Pigeon I-type paramyxovirus attenuated strain aQY as well as construction method and application thereof

Abstract

The invention relates to the technical field of poultry viruses, and discloses a pigeon I-type paramyxovirus attenuated strain aQY as well as a construction method and application thereof. According to the invention, a PPMV-1 QY strain genome is divided into five fragments A-E in full length by selecting a proper enzyme cutting site, a genetic marker is constructed through synonymous mutation based on a reverse genetic technology, the enzyme cutting site is added, the fragment A, the fragment B and the fragment E are properly modified, a full-length plasmid pOK-aQY is prepared, and then the full-length plasmid pOK-aQY and auxiliary plasmids pCI-NP, pCI-P and pCI-L are co-transfected to a BSR-T7 / 5 cell, so that the PPMV-1 QY strain is obtained. And after culture, taking cell supernatant, and inoculating the cell supernatant to a chick embryo to obtain the low virulent strain aQY of the pigeon type I paramyxovirus. The pigeon I-type paramyxovirus attenuated strain aQY strain has good stability, the MDT of the strain is 108 h, and the ICPI of the strain is 0. The method is suitable for research of PPMV-1 genome characteristics and preparation of pigeon Newcastle disease vaccines, and has important social benefits and outstanding application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of avian viruses, and specifically discloses a pigeon paramyxovirus type 1 attenuated strain aQY and its construction method and application. Background Art

[0002] Newcastle disease (ND) is an acute and highly contagious disease caused by Newcastle disease virus (NDV). NDV belongs to the order Mononegavirales, the family Paramyxoviridae, the subfamily Avulavirus, and the genus Avian paramyxovirus 1. Pigeon paramyxovirus type 1 (PPMV-1) is a variant of NDV with pigeons as the host, and its genomic characteristics and antigenic characteristics are similar to those of NDV. The genome of Pigeon paramyxovirus type 1 (PPMV-1) contains 6 genes in sequence: 3ʹ-NP-P-M-F-HN-L-5ʹ, encoding 8 proteins in total. Among them, the P gene can encode 3 proteins, namely P, V, and W, through RNA editing, and the other genes each encode one protein, namely NP, M, F, HN, and L. The F protein, also known as the fusion protein, is a surface glycoprotein that can mediate the fusion of the virus envelope and the host cell membrane. Among them, the cleavage site of the F protein is considered to be an important gene sequence determining the virulence of NDV. Research shows that PPMV-1 belongs to genotype VI of avian paramyxovirus type 1 (APMV-1) Class II. After PPMV-1 infects pigeons, the clinical manifestations are mostly neurological symptoms, such as paralysis, ataxia, and head and neck skew in the shape of "stargazing", causing huge economic losses to the pigeon breeding industry in China.

[0003] Reverse genetics adopts the research route of "gene" → "trait" to reveal the relationship between the phenotype and genotype of organisms. For negative-strand RNA viruses, their genomes cannot self-replicate and do not have infectious activity, and need to bind to nucleoproteins to form nucleocapsid complexes (RNPs) to replicate. At present, the prevention and control of PPMV-1 mainly uses the La Sota strain of Newcastle disease virus genotype II as the vaccine strain. There are significant antigenic differences between this vaccine strain and PPMV-1, and the antibodies induced by the vaccine are difficult to effectively neutralize the PPMV-1 strain, and cannot provide good protection for pigeons. Summary of the Invention

[0004] In view of the current situation that when the Newcastle disease vaccine prepared with the La Sota strain as the vaccine strain in the prior art is used for pigeons, due to the large antigenic difference between the vaccine strain and the epidemic strain, the protective effect on pigeons is not ideal. Based on reverse genetics technology, the present invention constructs a reverse genetic operating system for pigeon paramyxovirus type I QY strain, obtains the attenuated strain aQY of pigeon paramyxovirus type I, and this strain has good stability during passage and biological characteristics similar to those of the parental strain, laying a foundation for the research and development of a vaccine strain that matches the genotype of the epidemic strain.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions: In the first aspect, the present invention provides an attenuated strain aQY of pigeon paramyxovirus type I. The genes of the attenuated strain aQY of pigeon paramyxovirus type I include five fragments: A1, B1, C, D, and E1. Among them, the A1 fragment includes the NP gene and a part of the P gene, the B1 fragment includes the remaining part of the P gene, the M gene, and a part of the F gene, the C fragment includes the remaining part of the F gene, the HN gene, and the first part of the L gene, the D fragment includes the second part of the L gene, and the E1 fragment includes the remaining part of the L gene; Among them, in the B1 fragment, the cleavage site sequence of the NDV gene type VI F protein is mutated into the cleavage site sequence of the La Sota strain F protein, and a restriction enzyme site is introduced by synonymous mutation Nhe Ⅰ; The C fragment does not include the restriction enzyme site Nde Ⅰ, and includes the restriction enzyme site Kpn Ⅰ introduced by synonymous mutation; The D fragment includes the restriction enzyme site Nde Ⅰ.

[0006] According to the segmentation requirements, the present invention optimizes the restriction enzyme sites in the genomic sequence, and provides an attenuated strain aQY of pigeon paramyxovirus type I with good stability by rescuing the pigeon paramyxovirus type I QY strain. According to the virulence classification standard specified by the OIE, it is an attenuated strain, and the MDT of this attenuated strain is 108h, and the ICPI is 0.

[0007] The genetic markers in the attenuated strain aQY of pigeon paramyxovirus type I provided by the present invention are introduced by synonymous mutation. It has been verified that this genetic marker can stably exist after passage and can be used as a reliable genetic marker for identifying wild virus and rescued virus. In addition, in the present invention, genetic markers and two restriction enzyme sites ( Kpn Ⅰ and Nhe Ⅰ) are introduced by synonymous mutation. In this way, not only the integrity of the key functions of the virus is guaranteed, but also the accuracy and operation convenience of gene editing are optimized, the off-target risk is reduced, and it is beneficial for subsequent screening and application.

[0008] Among them, the A1 fragment includes the NP gene and a partial P gene, and the partial P gene is 1 to 1024 bases from the 5'-end; The B1 fragment includes the remaining part of the P gene, the M gene and a partial F gene, and the partial F gene is 1 to 1452 bases from the 5'-end; The C fragment includes the remaining part of the F gene, the HN gene and the first part of the L gene, and the first part of the L gene is 1 to 920 bases from the 5'-end; The D fragment includes the second part of the L gene, and the second part of the L gene is from the 905th base to the 4015th base from the 5'-end.

[0009] Furthermore, the 5'-end of the A1 fragment includes a T7 promoter sequence and a restriction site Asc I, and the 3'-end includes a restriction site Pac I.

[0010] Furthermore, a hepatitis D ribozyme sequence with self-cleavage function, a T7 terminator sequence and a restriction site are inserted at the 3'-end of the E1 fragment Not Ⅰ.

[0011] Furthermore, the nucleotide sequence of the A1 fragment is as shown in SEQ ID No.2; the nucleotide sequence of the B1 fragment is as shown in SEQ ID No.3; the nucleotide sequence of the C fragment is as shown in SEQ ID No.4; the nucleotide sequence of the D fragment is as shown in SEQ ID No.5; the nucleotide sequence of the E1 fragment is as shown in SEQ ID No.6.

[0012] Furthermore, the gene sequence of the pigeon paramyxovirus type 1 attenuated strain aQY is as shown in SEQ ID No.1.

[0013] In the second aspect, the present invention provides a method for constructing the above-mentioned pigeon paramyxovirus type 1 attenuated strain aQY, which is achieved by rescuing the pigeon paramyxovirus type 1 QY strain, and the construction method includes the following steps: Co-transfect the full-length genome plasmid of the QY strain and three helper plasmids into BSR-T7 / 5 cells, culture for 84h to 108h, repeatedly freeze-thaw the cells and take the cell supernatant, inoculate 9- to 11-day-old chicken embryos, and harvest the virus solution to obtain the pigeon paramyxovirus type 1 attenuated strain aQY; Among them, the three helper plasmids include plasmid pCI-NP, plasmid pCI-P and plasmid pCI-L.

[0014] Furthermore, the construction method of the full-length genome plasmid of the QY strain includes the following steps: S11. Extract the total genomic RNA of pigeon paramyxovirus type I QY strain, and prepare the genomic cDNA of QY strain by reverse transcription; Analyze the genomic cDNA sequence of QY strain, which is divided into fragment A to fragment E in sequence. According to the segmentation requirements, retain the restriction enzyme sites between the 2800nt and 3000nt of the genome Pac Ⅰ and the restriction enzyme sites between the 12300nt and 12500nt Nde Ⅰ, and eliminate the restriction enzyme sites between the 9200nt and 9300nt by synonymous mutation Nde Ⅰ; introduce a restriction enzyme site between the 5900nt and 6000nt by synonymous mutation Nhe Ⅰ, and introduce a restriction enzyme site between the 9200nt and 9400nt Kpn Ⅰ; add a restriction enzyme site at the 5ʹ end of fragment A Asc Ⅰ, and add a restriction enzyme site at the 3ʹ end of fragment E Not Ⅰ, to obtain the whole genome of QY strain -1; S12. Remove the T7 promoter sequence in plasmid pOK-12, and replace the multiple cloning site sequence of the vector with restriction enzyme sites Asc Ⅰ, Pac Ⅰ, Nhe Ⅰ, Kpn Ⅰ, Nde Ⅰ and Not I, to obtain plasmid pOK-12X; S13. Design corresponding primers according to the selected restriction enzyme sites, including five pairs of amplification primers SegA-F / SegA-R, SegB-F / SegB-R, SegC-R / SegC-F, SegD-F / SegD-R and SegE-F / SegE-R; S14. Using the whole genome of QY strain -1 cDNA as a template, and using the primers SegA-F / SegA-R, SegB-F / SegB-R, SegC-R / SegC-F, SegD-F / SegD-R and SegE-F / SegE-R, perform PCR amplification, detect and purify and recover the PCR amplification products to obtain five fragments A, B, C, D, and E; S15. Add a restriction enzyme site at the 5ʹ end Asc Ⅰ to fragment A, add a T7 promoter sequence, and add the base GGG after the T7 promoter sequence to enhance the transcription of downstream genes. The obtained nucleotide sequence is denoted as fragment A1; S16. Clone the B fragment into the plasmid pOK-12X, and by reverse PCR, mutate the F protein cleavage site in the B fragment from 112 RRQKRF 117Mutated into the cleavage site sequence of the F protein of the La Sota strain 112 GRQGRL 117 , and the obtained nucleotide sequence was denoted as the B1 fragment; S17. By means of inverse PCR, a hepatitis D ribozyme sequence and a T7 terminator sequence were introduced before the Not Ⅰ restriction site of the plasmid pOK-12X, denoted as the plasmid pOK-G, and the E fragment was cloned into the plasmid pOK-G by homologous recombination to obtain the plasmid pOK-E1; S18. The A1 fragment, B1 fragment, C fragment and D fragment were successively cloned into the plasmid pOK-E1 to obtain the full-length genome plasmid of the QY strain.

[0015] Further, in step S12, the primer pairs used for the modification of the plasmid pOK-12 were pOK-12-1-F / pOK-12-1-R and pOK-12-2-F / pOK-12-2-R; The nucleotide sequences of pOK-12-1-F / pOK-12-1-R are shown in SEQ ID No.7-SEQ ID No.8; the nucleotide sequences of pOK-12-2-F / pOK-12-2-R are shown in SEQ ID No.9-SEQ ID No.10.

[0016] Further, in step S17, when the plasmid pOK-12X was modified into the plasmid pOK-G, the primer pairs used were pOK-1-F / pOK-1-R, pOK-2-F / pOK-2-R and pOK-3-F / pOK-3-R in sequence; Among them, the nucleotide sequences of pOK-1-F / pOK-1-R are shown in SEQ ID No.11-SEQ ID No.12; The nucleotide sequences of pOK-2-F / pOK-2-R are shown in SEQ ID No.13-SEQ ID No.14; The nucleotide sequences of pOK-3-F / pOK-3-R are shown in SEQ ID No.15-SEQ ID No.16.

[0017] The present invention found that the target gene of the E1 fragment could not be obtained by direct amplification. It was necessary to clone the relevant gene into the vector plasmid pOK-G, and under the primer pairs PE-F / PE-R and E1-F / E1-R, the vector fragment and the E1 target gene fragment with homologous arms were amplified, and the plasmid pOK-E1 containing the E1 target gene fragment was obtained by homologous recombination technology.

[0018] Further, the construction methods of the three auxiliary plasmids include the following steps: According to the genomic sequence of QY strain, amplification primer pairs were designed for its NP gene, P gene and L gene for amplification, and restriction enzyme sites were introduced at the 5ʹ end and 3ʹ end of the gene fragments respectively Nhe Ⅰ and Sal Ⅰ to obtain the NP gene, P gene and L gene; The NP gene, P gene and L gene were respectively cloned into the eukaryotic expression vector pCI-neo to obtain plasmid pCI-NP, plasmid pCI-P and plasmid pCI-L.

[0019] Thirdly, the present invention provides the application of the above-mentioned pigeon paramyxovirus type I attenuated strain aQY strain or the construction method of the pigeon paramyxovirus type I attenuated strain aQY strain in the preparation of pigeon Newcastle disease vaccine.

[0020] The present invention successfully attenuated the pigeon paramyxovirus type I QY strain by reverse genetics technology to obtain the attenuated strain aQY strain. The aQY strain has an MDT of 108 h, an ICPI of 0, and good stability, laying a foundation for the study of the genomic characteristics of PPMV-1 epidemic strains and the development of genotype-matched vaccines. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 Schematic diagram of the construction strategy of the full-length cDNA plasmid of pigeon paramyxovirus type I QY strain in Example 1 of the present invention; Figure 2 Gel electrophoresis diagram of five fragments A1, B1, C, D and E1 in Example 1 of the present invention; Figure 3 Gel electrophoresis diagram of the NP gene, P gene and L gene in Example 2 of the present invention; Figure 4 Schematic diagram of the map of plasmid pCI-NP in Example 2 of the present invention; Figure 5 Schematic diagram of the map of plasmid pCI-P in Example 2 of the present invention; Figure 6 Schematic diagram of the map of plasmid pCI-L in Example 2 of the present invention; Figure 7 Fluorescence inverted microscope image of co-transfection of pCI-NP, pCI-P, pCI-L and pOK-Mini in Example 2 of the present invention; Figure 8This is the IFA result graph after the aQY strain F1 infects BHK-21 cells for 24 h in Example 3 of the present invention, where Figure 8 (a) is the blank control group, Figure 8 (b) is the aQY strain group; Figure 9 This is the sequencing result of the genetic marker and F protein cleavage site sequence of the aQY strain in Example 3 of the present invention, where Figure 9 (a) represents the genetic marker site sequence of the aQY strain, Figure 9 (b) represents the F protein cleavage site sequence of the aQY strain. Detailed implementation manners

[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from conventional biochemical reagent companies unless otherwise specified.

[0024] SPF chicken embryos are purchased from Repbio Corporation (Baoding); the virus RNA extraction kit is purchased from TransGen Biotech Co., Ltd.; the reverse transcription kit and high-fidelity enzyme are purchased from TaKaRa Bio Inc.; restriction enzymes, homologous recombination enzymes and T4 ligase are purchased from NEB Bio Inc.; agarose gel (Agarose Regular) is purchased from Shanghai Sangon Biotech Co., Ltd.; agarose gel purification kit and plasmid miniprep kit are purchased from OMEGA Company; 2×Taq Marker Mix is purchased from CW Biotech Co., Ltd.; Gold View nucleic acid dye is purchased from Saibaisheng Co., Ltd.; DMEM, trypsin, G418, and transfection reagent lipo2000 are purchased from Thermo Fisher Scientific; fetal bovine serum is purchased from Beijing Aoting Biotechnology Co., Ltd.

[0025] In the present invention, the EID 50 , MDT and ICPI of the virus are determined according to the OIE standards, and the specific determination methods are as follows.

[0026] Determination method of EID 50 : Dilute the virus solution 10-fold in PBS, take 10 -1 ~10 -9 9 dilutions of the virus are inoculated into 9-day-old SPF chicken embryos, 5 per group, 100 μL per embryo, and placed in an incubator for 3 d. Discard the chicken embryos that die within 24 h, and observe the death of the other chicken embryos every 12 h, record it, and store the dead chicken embryos at -20 °C. After 3 d, collect all allantoic fluids of the chicken embryos, measure the HA activity, and calculate the EID according to the Reed-Muench method50 。

[0027] Determination method of MDT: Dilute the virus solution 10-fold serially with PBS, and take 10 -1 ~10 -9 A total of 9 dilutions of the virus were inoculated into 9-day-old SPF chicken embryos, 5 embryos per group, 100 μL per embryo, and placed in an incubator for 5 days. Discard the chicken embryos that died within 24 hours, and observe the death of the other chicken embryos every 8 hours, record it, and store the dead chicken embryos at -20 °C. The highest dilution that caused 100% mortality was considered the minimum lethal dose. MDT was determined as the average death time (in hours) of the minimum lethal dose that killed all inoculated chicken embryos.

[0028] Determination method of ICPI: Intracerebrally inoculate 0.05 mL of freshly diluted virus allantoic fluid at a ratio of 1:10 into the brains of 10 1-day-old SPF chicks through the intracerebral route. Monitor the clinical symptoms and mortality of these chicks every 24 hours for 8 days. Each time of observation, score the birds as follows: 0 if normal, 1 if sick, and 2 if dead. ICPI is the average value of the scores of each chicken per observation within 8 days. The pathological types of ICPI are defined as follows: highly virulent strains, 1.50 - 2.00; moderately virulent strains, 0.70 - 1.50; weakly virulent strains, 0.00 - 0.70.

[0029] Example 1 The present invention provides a pigeon paramyxovirus type I attenuated strain aQY and its construction method, and the gene sequence of the aQY strain is shown in SEQ ID No.1. When constructing the pigeon paramyxovirus type I attenuated strain aQY, it is necessary to construct a full-length cDNA plasmid of the pigeon paramyxovirus type I QY strain. This example provides a construction method for the full-length cDNA plasmid of the pigeon paramyxovirus type I QY strain, and the schematic diagram of its construction strategy is as Figure 1 shown, and the specific construction process is as follows: S11. Extract the total RNA of the pigeon paramyxovirus type I QY strain genome, and prepare the QY strain genome cDNA by reverse transcription; Analyze the cDNA sequence of the QY strain genome, and divide it into fragments A to E in sequence. According to the segmentation requirements, select the original restriction enzyme sites Pac Ⅰ (2905 nt) and restriction enzyme site Nde Ⅰ (12390 nt); Eliminate Nde Ⅰ (9268 nt) by synonymous mutation, and this mutation can be used as a genetic marker; Introduce restriction enzyme sites Nhe Ⅰ (5990 nt) and Kpn Ⅰ (9296 nt) by synonymous mutation; Add a restriction enzyme site at the 5' end of fragment A Asc Ⅰ. Add a restriction enzyme site at the 3' end of fragment E Not Ⅰ. Obtain the whole genome of QY strain -1; S12. Remove the T7 promoter sequence in plasmid pOK-12, and replace the multiple cloning site sequence of the vector with a restriction enzyme site Asc Ⅰ. Pac Ⅰ. Nhe Ⅰ. Kpn Ⅰ. Nde Ⅰ and Not I. Obtain plasmid pOK-12X; S13. Design corresponding primers according to the selected restriction enzyme sites, including five pairs of amplification primers SegA-F / SegA-R, SegB-F / SegB-R, SegC-R / SegC-F, SegD-F / SegD-R, and SegE-F / SegE-R; S14. Using the cDNA of the whole genome of QY strain -1 as a template, and using the primers SegA-F / SegA-R, SegB-F / SegB-R, SegC-R / SegC-F, SegD-F / SegD-R, and SegE-F / SegE-R, perform PCR amplification, detect and purify and recover the PCR amplification products to obtain five fragments A, B, C, D, and E; S15. Add a restriction enzyme site at the 5' end Asc Ⅰ. Add a T7 promoter sequence to fragment A with a restriction enzyme site added at the 5' end, and add the base GGG after the T7 promoter sequence to enhance the transcription of downstream genes. The obtained nucleotide sequence is denoted as fragment A1; S16. Clone the B fragment into the plasmid pOK-12X, and by reverse PCR, mutate the F protein cleavage site in the B fragment from 112 RRQKRF 117 to the F protein cleavage site sequence of the La Sota strain 112 GRQGRL 117 , amplify under the primer pair SegB-F / SegB-R, and introduce a restriction enzyme site Nhe Ⅰ, The obtained nucleotide sequence is denoted as fragment B1; S17. By reverse PCR, introduce a hepatitis delta ribozyme sequence and a T7 terminator sequence in front of the Not Ⅰ restriction enzyme site of the plasmid pOK-12X, denoted as plasmid pOK-G, and clone the E fragment into the plasmid pOK-G by homologous recombination to obtain plasmid pOK-E1; S18. Clone the A1 fragment, B1 fragment, C fragment, and D fragment into the plasmid pOK-E1 in sequence to obtain the full-length genome plasmid of the QY strain, denoted as plasmid pOK-aQY.

[0030] Among them, the nucleotide sequence of the A1 fragment is shown in SEQ ID No. 2; The nucleotide sequence of the B1 fragment is shown in SEQ ID No. 3; The nucleotide sequence of the C fragment is shown in SEQ ID No. 4; The nucleotide sequence of the D fragment is shown in SEQ ID No. 5; The nucleotide sequence of the E1 fragment is shown in SEQ ID No. 6.

[0031] In the present invention, five fragments, namely A1, B1, C, D, and E1, are amplified respectively using PCR amplification primers. The results of 1% agarose gel electrophoresis are as Figure 2 shown, and their sizes are consistent with the expectations. The above 5 fragments are sequentially ligated to the modified vector pOK-12X to obtain the full-length plasmid pOK-QY. The plasmid is sent to Sangon for sequencing, and the sequencing results are consistent with the expectations.

[0032] Among them, in step S12, the primer pairs used for the modification of the plasmid pOK-12 are pOK-12-1-F / pOK-12-1-R and pOK-12-2-F / pOK-12-2-R; the nucleotide sequences of pOK-12-1-F / pOK-12-1-R are shown in SEQ ID No. 7 - SEQ ID No. 8; the nucleotide sequences of pOK-12-2-F / pOK-12-2-R are shown in SEQ ID No. 9 - SEQ ID No. 10.

[0033] In step S15, the mutant primer pairs used for the modification of the A1 fragment are A1-1-F / A1-1-R and A1-2-F / A1-2-R. The nucleotide sequences of A1-1-F / A1-1-R are shown in SEQ ID No. 21 - SEQ ID No. 22, and the nucleotide sequences of A1-2-F / A1-2-R are shown in SEQ ID No. 23 - SEQ ID No. 24.

[0034] In step S17, when the plasmid pOK-12X is modified to the plasmid pOK-G, the primer pairs used in sequence are pOK-1-F / pOK-1-R, pOK-2-F / pOK-2-R, and pOK-3-F / pOK-3-R; Among them, the nucleotide sequences of the pOK-1-F / pOK-1-R are shown in SEQ ID No. 11 - SEQ ID No. 12; The nucleotide sequences of the pOK-2-F / pOK-2-R are shown in SEQ ID No. 13 - SEQ ID No. 14; The nucleotide sequences of pOK-3-F / pOK-3-R are shown in SEQ ID No.15-SEQ ID No.16.

[0035] In step S17, when the plasmid pOK-1 was modified into the plasmid pOK-E1, the primer pairs used included PE-F / PE-R and E1-F / E1-R; Among them, the nucleotide sequences of PE-F / PE-R are shown in SEQ ID No.17-SEQ ID No.18; The nucleotide sequences of E1-F / E1-R are shown in SEQ ID No.19-SEQ ID No.20.

[0036] The amplification primers for the five fragments A, B, C, D, and E of pigeon paramyxovirus type I are shown in Table 1 below.

[0037] Table 1

[0038] Example 2 This example provides three methods for constructing helper plasmids and verifies the constructed helper plasmids. The three methods for constructing helper plasmids are as follows: According to the genomic sequence of the QY strain, amplification primer pairs were designed for the open reading frames (ORFs) of its NP, P, and L genes for amplification, and specific restriction enzyme site sequences were introduced at both ends of the primers. A restriction enzyme site Nhe Ⅰ was introduced at the 5ʹ end of the gene fragment, and a restriction enzyme site Sal Ⅰ was introduced at the 3ʹ end to obtain the amplification primer pairs SegNP-F / SegNP-R, SegP-F / SegP-R, and SegL-F / SegL-R; Using the genomic cDNA of the QY strain as a template, PCR amplification was performed using the above three pairs of primer pairs to obtain the NP gene, P gene, and L gene; the 1% agarose gel electrophoresis results of the NP gene, P gene, and L gene are as Figure 3 shown, and their fragments are 1470bp, 1188bp, and 6615bp respectively, which are consistent with the expected band sizes.

[0039] The NP gene, P gene, and L gene were respectively cloned into the eukaryotic expression vector pCI-neo to construct three helper plasmids, namely plasmid pCI-NP, plasmid pCI-P, and plasmid pCI-L.

[0040] Among them, the schematic diagram of the map of plasmid pCI-NP is as Figure 4 shown, the schematic diagram of the map of plasmid pCI-P is as Figure 5 shown, and the schematic diagram of the map of plasmid pCI-L is as Figure 6 shown.

[0041] Among them, the amplification primers for the NP gene, P gene, and L gene of pigeon paramyxovirus type I are shown in Table 2 below.

[0042] Table 2

[0043] In this example, the gene sequences and functions of the three constructed helper plasmids were also verified, as follows: The helper plasmids were co-transfected with the micro-genome plasmid containing the green fluorescent protein gene stored in our laboratory into BSR-T7 / 5 cells, and the expression of the green fluorescent protein was observed. The BSR-T7 / 5 cells were seeded in a 6-well plate. When the cell confluence reached 80%-90%, transfection was carried out according to the instructions of the transfection reagent Lipofectamine 2000. The total amount of transfected plasmids was 4 μg, and the mass ratio of the micro-genome plasmids pOK-Mini, pCI-NP, pCI-P, and pCI-L was 4:2:2:1.

[0044] (1) Gene verification The sequencing results of the constructed helper plasmids pCI-NP, pCI-P, and pCI-L by Sangon Biotech were consistent with the expected gene fragments.

[0045] (2) Function verification The plasmids pCI-NP, pCI-P, and pCI-L were co-transfected with the micro-genome plasmid pOK-Mini containing the green fluorescent protein gene into BSR-T7 / 5 cells. Observation under a fluorescence inverted microscope showed that when pCI-NP, pCI-P, pCI-L, and pOK-Mini were co-transfected, the green fluorescent protein gene could be normally expressed, while no green fluorescence appeared when pCI-NP, pCI-P, and pOK-Mini were co-transfected, indicating that the green fluorescent protein gene could not be normally expressed. The fluorescence inverted microscope images of the co-transfection of pCI-NP, pCI-P, pCI-L, and pOK-Mini are as Figure 7 shown.

[0046] It can be Figure 7 seen that green fluorescence can be observed under a fluorescence inverted microscope after co-transfection, indicating that the helper plasmids pCI-NP, pCI-P, and pCI-L can function normally.

[0047] Example 3 This example provides a method for rescuing pigeon paramyxovirus type I QY strain to obtain the attenuated strain aQY strain of pigeon paramyxovirus type I, and measures indicators such as the pathogenic index and median infectious dose of the aQY strain. The specific steps are as follows; (1) Obtaining the attenuated strain aQY strain of pigeon paramyxovirus type I BSR-T7 / 5 cells were cultured in a 6-well plate in advance. When the cells grew to 80%-90%, transfection was carried out. Using Lipofectamine 2000 as the transfection reagent, take two 1.5 mL clean centrifuge tubes, add 250 μL of Opti-MEM medium to each tube. Add the plasmid used for transfection (the mass of the plasmid is 10 μg, and the mass ratio of the four plasmids pOK-aQY: pCI-NP: pCI-P: pCI-L is 4:2:2:1) to one centrifuge tube, and add 30 μL of the transfection reagent Lipofectamine 2000 to the other centrifuge tube. Vortex and mix well, and incubate at room temperature for 5 min; mix the diluted plasmid with the diluted Lipofectamine 2000, gently mix well, and react at room temperature for 20 min. Discard the cell supernatant in the cell culture plate, and add Opti-MEM medium to the culture plate. Slowly drop the mixed solution into the cell culture medium, and place it in a cell culture incubator at 37 °C and 5% CO2. After 6 h, discard the cell supernatant, add the cell maintenance solution with 2% FBS, and place it in a cell culture incubator at 37 °C and 5% CO2 for 96 h. Freeze-thaw the cells repeatedly and take the cell supernatant; inoculate SPF chicken embryos through the allantoic cavity route at a dose of 200 μL / embryo. Observe the death of chicken embryos every 12 h, discard the chicken embryos that died within 24 h, collect the allantoic fluid of the remaining chicken embryos after 96 h of inoculation, detect it, collect the positive allantoic fluid to obtain the rescued virus (i.e., the attenuated strain aQY of pigeon paramyxovirus type I), label it as the F1 generation of the aQY strain, and store it in a -80 °C refrigerator.

[0048] (2)Determination of indicators such as replication ability and virulence The F1 generation virus solution was continuously passaged on chicken embryos for 5 generations, and the hemagglutination titer and genetic markers of each generation of virus solution were determined; verify whether the virus was successfully rescued by IFA; judge the replication and virulence of the strain by measuring the mean death time (MDT) of chicken embryos, the intracerebral pathogenicity index (ICPI) of 1-day-old chicks, and the 50% egg infectious dose (EID 50 ) of the rescued strain.

[0049] ① Infect BHK-21 cells with the F5 generation of the aQY strain, and perform an indirect immunofluorescence assay 24 h after infection. The IFA results are as Figure 8 shown, where Figure 8 (a) is the blank control group, Figure 8 (b) is the aQY strain group. In the blank control group, BHK-21 cells were not infected with the virus.

[0050] From Figure 8It can be seen that green fluorescence can be observed in the cell wells infected with the aQY strain, while the opposite is true for the cell wells in the uninfected group, proving the successful rescue of the virus.

[0051] ② The hemagglutination titers (i.e., HA titers) of the aQY strain virus solutions of F1 - F5 generations are shown in Table 3 as follows.

[0052] Table 3

[0053] As can be seen from Table 3, the HA titer of the F3 - generation aQY strain reached 2 10 and tended to be stable.

[0054] ③ In this example, the genetic markers and the F - protein cleavage site sequence of the F5 - generation aQY strain were sequenced, and the sequencing results are as Figure 9 shown, where Figure 9 (a) represents the genetic marker site sequence of the aQY strain; Figure 9 (b) represents the F - protein cleavage site sequence of the aQY strain. It can be Figure 9 seen that compared with the genomic sequence of the parental strain pigeon paramyxovirus type I QY strain, the genetic marker sites of the attenuated pigeon paramyxovirus type I aQY strain mutated, and at the same time, the F - protein cleavage site sequence mutated, further proving the successful rescue of the recombinant virus aQY strain and its good genetic stability.

[0055] ④ In the present invention, the MDT, ICPI, and EID 50 biological characteristic indexes of the F5 - generation virus solution of the aQY strain were measured, and the measurement results are shown in Table 4.

[0056] Table 4

[0057] As can be seen from Table 4, according to the virulence classification standard stipulated by the OIE, the parental strain QY strain belongs to the medium - virulence strain, and the rescued strain belongs to the attenuated strain; the EID 50 measurement results of the rescued strain aQY strain show that the rescued strain aQY strain has good replication ability on chicken embryos.

[0058] To sum up, in the present invention, by selecting appropriate restriction enzyme sites, the full - length genome of the PPMV - 1 QY strain was divided into 5 fragments, namely A - E. Based on the reverse genetics technology, a T7 promoter sequence was inserted at the 5ʹ - end of the A fragment, the F - protein cleavage site sequence of the B fragment was mutated into the F - protein cleavage site sequence of the La Sota strain, and a restriction enzyme site Nhe Ⅰ was introduced by synonymous mutation. A hepatitis delta virus ribozyme sequence with self - cleavage function and a T7 terminator sequence were inserted at the 3ʹ - end of the E fragment, and they were respectively labeled as A1, B1, and E1 fragments; at the same time, the restriction enzyme site in the C fragment was removed by synonymous mutation NdeThe restriction enzyme site introduced as a genetic marker Kpn Ⅰ. After constructing the full-length plasmid pOK-aQY and co-transfecting it with the helper plasmids pCI-NP, pCI-P, and pCI-L into BSR-T7 / 5 cells, the cell supernatant was collected after culturing and inoculated into chicken embryos to obtain the attenuated strain aQY of pigeon paramyxovirus type Ⅰ. Experiments showed that the attenuated strain aQY of pigeon paramyxovirus type Ⅰ had good stability, with an MDT of 108 h and an ICPI of 0.

[0059] For the above reasons, the attenuated strain aQY of pigeon paramyxovirus type Ⅰ provided by the present invention can be used as a vaccine strain for preventing pigeon Newcastle disease and further prepare a pigeon Newcastle disease vaccine.

[0060] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A pigeon type I paramyxovirus attenuated strain aQY strain, characterized in that: The genes of the pigeon type I paramyxovirus attenuated strain aQY strain include five segments: A1, B1, C, D and E1; wherein the A1 segment includes the NP gene and part of the P gene, the B1 segment includes the remaining part of the P gene, the M gene and part of the F gene, the C segment includes the remaining part of the F gene, the HN gene and the first part of the L gene, the D segment includes the second part of the L gene, and the E1 segment includes the remaining part of the L gene; The B1 fragment includes a mutation of the NDV gene type VI F protein cleavage site sequence to a La Sota strain F protein cleavage site sequence, and an enzyme cleavage site is introduced by a synonymous mutation. Nhe Ⅰ; The C fragment does not include a restriction site N Ⅰ, including restriction sites introduced by synonymous mutations Kpn Ⅰ.

2. The attenuated strain aQY of pigeon type I paramyxovirus according to claim 1, characterized in that The 5' end of the A1 fragment includes a T7 promoter sequence; and / or The 5' end of the A1 fragment includes a restriction site Asc I, 3ʹ end including restriction site Pac I; and / or The 3' end of the E1 fragment includes a hepatitis D ribozyme sequence, a T7 terminator sequence and a restriction site Not Ⅰ.

3. The pigeon type I paramyxovirus attenuated strain aQY strain according to claim 1 or 2, characterized in that The nucleotide sequence of the A1 fragment is shown in SEQ ID No. 2; The nucleotide sequence of the B1 fragment is shown in SEQ ID No. 3; The nucleotide sequence of the C fragment is shown in SEQ ID No.4; The nucleotide sequence of the D fragment is shown in SEQ ID No.5; The nucleotide sequence of the E1 fragment is shown in SEQ ID No.

6.

4. The attenuated strain aQY strain of pigeon type I paramyxovirus according to claim 3, characterized in that: The gene sequence of the pigeon type I paramyxovirus attenuated strain aQY strain is shown in SEQ ID No.

1.

5. The method for constructing the attenuated strain aQY strain of pigeon type I paramyxovirus according to any one of claims 1 to 4, characterized in that: The construction method is obtained by rescuing the pigeon type I paramyxovirus QY strain, and comprises the following steps: The full-length genome plasmid of QY strain and three auxiliary plasmids were co-transfected into BSR-T7 / 5 cells and cultured for 84h-108h. The cells were repeatedly frozen and thawed to obtain the cell supernatant, which was inoculated into 9-11-day-old chicken embryos to harvest the virus liquid, thus obtaining the attenuated strain of pigeon type I paramyxovirus aQY strain; Wherein, the three auxiliary plasmids include plasmid pCI-NP, plasmid pCI-P and plasmid pCI-L.

6. The method for constructing the attenuated strain aQY of pigeon type I paramyxovirus according to claim 5, characterized in that: The method for constructing the QY strain full-length genome plasmid comprises the following steps: S11, extracting total RNA of the pigeon type I paramyxovirus QY strain genome, and reversely transcribing it to prepare QY strain genome cDNA; The cDNA sequence of the QY strain genome was analyzed and divided into segments A to E in sequence. According to the segmentation requirements, the restriction sites between 2800nt and 3000nt in the genome were retained. Pac I and the restriction site between 12300nt~12500nt N Ⅰ, Eliminate the restriction site between 9200nt and 9300nt by synonymous mutation N Ⅰ; Through synonymous mutation, a restriction site was introduced at 5900-6000 nt Nhe Ⅰ, introduce restriction sites at 9200-9400 nt Kpn Ⅰ; Add restriction site at the 5ʹ end of fragment A Asc Ⅰ, add restriction site at the 3ʹ end of fragment E Not Ⅰ, obtain the complete genome of QY strain-1; S12, remove the T7 promoter sequence in the plasmid pOK-12 and replace the vector's multiple cloning site sequence with a restriction site Asc Ⅰ. Pac Ⅰ. Nhe Ⅰ. Kpn Ⅰ. N Ⅰ and Not I, to obtain plasmid pOK-12X; S13. According to the selected restriction sites, design corresponding primers, including five pairs of amplification primers SegA-F / SegA-R, SegB-F / SegB-R, SegC-R / SegC-F, SegD-F / SegD-R and SegE-F / SegE-R; S14, using the QY strain whole genome-1 cDNA as a template, using the primers SegA-F / SegA-R, SegB-F / SegB-R, SegC-R / SegC-F, SegD-F / SegD-R and SegE-F / SegE-R, performing PCR amplification, detecting and purifying the PCR amplification product to obtain five fragments A, B, C, D, and E; S15, added restriction site at the 5ʹ end Asc The T7 promoter sequence was added to the A fragment of Ⅰ, and the base GGG was added after the T7 promoter sequence. The resulting nucleotide sequence was recorded as the A1 fragment; S16, cloning the B fragment into the plasmid pOK-12X, and mutating the F protein cleavage site in the B fragment into the La Sota strain F protein cleavage site sequence by inverse PCR, and the obtained nucleotide sequence is recorded as B1 fragment; S17, by means of inverse PCR, Not Introduce the hepatitis D ribozyme sequence and T7 terminator sequence before the restriction site I, record it as plasmid pOK-G, and clone the E fragment into the plasmid pOK-G by homologous recombination to obtain plasmid pOK-E1; S18, clone the A1 fragment, B1 fragment, C fragment and D fragment into the plasmid pOK-E1 in sequence to obtain the full-length genome plasmid of QY strain.

7. The method for constructing the attenuated strain aQY of pigeon type I paramyxovirus according to claim 6, characterized in that: In step S12, the primer pairs used for the transformation of the plasmid pOK-12 are pOK-12-1-F / pOK-12-1-R and pOK-12-2-F / pOK-12-2-R; the nucleotide sequence of the pOK-12-1-F / pOK-12-1-R is shown in SEQ ID No.7-SEQ ID No.8; the nucleotide sequence of the pOK-12-2-F / pOK-12-2-R is shown in SEQ ID No.9-SEQ ID No.

10.

8. The method for constructing the attenuated strain aQY of pigeon type I paramyxovirus according to claim 6, characterized in that: In step S17, when the plasmid pOK-12X is transformed into plasmid pOK-G, the primer pairs used are pOK-1-F / pOK-1-R, pOK-2-F / pOK-2-R and pOK-3-F / pOK-3-R, respectively; Wherein, the nucleotide sequence of the pOK-1-F / pOK-1-R is shown as SEQ ID No.11-SEQ ID No.12; The nucleotide sequences of the pOK-2-F / pOK-2-R are shown in SEQ ID No.13-SEQ ID No.14; The nucleotide sequences of the pOK-3-F / pOK-3-R are shown in SEQ ID No.15 to SEQ ID No.

16.

9. The method for constructing the attenuated strain aQY of pigeon type I paramyxovirus according to claim 5, characterized in that: The construction method of the three auxiliary plasmids comprises the following steps: According to the genome sequence of QY strain, primers were designed to amplify the NP gene, P gene and L gene, and restriction sites were introduced at the 5ʹ end and 3ʹ end of the gene fragment. Nhe Ⅰ and Sal Ⅰ, get NP gene, P gene and L gene; The NP gene, P gene and L gene are cloned into the expression vector pCI-neo respectively, and plasmid pCI-NP, plasmid pCI-P and plasmid pCI-L are obtained accordingly.

10. Use of the construction method of the attenuated pigeon type I paramyxovirus strain aQY strain as described in any one of claims 1 to 4 or the attenuated pigeon type I paramyxovirus strain aQY strain as described in any one of claims 5 to 9 in the preparation of pigeon Newcastle disease vaccine.

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