CN114574452A

CN114574452A - Method for constructing recombinant Newcastle disease vaccine candidate strain VII-HNF by HN gene translocation and application

Info

Publication number: CN114574452A
Application number: CN202111649942.9A
Authority: CN
Inventors: 胡顺林; 刘秀梵; 朱珊珊; 王晓泉; 刘晓文; 陈玉
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2022-06-03
Anticipated expiration: 2041-12-29
Also published as: CN114574452B

Abstract

The invention discloses a method for constructing a candidate strain VII-HNF of a recombinant Newcastle disease vaccine by HN gene translocation and application thereof, wherein an established reverse genetic operation platform of a gene VII type Newcastle disease virus attenuated strain AI4 is utilized to translocate HN in an AI4 genome full-length transcription vector pNDV/AI4 to the front of an F gene, and a recombinant Newcastle disease virus genome full-length cDNA clone pNDV/rAI4-HNF subjected to F and HN gene translocation editing is obtained. The recombinant virus VII-HNF obtained by transfection has higher reproduction titer and weak virulence on chick embryos and can be used for preparing vaccines.

Description

Method for constructing recombinant Newcastle disease vaccine candidate strain VII-HNF by HN gene translocation and application

Technical Field

The invention relates to a method for constructing a recombinant Newcastle disease vaccine candidate strain VII-HNF by HN gene translocation, which is used for developing a vaccine, by applying a reverse genetic technology.

Background

Newcastle Disease is a virulent infectious Disease caused by a virulent strain of Newcastle Disease Virus (NDV) that presents a serious hazard to the poultry industry worldwide. The genome of NDV is nonsegmented, single-negative-strand RNA, and the genome structural pattern of NDV is 3 '-NP-P-M-F-HN-L-5', which sequentially encodes Nucleocapsid Protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), and macromolecular polymerase protein (L)6 structural proteins [ Yusoff, k., and Tan, w.s.newcastle disease virus: macromolecules and opportunities, avian Pathol, 2001, 30 (5): 439-55.]. The NP, P, and L proteins form together with the viral genomic RNA an RNP complex, which is the smallest infectious unit of the virus, efficiently initiates genome replication and transcription in cells and produces infectious virions [ Marcos, F., Ferreira, L., Cros, J., Park, M.S., Nakaya, T., Garcia-Sastre, A., and Villar, E.mapping of the RNA promoter of New case disease virus, virology, 2005, 331 (2): 396-406.]. The transcription of the NDV genome exhibits a polar effect, and the closer to the 3' end, the gene has a richer transcription product, so that the NP protein content in the virion is the highest.

During the pathogenic and immune processes of NDV, two envelope glycoproteins F and HN on the surface of the virus play important roles. The virulence differences between NDV strains are large [ Dimitrov, k.m., Ramey, a.m., Qiu, x, Bahl, j., and Afonso, c.l.temporal, geographic, and host distribution of average paramyxovirus 1(new castle disease virus). 22-34 ] NDV can be classified into virulent type, moderate virulent type and attenuated type according to the pathogenicity and toxicity of chicken and chick embryo. The F protein is a major determinant of NDV virulence and pathogenicity, mediating fusion of the virus and cell membrane during infection by the virus [ Panda, a., Huang, z., Elankumaran, s., rockmann, d.d., and Samal, s.k.role of fusion protein clearance in the virus of microbial pathway, 2004, 36 (1): 1-10.]. The cleavage site of the virulent strain F protein is a plurality of basic amino acids which are arranged in series and can be cleaved by a plurality of proteases of a plurality of tissues and organs of a body, so that systemic infection can be caused. The basic amino acids in the F protein cleavage region 112 and 115 of the attenuated strain are replaced by neutral amino acids, resulting in a G/E112-K/R-Q-G/E115-R-L117 sequence, which is cleaved by trypsin-like protease secreted from only limited tissues or organs, and is poorly or noninfectious [ Liu, H., Server de Almeida, R., Gil, P., and Albina, E.Cleavage site of New castle disease virus derivatives viral attachment in tissue infection cells.vet Microbiol, 2018, 216: 123-31.]. After a virulent F protein cleavage site sequence is mutated into a characteristic sequence that is attenuated by using a reverse genetic technique, the toxicity of the virulent F protein is significantly reduced [ Hu, s., Ma, h., Wu, y., Liu, w., Wang, x., Liu, y., and Liu, X.A vaccine candidate of attenuated gene VII new vaccine virus produced by reverse gene, vaccine, 2009, 27 (6): 904-10, it is well documented that the F protein is closely associated with the virulence of NDV, and thus reducing the amount of F protein in the virus effectively reduces the virulence of the virus. Another viral envelope glycoprotein HN has the function of recognizing sialic acid receptors of target cells, mediating the adsorption of the virus to target cells, and promoting the release of nascent virions from the surface of infected cells [ Adu-Gyamfi, e., Kim, l.s., Jardetzky, t.s., and Lamb, r.a. mutagenesis of Paramyxovirus-neuron Membrane-promoter strain-Receptor cascade strain, Receptor Binding, and Neuraminidase activity. j viro, 2016, 90 (17): 7778-88.]. The HN protein plays a very important role in the immune process of NDV, and the level of Hemagglutination Inhibition (HI) antibodies generated by the protein is high or low, which becomes a main index for evaluating the immune efficacy of the Newcastle disease vaccine at present. In 2017 and 2019, the replacement of HN protein of Vaccine Virus by HN of epidemic Strain was found by university of Yangzhou and Chinese agriculture university related subjects, respectively, and the recombinant Virus after HN replacement was found to have significantly enhanced ability to inhibit the detoxification of epidemic Strain compared with maternal Virus, thereby confirming that the degree of homology of HN protein between Vaccine Strain and epidemic Strain is closely related to the ability of Vaccine to inhibit infection and detoxification of epidemic Strain [ Liu, J.J., Zhu, J.Xu, H.X., Li, J.Hu, Z.L., Hu, S.L., Wang, X.Q.and Liu, X.F.Effects of the HN antiviral Difference between the Vaccine Strain and the Challenge Strain of New castle Disease Virus, Virus Strain, 2017, 2019: 8.]. Therefore, the content of HN protein in the NDV vaccine strain is increased, and the immune efficacy of the vaccine on the NDV epidemic virulent strain can be enhanced.

Vaccines are the main measure for ND control, and studies have shown that, using vaccines matched to the genotype of an epidemic strain, infection with an epidemic virulent strain can be effectively prevented, in which HN protein plays an important role [ Bu, y.w., Yang, h.m., Jin, j.h., Zhao, j.e., Xue, j.and Zhang, g.z.recombiant new cast disease virus (NDV) La Sota expression of the immunogenic protein-neuronal tissue protein of genetic VII NDV and shows improved efficacy against infection of infection NDV exchange. avian pathtool, 2019, 48 (2): 91-7.]. At present, the NDV virulent strain prevailing in China is gene VII, while the widely used attenuated live vaccine is gene II, so that the gene VII attenuated live vaccine suitable for immunizing 1-day-old chicks has wide application prospect in China. Hu et al obtained a genotype VII attenuated strain NDV/AI4 by way of mutation at the F-proteolytic site, but the attenuated strain still had some virulence [ Hu, Z., Hu, S., Meng, C., Wang, X., Zhu, J., and Liu, X.Generation of a genetic VII New castle disease virus vaccine with high mutated in attenuated cassette group of Avian Dis, 2011, 55 (3): 391-7.].

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a method for constructing a recombinant Newcastle disease vaccine candidate strain VII-HNF by HN gene translocation. The HN gene translocation code of the invention is a recombinant Newcastle disease vaccine candidate strain VII-HNF before the F gene, which is preserved in the China center for type culture Collection at 11/9/2021 with the preservation number V202177.

The invention also aims to provide the application of the HN gene translocation in constructing a candidate strain VII-HNF of the recombinant Newcastle disease vaccine.

The technical scheme is as follows: the method for constructing the recombinant Newcastle disease vaccine candidate strain VII-HNF by HN gene translocation comprises the following steps:

the genome of a weakening strain AI4 of Newcastle disease virus JS-5-05-Go is taken as a research object, HN gene in the genome is translocated to the front of F gene to obtain the total length of the translocated recombinant Newcastle disease virus genome, and a vector containing the total length of the genome and helper plasmid are cotransfected with cells to obtain the translocated recombinant virus VII-HNF.

Further, the construction method comprises the following steps: the full-length genome transcription vector pNDV/AI4 of gene VIId subtype newcastle disease virus JS-5-05-Go attenuated strain AI4 is used as a framework, HN gene in the vector is translocated to the front of F gene to obtain the full-length translocated recombinant newcastle disease virus genome cDNA clone pNDV/rAI4-HNF, and the plasmid and auxiliary plasmid are cotransfected with BSR-T7/5 cells to obtain the translocated recombinant virus VII-HNF.

Further, the construction method comprises the following steps:

1. construction of full Length clone pNDV/rAI4-HNF

The gene of gene VIId subtype Newcastle disease virus JS-5-05-Go attenuated strain AI4 is used as a research object, specific primers are designed according to the sequence of the gene group to respectively amplify partial sequence fragments of HN gene, upstream M gene and P gene, and the amplified gene fragments are connected with a linearized pCR2.1 vector by utilizing homologous recombination to obtain recombinant plasmid pCR2.1/P-M-HN; amplifying partial sequences of the F gene and the downstream L gene by the same method, and connecting the amplified sequences with a pCR2.1 vector to obtain a recombinant plasmid pCR2.1/F-L. Through a molecular cloning method, two fragments are connected in a pCR2.1 vector to obtain a recombinant plasmid pCR2.1/P-M-HN-F-L subjected to translocation editing. Plasmid pCR2.1/P-M-HN-F-L and transcription vector pNDV/AI4 containing NDVAI4 strain full-length cDNA clone are subjected to double enzyme digestion by restriction enzymes AgeI and FspAI respectively, a target fragment is recovered after electrophoresis and is subjected to enzyme digestion connection, so that the fragment P-M-HN-F-L subjected to translocation editing is replaced to the corresponding part in the AI4 strain genome to construct a transcription vector pNDV/rAI4-HNF subjected to translocation encoding of F and HN genes, and the genome structure after translocation editing is 3 '-NP-P-M-HN-F-L-5'.

2. Rescue of recombinant virus VII-HNF Strain

The full-length plasmid pNDV/rAI4-HNF and three auxiliary plasmids pCI-NP, pCI-P and pCI-L are co-transfected into BSR-T7/5 cells, after transfection, SPF chick embryo allantoic fluid is added, the supernatant is collected, SPF chick embryos are inoculated, chick embryo allantoic fluid after 96-120h is collected, the blood coagulation titer is determined, and the recombinant virus VII-HNF is obtained.

Further, the,

The primer sequence for amplifying the 2879-4503nt region of AI4 genome to obtain PMHN1 is as follows:

PMHN1-F：5′-AGTGTGCTGGAATTCGGCTTACCGGTGCAGCACCCTTC-3′(SEQ ID NO.1)；

PMHN1-R：5′-CTCTGACCGTTCTACCCGTGTTTTTTCTAATT-3′(SEQ ID NO.2)。

the primer sequence for amplifying 6327-8328nt region of AI4 genome to obtain PMHN2 is:

PMHN2-F：5′-AATTAGAAAAAACACGGGTAGAACGGTCAGAG-3′(SEQ ID NO.3)；

PMHN2-R：5′-ATATCTGCAGAATTCGGCTTTTTTTTCTTAATAAAGTGACAGTGGGCGAGAC-3′(SEQ ID NO.4)。

the primer sequence for amplifying the 4504-6259nt region of AI4 genome to obtain FL1 is as follows:

FL1-F：5′-AGTGTGCTGGAATTCGGCTTTTACTGGGAACAAGCAACCAAAGAGCAATGCACGGGTAGAAGAGTCTGGATCCC-3′(SEQ ID NO.5)；

FL1-R：5′-CCTTATATGTCAATGCTTCTTGTGTTTTTTCTTAAGTCTTC-3′(SEQ ID NO.6)。

the primer sequence for obtaining FL2 from the 8329-9527nt region of the amplification/AI 4 genome is as follows:

FL2-F：5′-GAAGACTTAAGAAAAAACACAAGAAGCATTGACATATAAGG-3′(SEQ ID NO.7)；

FL2-R：5′-ATATCTGCAGAATTCGGCTTGTGCGCACATCTGGCTCCT-3′(SEQ ID NO.8)。

the sequence of the primer for amplifying the fragment region of pCR2.1/P-M-HN is as follows:

PMHN1-F：5′-AGTGTGCTGGAATTCGGCTTACCGGTGCAGCACCCTTC-3′(SEQ ID NO.1)；

PMHNFL-R：5′-GGTTGCTTGTTCCCAGTAATTTTTTCTTAATAAAGTg-3′(SEQ ID NO.9)。

the sequence of the primer for amplifying the fragment region of pCR2.1/F-L is as follows:

PMHNFL-F：5′-CACTTTATTAAGAAAAAATTACTGGGAACAAGCAACC-3′(SEQ ID NO.10)；

FL2-R：5′-ATATCTGCAGAATTCGGCTTGTGCGCACATCTGGCTCCT-3′(SEQ ID NO.8)。

the recombinant virus VII-HNF obtained by transfection has higher propagation titer on chick embryos, is suitable for large-scale production of vaccines, and can be used for manufacturing vaccines.

The HN gene of the invention is easy to locate in the recombination Newcastle disease vaccine candidate strain before the F gene, relatively reduces the expression quantity of the F protein to reduce the virus virulence, and simultaneously increases the expression quantity of the HN protein to enhance the immunity effect of the attenuated strain to the epidemic strain, so that the invention is used for manufacturing a novel safe and efficient ND vaccine.

By modifying a full-length cloning vector pNDV/A14 containing AI4 genome and translocating and encoding HN gene to the front of F gene, the full-length cloning vector pNDV/rAI4-HNF is successfully constructed, and a translocating recombinant vaccine VII-HNF is obtained by reverse genetic operation, wherein the HA titer of allantoic fluid is 10log₂。

The research discloses a construction method of a recombinant Newcastle disease vaccine candidate strain VII-HNF with HN gene translocation coded in front of F gene, which is characterized in that: the full-length genome transcription vector pNDV/AI4 of gene VIId subtype newcastle disease virus JS-5-05-Go attenuated strain AI4 is used as a framework, HN gene in the vector is translocated and coded before F gene, and the translocated recombinant newcastle disease virus genome full-length cDNA clone pNDV/rAI4-HNF is obtained. After the recombinant plasmid and the helper plasmid transfect BSR-T7/5 cells together, the translocation recombinant Newcastle disease virus VII-HNF with higher reproductive performance is obtained, has weak virulence and is suitable for manufacturing vaccines.

In addition, the gene VIId subtype of the attenuated strain is consistent with the genotype of the current Newcastle disease epidemic strain, so that compared with the conventional vaccine, the translocation recombinant vaccine strain VII-HNF shows wide application prospect in the aspect of controlling the morbidity and the epidemic of the current Newcastle disease.

Has the advantages that: according to the polar effect of NDV genome transcription, on the premise of ensuring genome replication and transcription level, the position of an epidemic strain HN gene on the genome is moved forward, the expression quantity of F protein is relatively reduced to reduce virus virulence, meanwhile, the expression quantity of the HN protein is increased to enhance the immune efficacy of an attenuated strain on the epidemic strain, and a gene VII type attenuated vaccine candidate strain with better safety and stronger immune efficacy on the epidemic strain is developed.

Drawings

FIG. 1 is a schematic diagram of the construction of recombinant plasmid pNDV/rAI 4-HNF.

Detailed Description

HN gene translocation recombinant Newcastle disease vaccine candidate strain VII-HNF coded in front of F gene is preserved in the China center for type culture Collection (address: Wuhan university Collection) at 11 month and 9 days 2021, and is classified and named as Newcastle disease virus VII-HNF, and the preservation number is CCTCC NO: and V202177.

The method comprises the following construction steps: construction of full-Length expression clone in which HN Gene is easily located before F Gene

Preparing biological materials:

a full-length genome vector pNDV/A14(Hu, Z., Hu, S., Meng, C., Wang, X., Zhu, J., and Liu, X.Generation of a genetic VII New castle disease virus vaccine with high yield in infected chicken egg, Avian Dis, 2011, 55 (3): 391-7.) containing the Newcastle disease virus A14 strain was constructed and stored in an open laboratory for infectious diseases of Ministry of agriculture of Yangzhou.

pCR2.1 vector: purchased from Invitrogen corporation; AMV reverse transcriptase, High fidelity DNA polymerase, T4 DNA ligase, Agarose Gel DNA Extraction Kit from Roche; the transfection reagent SuperFect and a plasmid extraction Kit (QIAprep Spin miniPrep Kit) are products of QIAGEN company; fastpfu PCR SuperMix and T1 competent cells were purchased from Kyoto Biotech, Inc., Beijing; restriction enzymes were purchased from Thermo Scientific; seamless fastening Master Mix: purchased from bio-engineering (shanghai) incorporated; the other conventional reagents are all domestic analytical purifiers.

1. Obtaining of intermediate plasmid pCR2.1/P-M-HN-F-L

The full-length genome of gene VIId subtype NDV/JS-5-05-Go attenuated strain AI4 is used as research object, and primers are designed based on the whole genome sequence to amplify partial sequence segments of HN gene, upstream M gene and P gene, and partial sequence segments of F gene and downstream L gene.

The primer sequences are:

PMHN1-F：5′-AGTGTGCTGGAATTCGGCTTACCGGTGCAGCACCCTTC-3′(SEO ID NO.1)

PMHN1-R：5′-CTCTGACCGTTCTACCCGTGTTTTTTCTAATT-3′(SEQ ID NO.2)

PMHN2-F：5′-AATTAGAAAAAACACGGGTAGAACGGTCAGAG-3′(SEQ ID NO.3)

PMHN2-R：5′-ATATCTGCAGAATTCGGCTTTTTTTTCTTAATAAAGTGACAGTGGGCGAGAC-3′(SEQ ID NO.4)

FL1-F：5′-AGTGTGCTGGAATTCGGCTTTTACTGGGAACAAGCAACCAAAGAGCAATGCACGGGTAGAAGAGTCTGGATCCC-3′(SEQ ID NO.5)

FL1-R：5′-CCTTATATGTCAATGCTTCTTGTGTTTTTTCTTAAGTCTTC-3′(SEQ ID NO.6)

FL2-F：5′-GAAGACTTAAGAAAAAACACAAGAAGCATTGACATATAAGG-3′(SEQ ID NO.7)

FL2-R：5′-ATATCTGCAGAATTCGGCTTGTGCGCACATCTGGCTCCT-3′(SEQ ID NO.8)

homology arms are underlined; the above primers were synthesized by Nanjing Optimus Almotata Bio Inc.

And (3) PCR reaction: the following PCR reaction system was prepared using the transcription vector pNDV/AI4 as a template:

PCR cycling parameters: pre-denaturation at 95 ℃ for 2min, extension at 95 ℃ for 20s, extension at 57 ℃ for 20s, extension at 72 ℃ for 2min/kb, extension at 72 ℃ for 10min after 25 cycles, and storage at 4 ℃.

Amplifying 2879-4503nt region of AI4 genome by using primers PMHN1-F and PMHN1-R, recovering amplified segment by using Agarose Gel DNA Extraction Kit to obtain PMHN 1; amplifying 6327-8328nt regions of AI4 genome by using primers PMHN2-F and PMHN2-R to obtain PMHN 2; amplifying the 4504-6259nt region of AI4 genome by using primers FL1-F and FL1-R to obtain FL 1; the 8329-9527nt region of the/A14 genome was amplified with primers FL2-F and FL2-R to obtain FL 2.

Recovering fragments PMHN1, PMHN2 and pCR2.1 vector, connecting through homologous recombination, transforming to Escherichia coli T1 competent cell, extracting plasmid, and naming positive plasmid as pCR2.1/P-M-HN; recovering target fragments FL1 and FL2, connecting with pCR2.1 vector through homologous recombination, transforming to Escherichia coli T1 competent cells, extracting plasmid, and naming the positive plasmid as pCR2.1/F-L.

P-M-HN was ligated to both F-L fragments in pCR2.1 vector, again by homologous recombination.

The primer sequences are:

PMHN1-F：5′-AGTGTGCTGGAATTCGGCTTACCGGTGCAGCACCCTTC-3′(SEO ID NO.1)

PMHNFL-R：5′-GGTTGCTTGTTCCCAGTAATTTTTTCTTAATAAAGTG-3′(SEQ ID NO.9)

PMHNFL-F：5′-CACTTTATTAAGAAAAAATTACTGGGAACAAGCAACC-3′(SEQ ID NO.10)

FL2-R：5′-ATATCTGCAGAATTCGGCTTGTGCGCACATCTGGCTCCT-3′(SEQ ID NO.8)

homology arms are underlined; the above primers were synthesized by Nanjing Ongchongke Bio Inc.

Amplifying a fragment region of pCR2.1/P-M-HN with the length of 3627bp by using primers PMHN1-F and PMHNFL-R; the fragment region of pCR2.1/F-L was amplified with primers PMHNFL-F and FL2-R and was 3022bp in length. The amplified fragment is recovered and purified by an Agarose Gel DNAextraction Kit, then is connected with a pCR2.1 vector through homologous recombination, is transformed into an escherichia coli T1 competent cell, a plasmid is extracted, and the positive plasmid is named as pCR2.1/P-M-HN-F-L.

2. Obtaining of full-Length clone pNDV/rAI4-HNF

Plasmid pCR2.1/P-M-HN-F-L and pNDV/AI4 are double digested by AgeI and FspAI, and the digested large fragment pCR2.1/P-M-HN-F-L and the digested large fragment pNDV/AI4 are recovered. The pCR2.1/P-M-HN-F-L enzyme digestion product is connected with the pNDV/AI4 enzyme digestion product to construct a recombinant NDV genome full-length clone pNDV/rAI4-HNF (shown in figure 1).

Step two: rescue of recombinant Virus VII-HNF Strain

Biological material preparation

BSR-T7/5 cells stably expressing T7RNA polymerase: a gift from the high-minded researcher at the harbin veterinary institute of chinese academy of agricultural science. CEF cells were prepared by the laboratory on their own according to conventional methods.

Eukaryotic expression plasmids pCI-NP, pCI-P, pCI-L (Pichunlin, Zhanmei, Sunxiang, Liuyuliang, etc.. establishment of goose-derived Newcastle disease virus rescue system [ J ]. microbiological report.2007, 34 (3)): constructed and stored by a focus open laboratory of livestock and poultry infectious disease science of Yangzhou university department of agriculture. Among them, eukaryotic expression plasmids pCI-NP and pCI-P expressing NP and P genes are also disclosed in (Liuyuliang. (2005). Generation of infectious ZJ1 strains of goose-derived Newcastle disease from cDNA clones); eukaryotic expression plasmid pCI-L for expressing L gene is also disclosed in (picrine. (2007) establishment of goose-derived Newcastle disease virus reverse genetic technology platform and application thereof).

SPF hatching eggs were purchased from Lihua, Zhejiang and hatched in the major open laboratory of livestock and poultry infectious disease in Ministry of agriculture of Yangzhou university.

1. Rescue of recombinant Virus VII-HNF

Plasmids (pNDV/AI4-HNF, pCI-NP, pCI-P, and pCI-L) used for transfection were extracted with QIAprep SpinMiniPrepKit. Co-transfecting 3 auxiliary plasmids of pCI-NP, pCI-P and pCI-L and a full-length cDNA clone pNDV/rAI4-HNF of a recombinant NDV genome with BSR-T7/5 cells, adding allantoic fluid of SPF embryos with the final concentration of 10% after 18-24h, freezing and thawing a transfection sample for 3 times after 60h, harvesting and inoculating SPF embryos of 9-11 days old after 96h, collecting the allantoic fluid of the embryos, and measuring the hemagglutination titer according to OIE standard to obtain a translocation recombinant vaccine strain VII-HNF.

HA titres measured after 5 passages on chick embryosIs 10log₂Increased by 1 titer compared to the parental strain AI 4.

2. RT-PCR verification of rescued virus VII-HNF

Detecting positive allantoic fluid by erythrocyte agglutination test (HA) and erythrocyte agglutination inhibition test (HI), collecting total RNA of allantoic fluid extracted virus after 4 generations of SPF chick embryo, and performing reverse transcription to cDNA by 6nt random primer for RT-PCR reaction.

And amplifying the recombinant virus cDNA by using primers TEST-F and TEST-R, wherein the length is about 800bp, recovering a target fragment for sequencing identification, and the sequencing result of the amplified fragment shows that the HN gene is successfully translocated to the front of the F gene.

The primer sequences are:

TEST-F：5′-TCTTCGGGACGATGCTTGATGATGA-3′(SEQ ID NO.11)

TEST-R：5′-AAGAGGAGTGAGCAGAGTAGTCAGTG-3′(SEQ ID NO.12)

the above primers were synthesized by Nanjing Optimus Almotata Bio Inc.

Step three: recombinant viruses MDT, ICPI and EID₅₀Measurement of (2)

Chick embryo Mean Death Time (MDT) assay: 10 times (10 times) of recombinant viruses VII-HNF were separately added to sterilized physiological saline^-6、10^-7......10^-10) And (3) performing gradient dilution, inoculating 5 SPF (specific pathogen free) chick embryos of 9-11 days old to each dilution degree, wherein each chick embryo is 0.1mL, and meanwhile, setting 5 chick embryos inoculated with physiological saline as a control. Incubation at 37 ℃ and discarding dead chick embryos within 24h, followed by observation every 12h until 120h, and calculating the MDT of the virus according to the OIE standard method. As a result: the virus of 5 dilutions inoculated chick embryos did not die within 120h, and the MDT value of the strain was greater than 120 h.

Chick intracerebral inoculation pathogenicity index (ICPI) determination: taking HA titer more than 4log₂The allantoic fluid of fresh virus was subjected to aseptic examination, diluted 10-fold with physiological saline sterilized without antibiotic, and then inoculated with 10 SPF chickens 1 day old intracerebrally, each intracerebrally inoculated with 0.05mL of SPF chickens, and simultaneously 10 SPF chickens injected with physiological saline were used as controls. According to OIE standard method pairThe chickens were scored and the ICPI of the virus was calculated. Allantoic fluid does not die after being inoculated into SPF (specific pathogen free) chickens of 1 day old in the brain, and ICPI is only 0.19, which shows that the toxicity of the rescued viruses completely meets the weak toxicity standard.

Infection of half of the chick embryos (50% egg infection dose, EID)₅₀) And (3) determination: diluting fresh virus allantoic fluid with sterilized normal saline to 10 deg.C^-7、10^-8、10^-9、10^-104 dilution degrees, inoculating 5 SPF (specific pathogen free) chick embryos of 10 days old each, each embryo is 0.1ml, placing at 36-37 ℃ for continuous incubation, discarding the chick embryos dead before 48 hours, taking out the chick embryos dead after 48-120 hours at any time till 120 hours, taking out all live embryos, collecting chick embryo liquid one by one, respectively measuring the agglutination value of red blood cells, judging the chick embryos to be infected if the agglutination value is not less than 1: 128 (micro method), calculating EID (EID), and determining the number of the chick embryos to be infected if the chick embryos are not less than 1: 128₅₀. As a result: the virus content is 10 per 0.1ml^9.63EID₅₀Showing that the rescued viruses have higher reproduction titer on chicken embryos.

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Claims

1. A method for constructing a recombinant Newcastle disease vaccine candidate strain VII-HNF by HN gene translocation is characterized in that: the genome of a weakening strain AI4 of Newcastle disease virus JS-5-05-Go is taken as a research object, HN gene in the genome is translocated to the front of F gene to obtain the total length of the translocated recombinant Newcastle disease virus genome, and a vector containing the total length of the genome and helper plasmid are cotransfected with cells to obtain the translocated recombinant virus VII-HNF.

2. The method for constructing a candidate strain VII-HNF for a recombinant Newcastle disease vaccine by HN gene translocation as in claim 1, wherein: the full-length genome transcription vector pNDV/AI4 of gene VIId subtype newcastle disease virus JS-5-05-Go attenuated strain AI4 is used as a framework, HN gene in the vector is translocated to the front of F gene to obtain the full-length translocated recombinant newcastle disease virus genome cDNA clone pNDV/rAI4-HNF, and the plasmid and auxiliary plasmid are cotransfected with BSR-T7/5 cells to obtain the translocated recombinant virus VII-HNF.

3. The method for constructing a candidate strain VII-HNF for a recombinant Newcastle disease vaccine by HN gene translocation as in claim 2, wherein: the method comprises the following steps:

(1) construction of full-Length clone pNDV/rAI 4-HNF: with the gene of gene VIId subtype Newcastle disease virus JS-5-05-Go attenuated strain A14 as the target, designing specific primers according to the genome sequence to respectively amplify partial sequence segments of HN gene, upstream M gene and P gene, and connecting the partial sequence segments with pCR2.1 vector to obtain recombinant plasmid pCR2.1/P-M-HN; amplifying partial sequences of the F gene and the downstream L gene by the same method, and connecting the amplified fragment with a pCR2.1 vector to obtain a recombinant plasmid pCR2.1/F-L; through a molecular cloning method, two fragments are connected in a pCR2.1 vector to obtain a translocation-edited recombinant plasmid pCR2.1/P-M-HN-F-L; finally, replacing the translocation-edited fragment P-M-HN-F-L to the corresponding part in the genome of the A14 strain by a molecular cloning method to construct a transcription vector pNDV/rAI4-HNF of the translocation code of F and HN genes, wherein the genome structure after translocation editing is 3 '-NP-P-M-HN-F-L-5';

(2) rescue of recombinant virus VII-HNF Strain: co-transfecting BSR-T7/5 cells with full-length plasmids pNDV/rAI4-HNF and three auxiliary plasmids of pCI-NP, pCI-P and pCI-L, adding the allantoic fluid of SPF chick embryos after transfection, collecting the supernatant, inoculating the allantoic fluid of the SPF chick embryos, collecting the allantoic fluid of the chick embryos after 96-120h, and determining the hemagglutination titer to obtain the recombinant virus VII-HNF.

4. Use of the method for constructing a candidate strain VII-HNF for a recombinant Newcastle disease vaccine by HN gene translocation as in claim 1 in preparation of a vaccine.