CN111575247B

CN111575247B - Newcastle disease chimeric virus marked vaccine strain and construction method and application thereof

Info

Publication number: CN111575247B
Application number: CN202010441121.5A
Authority: CN
Inventors: 张凌云; 闫召璐; 谢田田; 张博; 郑杰; 马宁宁
Original assignee: Beijing Dingchi Biotechnology Co ltd; Zhejiang Dingzhi Biological Products Co ltd
Current assignee: Beijing Dingchi Biotechnology Co ltd; Zhejiang Dingzhi Biological Products Co ltd
Priority date: 2020-05-22
Filing date: 2020-05-22
Publication date: 2022-04-26
Anticipated expiration: 2040-05-22
Also published as: CN111575247A

Abstract

The invention relates to a Newcastle disease chimeric virus marked vaccine strain and a construction method and application thereof, belonging to the field of rescue and application of the Newcastle disease chimeric vaccine strain. The invention mutates the F protein cleavage site of Newcastle disease gene G VII strain into the cleavage site of low virulent strain, the F and HN genes of the mutated gene VII Newcastle disease strain and NP, P, M and L of gene II NDV La Sota strain construct full-length chimeric cDNA sequence, and 18 bp nucleotide marker sequence is inserted into the non-coding region between P and M. The transfected cells are rescued to obtain the Newcastle disease chimeric virus NDV DC strain. The chimeric strain contains envelope surface glycoprotein of the gene VII type Newcastle disease strain and contains a skeleton of the gene II type strain, and has the immunogenicity of the gene VII type Newcastle disease strain and the high propagation and high safety characteristics of the gene II type La Sota strain.

Description

Newcastle disease chimeric virus marked vaccine strain and construction method and application thereof

Technical Field

The invention relates to the technical field of veterinary biological products, in particular to a Newcastle disease chimeric virus marked vaccine strain and a construction method and application thereof.

Background

Newcastle disease, also known as asian fowl plague or pseudofowl plague, is an acute septic infectious disease caused by newcastle disease virus, which is highly contagious. The chickens can be infected with diseases, the chickens are the most susceptible groups, and the susceptibility of the chicks is higher than that of the adult chickens. The disease has high mortality rate, has no obvious seasonal characteristics, can be infected and ill in four seasons, but spring and autumn are high-incidence seasons of the disease.

Sick chickens have typical symptoms of dyspnea, diarrhea, listlessness and mucosal and serosal hemorrhage. At the beginning of the onset of the disease, respiratory symptoms appear firstly, which are manifested by unsmooth breathing of sick chickens, accompanying cough and wheezing, snore when breathing, sticky secretions in oral cavity and nasal cavity and shaking head. In the disease cycle, sick chickens die occasionally, most sick chickens recover gradually after 1 week of disease, but the death rate is increased if the treatment is not timely carried out. The excrement of the chicken with a long disease course is white or light green, and part of sick chicken can show nervous symptoms and shake when walking and raise the head frequently; laying hens are ill, the laying rate is affected, the egg quality is seriously reduced, and abnormal egg quantity is increased for part of sick hens. The disease brings huge economic loss to the poultry industry.

Recently, the NDV popular in China mostly belongs to genes VI, VII, VIII and IX, particularly more genes VII, and the attenuated vaccines used in China mostly belong to genes I and II. The existing vaccine in China has limited protection power to epidemic strains, and even has no protection basically. Therefore, there is a need to develop a new and effective vaccine to effectively prevent and treat the infectious diseases.

Disclosure of Invention

In order to solve the problems in the prior art, the invention designs a construction method of a Newcastle disease chimeric virus marker vaccine strain, which constructs the Newcastle disease chimeric virus vaccine strain by simultaneously combining a VII type F gene sequence and a HN gene sequence of a Newcastle disease gene with NP, P, M and L gene sequences of a Newcastle disease gene II type La Sota strain.

Based on the method, the invention designs a Newcastle disease chimeric virus marker vaccine strain NDV DC strain, the whole gene sequence of the NDV DC strain is shown as SEQ ID No.1, the NDV DC strain contains a VII type F gene sequence and an HN gene sequence of a Newcastle disease gene with mutant cracking sites, and NP, P, M and L gene sequences of a Newcastle disease gene II type La Sota strain, and the proposed classification name of the NDV DC strain is as follows: the newcastle disease virus is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is the microorganism research institute of China academy of sciences No.3, West Lu No.1 of Beijing, Chaoyang, and the preservation date is as follows: 3/5/2020, the accession number is: CGMCC No. 19298. At the 3170 th nucleotide of the nucleotide sequence of the NDV DC strain, a marker nucleotide sequence shown as SEQ ID No.3 is inserted into a non-coding region between a P gene sequence and an M gene sequence.

The construction method of the Newcastle disease chimeric virus marked vaccine strain comprises the following steps: (1) synthesizing a gene sequence of Newcastle disease gene VII type F and a gene sequence of HN after mutation of a cleavage site as shown in SEQ ID No.2, amplifying full-length cDNA plasmid pOK-rNDV of NP, P, M and L genes of the Newcastle disease gene II type La Sota strain, and cloning each genome into pOK12 vectors according to the sequence of NP (La Sota), P (La Sota), M (La Sota), F (Newcastle disease gene VII), HN (Newcastle disease gene VII) and L (La Sota);

(2) the marker sequence Tag Seq was inserted between genes P and M (at 3170 nt): TTTCGCGAGGCGCGCCAA, respectively; introducing a T7 promoter sequence 5 'upstream of the sequence, introducing a hepatitis D ribozyme sequence having self-cleaving function and a T7 terminator sequence having the function of terminating transcription of the T7 promoter at the 3' end of the sequence, ligating the cDNA fragments at a common restriction enzyme cleavage site, and assembling into pOK-rNDV-DC plasmid in a transcription plasmid pOK 12;

(3) respectively inserting full-length NP, P and L genes of a Newcastle disease gene II type La Sota strain into a pCI-neo vector to construct auxiliary plasmids pCI-NP and pCI-P, pCI-L;

(4) co-transfecting the plasmid pOK-rNDV-DC containing the label and auxiliary plasmids pCI-NP, pCI-P and pCI-L into BHK-T7 cells, culturing for 96h, repeatedly freezing and thawing the cells, taking cell supernatant, inoculating SPF (specific pathogen free) chick embryos of 9-11 days old, and harvesting virus liquid to obtain the Newcastle disease chimeric virus marked vaccine strain NDV DC.

The construction method of the BHK-T7 cell in the step (4) comprises the following steps: a. synthesizing a sequence based on a T7 RNA polymerase gene sequence recorded by an NCBI official website to construct a PMV-T7 RNA polymerase plasmid;

b. connecting the PMV-T7 RNA polymerase plasmid with a P1020 vector overnight, transforming DH5 alpha competent cells by a connecting product, and screening out a P1020-T7 RNA polymerase positive plasmid through PCR identification and sequencing identification;

c. extracting the recombinant P1020-T7 RNA polymerase plasmid by using a plasmid macroextraction kit, and performing subsequent experiments after the plasmid is confirmed to be correct;

d. transfecting P1020-T7 RNA polymerase plasmid to the cultured BHK-21 adherent cells based on an electroporation method; respectively culturing cell strains in a 96-well plate, a 24-well plate and a 6-well plate in sequence to complete the screening process of the cell strains;

e. amplifying the selected cell strain, and selecting a positive cell strain with high yield by using a WB detection mode, namely a cell strain which is successfully transfected, and naming the cell strain as BHK-T7 cell.

Further, the method for transfecting the BHK-T7 cells in the step (4) specifically comprises the following steps: BHK-T7 cells are inoculated in a six-hole plate, transfection is started when 80% -90% of monolayer cells grow, the cells are gently washed 3 times by PBS before transfection, the EAM01 culture medium is changed into opti-MEM culture medium, and the constructed full-length plasmid pOK-rNDV-DC and 3 auxiliary plasmids pCI-NP, pCI-P and pCI-L are transfected by using transfection reagent Lipofectamine^TM2000 BHK-T7 cells were co-transfected in appropriate ratios. The medium was changed after transfection to EAM01 medium. After mixing evenly, the mixture is cultured for 4 days, observed every day after transfection, and the solution is changed at the right time.

The culture solution and the cells are harvested, freeze thawing is carried out for 3 times repeatedly, then centrifugation is carried out, the supernatant is taken and inoculated into SPF (specific pathogen free) chick embryos (200 mu l/egg) with the age of 10 days, the inoculated SPF chick embryos are continuously cultured for 5 days, allantoic fluid is collected for HA detection, the allantoic fluid with HA positive is selected for subpackaging, RNA is extracted and sequencing is carried out, after correct identification, the rescued virus is named as NDV DC strain and is used as the first generation for rescuing the virus, and continuous passage is carried out for 10 times, so that the virus with stable HA activity is obtained. HA activity of each virus passage was determined, and the 1 st (F1), 2 nd (F2), 3 rd (F3), 5 th (F5) and 10 th (F10) viruses were selected for sequence determination. Sequencing the newly rescued virus genome into F (mutation cleavage site) and HN sequences of a gene VII type, and containing NP, P, M and L genes of a La Sota strain; and the virus contains a molecular label introduced during construction, so that the virus is successfully rescued.

Meanwhile, the Newcastle disease chimeric virus NDV DC strain obtained by the invention can be used for preparing a Newcastle disease vaccine, and the specific preparation method comprises the following steps: (1) inoculating the correctly identified Newcastle disease chimeric virus marked vaccine strain NDV DC into chick embryos, culturing for 72-96 h, and then harvesting allantoic fluid; inoculating 1% of allantoic fluid into BHK-21-SC suspension cells after harvesting the allantoic fluid, adding TPCK pancreatin to culture for 72h to obtain cell culture fluid, namely newcastle disease chimeric marker vaccine strain NDV DC strain cytotoxic antigen fluid; (2) adding formaldehyde, beta-propiolactone (BPL) or diethylene imine (BEI) inactivator into the harvested virus solution according to a proportion for inactivation; (3) mixing the inactivated antigen solution with one of oil adjuvant, aluminum adjuvant, propolis adjuvant and oil-water dual-phase adjuvant, and emulsifying to obtain uniform emulsion.

The method for clinically identifying the Newcastle disease chimeric virus marked vaccine strain NDV DC strain comprises the following steps: (1) designing and synthesizing a specific primer for identification, wherein an upstream primer JD-F: 5'-CACGCCCAATGCACCCGAGTTC-3' downstream primer JD-R: 5'-CAGCACTTGGGGTGCCTGCACTAC-3', respectively; (2) extracting RNA of the newcastle disease virus vaccine strain, and carrying out reverse transcription on the RNA into cDNA by using reverse transcriptase; (3) adding a specific primer to amplify a DNA fragment by using the cDNA as a template by using a PCR method, wherein the size of the amplified fragment is about 700 bp; (4) sending the PCR amplification product to a sequencing company for sequence determination, wherein the determined sequence contains TTTCGCGAGGCGCGCCAA labeled nucleotide sequence (located at 3170 nucleotides of the full-length sequence) which is the NDV DC strain of the Newcastle disease virus labeled vaccine strain; or performing gel recovery on the PCR amplification product, performing enzyme digestion on the gel recovery product by using NruI endonuclease, and judging that the gel recovery product can be cut into two strips of 500bp and 200bp according to an electrophoresis result, wherein the identified strain is the NDV DC strain of the Newcastle disease virus marker vaccine strain.

The invention further aims to provide application of the Newcastle disease chimeric virus marker vaccine strain NDV DC strain. And the application of the Newcastle disease virus marked vaccine strain NDV DC strain in the preparation of a live vector vaccine taking the Newcastle disease virus strain as a framework.

Compared with the prior art, the newcastle disease chimeric virus marker vaccine strain designed by the invention has the advantages that F gene (nucleotide sequence of position 334-351: GGGAGACAGGGGCGCCTT) and HN gene sequences of chicken newcastle disease gene VII are synthesized in the research, the sequences are sequences obtained by mutating the F gene (shown as SEQ ID No. 2), and the mutated sequences can reduce the toxicity of newcastle disease and ensure better virus strain safety.

In the recombinant Newcastle disease virus rescue system, the F (the cleavage site is mutated into the cleavage site of the low virulent strain) and HN sequences of the VII type gene strain of the epidemic strain Newcastle disease are used, so that the conformation and the epitope of the surface immunogenic protein of the rescued strain are consistent with those of the surface immunogenic protein of the epidemic strain, and the recombinant Newcastle disease virus rescue system has better immunogenicity; and NP, P, M and L genes of the chicken Newcastle disease gene II type classical vaccine strain La Sota strain are used as a framework, so that the characteristic of high propagation titer of the chicken Newcastle disease gene II type La Sota strain is reserved, and the strain shows excellent growth characteristic on BHK-21-SC suspension cells.

According to the invention, a marker sequence is added into the constructed recombinant Newcastle disease virus marker vaccine strain, and the strain can be identified by sequencing and enzyme digestion identification methods, so that the marker vaccine strain lays a foundation for identifying infection and vaccine immunity.

The marked vaccine strain has the biological characteristics of high growth titer and low pathogenicity in chick embryos, is stable in heredity and good in immunogenicity, and can be used for manufacturing a homotype vaccine for preventing and controlling the current epidemic gene VII type Newcastle disease.

Drawings

FIG. 1 is a schematic diagram of the assembly of full length pOK-rNDV-DC using a subgenomic overlapping cDNA fragment generated by high fidelity polymerase amplification;

FIG. 2 shows the sequencing comparison of the gene cleavage sites of the chimeric newcastle disease virus marker vaccine strain NDV DC strain at

generations

1, 2, 3, 5 and 10 with the gene type VII of newcastle disease gene.

FIG. 3 shows the insertion molecular tags Tag Seq of the Newcastle disease chimeric virus marker vaccine strain NDV DC strain at the 1 st, 2 nd, 3 rd, 5 th and 10 th generations: TTTCGCGAGGCGCGCCAA and La Sota strain.

FIG. 4 shows the HI antibody detection results 14 days after immunization of the Newcastle disease chimeric virus marker vaccine strain NDV DC strain with other vaccine strains; indicates that antibody levels were significantly higher than the other groups.

Detailed Description

The invention is further described with reference to the following figures and specific embodiments. The technical solutions in the embodiments of the present invention are clearly and completely described, and the described embodiments are only some embodiments, but not all embodiments of the present invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without any creative effort belong to the protection scope of the present invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all purchased from conventional biochemicals, unless otherwise specified.

Example 1

Construction of full-Length cDNA clone of recombinant Virus genome

The main experimental material culture medium is MEM culture medium containing 10% fetal calf serum, NDV vaccine strain La Sota is purchased from China veterinary medicine institute, BHK-21 cells are purchased from ATCC, SPF chick embryo and SPF chick and is provided by Bridgman animal center.

The main reagent plasmid extraction kit and the gel recovery kit are purchased from Tiangen corporation; restriction enzyme, T4 DNA ligase was purchased from NEB; the virus RNA extraction kit is purchased from Tiangen company; coli DH5 α competent cells were purchased from whole gold organisms; pMDl8-T Vector and

GXL DNA Polymerase was purchased from TaKaRa; lipofectamine^TM2000 transfection reagents were purchased from Invitrogen.

1. Designing and synthesizing a primer according to a VII type synthetic sequence of a newcastle disease gene of a chicken and a sequence of a II type LaSota strain of the newcastle disease gene, designing the primer, mutating a VII type F gene sequence of the gene after a cracking site, and constructing a full-length pOK-rNDV sequence chimeric strain with NP, P, M and L genes of the La Sota strain according to the sequence of the gene sequences. A marker sequence was inserted at 3170 th nucleotide of the full-length cDNA of the NDV DC strain, and the sequence was TTTCGCGAGGCGCGCCAA. Synthetic primers (shown in tables 1 and 2) were designed and synthesized by Biotechnology engineering (Shanghai) Inc.

2. Synthesis of the F and HN genes of Gene VII Newcastle disease

The F gene after the F protein cleavage site (RRQKRF) is designed and synthesized to be mutated into the F protein cleavage site (GRQGRL) of the LaSota low virulent strain and the HN gene of the VII gene type Newcastle disease RS strain, and 3602bp segments (shown as SEQ ID No. 2) are totally designed.

3. Amplification of genome sequence of Newcastle disease Gene II type La Sota Strain

(1) In order to facilitate cloning and ensure the unicity of each enzyme cutting site used by cloning, the full-length cDNA of the chimeric strain is divided into 4 fragments for amplification (shown in Table 1); (2) inserting the synthesized F and HN genes of the Newcastle disease gene VII type into the positions of the F and HN genes of the La Sota strain; (3) introducing a T7 promoter sequence 5' upstream of the sequence; (4) in order to ensure the accuracy of the tail sequence, a hepatitis D ribozyme (HDVRz) sequence with self-cutting function and a T7 terminator sequence with the function of terminating the transcription of the T7 promoter are introduced at the 3' end of the sequence; (5) connecting the obtained 4 fragments with pOK12 vectors, constructing pOK-Seg1, pOK-Seg2, pOK-Seg3 and pOK-Seg4 plasmids, screening positive, then carrying out sequencing verification, and storing the plasmids with correct sequencing for later use. (6) Each fragment was cloned into pOK12 vector in turn, and the constructed plasmid was named pOK-rNDV. And (3) after the cloning of the whole genome is completed, sequencing and enzyme digestion identification are carried out, and the constructed plasmid is confirmed to be correct.

TABLE 1 primers used for construction of full-Length cDNA sequences

Note: the underlined sections are the cleavage sites.

4. Introduction of marker genes

Primers P9 and P10 are respectively designed, a marker sequence TTCGCGAGGCGCGCCAA is introduced into the primers, and a pOK-rNDV-DC vector containing the marker sequence is constructed.

TABLE 2 primer sequences for introduction of marker genes

Note: the underlined sections are added marker sequences.

As shown in FIG. 1, is a pattern diagram of the assembly of full length pOK-rNDV-DC using a high fidelity polymerase amplification of the resulting subgenomic overlapping cDNA fragments; cloning NP (La Sota), P (La Sota), M (La Sota), F (Newcastle disease Gene VII), HN (Newcastle disease Gene VII), and L (La Sota) genes into pOK12 vector in this order; the marker sequence TagSeq is inserted between genes P and M: TTTCGCGAGGCGCGCCAA, respectively; adding a T7 promoter at the 3 'end of the gene sequence, and adding an HDVRz sequence and a T7 terminator at the 5' end of the gene sequence; the cDNA fragments were ligated at the consensus restriction sites and assembled into the pOK-rNDV-DC plasmid in the transcription plasmid pOK 12.

Example 2

Construction of helper plasmids

According to the full-length genome sequence of the La Sota strain, primers are designed to amplify the Open Reading Frames (ORFs) of the nuclear protein NP, the phosphoprotein P and the polymerase protein L which are coded by the primers, and the primers are shown in Table 3. And 3 ORFs were cloned downstream of the CMV promoter of eukaryotic expression vector pCI-neo, respectively. The constructed helper plasmids were named pCI-NP, pCI-P, and pCI-L, respectively.

TABLE 3 construction of helper plasmids primer sequences

Note: the underlined sections are the cleavage sites.

Example 3

Construction of T7 RNA polymerase-expressing BHK T7 cells

(1) Synthesizing a sequence based on a T7 RNA polymerase sequence (SEQ ID: FJ881694.1) recorded by NCBI official website to construct a PMV-T7 RNA polymerase plasmid; (2) connecting the PMV-T7 RNA polymerase plasmid with a P1020 vector overnight, transforming DH5 alpha competent cells by a connecting product, and screening out a P1020-T7 RNA polymerase positive plasmid through PCR identification and sequencing identification; (3) extracting the recombinant P1020-T7 RNA polymerase plasmid by using a plasmid macroextraction kit of Tiangen Biotechnology Ltd, and performing subsequent experiments after the plasmid is confirmed to be correct; (4) transfecting the plasmid to the cultured BHK-21 adherent cells based on an electroporation method; (5) respectively culturing cell strains in a 96-well plate, a 24-well plate and a 6-well plate to complete the screening process of the cell strains; (6) amplifying the selected cell strains, selecting positive cell strains with high yield by using a WB detection mode, namely successfully transfected cell strains, and selecting the optimal BHK-21 cell strain (named as BHK-T7) inserted with T7 RNA polymerase for virus rescue.

Example 4

Rescue and identification of recombinant viruses

1. Rescue of recombinant viruses

BHK-T7 cells are inoculated in a six-well plate, transfection is started when 80% -90% of monolayer cells grow, the cells are gently washed by PBS 3 times before transfection, a DMEM culture medium is changed into an opti-MEM culture medium, and the constructed full-length plasmid pOK-rNDV-DC and 3 auxiliary plasmids (3: (the)pCI-NP, pCI-P, and pCI-L) transfection reagent Lipofectamine^TM2000 BHK-T7 cells were co-transfected in the appropriate ratios. After transfection, the cells were gently washed 3 times with PBS and the medium was changed to EAM01 medium. After mixing evenly, incubation is continued for 4 days, observation is carried out every day after transfection, and liquid is changed at proper time. Harvesting culture solution and cells, repeatedly freezing and thawing for 3 times, centrifuging, taking supernatant, inoculating SPF (specific pathogen free) chick embryos (200 mu l/embryo) with the age of 9-11 days, continuously culturing the inoculated SPF chick embryos for 3-5 days, collecting allantoic fluid for HA detection, selecting HA-positive allantoic fluid for subpackaging, and storing in a refrigerator at-80 ℃ for freezing and reserving for later use.

2. Identification of recombinant viral molecular tags

(1) Designing and synthesizing a specific primer for identification, wherein an upstream primer JD-F: 5'-CACGCCCAATGCACCCGAGTTC-3' downstream primer JD-R: 5'-CAGCACTTGGGGTGCCTGCACTAC-3', respectively; (2) extracting RNA of the obtained newcastle disease virus vaccine strain, and carrying out reverse transcription on the RNA into cDNA by using reverse transcriptase; (3) adding specific primers JD-F and JD-R to amplify DNA fragments by a PCR method by taking the extracted cDNA as a template, sending PCR amplification products to a sequencing company for sequence determination to determine a labeled nucleotide sequence containing TTTCGCGAGGCGCGCCAA, wherein the size of the amplified fragment is about 700bp (4); (5) and (3) carrying out gel recovery on the PCR amplification product, carrying out enzyme digestion on the gel recovery product by using NruI endonuclease, and cutting the gel recovery product into two bands of 500bp and 200bp according to an electrophoresis result, thereby indicating that the identified virus strain is inserted into the labeled nucleotide sequence of TTTCGCGAGGCGCGCCAA.

3. Sequence identification and genetic stability of recombinant viruses

Selecting HA positive virus liquid to extract RNA and carrying out reverse transcription, then carrying out whole gene sequence determination, and if the newly rescued virus genome takes the La Sota strain as a framework, carrying out chimeric gene VII type F and HN genes; the virus containing the molecular tag introduced during construction is successfully rescued, and the rescued virus is named as a Newcastle disease chimeric virus marked vaccine strain NDV DC strain. The rescued viruses were passaged successively 10 times, and F and HN genes of the 1 st (F1), 2 nd (F2), 3 rd (F3), 5 th (F5) and 10 th (F10) viruses were selected for sequencing and sequenced by Biotechnology (Shanghai) Ltd. Sequencing results show that the sequences of F and HN of the NDV DC strains of the Newcastle disease virus at generation 1 (F1), generation 2 (F2), generation 3 (F3), generation 5 (F5) and generation 10 (F10) are consistent with the inserted sequences, and the virus is genetically stable during passage (the results are shown in tables 4 and 5); the F gene cleavage sites of the 1 st (F1), 2 nd (F2), 3 rd (F3), 5 th (F5) and 10 th (F10) viruses were all the cleavage sites of the attenuated strain (results are shown in fig. 2).

Selecting viruses of the 1 st generation (F1), the 2 nd generation (F2), the 3 rd generation (F3), the 5 th generation (F5) and the 10 th generation (F10) to identify primers JD-F by using molecular tags: 5'-CACGCCCAATGCACCCGAGTTC-3' and JD-R: 5'-CAGCACTTGGGGTGCCTGCACTAC-3' and sequencing the sequences by Biotechnology engineering (Shanghai) Inc., the sequencing result shows that the 1 st generation (F1), the 2 nd generation (F2), the 3 rd generation (F3), the 5 th generation (F5) and the 10 th generation (F10) Newcastle disease virus rNDV DC strains all contain TTTCGCGAGGCGCGCCAA labeled nucleotide sequences (the result is shown in figure 3). Carrying out enzyme digestion identification on products recovered after PCR amplification of the 1 generation (F1), the 2 nd generation (F2), the 3 rd generation (F3), the 5 th generation (F5) and the 10 th generation (F10) by using NruI endonuclease, wherein electrophoresis results show that two bands of 500bp and 200bp are obtained after enzyme digestion of the 1 generation (F1), the 2 nd generation (F2), the 3 rd generation (F3), the 5 th generation (F5) and the 10 th generation (F10) by using the endonuclease; strains without inserted molecular tags cannot be cut.

TABLE 4 genetic stability analysis of the F Gene of NDV DC Strain

TABLE 5 genetic stability analysis of HN Gene of NDV DC Strain

Example 5

Growth and pathogenic Properties of recombinant viruses

1. Culture Properties of recombinant viruses

BHK-21-SC suspension cell strain is inoculated into a shake flask when the cell density reaches 8X 10⁶cells/ml, according to MOThe allantoic fluid of rNDV DC strains of passage 1 (F1), passage 2 (F2), passage 3 (F3), passage 5 (F5) and passage 10 (F10) was inoculated at 0.005 for I, and inoculation medium and TPCK pancreatin were added. Sampling and counting 24h, 36h, 48h and 72h after virus inoculation, respectively, harvesting virus liquid 72h after virus inoculation, and determining HA titer which can reach 1: 2048 or more, and EID50 measurement of the harvested virus solution to obtain a virus titer of 10^9.5EID₅₀More than 0.1ml (the results are shown in Table 6).

TABLE 6 suspension culture Titers of NDV DC strains

2. Pathogenic Properties of recombinant viruses

And (3) determining the virulence indexes of the chicken embryo median lethal dose (MDT), the intracerebral inoculation pathogenic index (ICPI) and the intravenous inoculation pathogenic index (IVPI) of the La Sota strain, the Newcastle disease gene VII type strain RS strain and the chimeric strain NDV DC strain according to the OIE standard. The results are shown in Table 7. Experimental results show that the toxicity of the chimeric virus strain NDV DC strain rescued by the reverse genetic manipulation technology is obviously lower than that of the parent strain La Sota strain and the gene VII type strain RS strain, and the chimeric virus strain NDV DC strain proliferated in chick embryos has the biological characteristic of obviously weakening the toxicity of SPF chickens and SPF chick embryos.

TABLE 7 determination of the viral virulence index of the chimeric Virus strains NDV DC

Example 6

Inducing immune effects of protective antibodies in animals

1. Preparation of inactivated vaccine

Preparing an oil phase: taking 94 parts of high-quality white oil for injection, adding 2 parts of aluminum stearate while stirring until the white oil is completely transparent, adding 6 parts of span-80, fully and uniformly mixing, and carrying out autoclaving for later use.

Preparation of an aqueous phase: and (3) adding 96 parts of qualified semi-finished antigen liquid into 4 parts of sterilized Tween-80, and fully stirring until the Tween-80 is completely dissolved.

Emulsification: the water phase and the oil phase are in a ratio of 1:2, the oil phase is firstly input into an emulsification tank to be stirred, then the water phase is slowly added, the stirring is continued to ensure that the oil phase and the water phase are fully and uniformly mixed, and then the mixture is emulsified on line by a shearing machine. After emulsification, 10ml of vaccine is taken and added into a centrifuge tube, and the centrifuge tube is centrifuged at 3500rpm/min for 15 minutes, and the water separated out from the tube bottom is not more than 0.5ml correspondingly.

2. Immune challenge protection test protocol

(1) 50 healthy susceptible chickens of 30-60 days old are used for immunization and divided into 4 groups, the 1 st group is 20 chickens, and each vaccine is 0.2ml prepared by subcutaneous immunization of NDV DC; the 2 nd and 3 rd groups are 10 vaccines of NDV-GVIII-1 and NDV-GVIII-2 vaccines of 0.2 ml/vaccine respectively for subcutaneous immunization;

group

4, 10/group, was not immunized as a control (as shown in table 8).

TABLE 8 immunization and challenge information

(2) The HI titer of the antibodies was measured by blood collection on day 21 after the immunization of blood collection, and as a result, the antibodies in the immune group of the NDV DC strain were significantly higher than those in the immune groups of NDV-GVIII-1 and NDV-GVIII-2 (the results are shown in FIG. 4 and Table 9).

TABLE 9 day 21 antibody levels after immunization

(3) After blood collection, 10 RS strains 10 are respectively injected into the 1 st group to the 3 rd group through intramuscular injection⁵ELD₅₀In

group

1, 10 intramuscular injections of strain F48E8, strain 10, were administered⁵ELD₅₀(ii) a Control groups of 5 intramuscular RS strains 10 were each administered to each group⁵ELD₅₀Separately, 5 intramuscular injections of F48E8 strain 10 were made⁵ELD₅₀。

(4) The morbidity and mortality of the chicken flocks are observed through clinical observation, and the result shows that each immune group can provide 100% protection by using F48E8 strain and RS strain to challenge.

(5) Collecting anus and respiratory tract swabs 5 days after pathogen separation and challenge, and performing virus separation and titration, wherein the results show that 5 days after challenge, viruses are not separated from the NDV DC strain immunization group by using F48E8 strain and RS strain for challenge, and the NDV-GVIII-1 strain and the NDV-GVIII-2 strain respectively have toxin expelling rates of 20% and 50%; the NDV DC strain can inhibit detoxification more effectively and provide better immune protection (results are shown in table 10).

TABLE 10 protective results of challenge

The above description is only for the preferred embodiment of the present invention, and should not be taken as limiting the scope of the invention, which is defined by the appended claims and the description of the invention.

Claims

1. The newcastle disease chimeric virus marked vaccine strain is characterized in that the newcastle disease chimeric virus marked vaccine strain is named as NDV DC strain, the whole gene sequence of the newcastle disease chimeric virus marked vaccine strain NDV DC strain is shown as SEQ ID No.1, the newcastle disease gene VII type F gene sequence and HN gene sequence containing cracking site mutation, NP, P, M and L gene sequences of a newcastle disease gene II type La Sota strain are contained, the NDV DC strain is preserved in the common microorganism center of the China microorganism culture preservation management committee, and the preservation numbers are as follows: CGMCC No. 19298.

2. The newcastle disease chimeric virus marker vaccine strain according to claim 1, wherein a marker nucleotide sequence shown as SEQ ID No.3 is inserted into a non-coding region between a P gene sequence and an M gene sequence at 3170 th nucleotide of a nucleotide sequence of the NDV DC strain.

3. Use of the chimeric newcastle disease virus marker vaccine strain NDV DC strain of claim 2 in the preparation of a newcastle disease vaccine.

4. A method for preparing a newcastle disease vaccine by using the newcastle disease chimeric virus marked vaccine strain NDV DC strain of claim 2, which comprises the steps of (1) proliferating the newcastle disease chimeric virus marked vaccine strain NDV DC strain of claim 2 by means of chick embryo culture or cell culture, and harvesting a virus solution; (2) inactivating the harvested virus liquid by adopting one of formaldehyde, beta-propiolactone or diethylene imine; (3) the antigen liquid and the vaccine adjuvant are stirred and emulsified to obtain a uniform emulsion.

5. A method for clinically identifying the NDV DC strain of the chimeric newcastle disease virus marker vaccine strain according to claim 2, wherein (1) a specific primer for identification is designed and synthesized, and an upstream primer JD-F: 5'-CACGCCCAATGCACCCGAGTTC-3', respectively; downstream primer JD-R: 5'-CAGCACTTGGGGTGCCTGCACTAC-3', respectively; (2) extracting RNA of the newcastle disease virus vaccine strain, and carrying out reverse transcription on the RNA into cDNA by using reverse transcriptase; (3) adding a specific primer to amplify a DNA fragment by using the cDNA as a template by using a PCR method, wherein the size of the amplified fragment is about 700 bp; (4) sending the PCR amplification product to a sequencing company for sequence determination, wherein the determined sequence contains TTTCGCGAGGCGCGCCAA labeled nucleotide sequences located at 3170 nucleotides of the full-length sequence, and the sequence is a Newcastle disease virus labeled vaccine strain NDV DC strain; or performing gel recovery on the PCR amplification product, performing enzyme digestion on the gel recovery product by using NruI endonuclease, and judging that the gel recovery product can be cut into two strips of 500bp and 200bp according to an electrophoresis result, wherein the identified strain is the NDV DC strain of the Newcastle disease virus marker vaccine strain.