg tctggatgca 7020
gagatcactc acattcatat cagtatttag cacttggtgt gctccggaca tctgcaacag 7080
ggagggtatt cttttctact ctgcgttcca tcaacctgga cgacacccaa aatcggaagt 7140
cttgcagtgt gagtgcaact cccctgggtt gtgatatgct gtgctcgaaa gtcacggaga 7200
cagaggaaga agattataac tcagctgtcc ctacgcggat ggtacatggg aggttagggt 7260
tcgacggcca gtaccacgaa aaggacctag atgtcacaac attattcggg gactgggtgg 7320
ccaactaccc gggagtaggg ggtggatctt ttattgacag ccgcgtatgg ttctcagtct 7380
acggagggtt aaaacccaat tcacccagtg acactgtaca ggaagggaaa tatgtgatat 7440
acaagcgata caatgacaca tgcccagatg agcaagacta ccagattcga atggccaagt 7500
cttcgtataa gcctggacgg tttggtggga aacgcataca gcaggctatc ttatctatca 7560
aggtgtcaac atccttaggc gaagacccgg tactgactgt accgcccaac acagtcacac 7620
tcatgggggc cgaaggcaga attctcacag tagggacatc tcatttcttg tatcaacgag 7680
ggtcatcata cttctctccc gcgttattat atcctatgac agtcagcaaa aaaacagcca 7740
ctcttcatag tccttataca ttcaatgcct tcactcggcc aggtagtatc ccttgccagg 7800
cttcagcaag atgccccaac tcgtgtgtta ctggagtcta tacagatcca tatcccctaa 7860
tcttctatag aaaccacacc ttgcgagggg tattcgggac aatgcttgat ggtgtacaag 7920
caagacttaa ccctgcgtct gcagtattcg atagcacatc ccgcagtcgc attactcgag 7980
tgagttcaag cagtaccaaa gcagcataca caacatcaac ttgttttaaa gtggtcaaga 8040
ctaataagac ctattgtctc agcattgctg aaatatctaa tactctcttc ggagaattca 8100
gaatcgtccc gttactagtt gagatcctca aagatgacgg ggttagagaa gccaggtctg 8160
gctagttgag tcaattataa aggagttgga aagatggcat tgtatcacct atcctctgcg 8220
acatcaagaa tcaaaccgaa tgccggcgcg tgctcgaatt ccatgttgcc agttgaccac 8280
aatcagccag tgctcatgcg atcagattaa gccttgtcaa tagtctcttg atttagaaaa 8340
aatgtaagtg gcaatgagat acaaggcaaa acagctcatg gtaaataata cgggtaggac 8400
atggcgagct ccggtcctga aagggcagag catcagatta tcctaccaga gtcacacctg 8460
tcttcaccat tggtcaagca caaactactc tattactgga aattaactgg gctaccgctt 8520
cctgatgaat gtgacttcga ccacctcatt ctcagccgac aatggaaaaa aatacttgaa 8580
tcggcctctc ctgatactga gagaatgata aaactcggaa gggcagtaca ccaaactctt 8640
aaccacaatt ccagaataac cggagtgctc caccccaggt gtttagaaga actggctaat 8700
attgaggtcc cagattcaac caacaaattt cggaagattg agaagaagat ccaaattcac 8760
aacacgagat atggagaact gttcacaagg ctgtgtacgc atatagagaa gaaactgctg 8820
gggtcatctt ggtctaacaa tgtcccccgg tcagaggagt tcagcagcat tcgtacggat 8880
ccggcattct ggtttcactc aaaatggtcc acagccaagt ttgcatggct ccatataaaa 8940
cagatccaga ggcatctgat ggtggcagct aggacaaggt ctgcggccaa caaattggtg 9000
atgctaaccc ataaggtagg ccaagtcttt gtcactcctg aacttgtcgt tgtgacgcat 9060
acgaatgaga acaagttcac atgtcttacc caggaacttg tattgatgta tgcagatatg 9120
atggagggca gagatatggt caacataata tcaaccacgg cggtgcatct cagaagctta 9180
tcagagaaaa ttgatgacat tttgcggtta atagacgctc tggcaaaaga cttgggtaat 9240
caagtctacg atgttgtatc actaatggag ggatttgcat acggagctgt ccagctactc 9300
gagccgtcag gtacatttgc aggagatttc ttcgcattca acctgcagga gcttaaagac 9360
attctaattg gcctcctccc caatgatata gcagaatccg tgactcatgc aatcgctact 9420
gtattctctg gtttagaaca gaatcaagca gctgagatgt tgtgtctgtt gcgtctgtgg 9480
ggtcacccac tgcttgagtc ccgtattgca gcaaaggcag tcaggagcca aatgtgcgca 9540
ccgaaaatgg tagactttga tatgatcctt caggtactgt ctttcttcaa gggaacaatc 9600
atcaacgggt acagaaagaa gaatgcaggt gtgtggccgc gagtcaaagt ggatacaata 9660
tatgggaagg tcattgggca actacatgca gattcagcag agatttcaca cgatatcatg 9720
ttgagagagt ataagagttt atctgcactt gaatttgagc catgtataga atatgaccct 9780
gtcaccaacc tgagcatgtt cctaaaagac aaggcaatcg cacaccccaa cgataattgg 9840
cttgcctcgt ttaggcggaa ccttctctcc gaagaccaga agaaacatgt aaaagaagca 9900
acttcgacta atcgcctctt gatagagttt ttagagtcaa atgattttga tccatataaa 9960
gagatggaat atctgacgac ccttgagtac cttagagatg acaatgtggc agtatcatac 10020
tcgctcaagg agaaggaagt gaaagttaat ggacggatct tcgctaagct gacaaagaag 10080
ttaaggaact gtcaggtgat ggcggaaggg atcctagccg atcagattgc acctttcttt 10140
cagggaaatg gagtcattca ggatagcata tccttgacca agagtatgct agcgatgagt 10200
caactgtctt ttaacagcaa taagaaacgt atcactgact gtaaagaaag agtatcttca 10260
aaccgcaatc atgatccgaa aagcaagaac cgtcggagag ttgcaacctt cataacaact 10320
gacctgcaaa agtactgtct taattggaga tatcagacaa tcaaattgtt cgctcatgcc 10380
atcaatcagt tgatgggcct acctcacttc ttcgaatgga ttcacctaag actgatggac 10440
actacgatgt tcgtaggaga ccctttcaat cctccaagtg accctactga ctgtgacctc 10500
tcaagagtcc ctaatgatga catatatatt gtcagtgcca gagggggtat cgaaggatta 10560
tgccagaagc tatggacaat gatctcaatt gctgcaatcc aacttgctgc agctagatcg 10620
cattgtcgtg ttgcctgtat ggtacagggt gataatcaag taatagcagt aacgagagag 10680
gtaagatcag acgactctcc ggagatggtg ttgacacagt tgcatcaagc cagtgataat 10740
ttcttcaagg aattaattca tgtcaatcat ttgattggcc ataatttgaa ggatcgtgaa 10800
accatcaggt cagacacatt cttcatatac agcaaacgaa tcttcaaaga tggagcaatc 10860
ctcagtcaag tcctcaaaaa ttcatctaaa ttagtgctag tgtcaggtga tctcagtgaa 10920
aacaccgtaa tgtcctgtgc caacattgcc tctactgtag cacggctatg cgagaacggg 10980
cttcccaaag acttctgtta ctatttaaac tatataatga gttgtgtgca gacatacttt 11040
gactctgagt tctccatcac caacaattcg caccccgatc ttaatcagtc gtggattgag 11100
gacatctctt ttgtgcactc atatgttctg actcctgccc aattaggggg actgagtaac 11160
cttcaatact caaggctcta cactagaaat atcggtgacc cggggactac tgcttttgca 11220
gagatcaagc gactagaagc agtgggatta ctgagtccta acattatgac taatatctta 11280
actaggccgc ctgggaatgg agattgggcc agtctgtgca acgacccata ctctttcaat 11340
tttgagactg ttgcaagccc aaatattgtt cttaagaaac atacgcaaag agtcctattt 11400
gaaacttgtt caaatccctt attgtctgga gtgcacacag aggataatga ggcagaagag 11460
aaggcattgg ctgaattctt gcttaatcaa gaggtgattc atccccgcgt tgcgcatgcc 11520
atcatggatg caagctctgt aggtaggaga aagcaaattc aagggcttgt tgacacaaca 11580
aacaccgtaa ttaagattgc gcttactagg aggccattag gcatcaagag gctgatgcgg 11640
atagtcaatt attctagcat gcatgcaatg ctgtttagag acgatgtttt ttcctccagt 11700
agatccaacc accccttagt ctcttctaat atgtgttctc tgacactggc agactatgca 11760
cggaatagaa gctggtcacc tttgacggga ggcaggaaaa tactgggtgt atctaatcct 11820
gatacgatag aactcgtaga gggtgagatt cttagtgtaa gcggagggtg tacaagatgt 11880
gacagcggag atgaacaatt tacttggttc catcttccaa gcaatataga attgaccgat 11940
gacaccagca agaatcctcc gatgagggta ccatatctcg ggtcaaagac acaggagagg 12000
agagctgcct cacttgcaaa aatagctcat atgtcgccac atgtaaaggc tgccctaagg 12060
gcatcatccg tgttgatctg ggcttatggg gataatgaag taaattggac tgctgctctt 12120
acgattgcaa aatctcggtg taatgtaaac ttagagtatc ttcggttact gtccccttta 12180
cccacggctg ggaatcttca acatagacta gatgatggta taactcagat gacattcacc 12240
cctgcatctc tctacagggt gtcaccttac attcacatat ccaatgattc tcaaaggctg 12300
ttcactgaag aaggagtcaa agaggggaat gtggtttacc aacagatcat gctcttgggt 12360
ttatctctaa tcgaatcgat ctttccaatg acaacaacca ggacatatga tgagatcaca 12420
ctgcacctac atagtaaatt tagttgctgt atcagagaag cacctgttgc gattcctttc 12480
gagctacttg gggtggtacc ggaactgagg acagtgacct caaataagtt tatgtatgat 12540
cctagccctg tatcggaggg agactttgcg agacttgact tagctatctt caagagttat 12600
gagcttaatc tggagtcata tcccacgata gagctaatga acattctttc aatatccagc 12660
gggaagttga ttggccagtc tgtggtttct tatgatgaag atacctccat aaagaatgac 12720
gccataatag tgtatgacaa tacccgaaat tggatcagtg aagctcagaa ttcagatgtg 12780
gtccgcctat ttgaatatgc agcacttgaa gtgctcctcg actgttctta ccaactctat 12840
tacctgagag taagaggcct agacaatatt gtcttatata tgggtgattt atacaagaat 12900
atgccaggaa ttctactttc caacattgca gctacaatat ctcatcccgt cattcattca 12960
aggttacatg cagtgggcct ggtcaaccat gacggatcac accaacttgc agatacggat 13020
tttatcgaaa tgtctgcaaa actattagta tcttgcaccc gacgtgtgat ctccggctta 13080
tattcaggaa ataagtatga tctgctgttc ccatctgtct tagatgataa cctgaatgag 13140
aagatgcttc agctgatatc ccggttatgc tgtctgtaca cggtactctt tgctacaaca 13200
agagaaatcc cgaaaataag aggcttaact gcagaagaga aatgttcaat actcactgag 13260
tatttactgt cggatgctgt gaaaccatta cttagccccg atcaagtgag ctctatcatg 13320
tctcctaaca taattacatt cccagctaat ctgtactaca tgtctcggaa gagcctcaat 13380
ttgatcaggg aaagggagga cagggatact atcctggcgt tgttgttccc ccaagagcca 13440
ttattagagt tcccttctgt gcaagatatt ggtgctcgag tgaaagatcc attcacccga 13500
caacctgcgg catttttgca agagttagat ttgagtgctc cagcaaggta tgacgcattc 13560
acacttagtc agattcatcc tgaactcaca tctccaaatc cggaggaaga ctacttagta 13620
cgatacttgt tcagagggat agggactgca tcttcctctt ggtataaggc atctcatctc 13680
ctttctgtac ccgaggtaag atgtgcaaga cacgggaact ccttatactt agctgaaggg 13740
agcggagcca tcatgagtct tctcgaactg catgtaccac atgaaactat ctattacaat 13800
acgctctttt caaatgagat gaaccccccg caacgacatt tcgggccgac cccaactcag 13860
tttttgaatt cggttgttta taggaatcta caggcggagg taacatgcaa agatggattt 13920
gtccaagagt tccgtccatt atggagagaa aatacagagg aaagtgacct gacctcagat 13980
aaagcagtgg ggtatattac atctgtagtg ccctacaggt ctgtatcatt gctgcattgt 14040
gacattgaaa ttcctccagg gtccaatcag agcttactag atcaactagc tatcaattta 14100
tctctgattg ccatgcattc tgtaagggag ggcggggtag taatcatcaa agtgttgtat 14160
gcaatgggat actactttca tctactcatg aacttgtttg ctccgtgttc cacaaaagga 14220
tatattctct ctaatggtta tgcatgtcga ggagatatgg agtgttacct ggtatttgtc 14280
atgggttacc tgggcgggcc tacatttgta catgaggtgg tgaggatggc aaaaactctg 14340
gtgcagcggc acggtacgct tttgtctaaa tcagatgaga tcacactgac caggttattc 14400
acctcacagc ggcagcgtgt gacagacatc ctatccagtc ctttaccaag attaataaag 14460
tacttgagga agaatattga cactgcgctg attgaagccg ggggacagcc cgtccgtcca 14520
ttctgtgcgg agagtctggt gagcacgcta gcgaacataa ctcagataac ccagatcatc 14580
gctagtcaca ttgacacagt tatccggtct gtgatatata tggaagctga gggtgatctc 14640
gctgacacag tatttctatt taccccttac aatctctcta ctgacgggaa aaagaggaca 14700
tcacttaaac agtgcacgag acagatccta gaggttacaa tactaggtct tagagtcgaa 14760
aatctcaata aaataggcga tgtaatcagc ctagtgctta aaggcatgat ctccatggag 14820
gaccttatcc cactaaggac atacttgaag catagtacct gccctaaata cttgaaggct 14880
gtcctaggta ttaccaaact caaagaaatg tttacagaca cttctgtact gtacttgact 14940
cgtgctcaac aaaaattcta catgaaaact ataggcaatg cagtcaaagg atattacagt 15000
aactgtgact cttaacgaaa atcacatatt aataggctcc ttttttggcc aattgtattc 15060
ttgttgattt aatcatatta tgttagaaaa aagttgaacc ctgactcctt aggactcgaa 15120
ttcgaactca aataaatgtc ttaaaaaaag gttgcgcaca attattcttg agtgtagtct 15180
cgtcattcac caaatctttg tttggtgggt cggcatggca tctccacctc ctcgcggtcc 15240
gacctgggca tccgaaggag gacgcacgtc cactcggatg gctaagggag ggcgctagca 15300
taaccccttg gggcctctaa acgggtcttg aggggttttt tg 15342
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