CN113652406A - Recombinant virus strain for avian infectious laryngotracheitis and application thereof - Google Patents

Abstract

The invention provides a recombinant virus strain for avian infectious laryngotracheitis and application thereof, wherein the recombinant virus strain for avian infectious laryngotracheitis is obtained by deleting ORFC gene of virulent strain of avian infectious laryngotracheitis, rescuing by homologous recombination and screening. The specific infectious laryngotracheitis recombinant virus has a preservation number of CCTCC NO: C202158. the infectious laryngotracheitis recombinant virus provided by the invention is used for preparing vaccines. The ILTV gene provided by the invention is deleted with a low virulent strain, the pathogenicity is obviously weaker than that of a parent strain, the side reaction caused by immunization is obviously smaller than that of the existing vaccine strain K317, and the ILTV gene can provide a good protection effect on the currently popular ILTV.

Description

Recombinant virus strain for avian infectious laryngotracheitis and application thereof

Technical Field

The invention belongs to the field of biological products for livestock, and particularly relates to a recombinant virus strain for avian infectious laryngotracheitis and application thereof.

Technical Field

Infectious Laryngotracheitis (ILT) is an acute respiratory Infectious disease caused by Infectious laryngotracheitis virus (ILTV). The disease is characterized by high contact and infectivity, and is characterized by dyspnea, cough with exudation containing blood, swelling of throat and trachea mucosa, hemorrhage and formation of erosion. Serious economic losses are caused to the poultry industry due to death of infected chickens, egg drop and the like. ILT was first reported by May and Tittsler in the United states in 1925, and then reported to occur all over the world, with the disease occurring in China after 1950.

The infectious laryngotracheitis virus ILTV has a narrow host range, and the main infected host is chicken. Although the virus can infect chickens of all ages, it is most susceptible to adult chickens. Contact infection is the main mode of transmission of the disease, but viral fluid can enter the body of the chicken through the oropharyngeal route or the respiratory tract and eyes. The disease can occur all the year round, and is mainly characterized by less disease in summer, more disease in winter and spring, crowding feeding environment and poor feeding management conditions.

ILTV can be propagated on certain primary cells, such as chicken embryo liver Cells (CEL), chicken embryo kidney Cells (CEK), chicken kidney Cells (CK), chicken embryo lung cells, and the like. Studies have shown that chicken liver cancer cells (LMH) are able to adapt to ILTV and to reproduce well. LMH cells are obtained by screening liver cells of chicken suffering from hepatocellular carcinoma through mutation caused by diethylnitrosamine, and have been successfully used for constructing ILTV recombinant viruses. Compared with primary cells, the LMH cells have high growth speed, are easy to culture and passage, can be preserved by liquid nitrogen, and are free from pollution of other cells which are not suitable for ILTV proliferation. However, the proliferation efficiency of ILTV on chick embryos is highest, so that the currently common ILTV culture method is still chick embryos, and the ILTV is inoculated through an allantoic cavity or a chorioallantoic membrane (CAM), so that a plurality of central concave and opaque-edge pocks can be observed 48h after inoculation, and the chick embryos die generally within 72h-120 h.

After natural infection or vaccination, susceptible chickens have changed resistance to ILTV. Neutralizing antibodies were detected in serum 7 days after ILTV infection, with peak antibody levels around 21 days. Although humoral immunity is associated with infection, it is not the primary protective mechanism for infection with ILTV, but is dominated by cell-mediated immunity. The laryngotracheitis maternal antibodies can be transmitted to offspring through eggs, but cannot provide protection effect and cannot interfere with immunization of chickens. Therefore, the use of vaccines is the most effective method for the prevention of the disease.

The inactivated vaccine is simple to prepare, easy to prepare at any time, safe and free from the interference of maternal antibodies. However, the inactivated vaccine mainly induces humoral immunity, has weak effect of inducing cellular immunity and limited immune protection effect, and is hardly used for large-scale prevention and treatment of ILTV at present. Most used are attenuated vaccines of either chicken embryo or tissue origin.

The conventional attenuated live vaccine is generally prepared by attenuation through in vitro passage, but in actual use, the attenuation degree of the attenuated strains is difficult to control, the toxicity is unstable, and sometimes the attenuated vaccine strain can be restored to a virulent strain and become a new infection source. This is also a significant reason why ILTV has not been able to cure radically.

Disclosure of Invention

The invention aims to provide a recombinant virus strain for avian infectious laryngotracheitis and application thereof.

The invention firstly provides a strain of avian infectious laryngotracheitis virus attenuated strain, which is obtained by deleting ORFC gene of virulent strain of avian infectious laryngotracheitis, rescuing by homologous recombination and screening;

the nucleotide sequence of the ORFC gene is shown as SEQ ID NO: 1, the nucleotide sequence after the deletion of the ORFC gene is shown as SEQ ID NO: 2, respectively.

The invention also provides a construction method of the chicken infectious laryngotracheitis gene deletion strain, which comprises the following steps:

1) respectively connecting ICP4 protein gene and UL48 protein gene of a virulent strain of infectious laryngotracheitis to a vector to construct an auxiliary plasmid;

the carrier is particularly described as a PCI-neo carrier in one embodiment;

2) constructing a recombinant transfer vector containing an AcGFP expression cassette: respectively obtaining a left homologous arm ORFC-L and a right homologous arm ORFC-R at two sides of an infectious laryngotracheitis virus ORFC gene through PCR amplification, and obtaining AcGFP expression cassette fragments through PCR amplification; sequentially inserting the 3 fragments into a vector pBluescript II KS (+/-) to obtain a transfer vector L-AcGFP-R-pBluescript, wherein the upstream and downstream of an AcGFP expression cassette in the vector are respectively connected with a left homology arm ORFC-L and a right homology arm ORFC-R;

the nucleotide sequence of the left homology arm ORFC-L is shown as SEQ ID NO: 3, the nucleotide sequence of the right homology arm ORFC-R is shown as SEQ ID NO: 4, the nucleotide sequence of the AcGFP expression cassette is shown as SEQ ID NO: 5 is shown in the specification;

3) obtaining complete genome DNA of a virulent strain of the infectious laryngotracheitis virus;

4) respectively co-transfecting the obtained genome DNA of the virulent strain of the infectious laryngotracheitis virus, the helper plasmid constructed in 1) and the transfer vector constructed in 2) to host cells, and obtaining the infectious laryngotracheitis recombinant virus expressing the AcGFP fluorescent gene by a limiting dilution method.

The host cell is specifically described as an example, and is an LMH cell.

5) LMH cells are transfected with plasmid pBS513EF1alpha-cre expressing cre recombinase, and infectious laryngotracheitis recombinant viruses expressing AcGFP fluorescent genes are inoculated after 24 hours; after 96h of culture, the infectious laryngotracheitis recombinant virus without the fluorescent gene is obtained by screening by a limiting dilution method.

One of the infectious laryngotracheitis recombinant viruses is a chicken infectious laryngotracheitis virus rILTV/L strain which is preserved in China center for type culture Collection at Wuhan university and Wuhan university at 8-month and 4-month in 2021, with the preservation number of CCTCC NO: v202158;

the infectious laryngotracheitis recombinant virus provided by the invention is used for preparing a vaccine;

the invention further provides an ILTV ORFC gene deletion strain attenuated vaccine, and the used antigen is the attenuated strain of the infectious laryngotracheitis recombinant virus.

The ILTV gene provided by the invention is deleted with a low virulent strain, the pathogenicity is obviously weaker than that of a parent strain, the side reaction caused by immunization is obviously smaller than that of the existing vaccine strain K317, and the ILTV gene can provide a good protection effect on the currently popular ILTV.

Drawings

FIG. 1: PCR profiles of ILTV isolates,

FIG. 2: a map of the construction of the transfer vector,

FIG. 3: the construction of the helper plasmid is shown,

FIG. 4: a fluorescent screening profile of the recombinant virus,

FIG. 5: growth profiles of recombinant and parental viruses.

Detailed Description

The invention discloses an ORFC gene-deleted recombinant vaccine strain of chicken infectious laryngotracheitis virus, a construction method and application thereof, wherein gene deletion is carried out on ILTV virulent strains, ORFC genes of the ILTV virulent strains are deleted by using a homologous recombination technology, and attenuated strains with stable heredity are obtained by screening through a limit dilution method.

The ILTV recombinant attenuated strain constructed by the invention overcomes the defect of large side reaction after the traditional ILTV vaccine strain is immunized, effectively reduces the risk of the vaccine strain of strong virulence return, has no influence on immunogenicity, and is equivalent to the traditional vaccine strain.

The present invention will be described in detail with reference to examples.

Example 1 isolation and identification of virulent strains of infectious laryngotracheitis

Tissues such as trachea, larynx and the like of a sick chicken are obtained from a certain Shandong Weifang farm (without ILTV vaccine immunized) in 2019 and 10 months, and PBS solution (Penicilin 10000IU/mL, Streptomyces 10mg/mL, Geamicin 250 μ g/mL, Kanamycin 250 μ g/mL) with pH7.2 is added for grinding. Centrifuging at 8000r/min for 10min, collecting supernatant 0.2mL, inoculating to 10 day-old SPF chick embryo, and inoculating to allantoic membrane. The chick embryos which died within 48h after inoculation were discarded, and the chick embryos which died after 48h were placed in a refrigerator at 4 ℃ for temporary storage. Collecting the chick embryos which are not dead after 120h, placing the chick embryos in a refrigerator at 4 ℃ for 4h, collecting all allantoic membranes and allantoic fluid of the chick embryos which are dead after 48h and not dead after 120h, and observing the pathological changes of the allantoic membranes.

Collecting chorioallantoic membrane with pockmark, grinding, diluting with the collected allantoic fluid, grinding into tissue fluid, centrifuging at 7000rpm/min for 10min, and collecting supernatant. After dilution of virus supernatant, LMH cells were inoculated, virus was purified by plaque screening, and 3 passages were purified on the cells. Inoculating chick embryo allantoic membrane for passage, and taking the 5 th generation virus of the allantoic membrane for further analysis and detection.

According to an ILTV whole genome sequence (MF417808.1) published by GenBank, primers are designed, a TK gene, a gB1 gene and a gB2 gene are amplified (the gB gene is divided into 2 segments for amplification), and the sequence information of the primers is as follows:

TK-F：5′-AGCCACGCTCTCTCGAGTAAGAATGAGTAC-3′、

TK-R：5′-TCCTCCAGTGATCTGACGAGAGCTCGGT-3′、

gB-1F：5′-GATGCTTCTATGCCAAAACGACTGAAAGAT-3′、

gB-1R：5′-GAAGAGGCACAAAGACAAAATCATCTCCCG-3′、

gB-2F：5′-CAGTTGGCGGTTCTGAAGCTCACACCACGC-3′、

gB-2R：5′-GCGTCGATGACCGCGGGAAACTGGGTCCAG-3′。

taking 200 mul of virus liquid, extracting DNA according to the instruction of a DNA extraction kit of OMEGA company, taking the extracted virus DNA as an amplification template, and carrying out a PCR reaction system: 2 XGflex PCR Buffer, 25. mu.l; TK-F, 1. mu.l; TK-R, 1. mu.l; template, 2. mu.l; tks Gflex DNA Polymerase, 1. mu.l; sterile water, up to 50. mu.l.

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 1 min; 10sec at 98 ℃; 15sec at 55 ℃; 1min at 68 ℃ and 30 cycles of PCR; 10min at 68 ℃. After the PCR product is electrophoresed, a positive strip is recovered and sent to biological engineering Co. And comparing and analyzing the sequencing result at an NCBI website.

As a result, it was found that the TK gene, and the gB1 and gB2 gene bands (FIG. 1) were successfully amplified, and the band sizes were 1.2kb, 1.5kb and 1.5kb, respectively. And after the sequencing result is compared at an NCBI website, the separated strain is confirmed to be ILTV virus and named as ILTV/Q.

Example 2 animal regression test of ILTV/Q Strain

30 SPF chickens of 35 days old were randomly divided into 3 groups of 10, wherein 2 groups were virus inoculation groups, 1 group was PBS inoculation virus, 0.1ml of the virus was dripped into nose and spotted eye, and the inoculation dose and grouping condition are shown in Table 1. The 3 groups of animals were kept in isolation under the same conditions, continuously observed for 14 days after inoculation, and the disease states of the chickens including death, lacrimation, cough, watery nasal discharge, flail head and other symptoms were observed and recorded every day.

Table 1: ILTV/Q strain animal regression test table

The results indicated that the ILTV/Q strain was a virulent strain.

Example 3 construction of ORFC Gene-deleted ILTV recombinant Strain

Constructing a transfer vector by using a homologous recombination technology, co-transfecting the transfer vector, ICP4 and UL48 plasmids of transcriptional activator proteins and an ILTV genome with LMH cells, and screening by using a limiting dilution method to obtain the recombinant virus containing the fluorescence marker with ORFC gene deletion. Because Loxp sites are introduced when a transfer vector is constructed, sequences among the Loxp sites can be eliminated by utilizing cre recombinase, the purpose of deleting fluorescent genes is achieved, and the ORFC gene deletion recombinant virus without fluorescent markers is obtained.

The specific steps are described below.

(1) Construction of transfer vectors

According to ILTV whole genome sequence (MF417808.1) published by GenBank, primers are designed to amplify ORFC gene and genes on both wings, and the primer sequences are as follows:

ORFC-F1：5′-ATACAGGTGATGGAGGGAGTAACCGACAC-3′、

ORFC-R1：5′-ATCGGTTCAAGCGTTCGAACCACCTCG-3′、

ORFC-F2：5′-AGCTGATAAGTCAGCGCGCGGTTATTCTG-3′、

ORFC-R2：5′-TGTCTGCAGACAAGTGGAAAGATGGAGTCG-3′。

according to the sequencing result, the accurate sequences of the ORFC gene and the genes on both sides of the ORFC gene are obtained, and homologous arm primers are designed. AcGFP expression cassette primers were designed based on the gene sequence of plasmid pAcGFP 1-N1. The primer sequence information is as follows:

L-F：5′-accgcggtggcggccgctctagaactagtggatccGCCGTTGTGGTAACGCGTGGGGCAATTACAGAC-3′、

L-R：5′-TATCTGCAGTAAAGACCAATAAATAAATTATTGACAGTA-3′、

M-F：

M-R：

R-F：5′-TATGAATTCGCTGAGGTTCCGATCGAGAGAAG-3′、

R-R：5′-tgggtaccgggccccccctcgaggtcgacggtatcgataagcttGAAGAAACTCCCATGTGCGGCAAGTTTCTGTGCGCAGC-3′。

primer design follows

The requirement of the specification of the high-fidelity DNA assembly premix solution is that homologous arm sequences of about 30bp exist among all fragments. Wherein the lower case sequence is homologous to the flanking sequence in the vector pBluescript II KS (+/-), and the bold letters are the Loxp gene sequence.

The vector pBluescript II KS (+/-) was double digested with BamH I and Hind III, electrophoresed together with the 3 fragments amplified by PCR, and the positive band was recovered by gel cutting (FIG. 2). The 3 fragments recovered by PCR amplification are connected with the vector after enzyme digestion to construct a transfer vector, and the connection system is as follows: 2.5ul of each of the 3 recovered fragments; nebuilder HiFi DNA Assembly Master Mix 10 ul. Directly transforming escherichia coli competent cells after 1 hour of action at 50 ℃, completing the construction of a transfer vector after sequencing and identifying positive colonies, and naming the transfer vector as L-AcGFP-R-pBluescript.

(2) Construction of helper plasmids

Designing primers, amplifying an ICP4 gene and an UL48 gene, respectively recovering amplified products after electrophoresis (figure 3), respectively connecting with a pCI-neo vector digested by Nhe I/Xba I, and connecting a system: 6.6ul of PCR recovery product and 3.4ul of enzyme digestion product; nebuilder HiFi DNA Assembly Master Mix 10 ul. The reaction conditions were as above. The constructed helper plasmids were designated pCI-ICP4 and pCI-UL48, respectively. The primer sequences are as follows:

ICP4-F：

ICP4-R：5′-ctaaagggaagcggccgcccgggtcgactctagaTTACCACCAAGGGTCAGTGACAGTG-3′、

UL48-F：

UL48-R：5′-tcactaaagggaagcggccgcccgggtcgactctagaTTAGGGCATAGGTGTATCAAGG-3′。

wherein the lower case letter sequence is homologous to the flanking sequence in the vector pCI-neo, and the bold letters represent the Cozak sequence.

(3) Extraction of ILTV/Q genomic DNA

Inoculating ILTV/Q to a monolayer LMH cell with the length of 80%, discarding a culture medium when the cytopathic effect reaches more than 80%, adding 10mL of lysis solution (100mmol/L NaCl, 10mmol/L Tris-HCl, pH8.01mmol/L EDTA, 0.5% SDS and 200mg/L proteinase K) into each T75 cell bottle, acting at room temperature for 5-10 min, sucking the lysis solution into a 50mL centrifuge tube, and carrying out water bath at 37 ℃ for 2 h; equal volumes of phenol were added separately: chloroform and chloroform: extracting isoamyl alcohol once respectively, and centrifuging for 10min at 4000r/min each time; sucking supernatant, adding 2 times volume of precooled absolute ethyl alcohol, acting at-20 ℃ for 10min to precipitate genome DNA; washing the precipitate with 70% ethanol, drying, dissolving in 500 μ l TE solution, collecting a small amount of DNA, measuring nucleic acid concentration with spectrophotometer, packaging, and storing at 4 deg.C.

(4) Transfection and screening of recombinant viruses containing fluorescence marker genes

LMH cells were seeded in 6-well cell culture plates and transfected when 60% -80% of the cells had grown. 1.5ug of genomic DNA, 1. mu.g of transfer vector L-AcGFP-R-pBluescript, 0.5ug of helper plasmids pCI-ICP4 and pCI-UL48, respectively, were mixed and co-transfected, and the procedures were performed according to the Lipofectamine 3000 Lipofectase kit. The transfected cells were placed at 37 ℃ in 5% CO₂And (5) continuing culturing in the constant-temperature incubator. Observing fluorescence and CPE generation conditions under a fluorescence microscope after 72-96 h (figure 4), harvesting virus when CPE reaches about 80%, freezing and thawing for 3 times, and storing in a refrigerator at-70 ℃ for later use.

The harvested recombinant virus liquid is processed into 10 by serum-free DMEM^-1-10^-6Diluted by times, and inoculated to LMH cells grown to around 90%. 37 ℃ and 5% CO₂Culturing in a constant temperature incubator for 96h, observing fluorescence by a fluorescence microscope, selecting the largest dilution hole containing the fluorescence labeling virus, and continuously screening by the same method until the hole which can lead all CPE produced by the cells to have fluorescence is screened. And (3) taking 100 mu l of virus liquid, extracting virus DNA, detecting the ORFC gene of the recombinant virus by a fluorescence quantitative method, and detecting whether a purified recombinant strain is obtained. Until no ORFC gene was detected in the virus-containing wells examined. The screened recombinant virus was named rILTV-GFP. The sequence information of the fluorescent quantitative detection primers is as follows:

ORFC-DF1：5′-GCTGTCGTATAGAGTTTC-3′、

ORFC-DR1：5′-CCATCATTACCTTCAGTTAA-3′、

ORFC-DP1：5′-HEX-TCGTTCCTTGTCTACCATACTGTCG-BHQ1-3′。

(5) elimination of fluorescent marker genes

LMH cells were seeded in 6-well cell culture plates and transfected when 60% -80% of the cells had grown. 2.5. mu.g of plasmid pBS513EF1alpha-Cre expressing Cre recombinase was transfected according to the Lipofectamine 3000 Lipofectin transfection kit instructions. 24h after transfection, the transfected wells were inoculated with rILTV-GFP recombinant virus at a rate of 2%. Observing fluorescence and CPE generation conditions under a fluorescence microscope 72h after virus inoculation, harvesting the virus when CPE reaches about 80%, freezing and thawing for 3 times, and storing in a refrigerator at-70 ℃ for later use.

And (4) screening the recombinant virus without the fluorescent gene by using a limit dilution method, wherein the method is the same as the step (4). The recombinant virus obtained by screening was named rILTV/L.

Inoculating the recombinant virus rILTV/L allantoic membrane into SPF chick embryos of 10 days old, continuously passaging for 20 generations, and carrying out PCR detection and sequencing identification on the collected allantoic membrane virus samples every 5 generations, wherein the positions of gene deletion do not have any insertion, gene mutation and the like. The obtained recombinant strain can be stably inherited.

And (3) preserving the screened rILTV/L strains, wherein the preservation number is CCTCC NO: v202158.

EXAMPLE 4 growth curves of recombinant rILTV/L Strain on LMH cells

LMH cells were passaged and plated in 24-well cell culture plates and cells were counted by a cell counter. Respectively inoculating the recombinant virus rILTV/L strain and the parent strain ILTV/Q strain to a well-grown LMH cell monolayer at 0.01MOI, incubating for 2h at 37 ℃, discarding virus liquid, replacing with DMEM cell maintenance liquid containing 2% fetal calf serum, and culturing in a constant-temperature incubator at 37 ℃ with 5% CO 2. The virus was harvested at 0h (virus dilution before cell inoculation), 12h, 24h, 36h, 48h, 72h and 96h after inoculation, 3 replicates were performed at each time point, three replicates of repeated freeze-thaw at-70 ℃ and stored at-70 ℃ for future use. All samples were assayed for viral Titer (TCID) at the same time₅₀) The growth of 2 strains of virus was compared. The results show (fig. 5) that the virus titer of 2 strains of virus decreased at 12h after inoculation and then increased until 96h reached the maximum, and the growth trend of 2 strains of virus was similar, indicating that the recombinant ILTV virus had no effect on the virus proliferation ability after gene deletion.

Example 5 verification of recombinant rILTV/L Strain in the virulence Return test of SPF chickens

(1) Serial passage of ILTV on SPF chickens

20 SPF chickens 35 days old were randomly divided into 2 groups of 10, one group was a recombinant virus rILTV/L strain inoculated group, and one group was a commercial vaccine K317 strain inoculated group. When the primary generation is inoculated, the virus dose of each group of test chickens is 10^3.8EID₅₀Nasal drip, eye drop inoculation. On the 4 th day after inoculation, each group of test chickens was killed by dissection and tracheal tissue was harvested. Wherein 10 chicken tracheal tissues of the rILTV/L strain inoculated group were ground together and resuspended in 10ml PBS; 10 tracheal tissues of the K317 inoculated group were ground together and resuspended in 10ml PBS. Freezing and thawing for 3 times, centrifuging, collecting supernatant as virus source for the 2 nd generation of subcultured SPF chicken, and inoculating 0.2ml of the virus by dripping into nose and dripping into eyes. SPF chickens were passaged for 5 successive generations according to the method described above.

2 groups of virus solutions were continuously passaged to the 5 th generation on SPF chickens, and 10 SPF chick embryos were inoculated to each allantoic membrane. Collecting chick embryo allantoic membrane tissue and allantoic fluid after 120h, grinding with a tissue grinder, and filtering with a filter screen. For the collected rILTV/L strain virus liquid and K317 strain virusLiquid, separately determining the potency (EID)₅₀) The kit is used for detecting pathogenicity.

(2) Verification of virulence reinforcment test

Randomly dividing 20 SPF chickens 35 days old into 2 groups, inoculating rILTV/L strain virus solution and K317 strain virus solution with measured titer, dripping into nose, and dripping into eye with an inoculation dose of 10^3.8EID₅₀A/only. Keeping the chickens in isolation under the same condition, continuously observing for 14 days after inoculation, and observing and recording the morbidity states of the chickens, including symptoms of death, lacrimation, cough, running nose, head swinging and the like. The results are shown in the following table.

Table 2: ILTV toxicity back-strengthening test data table

Note: adverse reactions indicated that animals had mild ophthalmia or mild cough 3-5 days after inoculation, but returned to normal after 2-3 days; the number of healthy animals is the number of animals remaining after the removal of the number of deaths or the number of morbidity and side effects.

The result shows that the recombinant virus rILTV/L strain has no return of virulence after SPF chicken continuous passage for 5 generations. The K317 vaccine strain has the phenomenon of virulence reversion, and has 5 diseases, which are manifested by serious symptoms of ophthalmia, lacrimation, cough, dyspnea and the like. The recombinant virus rILTV/L strain is shown to have better stability and small probability of strong virulence reversion.

EXAMPLE 6 preparation of recombinant ILTV live vaccine

Inoculating recombinant virus rILTV/L strain to chick embryo, collecting chick embryo allantoic membrane tissue and allantoic fluid after 120h, grinding with tissue grinder, and filtering with filter screen for preparing vaccine. Adding 5% sucrose skim milk 1:1 into diluted virus solution, and quantitatively diluting to obtain virus solution with virus content of 10^3.8EID₅₀0.1mL, fully mixing, and freeze-drying to prepare the live vaccine. The prepared live vaccine is subjected to aseptic examination according to the appendix of Chinese animal pharmacopoeia 2010 edition, and the result meets the standard and has no bacterial pollution.

Example 7 safety testing of recombinant ILTV live vaccine

SPF chickens 7 days old, 21 days old and 53 days old were used for safety tests, and the K317 vaccine strain and/or the isolated parent strain ILTV/Q strain were selected as controls. Keeping the chickens under the same condition, wherein the inoculation mode is eye drop or nose drop, continuously observing for 14 days after inoculation, and observing and recording the morbidity states of the chickens, including symptoms of death, lacrimation, cough, watery nasal discharge, head swinging and the like. SPF chicken groups and results are shown in Table 3 below.

Table 3: safety test table for ILTV live vaccine

Note: "-" represents no relevant tests were performed; adverse reactions indicated that animals had mild ophthalmia or mild cough 3-5 days after inoculation, but returned to normal after 2-3 days; the number of healthy animals is the number of animals remaining after the removal of the number of deaths or the number of morbidity and side effects.

The results show that after rILTV/L is inoculated to animals, the rILTV/L only causes side reaction to individual chickens of 53 days old, and the safety is obviously superior to that of parent strains and the current vaccine strain K317. After the ORFC gene is deleted, the toxicity of ILTV is obviously reduced, and the constructed rILTV/L strain is an attenuated strain.

Example 8 evaluation of immune Effect of recombinant ILTV live vaccine

30 SPF chickens 35 days old were randomly divided into 3 groups of 10 chickens. The immune components are rILTV/L strain immune group and K317 immune group, and are dripped into nose or eyes, 10^2.8EID₅₀0.1ml each; the control group of 10 chickens was immunized with the same dose of PBS. Isolation breeding, 21 days after immunization, and injecting 0.2ml (containing 10) of ILTV virulent strain (Wanggangzhu strain) into trachea of each chicken^4.0EID₅₀) And observed for 14 days. The results are shown in Table 4 below.

Table 4: challenge protective test table for ILTV live vaccine

The result shows that the rILTV/L strain can provide 100% protection to virulent attack after immunizing animals, and the protection effect is the same as that of the K317 vaccine strain.

In conclusion, the ILTV ORFC gene deletion strain constructed by the invention has obviously better side reaction on chicken than the prior vaccine strain K317 strain after immunizing animals, has equivalent immune protection effect to the vaccine strain and can realize 100 percent of protection.

Sequence listing

<110> Qingdao Yibang bioengineering Co., Ltd

<120> an infectious laryngotracheitis recombinant virus strain and application thereof

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tggactcggt gcagagactc tctactattt tcagattact tgtttcgccg cggcaccatg 720

gatagtgccc ggcagcagtt gcaggttgga gtgcctaaga agggcggggt gtacagacag 780

ctatcacgtg atgatggaac ggtgttcgag gtcagtttgg atcctgcagt gtgcttcgag 840

ttctcagtga cgttaatttt accgggacat gatcttttct ggccggtagt gccgccgctg 900

caatttctcg agctgataag tcagcgcgcg gttattctgg cagaccagtt tatttcgtcc 960

tctatcatga agcgagtgag cttatctcca atcatgctat ttccgaagaa tacattcatg 1020

cccgggtact ggtccccgga tccccagcct ggtcggtacc gaccaaaatt tcagcctact 1080

cgatcagaac aacatttcat gaccgtcggc aagctggtgc caccgtttca aattgacttg 1140

catgggaaaa agaataattt catggctgga attgccgtgg gttttcacgg cccgccgacc 1200

ttgacgggca ctattcgcgc cctgactgaa caagcaattc acaacgctgt ggccgaggtg 1260

gttcgaacgc ttgaaccgat gactgtggtt ccgatcactc tgaaaaatgg aactggagct 1320

ctgatgggat tgacttcaga caacaagggt cttcggatat tgataaaacc ggcactagga 1380

gaattggcca ggtctccacg aagacggcgt tccgaatcgc gcggaagaga gtctgtttta 1440

ttataaattg aataaacgca gactttttat tactgtcaat aatttattta ttggtcttta 1500

<210> 4

<211> 1567

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gctgaggttc cgatcgagag aagcgcttgc ggcgtccaaa cggcatgaat ttgctcagcc 60

agcccatggt gtctcgccta gagagttctg agtttcagag caggctctcg cctagagagt 120

tctgagattc agagcagacc ctcgaacgcg ttcgagagct tttaggtgct tgttgcatgg 180

ggcgtgtact gggcgtggat ataagacatg tgcctcgacg cctgccacgc acgatttccg 240

cagacatgag ttccatggcc tgctctcgtt ttcagaaacc aaaagcgaag aatgatagtt 300

cgcccgaaaa atgtgaacgc actgccaagc aaattgtcgc agcaagtgat tattgcaatg 360

attcggaaaa attagaggaa gagatggtct tcgtcggagc tggaaagatt tcatattctt 420

cgtggatgta cgaggcaatt ttttcctgtg attgtattcc cgacggatca gcgtttgcag 480

ttgctaagca tgcagtgatc ctaaatcgcg taaacggagg cttgccatct tgtcttggga 540

gtgcacatgg gttgaccatt taccctgcca ggacaagcca gcgtggaagg ttcagccgtt 600

tagcatacag aaatgatatg tccccagctc cacaatcaga aaatactgtc gatgtggatg 660

gagagcttgg agcgtgcgcc atcggcgtgt tggagatggg cgaagaagtg tgggcacatg 720

aagaaatgtt ttttttaccg gtggttccta agttagatcg catgtggccg tgtgttccca 780

atcctgctct gttttggggc ctggcgcgcg cgcttaacaa tcttcccccc aagatactcg 840

tttcaggaaa acttgccaaa gggttcattt gccctctata ctatcttagc gtaccaactt 900

ttggagaaga gagcaaagaa actttccttc gcacgagaca gaagcagcga agtctatatg 960

accctttgtt tgaccctgct tcattacttg atcagccaca attttttgga tgtgatggta 1020

ttaaggattc taataacttt caagctgggc ttggcatagt tttccagcct caggatggat 1080

ggacggcaaa aacaaagctg ctcgagttgg acagagttcg gtcggaactt gtcacactat 1140

ttgcacgcca ggagcagtca aggcctatta ccgtgataat tactaggggg gcagtcagac 1200

gccggcttgt gttttctact gttggagggt ttaggttttc atcctcgttc gaaccctctg 1260

ggacacccct taaaacccac tcggtgactt tacatattta atggaacaca catacgaaat 1320

aaaagtttgt cagaaatgta tttgcgttta ttgatgttta tgtttcgggt aattatccaa 1380

acagatcagg cagcgtcgca attcccatgt tcttccatgc ttcggacact gctgtgcaag 1440

cagccgtctt aaaatcgcct atctcatcgg gggtccaatg agagtgcaat aaaatgccga 1500

gtctaggtgg atcgtacgcg tggcaaattg ctgcgcacag aaacttgccg cacatgggag 1560

tttcttc 1567

<210> 5

<211> 1582

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540

acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600

ccggactcag atccaccggt catggtgagc aagggcgccg agctgttcac cggcatcgtg 660

cccatcctga tcgagctgaa tggcgatgtg aatggccaca agttcagcgt gagcggcgag 720

ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 780

ctgcctgtgc cctggcccac cctggtgacc accctgagct acggcgtgca gtgcttctca 840

cgctaccccg atcacatgaa gcagcacgac ttcttcaaga gcgccatgcc tgagggctac 900

atccaggagc gcaccatctt cttcgaggat gacggcaact acaagtcgcg cgccgaggtg 960

aagttcgagg gcgataccct ggtgaatcgc atcgagctga ccggcaccga tttcaaggag 1020

gatggcaaca tcctgggcaa taagatggag tacaactaca acgcccacaa tgtgtacatc 1080

atgaccgaca aggccaagaa tggcatcaag gtgaacttca agatccgcca caacatcgag 1140

gatggcagcg tgcagctggc cgaccactac cagcagaata cccccatcgg cgatggccct 1200

gtgctgctgc ccgataacca ctacctgtcc acccagagcg ccctgtccaa ggaccccaac 1260

gagaagcgcg atcacatgat ctacttcggc ttcgtgaccg ccgccgccat cacccacggc 1320

atggatgagc tgtacaagtg agcggccgcg actctagatc ataatcagcc ataccacatt 1380

tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa 1440

aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag 1500

caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 1560

gtccaaactc atcaatgtat ct 1582

Claims (10)

1. The attenuated strain of the avian infectious laryngotracheitis virus is characterized in that the attenuated strain is prepared by deleting ORFC genes of virulent strains of the avian infectious laryngotracheitis virus, and screening after homologous recombination rescue.

2. The attenuated strain of claim 1 wherein the nucleotide sequence of the ORFC gene is SEQ ID NO: 1.

3. the attenuated strain of claim 1 wherein the ORFC gene is deleted and the nucleotide sequence after deletion is SEQ ID NO: 2.

4. the method for constructing the attenuated strain of claim 1, wherein the method comprises the following steps:

1) respectively connecting ICP4 protein gene and UL48 protein gene of a virulent strain of infectious laryngotracheitis to a vector to construct an auxiliary plasmid;

2) constructing a recombinant transfer vector containing an AcGFP expression cassette: respectively obtaining a left homologous arm ORFC-L and a right homologous arm ORFC-R at two sides of an infectious laryngotracheitis virus ORFC gene through PCR amplification, and obtaining AcGFP expression cassette fragments through PCR amplification; inserting the 3 fragments into a vector in sequence to obtain a transfer vector, wherein the upstream and the downstream of an AcGFP expression cassette in the vector are respectively connected with a left homology arm ORFC-L and a right homology arm ORFC-R;

3) obtaining complete genome DNA of a virulent strain of the infectious laryngotracheitis virus;

4) co-transfecting the obtained genome DNA of the virulent strain of the infectious laryngotracheitis virus with the helper plasmid constructed in 1) and the transfer vector constructed in 2) to obtain an infectious laryngotracheitis recombinant virus expressing an AcGFP fluorescent gene;

5) transfecting LMH cells with a plasmid expressing cre recombinase, and then inoculating infectious laryngotracheitis recombinant viruses expressing AcGFP fluorescent genes; and screening to obtain the infectious laryngotracheitis recombinant virus without the fluorescent gene after culture.

5. The method of claim 4, wherein said vector of step 1) is a PCI-neo vector.

6. The method of claim 4, wherein the nucleotide sequence of the left homology arm ORFC-L in step 4) is SEQ ID NO: 3, the nucleotide sequence of the right homology arm ORFC-R is SEQ ID NO: 4, the nucleotide sequence of the AcGFP expression cassette is SEQ ID NO: 5.

7. the method of claim 4, wherein said host cell of step 4) is an LMH cell.

8. The attenuated strain of claim 1, wherein the attenuated strain has a collection number of CCTCC NO: v202158.

9. Use of the attenuated strain of claim 1 or claim 8 in the preparation of a vaccine.

10. An attenuated vaccine, wherein the antigen used in said attenuated vaccine is the attenuated strain of claim 1 or claim 8.

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