Construction method and application of Newcastle disease virus F protein labeled vaccine strain

Abstract

The invention discloses a construction method and use of a Newcastle disease virus F protein-marked vaccine strain. Using a Newcastle disease virus reverse genetics operation platform, the corresponding part of the NDV genome is replaced with the F protein extracellular region of type 2 paramyxovirus and the HN protein rod region that interacts therewith, and the recombinant chimeric virus rAI4-TF is rescued. After the vaccine is made into SPF chickens for 3 weeks after immunization, they are infected with the NDV virulent strain JS-5-05. The immune chickens can resist the attack of a lethal dose of NDV virulent, and the infected animals and the immune animals can be distinguished by a serological method on the 7th day after infection. The recombinant virus can be used for the research and development of NDV marker vaccines.

Description

Technical Field

[0001] The invention relates to the application of reverse genetics technology and recombinant DNA technology, and in particular to a construction method and application of a Newcastle disease virus F protein-marked vaccine strain. Background Art

[0002] RNA virus reverse genetic manipulation technology refers to a research technology that reverse transcribes viral genome RNA into cDNA in vitro, performs various in vitro artificial operations on it at the DNA molecular level, and finally assembles a new RNA virus from the viral genome cDNA and the corresponding auxiliary plasmid [Leyrer S, Neubert WJ, Sedlmeier R. Rapid and efficient recovery of Sendai virus from cDNA: factors influencing recombinant virus rescue. J Virol Methods 1998, 75 (1): 47-58.], which is often referred to as "virus rescue". Since the RNA virus that is finally "rescued" is derived from the cDNA clone, the RNA virus genome can be subjected to various in vitro artificial operations at the DNA level, such as gene mutation, gene insertion, gene knockout (deletion) and other modifications. This not only solves the problem of difficulty in manipulating RNA virus genomes, but also facilitates research on the functions of viral genes, pathogenic mechanisms and strategies for viral disease prevention and control. At the same time, it provides new ideas for the research and development of new vaccines [Huang Z, Elankumaran S, Panda A, et al. Recombinant Newcastle disease virus as a vaccine vector. Poultry Science, 2003, 82: 899-906.].

[0003] Newcastle disease (ND) is an acute, septicemic, highly contagious disease caused by virulent strains of Newcastle disease virus (NDV) that mainly infects poultry and wild birds such as chickens, pigeons, quails, and turkeys. According to pathogenicity indicators, NDV can be divided into rapid-type (strong), intermediate-type (medium virulence) and slow-type (weak) strains. NDV is a single-stranded, negative-strand, non-segmented RNA virus with a genome length of about 15.2 kb. The NDV genome structure is 3'--leader-NP-PMF-HN-L-trailer-5', which encodes 6 structural proteins in sequence: nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and large protein (Large protein, L). Among them, F and HN proteins are envelope proteins of NDV, which can stimulate the body to produce protective antibodies and are the main protective antigens of NDV. Among them, F protein has the function of inducing cell fusion, and the F protein cleavage site sequence is the main factor determining the virulence of NDV [Peeters BP, Deleeuw OS, Koch G, et al. Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence [J]. Journal of Virology, 1999, 73 (6): 5001-5009.]. The mature HN protein is a tetramer, in which the 1st to 26th amino acid residues are located in the intracellular region, the 27th to 48th amino acid residues are the transmembrane region, and the 49th to 571st amino acid residues are the HN protein extracellular region, which is also the main functional region of the HN protein, which can be further divided into a rod region and a globular region.The rod region of the HN protein interacts with the F protein to promote the fusion of the virus and the host cells, and plays an important role in the process of virus infection of cells [Huang Z, Panda A, Elankumaran S, et al. The hemagglutinin-neuraminidase protein of Newcastle desease virus determines tropism and virulence [J]. Journal of Virology, 2004, 78 (8): 4176-4184.].

[0004] Marker vaccines are a new generation of recombinant vaccines that use genetic engineering technology to introduce molecular markers into the viral genome. After immunizing animals, the corresponding serological detection methods can distinguish infected animals from immune animals [Van Oirschot J T. Diva vaccines that reduce virus transmission [J]. Journal of Biotechnology, 1999, 73.2-3: 195-205.]. The prevention of ND mainly relies on vaccination with attenuated live vaccines or inactivated vaccines. The use of vaccines has effectively controlled the occurrence and spread of ND, but sporadic cases still exist in some areas, and it is impossible to distinguish by serological methods whether the ND antibodies produced by chickens are caused by immune strains or wild strains, which affects the rapid diagnosis and epidemiological investigation of ND.

[0005] At present, there are at least 15 serotypes of avian paramyxovirus (APMV) that have been identified [Thomazelli LM, Jansende Araújo, Fabrizio T, et al. Novel avian paramyxovirus (APMV-15) isolated from amigratory bird in South America [J]. Plos One, 2017, 12 (5): e0177214.], among which NDV belongs to APMV-1 type. The results of cross-hemagglutination inhibition test and virus neutralization test show that the three serotypes of APMV-3, APMV-8 and APMV-9 are closest to APMV-1 in antigenicity and have cross-reaction in serology, while the antigenicity of other serotypes of APMV is relatively distant from APMV-1 [Tsunekuni R, Hikono H, Saito T. Evaluation of avian paramyxovirus serotypes 2 to 10 as vaccine vectors in inchickens previously immunized against Newcastle disease virus [J]. Veterinary Immunology and Immunopathology, 2014, 160: 184-191.]. APMV-2 virus cannot cause obvious clinical symptoms when it infects chickens alone and is non-pathogenic to chickens. A large number of experiments have shown that the cross protection rate between NDV and APMV-2 is low, and the replication efficiency of APMV-2 in chickens is high [Warke A, Stallknecht D, Williams SM. Comparative study on the pathogenicity and immunogenicity of wild bird isolates of avian paramyxovirus 2, 4, and 6 in chickens [J]. Avian Pathology, 2008, 37 (04): 429-434.]. In addition, APMV-2 has a genome structure and transcription control elements similar to NDV. Therefore, the extracellular region of the NDV F protein is replaced with the corresponding part of the APMV-2 type. The obtained recombinant virus will not produce antibodies to the NDV F protein after immunization. By detecting whether there are antibodies to the Newcastle disease virus F protein in the serum of the immune birds, the purpose of distinguishing the immune animals from the infected animals can be achieved.When NDV proliferates, the M protein binds to the two envelope proteins F and HN, and also binds to the nucleocapsid of the virus to complete the packaging of the virus. In this process, the M protein mainly interacts with the intracellular region of the F protein. Therefore, in the construction of the chimeric F protein, only the extracellular region sequence of the F protein is replaced, and the interaction between the M protein and the chimeric F protein will not be significantly affected. In addition, studies have shown that the rod region of the HN protein can only promote the fusion function of the F protein when it interacts with its homologous extracellular region of the F protein [Shin-Hee K, Madhuri S, Samuel AS, et al. Roles of the fusion and hemagglutinin-neuraminidase proteins in replication, tropism, and pathogenicity of avian paramyxoviruses [J]. Journal of Virology, 2011, 85 (17): 8582-8596.], so when replacing the extracellular region sequence of the F protein, the rod region sequence of HN should also be replaced accordingly to ensure the infectivity of the recombinant virus.

[0006] In 2011, Hu Zenglei et al. successfully constructed the full-length cloning vector pNDV / rAI4 by mutating the F protein cleavage site of the VIId subtype NDV strain JS-5-05 isolated in their laboratory, and obtained the attenuated virus AI4 through reverse genetic manipulation. The HA titer of its allantoic fluid was 10log2 [Hu Z, Hu S, Meng C, et al. Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with High Yield in Embryonated Chicken Eggs. Avian Diseases, 2011, 55(3): 1759-1769]. Summary of the invention

[0007] Purpose of the invention: The present invention provides a method for constructing a Newcastle disease virus F protein-labeled vaccine strain and its use.

[0008] Technical solution: The construction method of the Newcastle disease virus F protein marker vaccine strain is to use the Newcastle disease virus AI4 strain as the skeleton, replace the gene fragment encoding the extracellular region of the Newcastle disease virus F protein with the corresponding sequence of the paramyxovirus type 2 strain, and obtain the plasmid B-TF-ECD containing the chimeric F gene; replace the gene fragment encoding the rod region of the HN protein with the corresponding sequence of the paramyxovirus type 2 strain, and obtain the plasmid B-THN-S containing the chimeric HN gene. After connecting the two chimeric gene fragments, replace the corresponding part of the Newcastle disease virus AI4 strain genome to obtain the recombinant plasmid pNDV / rAI4-TF. Using the established reverse genetic operation platform of the NDVAI4 strain, the recombinant plasmid and three auxiliary plasmids were co-transfected into BSR-T7 / 5 cells to obtain the NDV marker vaccine strain rAI4-TF with higher reproduction performance and weaker virulence.

[0009] This vaccine strain cannot produce antibodies against the Newcastle disease virus F protein after immunization. By detecting whether there are antibodies to the Newcastle disease virus F protein in the serum of the immune birds, the purpose of distinguishing infected and immune animals can be achieved.

[0010] The specific method includes the following steps:

[0011] 1 Construction of full-length clone pNDV / rAI4-TF

[0012] 1) Construction of intermediate plasmid B-TF

[0013] Six pairs of primers, primer1 to primer12, were designed according to the genomic sequences of AI4 and APMV-2 strains. Primers primer1 to primer6 were used to construct chimeric F protein, primers primer7 to primer12 were used to construct chimeric HN protein, and a PacI restriction site was added to primer primer7. PCR amplification of each target fragment was performed using plasmid pNDV / rAI4 and APMV-2 cDNA as templates, and the PCR products were subjected to Overlap PCR, and the Overlap PCR products were connected to the vector to obtain chimeric plasmids B-TF-ECD and B-THN-S, respectively.

[0014] The two chimeric plasmids B-TF-ECD and B-THN-S were double-digested with Pac I and SacII, respectively. The target fragments were recovered and ligated with T4 DNA ligase at 16°C overnight. The correct plasmid was identified as the intermediate plasmid, named B-TF.

[0015] 2) Obtaining the full-length plasmid pNDV / rAI4-TF

[0016] Plasmids B-TF and AI4 were digested with PacI and SpeI restriction endonucleases at the same time, and the 4808 bp fragments of B-TF and the 13413 bp fragments of AI4 were recovered respectively. After purification, they were ligated with a DNA Ligation Kit (Mighty Mix) to construct the full-length recombinant NDV genome pNDV / rAI4-TF. The specific construction method is shown in Figure 1 .

[0017] 2. Rescue of recombinant virus rAI4-TF

[0018] The recombinant full-length plasmid pNDV / rAI4-TF and three auxiliary plasmids pCI-NP, pCI-P and pCI-L were co-transfected into BSR-T7 / 5 cells. After 60 hours, the cells and supernatant suspension were inoculated into 9-10 day old SPF chicken embryos to obtain the recombinant virus rAI4-TF.

[0019] Establishment of 3DIVA detection method

[0020] A eukaryotic expression plasmid expressing the NDV F gene was constructed and transfected into 293T cells. An indirect immunofluorescence detection method was established to detect antibodies against the F protein in chicken serum. Based on the detection of F antibodies, infected and immune animals were distinguished. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Construction diagram of the recombinant plasmid pNDV / rAI4-TF;

[0022] Figure 2 Electrophoresis diagram of RT-PCR identification of recombinant virus rAI4-TF;

[0023] Figure 3 Results of indirect immunofluorescence detection of the eukaryotic expression product of F protein;

[0024] Figure 4 Indirect immunofluorescence method specificity test;

[0025] Figure 5 Indirect immunofluorescence was used to detect the serum samples of infected and immunized animals, where A, B: chicken serum 7 days after rAI4-TF immunization infection; C: negative serum; D, E: chicken serum 9 days after rAI4-TF immunization infection; F: AI4 positive serum. DETAILED DESCRIPTION

[0026] Step 1: Construction of recombinant full-length plasmid pNDV / AI4-TF

[0027] The transcription vector pNDV / rAI4 containing the full-length genomic cDNA of NDV AI4 strain [Hu Z, Hu S, Meng C, et al. Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with High Yield in Embryonated Chicken Eggs [J]. Avian Diseases 2011, 55 (3): 1759-1769] was constructed and preserved by the Key Open Laboratory of Livestock and Poultry Infectious Diseases of the Ministry of Agriculture of Yangzhou University. The genomic cDNA of APMV-2 virus T4 strain was constructed by the Key Open Laboratory of Livestock and Poultry Infectious Diseases of the Ministry of Agriculture of Yangzhou University (Liu Jingjing. Construction of recombinant chimeric Newcastle disease virus vector and its application in the development of H7N9 and H9N2 subtype avian influenza vaccines. Doctoral dissertation of Yangzhou University, 2018).

[0028] Blunt-zero vector, 2×TransStart FastPfu PCR SuperMix, Basic Seamless Cloning and Assembly KIT and T1 competent cells were products of Beijing Quanshijin Biotechnology Co., Ltd.; SacII, Pac I and SpeI restriction endonucleases were purchased from NEB and Thermo fisher; AMV reverse transcriptase and transfection reagent SuperFect were purchased from Roche; plasmid extraction kit (QIAprep Spin MiniPrep Kit) was a product of QIAGEN; T4 DNA ligase, Agarose Gel DNA ExtractionKit and DNA Ligation Kit (Mighty Mix) were purchased from Takara; other conventional reagents were of domestic analytical grade.

[0029] 1 Construction of intermediate plasmid B-TF

[0030] Six pairs of primers primer1 to primer12 were designed according to the genome sequences of plasmid AI4 and APMV-2. Primers primer1 to primer6 were used to construct chimeric F gene, and primers primer7 to primer12 were used to construct chimeric HN gene. The primer sequences are as follows:

[0031]

[0032] The wavy line and bold part in the primer sequence are PacI restriction sites, and the black bold part is the overlapping part required for Overlap PCR. The above primers were synthesized by Beijing Qingke Technology Biological Co., Ltd. (Nanjing).

[0033] 1.1 Construction of the chimeric F gene B-TF-ECD

[0034] Using the cDNA of AI4 strain and APMV-2 / T4 strain as templates, primer1 and primer2 were used to amplify the gene sequence between the PacI restriction site (3250 bp) on the AI4 genome and the NDV F protein start codon ATG (4550 bp), named fragment 12; Primer3 and primer4 were used to amplify the gene sequence encoding the extracellular region of the APMV-2 / T4 strain F protein (4371 bp-5810 bp), named fragment 34; primer5 and primer6 were used to amplify the gene sequence between the transmembrane region of the F protein of the AI4 genome (6050 bp) and the SacII restriction site (6539 bp) on the HN protein, named fragment 56. The PCR reaction system and conditions are as follows:

[0035]

[0036]

[0037] PCR reaction conditions:

[0038] The PCR product was electrophoresed on 1% agarose gel, and the band that matched the size of the desired target fragment was cut out. The PCR product was recovered according to the instructions of Agarose Gel DNA Extraction Kit.

[0039] Overlap PCR reaction system and conditions are as follows:

[0040] The PCR products of fragment 34 and fragment 56 were connected by Overlap PCR using Primer3 and Primer6. After recovery and purification using a gel recovery kit, Overlap PCR was performed with fragment 12 using primers Primer1 and Primer6.

[0041] Taking fragment 34 and fragment 56 as examples, the reaction system and conditions for Overlap PCR are as follows:

[0042]

[0043] PCR reaction conditions were the same as above.

[0044] The Overlap PCR product was recovered and purified, connected to the Blunt vector, and transformed into T1 competent cells. The operation steps are as follows:

[0045] Blunt ligation system: Overlap PCR product 2μL, Blunt 1μL, supplement SW to 5μL. Flick to mix, spin briefly, and connect in a 25℃ metal bath for 0.5h. After the connection, place the reaction tube on ice and transform into T1 competent cells. Pick a single colony for culture, extract the plasmid, and perform enzyme digestion identification. The plasmid with the correct enzyme digestion identification was sent to Beijing Qingke Technology Biological Co., Ltd. (Nanjing) for sequencing. The plasmid with the correct sequencing was named B-TF-ECD.

[0046] 1.2 Construction of the chimeric HN gene B-THN-S

[0047] Primers primer7 and primer8 were used to amplify the gene sequence of the intracellular region of plasmid AI4 HN protein (6417-6552, containing SacII restriction site), primer9 and primer10 were used to amplify the gene sequence of the rod region of APMV-2 / T4 strain HN protein (6277-6576), and primer11 and primer12 were used to amplify the gene sequence of the globular region of AI4 HN protein (6851-8133, with Spe I restriction site at position 8100). After 1% agarose gel electrophoresis, each target band was recovered and Overlap PCR was performed in sequence (PCR and Overlap PCR reaction systems and conditions were the same as above). After recovery and purification, the Overlap PCR product was connected to the Blunt vector, connected in a 25°C metal bath for 0.5h, and transformed into T1 competent cells. After a single colony was selected for culture, the plasmid was extracted and identified by enzyme digestion. The plasmid with the correct enzyme digestion was sent to Beijing Qingke Technology Biological Co., Ltd. (Nanjing) for sequencing. The plasmid with the correct sequencing was named B-THN-S.

[0048] 1.3 Obtaining the intermediate plasmid B-TF

[0049] The two chimeric plasmids B-TF-ECD and B-THN-S were double-digested with Pac I and SacII, respectively, and the target fragments of 3229 bp and 5535 bp were recovered, respectively, and then ligated with T4 DNA ligase at 16°C overnight, transformed into Escherichia coli T1 competent cells, and a single colony was picked for plasmid extraction. The plasmid identified by enzyme digestion was the intermediate plasmid, named B-TF.

[0050] 2 Obtaining the full-length clone pNDV / AI4-TF

[0051] Plasmids B-TF and AI4 were digested with PacI and SpeI restriction endonucleases at the same time. The former was digested into 4808 bp and 4054 bp, and the latter was digested into 4850 bp and 13413 bp. The fragments of 4808 bp and 13413 bp were recovered respectively, purified and connected with DNA Ligation Kit (Mighty Mix). The ligation products were transformed into E. coli DH5α competent cells, and a single colony was picked and cultured for plasmid extraction. The recombinant plasmid was digested and identified with restriction endonuclease HindIII. The correctly identified plasmid was the recombinant full-length plasmid pNDV / AI4-TF.

[0052] Connection system: Step 2: Rescue of recombinant virus rNDV / AI4-TF

[0053] Biological material preparation:

[0054] BSR-T7 / 5 cells that stably express T7 RNA polymerase were kindly provided by Dr. Zhigao Bu from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0055] The eukaryotic expression plasmids pCI-NP, pCI-P, and pCI-L (Hu Shunlin, Zhang Yanmei, Liu Yuliang, et al. Establishment of a rescue system for Newcastle disease virus from geese [J]. Bulletin of Microbiology. 2007, 34(3): 426-429.) were constructed and preserved by the Key Open Laboratory of Livestock and Poultry Infectious Diseases of the Ministry of Agriculture, Yangzhou University.

[0056] SPF breeder eggs were purchased from Zhejiang Lihua Agricultural Science and Technology Co., Ltd. and incubated to the required age in the Key Open Laboratory of Animal and Poultry Infectious Diseases of the Ministry of Agriculture of Yangzhou University; 2-week-old SPF chickens were purchased from Shandong Jinan Spafas Poultry Co., Ltd.

[0057] 1. Rescue of recombinant viruses

[0058] All plasmids used in transfection (pNDV / AI4-TF, pCI-NP, pCI-P and pCI-L) were extracted using QIAprep Spin MiniPrep Kit. BSR-T7 / 5 cells were grown to about 80% in a 35 mm cell culture dish, and three auxiliary plasmids, pCI-NP, pCI-P and pCI-L, were co-transfected with the recombinant plasmid pNDV / AI4-TF. The transfection amount ratio of pNDV / AI4-TF: pCI-NP: pCI-P: pCI-L was 1: 1: 0.5: 0.5. SPF chicken embryo allantoic fluid was added 20 h after transfection to make a final concentration of 10%. After 60 h, the transfected sample was repeatedly frozen and thawed 3 times. The cells and the supernatant suspension were harvested and inoculated into 9-day-old SPF chicken embryos. The embryos that died within 24 h were discarded. After 96 h, the hemagglutination titer was determined according to the OIE standard, and the chicken embryo allantoic fluid that was positive in the hemagglutination test (HA) was collected.

[0059] 2RT-PCR verification of rescued virus rAI4-TF

[0060] The allantoic fluid that tested positive for HA was collected after being passed through SPF chicken embryos for three generations, and the viral RNA was extracted. The RNA was reverse transcribed into cDNA using 6nt random primers and then used for PCR identification.

[0061] The chimeric F and HN genes were amplified using primers FF and HN-R, and the target fragment with a length of about 3.5 kb was amplified (see Figure 2 ), recovered the target fragment for sequencing, and the sequencing results showed that the gene fragment related to the APMV-2 envelope protein had successfully replaced the corresponding part of the AI4 strain virus genome.

[0062] FF: 5'-CATTCAGGACACAGCATGATACC-3' (SEQ ID NO.13)

[0063] HN-R: 5'-GTGTCGTCTTCCTAACCATCCTATG-3' (SEQ ID NO.14)

[0064] Step 3: Identification of biological characteristics of recombinant viruses

[0065] 1EID 50 , MDT and ICPI determination

[0066] Embryo infectious dose (EID) 50 ) determination: the recombinant virus rAI4-TF was diluted 10 times in a row with sterile PBS, and 10 -6 ~10 -109-day-old SPF chicken embryos were inoculated with 5 dilutions (0.1 mL / egg), and the embryos were placed in an incubator at 37°C. The embryos that died within 24 hours were discarded. After 96 hours, the hemagglutination was tested with the HA test, and the half-infectious dose of the embryos was calculated by the Reed-Muench method.

[0067] Determination of the mean death time (MDT) of the minimum lethal dose of chicken embryos: The recombinant virus rAI4-TF was diluted 10 times in a row with sterile PBS, and 10 -6 ~10 -10 The SPF chicken embryos were inoculated with 5 dilutions (0.1 mL / egg) and placed in a 37°C incubator. The chicken embryos that died within 24 hours were discarded. After inoculation, the embryos were checked for death at regular intervals every day and the specific death time was recorded. The average time of the minimum dose that killed the chicken embryos was calculated after 168 hours of observation. Results: The MDT value of the strain was greater than 120 hours.

[0068] Determination of the intracerebral inoculation pathogenicity index (ICPI) of 1-day-old chicks: The recombinant virus rAI4-TF was diluted 10 times with sterile saline and inoculated intracerebrally into 10 1-day-old SPF chickens (0.05 mL / chicken). The chickens were observed for 8 consecutive days and scored according to the corresponding standards, with normal chickens scored as 0 points, diseased chickens scored as 1 point, and dead chickens scored as 2 points. The final results were calculated according to the OIE standard. The results showed that no one-day-old SPF chickens became ill or died after the allantoic fluid was inoculated intracerebrally, and the ICPI was 0, indicating that the virulence of the rescued virus met the standard of attenuated toxicity.

[0069] Step 4: Vaccine strain immune efficacy test

[0070] Two-week-old SPF chickens were randomly divided into two groups, 10 in the immunization group and 5 in the control group, and kept in isolation. The recombinant virus rAI4-TF was inactivated and made into an oil emulsion inactivated vaccine, which was inoculated into SPF chickens, 0.3 mL per chicken, and a PBS vaccination group was set as a negative control. Blood was collected and serum was separated at 7d, 14d, and 21d after immunization, and the serum antibody conversion level against the immune strain was detected. 21 days after immunization, the strong strain JS-5-05 was used at 10 6 EID 50 / 0.1mL was used for the challenge test, and the mental state of the chickens was observed every day. Blood was collected and serum was separated on the 3rd, 5th, 7th and 9th days after the challenge, and the serum was tested using the corresponding indirect immunofluorescence method.

[0071] The results showed that no clinical symptoms were observed in 2-week-old SPF chickens after inoculation with the recombinant virus rAI4-TF inactivated vaccine. The inactivated vaccine rAI4-TF was able to produce high antibody titers in the body. Three weeks after immunization, the specific hemagglutination inhibition (HI) antibody titer against NDV HN could reach 8-9log2; while the control group could not produce HN antibodies against NDV. The NDV JS-5-05 strain of gene VIId subtype was used at 10 6 EID 50 The chickens in the immunized group showed no obvious clinical symptoms, while all the chickens in the PBS control group died on the 4th day after the challenge. This indicates that the vaccine prepared by the recombinant virus can provide good immune protection for the experimental chickens.

[0072] Step 5: Establishment of DIVA detection method

[0073] Construction of 1pCAGGS-AI4-F eukaryotic expression plasmid

[0074] The F gene of AI4 strain was amplified by PCR using the full-length clone of AI4 as a template and then ligated to the eukaryotic expression vector pCAGGS which had been digested with EcoR I and Xho I by homologous recombination.

[0075] The PCR reaction system is as follows:

[0076]

[0077] PCR reaction as described in step 1.1

[0078] The linearization restriction enzyme system of pCAGGS eukaryotic expression vector is as follows:

[0079]

[0080] After the enzyme digestion system is configured, flick the tube to separate instantly. Digest in a 37℃ water bath for 45 minutes.

[0081] The PCR and enzyme digestion products were electrophoresed on 1% agarose gel, and the target fragments were cut out and purified using a gel recovery kit before use in homologous recombination.

[0082] The homologous recombination system is as follows:

[0083] PCR fragment 1 μL

[0084] pCAGGS linearized vector 2.5 μL

[0085] Basic Seamless Cloning and Assembly MIX 1μL

[0086] The metal bath was connected for 0.5 h, and the ligation product was transformed into T1 competent cells. After culturing a single colony, the plasmid was extracted and digested with EcoRI and XhoI for identification. The correct plasmid was sent to Beijing Qingke Technology Biological Co., Ltd. (Nanjing) for sequencing, and the positive plasmid was named pCAGGS-AI4-F.

[0087] Verification of 2F protein expression

[0088] 293T cells were seeded in a 24-well cell plate, and the eukaryotic expression plasmid pCAGGS-AI4-F was transfected when the cells grew to 80%-90%. 48 hours after transfection, NDV AI4 strain positive serum diluted 1:200 was used as the primary antibody, and FITC-labeled rabbit anti-chicken IgG was used as the secondary antibody for indirect immunofluorescence test. The results showed that green fluorescence could be observed in the cells transfected with the eukaryotic expression plasmid pCAGGS-AI4-F, but no green fluorescence was observed in the cells transfected with the empty vector pCAGGS, indicating that the recombinant eukaryotic expression plasmid pCAGGS-AI4-F can successfully express F protein in 293T cells. The results are shown in Figure 2. Figure 3 .

[0089] 2.1 Indirect immunofluorescence specificity test

[0090] 48 hours after transfection of the eukaryotic expression plasmid pCAGGS-AI4-F, the H9 avian influenza virus serum, ND negative serum, ND positive serum and recombinant virus rAI4-TF serum were used for detection to verify the specificity of the method. The results showed that the expressed NDV F protein did not react with H9 avian influenza virus serum, recombinant virus rAI4-TF positive serum and NDV negative serum, but had good reactivity with NDV positive serum. The results are shown in Figure 4 .

[0091] 2.2 Detection of serum samples

[0092] The chicken serum collected after immunization with the recombinant virus rAI4-TF and infection with the strong NDV strain JS-5-05 in step 4 was tested by indirect immunofluorescence method according to the above method, and NDV positive and negative serum were set as controls. The results showed that the serum after immunization with the recombinant virus rAI4-TF did not react with the F protein; the reactivity of the serum on the 3rd and 5th day after infection was not significantly different from that of the immunized group serum, but on the 7th day after infection, the serum of the infected group chickens could react with the F protein, and strong green fluorescence could be observed under a fluorescence microscope (such as Figure 5 ), indicating that the chickens produced antibodies against NDV F protein after infection. Therefore, this method can distinguish infected animals from immune animals on the 7th day after NDV virulent infection.

Claims

1. A method for constructing a Newcastle disease virus F protein-labeled vaccine strain, characterized in that: The Newcastle disease virus AI4 strain was used as the backbone, and its F protein extracellular domain and the HN protein rod region that interacted with it were replaced with the corresponding parts of avian paramyxovirus type 2 to obtain the recombinant plasmid pNDV / rAI4-TF. The Newcastle disease virus F protein-labeled vaccine strain rAI4-TF was obtained by co-transfecting the plasmid with the helper plasmid into cells. Specifically, cDNA of AI4 strain and APMV-2 / T4 strain were used as templates, and primer1 and primer2 were used to amplify the cDNA of AI4 genome. Pac The gene sequence between 3250 bp of the I restriction site and 4550 bp of the NDV F protein start codon ATG was named segment 12; Primer3 and primer4 were used to amplify the gene sequence encoding the extracellular region of the APMV-2 / T4 strain F protein, 4371 bp-5810 bp, named segment 34; primer5 and primer6 were used to amplify the transmembrane region of the AI4 genome F protein from 6050 bp to the HN protein. Sac The gene sequence between 6539 bp of the II restriction site is named fragment 56; the primer sequences are shown in SEQ ID NOs. 1 to 6; Primers primer7 and primer8 were used to amplify the gene sequence between 6417-6552 bp of the intracellular region of plasmid AI4 HN protein, primer9 and primer10 were used to amplify the gene sequence between 6277-6576 bp of the rod region of APMV-2 / T4 strain HN protein, and primer11 and primer12 were used to amplify the gene sequence between 6851-8133 bp of the globular region of AI4 HN protein; the primer sequences are shown in SEQ ID NO.7~12.

2. The method for constructing a Newcastle disease virus F protein-labeled vaccine strain according to claim 1, wherein: The gene fragment encoding the extracellular region of the Newcastle disease virus F protein was replaced with the corresponding sequence of the avian paramyxovirus type 2 strain to obtain a plasmid B-TF-ECD containing a chimeric F gene; the gene fragment encoding the rod-shaped region of the HN protein was replaced with the corresponding sequence of the avian paramyxovirus type 2 strain to obtain a plasmid B-THN-S containing a chimeric HN gene, and after connecting the two chimeric gene fragments, they replaced the corresponding part of the Newcastle disease virus AI4 strain genome to obtain a recombinant plasmid pNDV / rAI4-TF.

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