CN116926022A - PiNew castle disease virus mGZ VI, application, preparation method, culture method and vaccine - Google Patents

PiNew castle disease virus mGZ VI, application, preparation method, culture method and vaccine Download PDF

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CN116926022A
CN116926022A CN202310896009.4A CN202310896009A CN116926022A CN 116926022 A CN116926022 A CN 116926022A CN 202310896009 A CN202310896009 A CN 202310896009A CN 116926022 A CN116926022 A CN 116926022A
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gene
pigeon
newcastle disease
disease virus
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CN116926022B (en
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任涛
任金莲
许芬芬
刘兆洁
陈礼斌
林秋燕
董志轩
陈爱华
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Guangdong Huasheng Biotechnology Co ltd
South China Agricultural University
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Abstract

The application relates to the technical field of vaccine production, and discloses a pigeon source gene VI newcastle disease virus mGZ with a preservation number of CCTCC NO: v202253; the preservation date is 2022, 7 and 5; the preservation unit is China center for type culture Collection, and meanwhile, the application also provides a preparation method of pigeon source gene VI type newcastle disease virus mGZ, which can not cause random homologous recombination of virus genes and can accurately obtain target genotypes; in addition, the application optimizes the culture condition during culture, thereby further improving the virus titer; finally, experiments prove that the inactivated vaccine prepared from the pigeon source gene VI type newcastle disease virus mGZ has more excellent protection rate compared with the traditional newcastle disease vaccine on the market.

Description

PiNew castle disease virus mGZ VI, application, preparation method, culture method and vaccine
Technical Field
The application relates to the technical field of vaccine production, in particular to pigeon source gene VI type newcastle disease virus mGZ, application, a preparation method, a culture method and a vaccine.
Background
There is a long history of pigeon keeping in China, and pigeons are classified into carrier pigeons, meat pigeons and ornamental pigeons by human domestication. In recent years, with the development of economy and the improvement of cultural level of people in China, the carrier pigeon industry in China is developed vigorously, racing pigeons are derived, racing pigeons are more and more popular as a sports, and the national sports administration formally lists the racing pigeons as 93 rd sports items in China in 1984. The carrier pigeon is also called as a 'communication pigeon', is a variety developed and cultivated by common pigeons, and the excellent flying ability of the carrier pigeon is utilized and trained by the domestication of human beings for many years, so that the carrier pigeon is separated from the common pigeons to form an independent variety.
However, with the rapid development and update of modern communication technology, the carrier pigeons are no longer used for transmitting information, and their competitive activities are more and more favored by people, and the national public sports organization, china carrier pigeon society, is established by China national sports Congress. In recent years, as the wide mass foundation makes the racing pigeons rapidly develop, the industrialization and scale steps of the racing pigeons are continuously accelerated, more and more related industries are emerging, such as the emergence of the new industry of the pigeon show auction, pigeon grain, pigeon medicines, racing pigeon import and export companies, racing pigeon sheds and the like, and the scale and intensification of the racing pigeon breeding are rapidly developed. According to statistics of China carrier pigeon association, more than 150 ten thousand racing pigeons are found in China in 2015, about 2000 ten thousand young pigeons are newly added each year, and various competition institutions are close to 6000 (club, public shed, association and the like). To the present, the nationwide racing pigeon sheds are nearly thousands of, more than 100 sheds exist in the Hebei area, the number of pigeons in each shed is seven eight thousands, more than ten thousands of racing pigeon industry annual output values are nearly billions yuan. The breeding mode of the pigeon racing industry is to collect pigeons for centralized management from all regions of the country, but because of poor epidemic disease prevention and control consciousness, different pigeon racing pigeons with different immune conditions, large immune level difference, stress reaction caused in the transportation process and possible toxic phenomenon of part of pigeons, the pigeons are mutually infected after pigeon racing, epidemic disease frequently occurs, and immeasurable economic loss is caused for pigeon racing industry. However, the newcastle disease of pigeons is one of the important causes among many factors causing the death of racing pigeons. In the current production practice, the racing pigeon newcastle disease is mainly controlled by vaccine immunization and comprehensive sanitary epidemic prevention measures. However, due to the lack of vaccines against pigeon-derived newcastle disease virus, the vaccine used in clinical vaccination is mainly newcastle disease vaccine. Although the pigeon newcastle disease virus belongs to the avian type I paramyxovirus and belongs to the same genus virus as the chicken newcastle disease virus, the two viruses are different in pathogenic characteristics and reaction characteristics with monoclonal antibodies due to the difference of genotypes, so that the chicken newcastle disease vaccine cannot effectively protect and control the infection and the epidemic of the pigeon newcastle disease virus. Therefore, research and development of the vaccine against the pigeon origin newcastle disease is urgent for preventing and treating the pigeon newcastle disease.
Chinese patent application 202010872170.4 discloses a pigeon paramyxovirus type 1 PPMV-1/BJ-C strain and application thereof, and the preservation number of the pigeon paramyxovirus type 1 PPMV-1/BJ-C strain disclosed by the scheme is CGMCC No.19989; the strain is obtained by performing weakening mutation on F protein cleavage site mutation of a virulent strain BJ-C strain into a corresponding site of a LaSota strain, and then saving the weakened pigeon paramyxovirus type 1 PPMV-1/BJ-C strain;
meanwhile, the scheme also carries out a comparison test on the inactivated vaccine prepared by the paramyxovirus 1 type PPMV-1/BJ-C and the chicken newcastle disease inactivated vaccine (LaSota strain) on chickens, and the test result shows that the protection rate of the inactivated vaccine and the chicken newcastle disease inactivated vaccine is not obviously different, so that the protection capability of the inactivated vaccine prepared by the paramyxovirus 1 type PPMV-1/BJ-C is equivalent to that of the chicken newcastle disease inactivated vaccine (LaSota strain) when the chickens are receptors; it should be noted, however, that this approach does not address what viruses are used for the above-described tests to attack.
Jiang Yanyu, zhao Shasha, sun Nana et al in the Chinese Protect veterinary journal, volume 40, 11, disclose that pigeon Newcastle disease virus virulence attenuated strains rescue and immunogenicity analysis indicate that pigeon-derived NDV is of paramyxovirus serotype I, which is the same genus as chicken NDV, but that pigeon NDV evolutionarily forms independent branches: the ClassII type gene VIb has larger antigenicity difference with the current common vaccine strains (ClassII type genes I and II type), so that the current vaccine has poor immune effect on pigeons, and causes high toxicity rate and morbidity of the pigeons; therefore, the existing newcastle disease vaccine does not necessarily have a good protection effect on the pigeon newcastle disease virus, and the same pigeon newcastle disease vaccine does not necessarily have a good protection effect on the chicken newcastle disease virus, and in most cases, the pigeon newcastle disease vaccine is more suitable for pigeons infected with the pigeon newcastle disease virus;
the above document uses primers FF1/FR1 and FF2/FR2 of pigeon NDVPi/CH/LHLJ/110822 to carry out point mutation on the F gene of 0822 virus strain by overlap PCR, and the F protein cleavage site sequence is defined by 112 RRQKRF 117 Mutant to LaSota strain 112 GRQGRL 117 Obtaining attenuated strains, preparing the attenuated strains into attenuated seedlings and inactivated seedlings respectively, and testing, although the attenuated strains have effect after being used as the inactivated seedlings, it is noted that the receptor vaccinated in the experiment is chicken instead of pigeon, and the document mentionsThe virus strain obtained by reverse genetics has random homologous recombination with the L gene of the auxiliary plasmid in the rescue process, so that small deviation of the L gene is generated.
Liyao of the university of Hebei North and North in the 'Saigan New castle disease virus separation and identification and HB-strain inactivated vaccine research and application' mentions that a new castle disease virulent HB strain is determined, meanwhile, sequencing is carried out on the HB strain, the sequencing result is submitted to GenBank, the serial number is MK973060, and the genotype of the HB strain is obtained by analysis through the drawing of an evolutionary tree, the similarity with the Pigeon/Qinghai/1344 reaches 98.45%, and the HB strain has obvious difference with the current domestic genotype, so that the domestic traditional chicken Newcastle disease vaccine cannot provide a good enough protection effect;
then, the authors inactivate the HB strain by formaldehyde and prepare an inactivated vaccine after inactivation, of course, the water adjuvant and the oil adjuvant are respectively added into the inactivated vaccine, the optimal immunity dose, the antibody production time, the immunity duration and the like of the water adjuvant inactivated vaccine and the oil adjuvant inactivated vaccine are respectively tested, and the protection level of the vaccine prepared after the HB strain inactivation and the protection level of the inactivated vaccine prepared by LaSota strain are compared, so that the protection capability of the inactivated vaccine prepared by the HB strain is better than that of the inactivated vaccine prepared by the LaSota strain;
however, it should be noted that the above-mentioned document does not make the HB strain a virulent strain which is attenuated in the process of preparing an inactivated vaccine by using the HB strain, and there is a risk that the strain will be infected with a virulent strain virus if the inactivation is not complete, and the probability of occurrence of the above-mentioned phenomenon is not negligible although it is not great.
The problem that this scheme needs to solve: how to provide a attenuated pigeon source gene VI type newcastle disease virus and apply the same to a pigeon source gene VI type newcastle disease vaccine.
Disclosure of Invention
The application aims to provide a attenuated pigeon source gene VI type newcastle disease virus and a method for preparing the attenuated pigeon source gene VI type newcastle disease virus, and the attenuated pigeon source gene VI type newcastle disease virus is applied to a pigeon source gene VI type newcastle disease vaccine.
The application is not specifically described: nM represents nanomole/liter, μM represents micromoles/liter, mM represents millimoles/liter, and M represents moles/liter;
in order to achieve the aim, the application discloses a pigeon source gene VI type newcastle disease virus mGZ with a preservation number of CCTCC NO: v202253; the preservation date is 2022, 7 and 5; the collection unit is China center for type culture Collection.
In addition, the application also discloses a preparation method of the pigeon source gene VI type newcastle disease virus mGZ, which adopts gene segments H-P1, P2-Las, P3 and P4 to obtain the pigeon source gene VI type newcastle disease virus mGZ by homologous recombination;
the gene sequence of the gene fragment H-P1 is shown in SEQ ID NO:1 is shown in the specification;
the gene sequence of the gene fragment P2-Las is shown in SEQ ID NO:2 is shown in the figure;
the gene sequence of the gene fragment P3 is shown in SEQ ID NO:3 is shown in the figure;
the gene sequence of the gene fragment P4 is shown in SEQ ID NO: 4.
Preferably, the gene segment H-P1, the gene segment P2-Las, the gene segment P3, the gene segment P4 and the vector TVT which is subjected to linearization treatment are connected to obtain a target plasmid, and the target plasmid is transfected into cells to obtain the pigeon source gene VI type newcastle disease virus mGZ.
Preferably, the preparation method of the gene fragment P2-Las comprises the following steps:
step 1: ligating the P2 fragment to the pCI-neo vector to yield plasmid pCI-P2;
step 2: taking plasmid pCI-P2 as a template, and mutating the plasmid pCI-P2 by using primers P2-Las F and P2-Las R to obtain pCI-P2-Las;
the gene sequence of the P2 fragment is shown as SEQ ID NO:5 is shown in the figure;
the gene sequence of the primer P2-Las F is shown as SEQ ID NO:6 is shown in the figure;
the gene sequence of the primer P2-Las R is shown in SEQ ID NO: shown at 7.
In addition, the application also discloses a suspension culture method of the pigeon source gene VI type newcastle disease virus mGZ, which comprises the following steps:
step A1: the cell density was taken to be 6.0X10 6 Mixing the cell/mL serum-free full suspension culture type BHK-21 cell suspension with a serum-free full suspension culture medium to prepare a culture medium, wherein the volume ratio of the serum-free full suspension culture type BHK-21 cell suspension to the serum-free full suspension culture medium is 1:1, a step of;
step A2: and (3) inoculating the pigeon source gene VI type newcastle disease virus mGZ into the culture medium prepared in the step A1 for culture to obtain the pigeon source gene VI type newcastle disease virus mGZ08.
Preferably, in the step A2, the addition amount of the pigeon source gene VI type newcastle disease virus mGZ is 1-2 per mill of the volume of the culture medium.
Preferably, in the step A2, the culture medium after the pigeon-derived gene VI newcastle disease virus mGZ is inoculated has a culture temperature of 34.5-35.5 ℃.
In addition, the application also discloses application of the pigeon source gene VI type newcastle disease virus mGZ08 in preparing a pigeon newcastle disease vaccine.
In addition, the application also discloses a pigeon newcastle disease vaccine which contains inactivated pigeon source gene VI newcastle disease virus mGZ08.
The beneficial effects of the application are as follows:
the pigeon source gene VI type newcastle disease virus provided by the application is the pigeon source gene VI type newcastle disease virus after weakening, so that the vaccine prepared by using the pigeon source gene VI type newcastle disease virus has higher safety compared with the pigeon source gene VI type newcastle disease virus which is not subjected to weakening treatment;
meanwhile, the preparation method of the pigeon source gene VI type newcastle disease virus provided by the application can not cause random homologous recombination of virus genes and can accurately obtain the target genotype;
in addition, the application optimizes the culture condition during culture, thereby further improving the virus titer;
finally, experiments prove that the inactivated vaccine prepared from the pigeon source gene VI type newcastle disease virus mGZ has more excellent protection rate compared with the traditional newcastle disease vaccine on the market.
Description of the drawings:
FIG. 1 is a schematic diagram showing the amplification results of GZ08 strain fragments P1-1, P1-2 and P1-3;
FIG. 2 is a schematic diagram showing the result of amplification of GZ08 strain fragment P2, P3 gene;
FIG. 3a is a schematic diagram of the point mutation amplification of pCI-P2 plasmid, and FIG. 3b is a schematic diagram of the result of the amplification of GZ08 mutant fragment P2-Las;
FIG. 4 is a schematic diagram of the sequencing result of the mutation site of the rescue virus F gene;
FIG. 5 is a schematic diagram showing the result of amplification of GZ08 strain fragment H-P1-1 gene;
FIG. 6 is a schematic diagram of the result of amplification of GZ08 strain fragment H-P1-1 gene;
FIG. 7 is a schematic diagram showing the result of amplification of the GZ08 strain P3 gene;
FIG. 8a is a schematic diagram showing the results of amplification of the GZ08 strain fragments P4-1 and P4-2 genes, and FIG. 8b is a schematic diagram showing the results of amplification of the GZ08 strain P4 genes;
FIG. 9 is a schematic diagram showing the result of TVT vector linearization gene amplification;
FIG. 10 is a schematic representation of the results of first generation allantoic fluid agglutination of red blood cells;
FIG. 11 shows the results of indirect immunofluorescence assay of recombinant viruses, wherein FIG. 11a and FIG. 11b show the results of IFA of recombinant viruses, FIG. 11c and FIG. 11d show the results of IFA of negative control, and FIG. 11e and FIG. 11f show the results of IFA of parent viruses;
FIG. 12 shows sequencing results of each generation of mGZ strain, wherein FIG. 12a shows modification of the cleavage site of the 5 th generation F gene, FIG. 12b shows modification of the cleavage site of the 10 th generation F gene, and FIG. 12c shows modification of the cleavage site of the 15 th generation F gene.
Detailed Description
In the description of the present application, it is to be noted that the specific conditions are not specified in the examples, and the description is performed under the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The present application now will be described more fully hereinafter with reference to the accompanying drawings, in which it is shown, however, to illustrate some, but not all embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The raw materials used in the examples and the suppliers or sources of the raw materials are shown in Table 1:
table 1: raw material information table
Example 1
1. Proliferation of viruses
The original strain GZ08 is diluted by sterilized PBS according to a ratio of 1:1000, inoculated into allantoic cavities of SPF chick embryos of 9 days old, each 0.1mL, and placed in a constant temperature incubator at 37 ℃ for culture. Collecting chick embryo allantoic fluid within the death time of 48-72 hours, measuring the HA titer of the chick embryo allantoic fluid, sub-packaging the chick embryo allantoic fluid, and preserving the chick embryo allantoic fluid at the temperature of-80 ℃;
2. viral genome RNA extraction
Reverse transcription is carried out on the virus nucleic acid by utilizing reverse transcriptase Superscript III transcriptase, the virus nucleic acid acts for 5min at 25 ℃, and water bath is carried out for 60min at 55 ℃ to obtain cDNA;
3. preparation of full-Length plasmid:
dividing the full-length cDNA sequence into P1 segment, P2 segment, P3 segment and P4 segment for amplification; wherein the P1 section is divided into a P1-1 section, a P1-2 section and a P1-3 section, and the P4 section is divided into a P4-1 section and a P4-2 section; designing primers as shown in Table 2, and amplifying the P1-1 section, the P1-2 section, the P1-3 section, the P2 section, the P3 section, the P4-1 section and the P4-2 section by using the primers in Table 2;
table 2: primer sequences of intermediate plasmids
3.1 preparation of intermediate plasmid pCI-H-P1:
synthesizing a HamRz fragment, adding two guanines at the 5' end of the HamRz fragment, and preparing an H-P1-1 fragment from the HamRz fragment added with the guanines and the P1-1 fragment in an overlap mode;
sequentially connecting the H-P1-1 fragment, the P1-2 fragment and the P1-3 fragment to a pCI-neo carrier, and sequencing a positive sample after bacterial liquid PCR identification to obtain an intermediate plasmid pCI-H-P1 after sequencing without errors;
3.2 preparation of intermediate plasmid pCI-P2-Las:
connecting the P2 fragment to a pCI-neo vector to obtain a plasmid pCI-P2, then carrying out point mutation by using the constructed plasmid pCI-P2 as a template and using primers P2-Las F and P2-Las R, replacing the F cleavage site amino acid of GZ08 with the corresponding site of a attenuated strain LaSota, carrying out bacterial liquid PCR identification, sequencing a positive sample, and obtaining an intermediate plasmid pCI-P2-Las after sequencing without errors, wherein the sequence of the point mutation primer is shown in the table 3;
table 3: point mutation primer sequence
3.3 preparation of intermediate plasmid pCI-P3:
connecting the P3 fragment to a pCI-neo vector, and sequencing a positive sample after bacterial liquid PCR identification to obtain an intermediate plasmid pCI-P3 after sequencing without errors;
3.4 preparation of intermediate plasmid pCI-P4:
sequentially connecting the P4-1 fragment and the P4-2 fragment to a pCI-neo vector, and sequencing a positive sample after bacterial liquid PCR identification to obtain an intermediate plasmid pCI-P4 after sequencing without errors;
3.5 preparation of full Length plasmid:
amplifying complete fragments from plasmids pCI-H-P1, pCI-P2-Las, pCI-P3 and pCI-P4, cloning 4 fragments onto a vector TVT-7R according to the sequence of H-P1-P2-Las-P3-P4 by using a homologous recombination method to obtain a full-length plasmid;
the primers used in the homologous recombination process are shown in Table 4;
table 4: primer sequences for constructing full-length plasmids
Homologous recombination of GZ08 strain gene fragment:
recombination reaction: determining the concentration of the recovered fragment DNA; in the multi-fragment homologous recombination reaction, the optimal cloning vector and the insert are used in an amount of 0.03pmol each; 2 XClonexpress Mix 5. Mu.L was added, the total amount of fragments and vector was controlled to be within 5. Mu.L, and less than 5. Mu.L was aseptically filled. (Multi-fragment recombination reaction, 50 ℃, 15-30 min, and rapid ice cooling after the reaction is completed.)
Conversion of recombinant products: the recombinant product was transferred into HB101 competent cells according to the conventional transformation method, and the coated plates were placed in a biochemical incubator at 30℃for 20h.
According to a conventional monoclonal colony picking method, after the bacterial picking is finished, shaking the bacteria for 6 hours at 30 ℃, and carrying out PCR amplification identification through bacterial liquid, wherein the reaction procedure is as follows: 95℃for 3min,95℃for 15s,60℃for 15s,72℃for 30s/kb,35 cycles, 72℃for 5min,4 ℃. The bacterial liquid PCR amplification reaction system is shown in Table 5, amplified products are subjected to gel electrophoresis analysis, samples with correct target bands are screened for amplification culture, and plasmids are extracted strictly according to the specification of a QIAGEN plasmid extraction kit;
table 5: bacterial liquid PCR system configuration
Component (mu L) Dosage (mu L)
Green taq Mix 12.5
Primer 1 1
Primer 2 1
Bacterial liquid 1
Sterile ultrapure water 2.5
Total volume of 25
5. Rescue of pigeon-derived newcastle disease recombinant viruses
BSR-T7/5 cells were inoculated in 35mm dish/6 well plates, incubated overnight with DMEM without anti-10% FBS, and used for transfection when monolayers grew to 60% -80%; before transfection, MVA was diluted 500-fold with DMEM, cells were washed 3 times with PBS, 600. Mu.L of the dilution was added to each well of cells, CO at 37 ℃ 2 Incubating the incubator for 30min; during the incubation of the poxvirus, 2 1.5mL finger tubes were taken, 100. Mu.L of LOpti-MEM was added, respectively, and helper plasmids pCI-NP, pCI-P, pCI-L and full length plasmid were added in sequence in a 4:2:1:10 ratio in tube A, with a total plasmid mass of 8.5. Mu.g/well; adding 10 mu L Lipo-2000 into the finger-type tube B, and allowing the mixture to act at room temperature for 5min; carefully transferring the liquid in the pipe B into the pipe A, blowing uniformly by using a gun head, and incubating for 15min at room temperature; the MVA incubated cells were removed, the supernatant was discarded, washed 3 times with PBS, opti1mL was added to each well, and the biosafety cabinet was placed for transfection. After 15min of the transfection complex at room temperature, the cells were added dropwise, gently shaken well, and placed in an incubator containing 5% CO2 at 37 ℃.
After 4h of transfection, the transfected cells were removed, the medium was discarded, washed 3 times with sterile PBS and placed in an incubator containing 5% CO2 at 37℃for 72h with the addition of 2% cell maintenance solution.
NDV attenuated infected cells require the aid of pancreatin; after 24h of transfection, the cells were removed and the culture was continued with the addition of TPCK pancreatin to a final concentration of 1. Mu.g/mL.
Observing the cell state 24h after transfection, repeatedly freezing and thawing the cells after 48-72 h, taking the mixed solution of the cells and the supernatant, inoculating 9-day-old SPF chick embryos according to 0.2 mL/piece, collecting chick embryo allantoic fluid cultured for 24-72 h, and measuring the hemagglutination value of the chick embryo allantoic fluid. Allantoic fluid with hemagglutination activity was collected in its entirety and subjected to HI test. The allantoic fluid with hemagglutination activity (as shown in fig. 10) was continuously passaged, 1 part of virus allantoic fluid was taken to extract mGZ strain RNA according to the above method when passing to 5 generations, F gene thereof was amplified after reverse transcription, amplified product was sent to Shanghai Biotechnology company for sequencing, sequencing result was analyzed by Snap Gene and presence of mutation site (molecular tag) was determined. The successfully rescued virus was named mGZ08.
Example 2
Identification of pigeon source gene VI newcastle disease virus mGZ and biological property determination:
1. indirect immunofluorescence identification of pigeon source gene VI type newcastle disease virus mGZ strain:
preparation of CEF: preparing an autoclave, a plate and tweezers, and pouring sterilized PBS; taking 9-day-old SPF chick embryo, sterilizing the top end of an eggshell air chamber by iodine tincture, deiodizing by alcohol, removing shell membrane, air chamber membrane, allantoic cavity membrane and amniotic membrane by forceps, taking out embryo bodies, placing the embryo bodies on a plate, removing heads, limbs, viscera and the like by forceps, and washing the embryo bodies on another plate; placing the embryo body in a 50mL centrifuge tube subjected to high-pressure sterilization, placing the embryo body slightly horizontally, and fully cutting the embryo body by using small scissors; adding 3mL pancreatin, digesting for about 5min in a water bath at 37 ℃ until muscles are villous, and discarding the digestive juice; digestion was terminated by adding 3mL FBS, 30mL DMEM was added, gently and thoroughly blown, left to stand for a moment, and the mixture was filtered into a conical flask.
Indirect immunofluorescence technique: CEF is plated in 24-well plates, and virus inoculation can be prepared after cells grow to 90%; the culture medium in each cell culture well was discarded with a pipette and washed 3 times with sterile PBS; diluting GZ08 and mGZ virus solution with DMEM, taking 100 μl CEF infected with 24-well plate at 1MOI, and setting positive control and negative controlSex control. The cell plates were subjected to 37℃5% CO 2 Is incubated for 1h; after 1h incubation of the virus, the GZ08 group, the negative control group and the positive control group are changed into 2% maintenance solution, the mGZ group is changed into DMEM containing 1 mug/mL TPCK, and the culture is continued; 24h after inoculation, the medium is discarded, washed 3 times with PBS and fixed with 250. Mu.L paraformaldehyde for 5min at room temperature; the paraformaldehyde was aspirated off and the cells were washed 3 times with 500. Mu.L of PBST and permeabilized with 250. Mu.L of PBST containing 0.2% Triton X-100 per well for 5min; closing: washing the cells 3 times with 500 μl of PBST, adding a blocking solution to each well, and blocking at room temperature for 1h; adding primary antibody: removing the blocking solution, washing with PBST for 3 times, taking rabbit anti-NDV-NP hyperimmune serum diluted by 1:200 as primary antibody, taking rabbit negative serum with the same dilution as control, and incubating at 37 ℃ for 60min; adding a secondary antibody: primary antibody was blotted off, PBST washed 3 times, and 1:200, taking the fluorescence-labeled goat anti-rabbit Fluro 488 as a secondary antibody, and incubating for 30min at the temperature of 37 ℃ in a dark place; the secondary antibody was blotted off and washed 3 times with PBST for 5min each, and after washing was completed, observed under a fluorescence microscope.
The NDV NP hyperimmune serum detection mGZ strain and GZ08 strain infected CEF both showed green fluorescence (as shown in fig. 11-b and 11-f), while it can be seen that the virus proliferated mainly in the cytoplasm, conforming to the replication characteristics of NDV, while no significant green fluorescence signal was observed in the cell background of the negative control group, while it can be seen that the virus proliferated mainly in the cytoplasm, conforming to the proliferation characteristics of NDV. The full-length cDNA infectious molecular clone of GZ08 strain constructed by reverse genetic technology is successfully saved for infectious newcastle disease recombinant virus.
2. Passage stability of pigeon-derived gene vi newcastle disease virus mGZ:
the rescue virus mGZ strain is blindly transferred in the allantoic cavity of the SPF chick embryo for 15 generations, 5 chick embryos are inoculated with the diluted sterile virus allantoic fluid each time, 0.2 mL/piece of the sterile virus allantoic fluid is used, and the hemagglutination price of the virus allantoic fluid of each generation is recorded. The mutation positions (molecular markers) and the whole gene sequences were determined every 5 th generation from the 5 th generation.
Referring to FIG. 12, the results show that the amino acid at the cleavage site on the rescue virus F gene is "G-R-Q-G-R-L", and no back mutation occurs. The other gene sequences are the same as the parent strain, and the basic groups are not changed, so that the rescue strain has good genetic stability.
3. Major biological properties of recombinant viruses:
ICPI and MDT disease indices were determined for rescue toxins as required by OIE standards. The results show that the parent strain GZ08 ICPI is 0.8125, the MDT is 102h, the pigeon NDV MDT and ICPI values generally show weak virulence or medium virulence characteristics, but show stronger pathogenicity to pigeons, and the rescue virus mGZ strain ICPI is 0, the MDT is 141h, which indicates that the rescue virus is relatively weak compared with the parent virus.
Example 3
1. Determination of optimal virus receiving density of pigeon source gene VI type newcastle disease virus mGZ08
The cell density was taken to be 6.0X10 6 Taking a cell/mL serum-free full suspension culture type BHK-21 cell suspension, and taking a serum-free full suspension culture medium according to the volume ratio of the cell suspension to the serum-free full suspension culture medium of 1:1,1:2 and 2:1, and simultaneously inoculating pigeon newcastle disease virus (mGZ strain, ha=8 Log 2 ,EID 50 =10 -8.17 0.1 mL), rotating at 90 rpm, at 35 ℃, dissolved oxygen at 40%, and pH at 7.1+ -0.1 for 72 hours, and detecting virus titer HA and virus content EID 50
The results are shown in Table 6: when the volume ratio is 1:1, after dilution, the pigeon newcastle disease virus is inoculated, and the virus titer is highest and is 1:512, EID 50 =10 -8.83 0.1mL; when the volume ratio is 1:2, after dilution, the pigeon newcastle disease virus is inoculated, and the virus titer is lower, namely 1:256, EID 50 =10 -7.83 0.1mL; when the volume ratio is 2:1 the virus titer after dilution was 1:512, EID 50 =10 -8.5 0.1mL; it can be seen that the volume ratio is 1:1, the effect is best.
Table 6: effects of different virus-receiving densities on viral proliferation
2. Optimal virus receiving amount of pigeon source gene VI type newcastle disease virus mGZ08
The cell density was taken to be 6.0X10 6 Taking a serum-free full-suspension culture medium from a cell/mL serum-free full-suspension culture type BHK-21 cell suspension, wherein the volume ratio of the cell suspension to the culture medium is 1:1, and simultaneously inoculating pigeon newcastle disease virus (mGZ strain, HA=8 Log) with 0.5%o, 1%o, 2%o, 5%o of the total volume of the cell suspension and the serum-free total suspension medium 2 ,EID 50 =10 -8.17 0.1 mL), the culture was carried out at a rotation speed of 80 rpm at 35℃and a dissolved oxygen of 40% and at a pH of 7.1.+ -. 0.1 for 72 hours, and virus titers HA and EID were detected 50
The results are shown in Table 7: the virus proliferation effect is better after 1 millinoculation, and the virus titer HA is 1:512 virus content EID 50 =10 -8.83 0.1mL; after the 5%o of the sample is inoculated for 72 hours, the cell death speed is high due to the large inoculation amount, so that the number of live toxins is low in the 72 hour sample collection, and the EID is low 50 =10 -6.67 0.1mL; and after inoculation with 0.5 per mill of the inoculation dose, the virus titer HA is the lowest at 72 hours, and is 1:256, EID 50 =10 -7.17 0.1mL; and after inoculation with 2% 50 =10 -8.17 0.1mL; it follows that the optimal toxin-receiving agent amount is determined to be 1 per mill.
Table 7: effect of different viral inoculum sizes on viral proliferation
3. Determination of optimal culture temperature of pigeon source gene VI type newcastle disease virus mGZ08
The cell density was taken to be 6.0X10 6 Taking a serum-free full-suspension culture medium from a cell/mL serum-free full-suspension culture type BHK-21 cell suspension, wherein the volume ratio of the cell suspension to the culture medium is 1:1, and simultaneously inoculating pigeon newcastle disease virus (mGZ strain, ha=8 Log) in an amount of 1% 2 ,EID 50 =10 -8.17 0.1 mL), rotation speed 80 rpmThe virus content was measured by culturing at 35℃and 40% dissolved oxygen and pH 7.1.+ -. 0.1 for 72 hours, and the results are shown in Table 8: at 35 deg.C, the virus content is high, and EID is high 50 =10 -8.83 0.1mL, and the cells react slowly with the virus in culture at 33 ℃, EID 50 =10 -7.62 0.1mL; at 37deg.C and 39deg.C, the virus activity is high, the cell death rate is high, and the virus dies in 72 hr, resulting in low virus content and EID 50 ≤10 -7.5 0.1mL; thus, 35℃is the optimal virus culture temperature.
Table 8: effects of different temperatures on viral proliferation
Example 4
Method for culturing pigeon source gene VI type newcastle disease virus mGZ08
Resuscitate the frozen BHK-21 suspension seed cells from the liquid nitrogen tank, add the cell fluid into 250mL triangular shake flask with a straw, add the filtration degerming serum-free whole suspension culture medium with pH of 7.1+ -0.1 to 100mL; after culturing the cells for about 48 hours, the cell density reaches 6.0X10 6 The virus inoculation is carried out when the cell/mL is about;
the BHK-21 cells after 48h culture are cultured by adopting a fresh serum-free culture medium according to the volume ratio of the cell suspension to the culture medium of 1:1, diluting, and placing the mixture at 35 ℃ after the inoculation, wherein the volume ratio of the inoculation amount is 1% 2 Suspension culturing in an incubator for 72h;
the above method for culturing pigeon newcastle disease virus was carried out for 3 batches with virus titers of HA and EID 50 The results are shown in Table 9: the virus titer HA can reach 1:512, EID 50 =10 -8.83 0.1mL, up to 10 -9.0 /0.1mL。
Table 9: results of different batches of cultured pigeon Newcastle disease Virus (mGZ strain 08)
Vaccine protection capability and safety test
Safety comparison:
taking 3 batches (P20001, P20002 and P20003) of vaccine prepared from inactivated pigeon source gene VI type newcastle disease virus mGZ and 1 batch (202104) of newcastle disease inactivated vaccine, subcutaneously inoculating 10 30-60 day old pigeons on each neck, continuously observing for 14 days after injection, and recording the reaction condition of the test pigeons in detail, wherein the results are shown in Table 10;
table 10: safety comparison result
Comparison of immunopotentiation
Taking laboratory prepared vaccine (batch number P20003) and chicken newcastle disease inactivated vaccine, respectively immunizing 20 meat pigeons 30-60 days old under the skin of the neck, taking blood together with 20 control pigeons after 0.3 ml/meat, and separating serum, and measuring the blood coagulation inhibition antibody titer of the pigeon newcastle disease virus and chicken newcastle disease virus, wherein the result is shown in Table 11;
table 11: results of virus attack protection test of inactivated vaccine for pigeon Newcastle disease and inactivated vaccine for chicken Newcastle disease
Analysis of results:
the two vaccines are injected into 30-60 day old pigeons at a dose of 0.6 ml/dose, no adverse reaction occurs in local and whole bodies within 14 days after injection, and the safety of the two vaccines is good. The two vaccines are respectively immunized with 30-60 days old meat pigeons, and immune efficacy comparison is carried out, and the results show that serum HI antibodies of two groups of test pigeons are detected by taking the pigeon newcastle disease virus hemagglutination inhibition antigen as a detection antigen, the HI antibody titer of the pigeon newcastle disease inactivated vaccine group is higher than that of the chicken newcastle disease inactivated vaccine group, and the immune protection results after 21 days of immunization show that the protection rate of the pigeon newcastle disease inactivated vaccine group reaches 90%, and the attack protection rate of the chicken newcastle disease inactivated vaccine group is 50%. The immune effect of the pigeon Newcastle disease inactivated vaccine on the pigeons is superior to that of the chicken Newcastle disease inactivated vaccine.

Claims (9)

1. A pigeon source gene VI newcastle disease virus mGZ, which is characterized in that the preservation number is CCTCCNO: v202253; the preservation date is 2022, 7 and 5; the collection unit is China center for type culture Collection.
2. The method for preparing the pigeon-derived gene VI type newcastle disease virus mGZ according to claim 1, wherein the pigeon-derived gene VI type newcastle disease virus mGZ is obtained by homologous recombination of a gene segment H-P1, a gene segment P2-Las, a gene segment P3 and a gene segment P4;
the gene sequence of the gene fragment H-P1 is shown in SEQ ID NO:1 is shown in the specification;
the gene sequence of the gene fragment P2-Las is shown in SEQ ID NO:2 is shown in the figure;
the gene sequence of the gene fragment P3 is shown in SEQ ID NO:3 is shown in the figure;
the gene sequence of the gene fragment P4 is shown in SEQ ID NO: 4.
3. The method for preparing pigeon-derived gene VI type newcastle disease virus mGZ according to claim 2, wherein the vector TVT is obtained by connecting gene segment H-P1, gene segment P2-Las, gene segment P3, gene segment P4 and vector TVT which is subjected to linearization treatment, and the target plasmid is transfected into cells to obtain pigeon-derived gene VI type newcastle disease virus mGZ08.
4. The method for preparing pigeon-derived gene VI newcastle disease virus mGZ08 according to claim 2, wherein the method for preparing the gene fragment P2-Las comprises the following steps:
step 1: ligating the P2 fragment to the pCI-neo vector to yield plasmid pCI-P2;
step 2: taking plasmid pCI-P2 as a template, and mutating the plasmid pCI-P2 by using primers P2-Las F and P2-Las R to obtain pCI-P2-Las;
the gene sequence of the P2 fragment is shown as SEQ ID NO:5 is shown in the figure;
the gene sequence of the primer P2-Las F is shown as SEQ ID NO:6 is shown in the figure;
the gene sequence of the primer P2-Las R is shown in SEQ ID NO: shown at 7.
5. A suspension culture method of pigeon-derived gene vi type newcastle disease virus mGZ08 according to claim 1, comprising the steps of:
step A1: the cell density was taken to be 6.0X10 6 Mixing the cell/mL serum-free full suspension culture type BHK-21 cell suspension with a serum-free full suspension culture medium to prepare a culture medium, wherein the volume ratio of the serum-free full suspension culture type BHK-21 cell suspension to the serum-free full suspension culture medium is 1:1, a step of;
step A2: and (3) inoculating the pigeon source gene VI type newcastle disease virus mGZ into the culture medium prepared in the step A1 for culture to obtain the pigeon source gene VI type newcastle disease virus mGZ08.
6. The method for suspension culture of pigeon-derived gene VI type newcastle disease virus mGZ08 as claimed in claim 5, wherein the addition amount of pigeon-derived gene VI type newcastle disease virus mGZ08 in the step A2 is 1-2 per mill of the volume of the culture medium.
7. The method for suspension culture of pigeon-derived gene VI type newcastle disease virus mGZ08 according to claim 5, wherein in the step A2, the culture medium after inoculation of pigeon-derived gene VI type newcastle disease virus mGZ08 is cultured at a temperature of 34.5-35.5 ℃.
8. Use of the pigeon-derived gene vi newcastle disease virus mGZ08 according to claim 1 for the preparation of a pigeon newcastle disease vaccine.
9. A pigeon newcastle disease vaccine comprising an inactivated pigeon-derived gene vi newcastle disease virus mGZ08 according to claim 1.
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