CN117568288A

CN117568288A - Method for efficiently rescuing avian coronavirus and application thereof

Info

Publication number: CN117568288A
Application number: CN202311330504.5A
Authority: CN
Inventors: 刘胜旺; 孙军峰; 韩宗玺
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2023-10-16
Filing date: 2023-10-16
Publication date: 2024-02-20
Anticipated expiration: 2043-10-16
Also published as: CN117568288B

Abstract

The invention relates to a method for efficiently rescuing avian coronavirus and application thereof, belonging to the technical field of coronavirus reverse genetics. In order to establish a reverse genetics technical method capable of rapidly and efficiently rescuing avian coronavirus, the invention optimizes and constructs a chimeric transcription element group, adopts CPER technology to connect the chimeric transcription element group sequence and the avian coronavirus genome sequence from head to tail in the 5 'to 3' direction, rapidly constructs a circular clone containing the chimeric transcription element group and the full-length cDNA of the avian coronavirus genome, and achieves transcription of the viral genome and completion of viral packaging in cells by co-transfecting cells with an eukaryotic expression vector of avian coronavirus nuclear protein, thereby rapidly rescuing and obtaining avian coronavirus. The method is simple and efficient to operate, can construct reverse genetic recombinant viruses which take avian coronaviruses as vectors to chimeric express exogenous genes, and provides efficient technical tools for researching pathogenic and immune mechanisms of the viruses, developing novel chimeric vaccines and the like.

Description

Method for efficiently rescuing avian coronavirus and application thereof

Technical Field

The invention belongs to the technical field of coronavirus reverse genetics, and particularly relates to a method for efficiently rescuing avian coronavirus and application thereof.

Background

Coronaviruses are a single-stranded positive-strand RNA virus with a capsule membrane belonging to the order of the family Coronaviridae, and seriously threaten animal health and public health safety. Coronaviruses are classified into the genus alpha, beta, delta and gamma coronaviruses, and include a variety of viruses capable of infecting humans, pigs, chickens, cattle, horses, dogs, cats, turkeys, ducks, and a wide variety of wild animals. The gamma coronavirus is represented by avian coronavirus, including infectious bronchitis virus, pheasant coronavirus, turkey coronavirus, etc., and can cause respiratory system diseases of chicken and pheasant and digestive system diseases of turkey.

A typical representative of avian coronaviruses is avian infectious bronchitis virus (Infectiousbronchitis viru, IBV), which is also the first coronavirus discovered and causes avian infectious bronchitis disease (IB) which is widely prevalent in chicken raising areas worldwide, mainly damaging the respiratory tract, kidneys and reproductive system, and can infect chickens of all ages and different types, causing serious economic losses. The pheasant coronavirus (PhCoV) in avian coronaviruses is another avian coronavirus closely related to IBV. Mainly causes respiratory diseases of pheasants, reduces egg yield, reduces eggshell quality and has kidney lesions. In recent years, with the increase of the breeding quantity of pheasants in China, the infectious disease of chickens caused by PhCoV has great loss to the breeding industry.

The control of avian coronavirus disease IB is primarily dependent on biosafety and vaccine immunization, which is the most effective measure for preventing epidemic disease in most countries. Although vaccines are widely used for IB control, IB is still frequent in farms and has become a serious threat to the poultry farming industry worldwide. The biggest problem faced by the disease prevention and control is that the virus lacks a correction function in the replication and proliferation process of the virus in host cells, so that the virus genome is easy to generate mutation such as point mutation, deletion, insertion, recombination and the like, further, the virus is continuously evolved in the genetic evolution process, new variant strains or serotypes are continuously generated, and protective immunity induced by strains of specific serotypes cannot provide effective protection for other serotypes, so that IB is one of the most serious viral diseases in the poultry farming industry worldwide.

The functions of various proteins of the coronavirus in the life cycle of virus infection, the influence on pathogenicity, antigenicity and the like of the virus and the function in screening antiviral drugs are clarified, and a solid foundation can be laid for deep understanding of the infectious pathogenicity mechanism of the coronavirus, excitation of the immune mechanism, screening of the antiviral drugs and development of novel vaccines. Reverse genetics techniques, which allow modification and engineering of viral genes at the molecular level to explore the biological functions of the proteins and their role in viral pathogenicity, have proven to be effective methods of studying the biological properties of RNA viruses, including coronaviruses. The main technical principle of coronavirus reverse genetics technology is as follows: firstly, constructing a full-length cDNA clone of a viral RNA genome; on this basis, infectious RNA transcripts are prepared; the final transfection of cells to obtain infectious virus is often referred to as "rescuing" the virus. The construction of full-length cDNA clones of viral RNA genomes is a key core problem in coronavirus reverse genetics technology, and is also a key link for rescuing whether viruses succeed or not and success efficiency. The coronavirus genome is single-stranded positive-strand RNA, and has a length of about 27-32 kb, and is the largest genome among currently known RNA viruses. Conventional molecular cloning techniques have difficulty in assembling such large full-length cDNA fragments, and partial replicase genes of the coronavirus genome are unstable in bacteria, making conventional E.coli-based cloning methods unsuitable for construction of coronavirus full-length cDNA, which also results in difficulty in establishment of coronavirus reverse genetic technology systems.

Reported systems that were successful for full-length cDNA cloning of animal coronaviruses are: (1) a traditional in vitro enzyme digestion ligation cloning method; (2) Bacterial Artificial Chromosome (BAC) system cloning; (3) Yeast Artificial Chromosome (YAC) system cloning; (4) cloning of poxvirus vector System. Wherein the traditional in vitro enzyme digestion ligation cloning method, bacterial artificial chromosome system cloning method and poxvirus vector system cloning method have been applied to the rescue of avian coronaviruses. The traditional in vitro enzyme cutting and connecting cloning method has the strategy that each segment of IBV genome is obtained through RT-PCR amplification, is connected into full-length cDNA one by one through DNA ligase in vitro, uses the full-length cDNA as a template to synthesize viral RNA transcripts through T7RNA polymerase, and introduces the transcripts into cells through electroporation, so that the recombinant viruses are finally obtained through rescue. The method avoids cloning viral genome in bacteria, but has more restriction of enzyme cutting site selection in the construction process, low efficiency of in vitro large fragment connection and heterogeneity of transcripts obtained by in vitro transcription; and the process from cloning of full-length cDNA to transfection of cells is cumbersome, time-consuming, and inefficient in virus rescue. The bacterial artificial chromosome system cloning method adopts enzyme cutting connection or homologous recombination method to clone the genome full-length cDNA of IBV into BAC vector. Compared with the conventional cloning vector, the BAC vector has the characteristics of large capacity and low copy number, and avoids instability caused by coronavirus genotoxic sequences with high copy number. And the BAC plasmid containing a Cytomegalovirus (CMV) promoter can be used for completing RNA transcription by using RNA polymerase II of the cell after the cell is transfected, thereby realizing the packaging of the virus. However, the method needs to construct a plurality of intermediate plasmids, and the sequence of the intermediate plasmids constructed in each step is ensured to be accurate, so that the complete full-length cDNA clone can be finally assembled, the workload is high, and the gene sequencing cost is high. In addition, the need to reconstruct full-length cNDA and the susceptibility to toxic or unstable sequences results in construction failures when modifying the gene. Another system that has application in IBV rescue is based on poxvirus vectors. The method is to introduce a T7 promoter sequence at the 5 'end of an IBV genome, a hepatitis delta virus ribozyme (Hdvrz) sequence with self-shearing activity and a T7 terminator sequence at the 3' end, clone and recombine the full-length cDNA into the genome of the poxvirus in a segmented way, and finally obtain the recombined poxvirus containing the full-length IBV cDNA through drug screening. DNA of the recombinant poxvirus is extracted and transfected into chicken kidney cells inoculated with the chicken poxvirus expressing T7 polymerase, infectious IBV RNA is generated in the cells, and the IBV is finally obtained by rescue. Another method for rescuing IBV based on poxvirus vector is to introduce cleavage sites and T7 promoter sequence, hdvrz sequence and T7 terminator sequence on both sides of viral genome, construct recombinant poxvirus containing IBV full-length cDNA, enzyme cleave IBV full-length cDNA from poxvirus genome by restriction enzyme cleavage, in vitro transcribe RNA and electrically transform it into cells, and complete the packaging of the virus therein. The IBV full-length cDNA constructed by the poxvirus vector system can stably exist in poxvirus genome, and the IBV genome can be conveniently subjected to mutation, insertion and deletion operation by a homologous recombination method. However, the process of constructing the full-length IBV cDNA based on the poxvirus vector cloning method is very complicated, the acquisition of infectious viruses depends on the recombinant viruses expressing T7 polymerase or an in vitro transcription system to acquire infectious RNA, a series of auxiliary tools and extremely high experimental operation technology are needed, and the practical operability is not strong.

At present, the construction of full-length cDNA clones, the acquisition of infectious RNA and the improvement of virus rescue efficiency which are convenient to operate are important problems which are urgently needed to be solved in the technical research field of IBV reverse genetics. In addition, almost all IBV isolates cannot proliferate in vitro cultured passaged cell lines relative to other coronaviruses. Only one Beaudette attenuated strain obtained after high passage adaptation in Vero cells can replicate in Vero cells, but the strain loses the ability to replicate in chickens and is pathogenic for chickens, and is not suitable for researching the pathogenic mechanism of IBV. The development of IBV reverse genetics technology and the application of IBV pathogenic mechanism, immune mechanism, drug screening, novel chimeric vaccine and the like are greatly limited by the existence of the problems.

Disclosure of Invention

In order to establish a reverse genetics technical method capable of rapidly and efficiently rescuing the avian coronavirus, the invention overcomes the defects of complicated construction process and low virus rescuing efficiency of the prior method.

In order to solve the technical problems and achieve the corresponding technical effects, the invention provides the following technical scheme:

the first object of the invention is to provide a method for efficiently rescuing avian coronavirus, which comprises the following steps:

s1, cloning a chimeric transcription regulatory sequence TRS with a nucleotide sequence shown in any one of SEQ ID NO. 1-SEQ ID NO.9 into a pUC57 vector for sequencing and identification to obtain a plasmid pUC-TRS;

s2, respectively designing primers at two ends of the TRS sequence by taking the TRS sequence in the S1 as a template, wherein the upstream primer comprises a sequence of about 30nt at the 5 'end of the TRS sequence, and the downstream primer comprises a sequence of about 30nt at the 3' end of the TRS sequence;

s3, randomly dividing the genome sequence of the avian coronavirus into N fragments with approximate sizes, respectively designing primers at two ends of each fragment, wherein the 5 '-end primer of the fragment 1 comprises 50-60 nt homologous to the 3' -end of the TRS sequence in S1, the 3 '-end primer of the fragment N comprises 50-60 nt homologous to the 5' -end of the TRS sequence in S1, in addition, the sequence of the head and tail parts of each fragment is repeated with the adjacent fragments by about 40-60 nt, and the annealing temperature (tm) value of the primer pair between the fragments is similar;

s4, taking the plasmid pUC-TRS in the S1 as a template, utilizing an upstream primer and a downstream primer of a TRS sequence designed and synthesized in the S2, and amplifying by using high-fidelity DNA polymerase to obtain a TRS fragment;

S5, extracting avian coronavirus RNA, transcribing the RNA into cDNA, and respectively amplifying the cDNA by using a high-fidelity DNA polymerase by adopting a primer designed by S3 to obtain N genome fragments, which are sequentially named as AVCov1 and AVCov 2-AVCovN;

s6, designing a primer according to the avian coronavirus nucleoprotein gene sequence, using the cDNA of S5 as a template, amplifying the coding sequence of the nucleoprotein gene, cloning the coding sequence into a eukaryotic expression vector, and constructing and obtaining an avian coronavirus nucleoprotein eukaryotic expression vector pCAGGS-N;

s7, performing a circular polymerase extension reaction by using the TRS fragment obtained in the S4 and the N genome fragments obtained in the S5 as templates and using high-fidelity DNA polymerase to obtain a circular product containing a transcription regulatory sequence and an avian coronavirus genome sequence;

s8, co-transfecting the annular product obtained in the S7 and the avian coronavirus nucleoprotein eukaryotic expression vector obtained in the S6 into a cell line, and culturing for 4 days after transfection to collect cells and supernatant;

s9, repeatedly freezing and thawing the cells and the supernatant in the S8 for 3 times, inoculating 8-9-day-old SPF chick embryo allantoic cavity, continuously culturing for 96-120 hours, and collecting chick embryo allantoic fluid to obtain the avian coronavirus reverse genetics rescue strain.

In one embodiment of the invention, the chimeric transcription regulatory sequence of S1 consists of a ribozyme sequence, a transcription termination signal sequence, an enhancer sequence and a promoter sequence, as shown in SEQ ID NO. 2.

In one embodiment of the present invention, S3 is 5 to 12.

In one embodiment of the invention, the cell line of S8 is hamster kidney cells (BHK-21), african green monkey kidney cells (VERO) or chick embryo fibroblasts.

A second object of the present invention is to provide a reverse genetics rescue strain of avian coronavirus obtained by the above method.

The third object of the invention is to provide the application of the avian coronavirus reverse genetics rescue strain obtained by the method in researching biological functions and effects of avian coronavirus genes, bases and amino acids.

The fourth object of the present invention is to provide a method for rescuing recombinant avian coronavirus expressing exogenous gene, comprising the steps of:

S1-S5 are as in claim 1S 1-S5;

s6, replacing the 5a or 3a gene in the genome segment where the 5a or 3a gene in the S5 is located with an exogenous gene;

s7 is the same as S6 in claim 1;

s8, taking the TRS fragment obtained in S4, the genome fragment obtained in S6 and the genome fragment except the genome fragment where the 5a or 3a gene is located in S5 as templates, and carrying out a circular polymerase extension reaction by using high-fidelity DNA polymerase to obtain a circular product of the avian coronavirus genome sequence containing a transcription regulatory sequence and a chimeric fragment gene;

S9, co-transfecting the annular product obtained in the S8 and the avian coronavirus nucleoprotein eukaryotic expression vector obtained in the S7 into a cell line, and culturing for 4 days after transfection to collect cells and supernatant;

s10, repeatedly freezing and thawing the cells and the supernatant in the S9 for 3 times, inoculating 8-9-day-old SPF chick embryo allantoic cavity, continuously culturing for 96-120 hours, and collecting chick embryo allantoic fluid to obtain the avian coronavirus reverse genetic rescue strain expressing the exogenous gene.

In one embodiment of the present invention, the exogenous gene in S6 is EGFP gene or NDV-F gene, the nucleotide sequence of EGFP gene is shown as SEQ ID NO.11, the nucleotide sequence of NDV-F gene is shown as SEQ ID NO.12, and the nucleotide sequence of 5a gene is shown as SEQ ID NO. 10.

A fifth object of the present invention is to provide a recombinant avian coronavirus reverse genetics rescue strain expressing a foreign gene obtained by the above-mentioned method.

The sixth object of the invention is to provide the application of the recombinant avian coronavirus reverse genetics rescue strain expressing exogenous genes obtained by the method in screening antiviral drugs of avian coronavirus report viruses.

The seventh object of the invention is to provide the application of the recombinant avian coronavirus reverse genetics rescue strain expressing the exogenous gene obtained by the method in preparing the avian infectious bronchitis virus vector vaccine.

The invention has the beneficial effects that:

the invention constructs and obtains the annular product containing the full-length cDNA of the IBV genome and the transcription regulatory elements by optimizing and combining the transcription regulatory elements such as promoters, ribozyme sequences, transcription termination signals and the like and applies CPER technology, directly transfects the annular product into cells, completes transcription of infectious RNA, translation of proteins and packaging of viruses in the cells, and inoculates the cells and a culture medium with SPF chick embryos to obtain IBV reverse genetic rescue strains, thereby establishing a rapid and efficient IBV rescue method. The transcription regulating element capable of efficiently saving the IBV is preferably obtained, the cloning of the avian coronavirus genome is further simplified, the construction method is simple and convenient to operate, the construction efficiency of CPER technology is remarkably improved, and the obtained IBV reverse genetic rescue strain can be stably subjected to passage proliferation. The invention provides an effective tool for the research of IBV infection, pathogenicity and immune mechanism and the application of the rescued recombinant virus in the aspects of antiviral drug screening and preparation of infectious bronchitis virus vector vaccines, solves the technical problems of IBV foundation and application research, and has important practical application value.

Drawings

FIG. 1 is a schematic diagram of a method for efficiently rescuing avian coronaviruses according to the present invention;

FIG. 2 is an electron micrograph of the rLDT3/03 strain obtained by rescue in example 1 after negative staining of allantoic fluid

FIG. 3 is a graph showing the result of AVCov7 fragment sequencing of allantoic fluid of rLDT3/03 strain obtained by rescue in example 1;

FIG. 4 is a graph showing the result of AVCov7 fragment sequencing of allantoic fluid of each generation obtained after 5 serial passages of rLDT3/03 strain obtained in the rescue of example 1 in chick embryo;

FIG. 5 is a graph showing the results of Western blot detection of N protein in allantoic fluid of rLDT3/03 strain obtained by rescue in example 1; wherein M is Marker,1 is negative allantoic fluid, 2 is rLDT3/03 strain allantoic fluid;

FIG. 6 is an immunofluorescence of rCEA-. DELTA.5a-EGFP strain after infection of CEF cells for various times;

FIG. 7 is an immunofluorescence of CEF cells infected with rCEA-. DELTA.5a-EGFP strain treated with different substances.

Detailed Description

The present invention is described in further detail below with reference to specific examples and figures, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions are within the scope of the present invention. The procedures, conditions, experimental methods, reagents and the like for carrying out the present invention are common knowledge in the art and conventional products in the market except for those specifically mentioned below, and the present invention is not particularly limited.

The avian coronavirus saved in the embodiment of the invention is divided into two types, one type is coronavirus with infectious host chicken, the strain is avian infectious bronchitis virus tl/CH/LDT3/03 (LDT 3/03) strain with strong pathogenicity to chicken, and the attenuated strain LDT3/03CEF adapted (CEA) strain of avian infectious bronchitis virus which is subjected to adaptive subculture of primary Chick Embryo Fibroblasts (CEFs) to the virulent strain; another type is coronavirus which infects pheasants as the host.

The avian coronavirus strain information is specifically as follows:

the infectious bronchitis virus tl/CH/LDT3/03 strain has been described in Shengwang Liu, jianfei Chen, jinding Chen, xiangang Kong, yuhao Shao, zongxi Han, li Feng, xuehui Cai, shoulin Gu andMing Liu. Isolation ofavian infectiousbronchitis coronavirus from domestic peafowl (Pavo crystals) and teal (Anas) Journal ofGeneral Virology,2005,86 (3): 719-725, genBank accession number KT852992.1.

The low virulent strain LDT3/03CEF adapted (CEA) of the infectious bronchitis virus is disclosed in patent document ZL202111487158.2, the name of the low virulent strain is an infectious bronchitis virus suitable for cell replication and proliferation, and the application of the low virulent strain is Genbank accession number is ON036185.

The strain I0623/17 of the pheasant cockscomb virus has been described in the literature Zongxi Han, liwen Xu, ringing Ren, jie shaping, tianxin Ma, junfeng Sun, yan Zhao, shingwang Liu. Genetics, antigenic and pathogenic characterization ofavian coronaviruses isolated from pheasants (Phasianus colchicus) in China, vector Microbiology 2020,240:108513, genbank accession MK423877.

Mab 4F10 is described in literature: zongxi Han, fei Zhao, yuhao Shao, xaoli Liu, xangang Kong, yang Song, shengwang Liu.fine level epitope mapping and conservation analysis of two novel linear B-cell epitopes of the avian infectious bronchitis coronavirus nucleic acids Research,2013,171:54-64.

Mab 3a11 is described in literature: cheng Jie construction of ILT recombinant virus expressing IBV fiber protein and evaluation of its immune effect, national academy of agricultural sciences' Shuoshi treatises in 2020.

A schematic diagram of the efficient rescue method of avian coronavirus provided in the following examples is shown in FIG. 1.

Example 1: rescue of virulent avian infectious bronchitis Virus tl/CH/LDT3/03 strain

(1) A chimeric TRS combination (SEQ ID NO. 2) containing 1041nt in total of a hepatitis delta virus ribozyme (Hdvrz) sequence, a monkey vacuole virus 40 (SV 40) polyA sequence, a Cytomegalovirus (CMV) enhancer and a promoter sequence was synthesized, and cloned into a pUC57 vector for sequencing and identification, thereby obtaining a plasmid pUC-TRS.

(2) And (2) taking the TRS sequence in the step (1) as a template, respectively designing primers at two ends of the TRS sequence, wherein the upstream primer comprises a sequence of about 30nt at the 5 'end of the TRS sequence, as shown in SEQ ID NO.13, and the downstream primer comprises a sequence of about 30nt at the 3' end of the TRS sequence, as shown in SEQ ID NO. 14.

(3) The genome sequence of LDT3/03 strain is randomly divided into 8 fragments (named as AVCov1, AVCov2 and … … AVCov8 respectively), primers are designed at two ends of each fragment (the sequences of the upstream and downstream primers AVCov1-F and AVCov1-R of the AVCov1 are shown as SEQ ID NO.17 and SEQ ID NO.18 respectively, the sequences of the upstream and downstream primers AVCov2-F and AVCov2-R of the AVCov2 are shown as SEQ ID NO.19 and SEQ ID NO.20 respectively, the sequences of the upstream and downstream primers AVCov8-F and AVCov8-R of … … are shown as SEQ ID NO.31 and SEQ ID NO.32 respectively), wherein the 5 'end primer AVCov1-F of the fragment 1 comprises 50-60 nt homologous to the 3' end of the TRS sequence in (1), the 3 'end primer AVCov8-R of the fragment 8 comprises 50-60 nt homologous to the 5' end of the TRS sequence in (1), and the other repeats of the fragment between adjacent fragments of about 50% and the temperature values of each other fragment pair.

The 3 'end of the TRS is composed of AVCov1-F base 1-44nt, AVCov2-F base 2841-2888nt, AVCov3-F base 6989-7039nt, AVCov4-F base 10428-104 nt, AVCov5-F base 13399-13447nt, AVCov6-F base 16585-16636nt, AVCov7-F base 19726-19774nt, and AVCov8-F base 23358-2340 nt, and the reverse complement of the LDT3/03 base 27667-2799+5' end of the TRS.

(4) And (3) taking the pUC-TRS constructed in the step (1) as a template, and utilizing the designed and synthesized TRS sequence upstream and downstream primers in the step (2) to amplify by using high-fidelity DNA polymerase so as to obtain a TRS fragment.

(5) Extracting LDT3/03 strain RNA, transcribing the RNA into cDNA by using random primers, and amplifying the cDNA by using high-fidelity DNA polymerase according to the 8 primer pairs designed in the step (3) to obtain 8 DNA fragments (L1-L8).

(6) Designing a primer according to the gene sequence of the LDT3/03 plant nucleoprotein, amplifying the coding sequence of the nucleoprotein gene by taking the cDNA obtained in the step (5) as a template, cloning the coding sequence into a eukaryotic expression vector, and constructing and obtaining an LDT3/03 plant nucleoprotein eukaryotic expression vector pCAGGS-N; the upstream primer of the gene sequence of the LDT3/03 strain nucleoprotein is shown as SEQ ID NO.15, and the upstream primer is shown as SEQ ID NO. 16.

(7) Using the TRS fragment obtained in (4) and 8 DNA fragments obtained in (5) as templates, and performing a Circular Polymerase Extension Reaction (CPER) with high-fidelity DNA polymerase PrimeSTAR GXL DNAPolymerase to obtain a circular product pTRS-LDT3/03 containing TRS and LDT3/03 strain genome sequences. The reaction system is as follows: 5X PrimeSTAR GXLBuffer, 10. Mu.l; dNTP mix (2.5 mM each), 4. Mu.l; templates (TRS fragment and AVCov1 to AVCov8 fragment) of 0.1pmol each; primeSTAR GXLDNAPolymerase,2 μl; sterilized distilled water was added to 50. Mu.l. The reaction procedure was as follows: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 15s, elongation at 68℃for 15min, and performing 35 cycles; extending at 68℃for 15min.

(8) Mu.l of the circular product pTRS-LDT3/03 obtained in (7) and 2. Mu.g of the expression vector pCAGGS-N obtained in (6) were co-transfected into chick embryo fibroblasts (DF-1) with TransIT LT-1 transfection reagent, and the cells and supernatant were collected 4 days after transfection.

(9) Repeating freezing and thawing of the cells and the supernatant obtained in the step (8) for 3 times, inoculating an allantoic cavity of an 8-day-old SPF chick embryo, continuously culturing for 96 hours, collecting allantoic fluid, and obtaining the LDT3/03 reverse genetics rescue strain named rLDT3/03, wherein the chick embryo can see typical symptoms of IBV infection, and is delayed and crimped.

Identification and passaging of rLDT3/03 strain:

the obtained rLDT3/03 strain was negatively stained and subjected to electron microscopic observation, whereby spherical coronavirus particles having fibrils were observed (see FIG. 2). RNA of allantoic fluid of rLDT3/03 strain is extracted, RT-PCR detection is carried out by using primers AVCov7-F and AVCov7-R, a 3683bp target band is found by electrophoresis, and sequencing results show that the sequence accords with the theoretical sequence of AVCov7 fragment of rLDT3/03 strain (see figure 3). The rLDT3/03 strain is continuously passaged in chick embryo for 5 times (F1-F5), RNA of allantoic fluid of each generation is extracted, RT-PCR amplification is carried out by using primers AVCov7-F and AVCov7-R, and sequencing is carried out, so that the result shows that the F1-F5 generation rLDT3/03 strain can be amplified into an AVCov7 fragment and is consistent with the wild strain sequence, and the rLDT3/03 strain has good genetic stability (see figure 4). N protein in allantoic fluid of rLDT3/03 strain was detected by Western blot using monoclonal antibody 4F10 of IBV N protein, and the result shows that N protein can be detected in allantoic fluid of rLDT3/03 strain (see FIG. 5).

Example 2: rescue of avian infectious bronchitis Virus cell passage-attenuated Virus LDT3/03CEF adapted (CEA) Strain

(1) Randomly dividing the genome sequence of the CEA strain into 9 fragments (named AVCov1', AVCov2', … … AVCov9 ') with similar sizes, designing primers at two ends of each fragment (the sequences of the upstream primer AVCov1' -F and the downstream primer AVCov1'-R of the AVCov1' are shown as SEQ ID NO.33 and SEQ ID NO.34 respectively, the sequences of the upstream primer AVCov2'-F and the downstream primer AVCov2' -R of the AVCov2 'are shown as SEQ ID NO.35 and SEQ ID NO.36 respectively, the sequences of the upstream primer AVCov9' -F and the AVCov9'-R of the … … AVCov9' are shown as SEQ ID NO.49 and SEQ ID NO.50 respectively), wherein the 5 'primer AVCov1' -F of the fragment 1 comprises 50-60 nt homologous to the 3 'end of the TRS sequence in step (1) of example 1, the 3' primer AVCov9'-R of the fragment 9 comprises 50-60 homologous end portions of the TRS sequence in step 1) of example 1 and the adjacent fragment pairs of the sequences of the 5' -end pairs are annealed at about the same temperature as each other fragment.

The 3 'end of the TRS has 1-30nt of the AVCov1' -F base composition, 2473-2526nt of the CEA genome, 5258-5317nt of the CEA genome, 8066-8121nt of the CEA genome, 10686-10735nt of the CEA genome, 13727-13781nt of the CEA genome, 17005-17054nt of the CEA genome, 20132-20178nt of the CEA genome, 23762-231 nt of the CEA genome, 23762-23nt of the CEA genome, 27457-274816 nt of the CEA genome, and the 5 'end of the TRS has a sequence of AVCov7' -F base composition, 17005-17054nt of the CEA genome, AVCov8'-F base composition, 20132-20178nt of the CEA genome, 23762-231 nt of the CEA genome, and AVCov9' -R base composition.

(2) CEA RNA was extracted, transcribed into cDNA using random primers, and amplified using high-fidelity DNA polymerase according to the 9 primer pairs designed in (1) to obtain 9 DNA fragments (AVCov 1 'to AVCov 9').

(3) Designing a primer according to the sequence of the CEA plant nucleoprotein gene (the primer is the same as in example 1), using the cDNA obtained in (2) as a template, amplifying the coding sequence of the nucleoprotein gene, cloning the coding sequence into a eukaryotic expression vector, and constructing and obtaining the CEA plant nucleoprotein eukaryotic expression vector pCAGGS-N.

(4) CPER was performed with high-fidelity DNA polymerase PrimeSTAR GXL DNAPolymerase using the 9 genomic fragments obtained in (2) and the TRS fragment obtained in step (4) of example 1 as templates to obtain a circular product pTRS-CEA containing the genomic sequences of the TRS and CEA strains. The reaction system and procedure were the same as in example 1, except that the template used was different.

(5) Co-transfecting chicken embryo fibroblasts (DF-1) with 25. Mu.l of the circular product pTRS-CEA obtained in (4) and 2. Mu.g of the expression vector pCAGGS-N obtained in (3) using a TransIT LT-1 transfection reagent; cells and supernatant were collected 4 days after transfection.

(6) Repeatedly freezing and thawing the cells and the supernatant obtained in the step (5) for 3 times, inoculating 8-day-old SPF chick embryo allantoic cavity, continuously culturing for 96 hours, and collecting allantoic fluid; the typical symptoms of IBV infection are seen in chick embryos, the development is delayed and the strain is contracted, and the obtained CEA reverse genetics rescue strain is named rCEA strain.

Identification and passaging of rCEA strain was performed as described in example 1:

the allantoic fluid of the rCEA strain obtained by rescue was negatively stained and observed by electron microscopy, and spherical coronavirus particles having fibers were observed. The RNA of the allantoic fluid of rCEA strain is extracted, RT-PCR detection is carried out by using the primer pair AVCov8'-F/R in the invention, a 3679bp target band is found by electrophoresis, and a sequencing result shows that the sequence accords with the theoretical sequence of the AVCov8' fragment of rCEA strain. The rCEA strain is continuously passaged for 5 times in chick embryo, RNA of allantoic fluid of each generation is extracted, RT-PCR amplification is carried out on AVCov8'-F/R by using the primer pair and sequencing is carried out, and the result shows that the AVCov8' fragment can be amplified in the 5-generation rCEA strain and accords with the wild strain sequence, thus indicating that the rCEA strain has good genetic stability. N protein was detected in rCEA strain allantoic fluid by Westernblot using monoclonal antibody 4F10 to IBV N protein, and the results showed that N protein could be detected in rCEA strain allantoic fluid.

Example 3: rescue of recombinant rCEA-LS virus of avian infectious bronchitis virulent LDT3/03 strain S gene chimeric avian infectious bronchitis virus virulent CEA

(1) Construction of plasmid containing LDT3/03 Strain S Gene chimeric fragment

(1) The 20405-20424nt sequence in the LDT3/03 strain genome is used as a primer S-F (SEQ ID NO. 51), and the reverse complementary sequence is used as a complementary primer S-R (SEQ ID NO. 52); using AVCov8'-F and S-R of example 2, using the avirulent CEA strain AVCov8' fragment of example 2 as a template, obtaining fragment 1 of 293nt by PCR amplification with high fidelity DNA polymerase KOD-Plus-Neo; using the primers S-F and AVCov8' -R of example 2, 3407nt of fragment 2 was obtained by PCR amplification using the high-fidelity DNA polymerase KOD-Plus-Neo, using the LDT3/03 strain cDNA obtained in step (5) of example 1 as a template.

(2) And (3) carrying out fusion PCR on the fragments 1 and 2 obtained in the step (1) by using a attenuated CEA strain primer AVCov8' -F/R, cloning the fragments into a pGEM-T Easy vector, and carrying out sequencing identification to obtain a plasmid pGEM-LS containing the LDT3/03 strain S gene chimeric fragment.

(2) The primers AVCov8'-F and AVCov8' -R were used to obtain LS fragments by PCR amplification using the constructed pGEM-LS plasmid as template and the high fidelity DNA polymerase KOD-Plus-Neo.

(3) CPER was performed with high-fidelity DNA polymerase PrimeSTAR GXLDNAPolymerase using the TRS fragment obtained in example 1, the AVCov1' -AVCov 7' and AVCov9' fragments of CEA obtained in example 2, and the LS fragment obtained in step (2) of this example as templates to obtain a circular product pCMV-CEA-LS comprising the transcription element and the CEA strain genomic sequence of the chimeric LDT3/03 strain S gene. The reaction system and the reaction procedure were carried out as in example 1, except that the templates were different.

(4) 25. Mu.l of the circular product pCMV-CEA-LS obtained in (3) and 2. Mu.g of the expression vector pCAGGS-N were co-transfected with DF-1 cells using TransIT LT-1 transfection reagent as in example 1; cells and supernatant were collected 4 days after transfection.

(5) Repeatedly freezing and thawing the cells and the supernatant obtained in the step (4) for 3 times, and inoculating 8-day-old SPF chick embryo allantoic cavity; after continuous culture for 96 hours, collecting chick embryo allantoic fluid to obtain recombinant CEA strain of chimeric LDT3/03 strain S gene, which is named rCEA-LS strain.

Identification of rCEA-LS strains was performed as in example 1:

the spherical virus particles with fibers exist in allantoic fluid of the rCEA-LS strain obtained by rescue can be observed by an electron microscope; extracting RNA of allantoic fluid of rCEA-LS strain, carrying out RT-PCR detection on AVCov8' -F/R by using a primer pair, and detecting a 3680bp target band through electrophoresis, wherein a sequencing result shows that the sequence of the fragment is different from CEA and is consistent with the corresponding sequence of LDT3/03 strain; n protein was detected in the allantoic fluid of rCEA-LS strain by Westernblot using mAb 4F10 to IBVN protein, and the result showed that N protein could be detected in the allantoic fluid of rCEA-LS strain.

Example 4: rescue of recombinant rLDT3/03-CS virus of avian infectious bronchitis attenuated CEA strain S gene chimeric avian infectious bronchitis virulent LDT3/03

(1) Construction of plasmid containing coding region of CEA Strain S Gene

A3680 nt fragment containing the CEA strain S gene coding region was obtained by PCR amplification using the primer set AVCov8' -F/R of example 2 and cDNA of the CEA strain as a template, and cloned into pGEM-T Easy vector for sequencing and identification, thereby obtaining plasmid pGEM-CS containing the CEA S gene fragment.

(2) The primer pair AVCov8' -F/R of example 2 was used to obtain CS fragments by PCR amplification using the constructed pGEM-CS plasmid as a template and the high-fidelity DNA polymerase KOD-Plus-Neo.

(3) CPER was performed using the TRS fragment obtained in example 1, the AVCov1 to AVCov6 and AVCov8 fragments of LDT3/03 and the CS fragment of this example as templates, and a circular product pCMV-LDT3/03-CS containing a transcription element and the genomic sequence of LDT3/03 strain of chimeric CEA S gene was obtained by using high-fidelity DNA polymerase PrimeSTAR GXL DNAPolymerase, and the reaction system and procedure were performed as in example 1 except that the templates were different.

(4) Co-transfecting DF-1 cells with 25. Mu.l of the circular product pCMV-LDT3/03-CS obtained in (3) and 2. Mu.g of the expression vector pCAGGS-N using TransIT LT-1 transfection reagent as in example 1; cells and supernatant were collected 4 days after transfection.

(5) Repeatedly freezing and thawing the cells and the supernatant obtained in the step (4) for 3 times, and inoculating 8-day-old SPF chick embryo allantoic cavity; and (3) continuously culturing for 96 hours, and collecting allantoic fluid of the chick embryo to obtain a recombinant LDT3/03 strain of the S gene of the chimeric CES strain, which is named rLDT3/03-CS strain.

Identification of rLDT3/03-CS strain was performed as described in example 3:

the spherical virus particles with fibers exist in allantoic fluid of the rLDT3/03-CS strain obtained through rescue can be observed by an electron microscope; extracting RNA of allantoic fluid of rLDT3/03-CS strain, carrying out RT-PCR detection on AVCov8-F/R by using a primer pair of LDT3/03, and detecting 3680bp target band by electrophoresis, wherein a sequencing result shows that the sequence of the fragment is different from LDT3/03 and is consistent with the theoretical sequence of CEA strain; n protein in allantoic fluid of rLDT3/03-CS strain was detected by Westernblot with monoclonal antibody to IBVN protein, and the result shows that N protein can be detected in allantoic fluid of rLDT3/03-CS strain.

Example 5: rescue of the Phasian cockscomb Virus I0623/17 Strain

(1) The genomic sequence of strain I0623/17 was randomly divided into 10 fragments of similar size (designated as AVCov1 ', AVCov2 ', … … AVCov10 '), primers were designed at both ends of each fragment, wherein 5' end primer AVCov1 ' -F of fragment 1 contained 50-60 nt homologous to 3' end of TRS sequence in step (1) of example 1, 3' end primer AVCov10 ' -R of fragment 10 contained 50-60 nt homologous to 5' end of TRS sequence in (1) of example 1, and the head-tail portion sequence of each fragment was repeated about 50nt with the adjacent fragments, and the annealing temperature (tm) value of primer pair between fragments was similar.

(2) Extracting I0623/17 strain RNA, transcribing the RNA into cDNA by using random primers, and amplifying 10 DNA fragments (I1-I10) by using high-fidelity DNA polymerase according to the 10 primer pairs designed in (1).

(3) Designing a primer according to the gene sequence of the I0623/17 plant nucleoprotein, amplifying the coding sequence of the nucleoprotein gene by taking the cDNA obtained in the step (2) as a template, cloning the coding sequence into a eukaryotic expression vector, and constructing and obtaining an I0623/17 plant nucleoprotein eukaryotic expression vector pCAGGS-N'; the upstream primer of the gene sequence of the nucleoprotein of the I0623/17 strain is shown as SEQ ID NO.53, and the upstream primer is shown as SEQ ID NO. 54.

(4) CPER was performed using the 10 genomic fragments obtained in (2) and the TRS fragment obtained in step (4) of example 1 as templates with high-fidelity DNA polymerase PrimeSTAR GXLDNAPolymerase to obtain a circular product pTRS-I0623/17 containing TRS and genomic sequences of strain I0623/17. The reaction system and procedure were the same as in example 1, except that the template was different.

(5) Mu.l of the circular product pTRS-I0623/17 obtained in (4) and 2. Mu.g of the expression vector pCAGGS-N' obtained in (3) were co-transfected into chick embryo fibroblasts (DF-1) with TransIT LT-1 transfection reagent, and the cells and supernatant were collected 4 days after transfection.

(6) Repeatedly freezing and thawing the cells and the supernatant obtained in the step (5) for 3 times, inoculating 8-day-old SPF chick embryo allantoic cavity, continuously culturing for 96 hours, and collecting allantoic fluid; the typical symptoms of IBV infection can be seen in the chick embryo, the development is delayed and the chick embryo is contracted, and the obtained I0623/17 reverse genetics rescue strain is named rI0623/17 strain.

Identification and passaging of rI0636/17 strain:

the obtained rI0623/17 strain allantoic fluid was negatively stained and observed by electron microscopy, and spherical coronavirus particles having fibrils were observed. RNA of rI0623/17 strain allantoic fluid is extracted, RT-PCR detection is carried out by using primers AVCov9 ' -F and AVCov9 ' -R, a 3680bp target band is found by electrophoresis, and a sequencing result shows that the sequence accords with the theoretical sequence of the AVCov9 ' fragment of rI0623/17 strain. The rI0623/17 strain is continuously passaged in chick embryo for 5 times, RNA of allantoic fluid of each generation is extracted, primers AVCov9 ' -F and AVCov9 ' -R are used for RT-PCR amplification and sequencing, and the result shows that the rI0623/17 strain of 5 generations can be amplified to AVCov9 ' fragments and is consistent with wild strain sequences, so that the rI0623/17 strain has good genetic stability. N protein in rI0623/17 strain allantoic fluid was detected by Western blot using monoclonal antibody 4F10 of IBV N protein, and the result shows that N protein can be detected in rI0623/17 strain allantoic fluid.

Example 6: evaluation of pathogenicity and immunogenicity of avian infectious bronchitis Virus S Gene chimeric Virus

The virulent rLDT3/03 strain of the infectious bronchitis of the chicken, which is obtained in the example 1, has the genome consistent with that of the isolated wild strain, and has strong pathogenicity for SPF chickens of 1 day old, and the morbidity is 100 percent and the mortality is 40 percent; the infectious bronchitis attenuated rCEA strain obtained in example 2 has the same genome as the LDT3/03 strain cell-adaptive attenuated CEA strain, and has no disease for 1 day-old SPF chicken, and has a morbidity of 0% and a mortality of 0%.

In order to investigate the pathogenic mechanism of main epidemic infectious bronchitis strain types in China, according to the potential effect of coronavirus S genes, in order to confirm the effect of the genes in the viral pathogenic process, recombinant rCEA-LS virus of chicken infectious bronchitis virulent LDT3/03 strain S genes chimeric chicken infectious bronchitis virus attenuated CEA obtained in example 3 and recombinant rLDT3/03-CS virus of chicken infectious bronchitis virulent LDT3/03 strain S genes chimeric chicken infectious bronchitis attenuated CEA obtained in example 4 are constructed, and rLDT3/03 strain described in example 1 and rCEA strain described in example 2 are used as pathogenic virulent strain and non-pathogenic attenuated strain control. The pathogenic phenotype of each virus is verified by grouping infection of SPF chickens of 1 day old, and the exact role of the S genes of the viruses in pathogenic biological functions and effects can be clarified by combining different virus gene structures.

In view of the above, 50 SPF chickens of 1 day old were randomly divided into 5 groups (10 chickens per group), and the 5 groups of chickens were respectively fed into 5 negative pressure isolators, with the chicks freeTaking food and drinking water. rLDT3/03 strain (10) for group 1 chicks ^5.5 EID ₅₀ ) Inoculation is carried out, and each nose drops is 100 mu L; rCEA strain (10) for group 2 chicks ^5.5 EID ₅₀ ) Inoculation is carried out, and each nose drops is 100 mu L; rLDT3/03-CS strain (10) for group 3 chicks ^5.5 EID ₅₀ ) Inoculation is carried out, and each nose drops is 100 mu L; rCEA-LS strain (10) for group 4 chicks ^5.5 EID ₅₀ ) Inoculation is carried out, and each nose drops is 100 mu L; group 5 chicks served as a blank control group, with 100 μl of SPF chick embryo culture per drop nose. Starting from the day of inoculation, the morbidity and mortality of the inoculated chicken flock are observed and recorded daily, and the killed chicken is subjected to a split examination. The result shows that the rLDT3/03-CS strain, the rCEA strain and the blank control group do not have obvious morbidity and mortality, and the specific pathological changes such as tracheal bleeding, kidney swelling and the like are not seen after the observation and the finishing of the sectioning; the morbidity of SPF chicks by rLDT3/03 strain and rCEA-LS strain virus-attack groups is 100%, and the mortality is 4/10. The sick chickens are characterized by symptoms such as depression, neck contraction, back arch, coarse and disordered fur, sagging of two wings, open mouth and breathing and the like. The section of the dead chicken can be seen to have the bleeding of the trachea, the swelling of the kidneys on two sides, clear texture and typical 'plaque kidneys' appearance, and has the deposition of urate, and partial swelling and bleeding of tonsils of the cecum of the chicken (see table 1).

TABLE 1 pathogenicity of different strains on SPF chicks

By constructing recombinant viruses of chimeric S genes, animal infection models are utilized, so that the virulence of the virulent strain can be obviously enhanced after the S genes of the virulent strain are replaced by the S genes of the virulent strain, otherwise, the virulence of the virulent strain can be obviously reduced after the S genes of the virulent strain are replaced by the S genes of the virulent strain, and the S genes are proved to play a key role in the attenuation process of infectious bronchitis viruses.

Similarly, by replacing the S genes of strains with completely different serotypes, the effect of the S genes of the viruses on the level of the elicited antibodies can be verified, the exact relationship between the S genes and the immunological cross-reactivity of the strains can be further verified through a virus cross-neutralization test, and the specific mechanism of the serotype diversity of the infectious bronchitis viruses is explained by combining the change characteristics of the genes.

Example 7: construction of chicken infectious bronchitis virus chimeric EGFP tracing recombinant virus and antiviral agent screening

1. Construction of chicken infectious bronchitis virus chimeric EGFP tracing recombinant virus

(1) Constructing a plasmid deleted 5a and replaced with a chimeric fragment containing the EGFP gene: the primers were designed using the AVCov9' fragment of the attenuated CEA strain of example 2 as a template, replacing the coding region of the 5a gene with EGFP gene by fusion PCR using high-fidelity DNA polymerase KOD-Plus-Neo, and cloning into pGEM-T Easy vector for sequencing and identification to obtain plasmid pGEM- Δ5a-EGFP containing EGFP gene chimeric fragment. The CEA strain 5a gene sequence is shown in SEQ ID NO.10, and the EGFP gene sequence is shown in SEQ ID NO.11.

(2) The constructed pGEM-delta 5a-EGFP plasmid is used as a template, and the delta 5a-EGFP fragment is obtained by PCR amplification by using high-fidelity DNA polymerase KOD-Plus-Neo.

(3) CPER was performed using the TRS fragment obtained in example 1, the AVCov1 '-AVCov 8' fragment of CEA obtained in example 2, and the Δ5a-EGFP fragment of this example (2) as templates, and using high-fidelity DNA polymerase PrimeSTAR GXLDNAPolymerase, a circular product pCMV-CEA- Δ5a-EGFP containing a transcription element and the CEA strain genome sequence of the chimeric EGFP gene was obtained, and the reaction system and the reaction procedure were the same as in example 1 except for the templates.

(4) Co-transfecting DF-1 cells with 25. Mu.l of the circular product pCMV-CEA-. DELTA.5a-EGFP obtained in (3) and 2. Mu.g of the expression vector pCAGGS-N using TransIT LT-1 transfection reagent as described in example 1; cells and supernatant were collected 4 days after transfection;

(5) Repeatedly freezing and thawing the cells and the supernatant obtained in the step (4) for 3 times, and inoculating 8-day-old SPF chick embryo allantoic cavity; continuously culturing for 96 hours, and collecting chick embryo allantoic fluid to obtain a recombinant CEA strain which is deleted of 5a and embedded with EGFP genes and is named rCEA-delta 5a-EGFP strain;

identification of rCEA-. DELTA.5a-EGFP strain was performed as in example 1:

the spherical virus particles with fibers exist in allantoic fluid of the rCEA-delta 5a-EGFP strain obtained by rescue can be observed by an electron microscope; extracting RNA of allantoic fluid of rCEA-delta 5a-EGFP strain, carrying out RT-PCR detection on AVCov9' -F/R by using the primer pair in example 2, and detecting 4225bp target bands by electrophoresis, wherein sequencing results show that the sequence of the fragment is different from CEA, and the 5a region sequence of the fragment is consistent with the corresponding sequence of EGFP; green fluorescence was observed 48 hours after infection of CEF cells under a fluorescence microscope.

2. Screening of anti-avian infectious bronchitis virus pharmaceutical preparation

(1) Virus infected cell model: CEF cells were cultured at 1X 10 ⁶ Cell number of wells/cell number of wells was plated in 6 well plates. When the virus is inoculated, one hole is taken, and the cell number is counted after pancreatin digestion to determine the virus loading amount. The six well plates were washed three times with PBS. 2ml of serum-free medium containing 1MOI rCEA-. DELTA.5a-EGFP strain virus was added to each well. Incubating the 6-well plate at 37 ℃ for 1h, discarding the virus liquid, washing the solution 3 times by PBS, and replacing the culture medium containing 10% of fetal calf serum for continuous culture. The control group was not infected with virus, and other procedures were consistent with the infected group. Observing the green fluorescent lesion plaque numbers of the cells at 48h and 72h after virus inoculation respectively, and evaluating the severity of virus infected cells; as a result, green fluorescence was observed at 48 hours, and significantly increased at 72 hours (see FIG. 6).

(2) Screening of virus inhibition pharmaceutical preparation: after CEF cells were infected with rCEA- Δ5a-EGFP strain virus, the cells were treated with monoclonal antibody 3A11 strain, diltiazem, paxillin reagent, respectively, with positive serum treatment group of infectious bronchitis virus as positive control, untreated infected group as negative control, and the number of green fluorescent lesions plaques was observed 48h after the treatment. Results the number of green fluorescent lesions in the infectious bronchitis virus positive serum, monoclonal antibody 3A11 strain and drug diltiazem treated group was significantly reduced compared to the untreated infected group, while the number of green fluorescent lesions in the Paxillin reagent treated group was not significantly reduced (see FIG. 7).

Example 8: construction and immune protection evaluation of chicken infectious bronchitis virus chimeric newcastle disease virus F gene recombinant vaccine strain

1. Construction of chicken infectious bronchitis virus chimeric newcastle disease virus F gene recombinant vaccine strain

(1) Constructing a plasmid which is deleted of EGFP gene and replaced by a chimeric fragment containing the F gene of newcastle disease virus: the primer is designed by taking the delta 5a-EGFP fragment in the example 7 as a template, using high-fidelity DNA polymerase KOD-Plus-Neo to replace EGFP with a newcastle disease virus F gene through fusion PCR, cloning the EGFP into a pGEM-T Easy vector for sequencing and identification, and obtaining a plasmid pGEM-delta 5a-NDV-F containing the EGFP gene chimeric fragment. The F gene sequence of the newcastle disease virus is shown in SEQ ID NO.12.

(2) The constructed pGEM-delta 5a-NDV-F plasmid is used as a template, and the delta 5a-NDV-F fragments are obtained by PCR amplification by using high-fidelity DNA polymerase KOD-Plus-Neo.

(3) The PCR was performed using the TRS fragment obtained in example 1, the AVCov1 '-AVCov 8' fragment of CEA obtained in example 2, and the Δ5a-NDV-F fragment of this example (2) as templates, and CPER was performed using high fidelity DNA polymerase PrimeSTAR GXLDNAPolymerase to obtain a circular product pCMV-CEA-. DELTA.5a-NDV-F, which contains a transcription element and the genomic sequence of the CEA strain of the chimeric Δ5a-NDV-F fragment gene, and the reaction system and procedure were the same as in example 1 except for the templates.

(4) Co-transfecting DF-1 cells with 25. Mu.l of the circular product pCMV-CEA-. DELTA.5a-NDV-F obtained in (3) and 2. Mu.g of the expression vector pCAGGS-N using TransIT LT-1 transfection reagent as in example 1; cells and supernatant were collected 4 days after transfection;

(5) Repeatedly freezing and thawing the cells and the supernatant obtained in the step (4) for 3 times, and respectively inoculating 8-day-old SPF chick embryo allantoic cavities; after continuous culture for 96 hours, the chick embryo allantoic fluid is collected to obtain a recombinant CEA strain which is deleted of 5a and embedded with the newcastle disease virus F gene, and the recombinant CEA strain is named rCEA-delta 5a-NDV-F.

Identification of rCEA-. DELTA.5a-NDV-F strain was carried out as in example 1:

the spherical virus particles with fibers exist in allantoic fluid of the rCEA-LS strain obtained by rescue can be observed by an electron microscope; extracting RNA of rCEA-delta 5a-NDV-F strain allantoic fluid, carrying out RT-PCR detection on AVCov9' -F/R by using the primer pair in example 2, and detecting 5208bp target bands by electrophoresis, wherein sequencing results show that the sequence of the fragment is different from CEA, and the sequence of the fragment in the 5a region is consistent with the sequence of Newcastle disease virus F gene; the presence of newcastle disease virus F protein in CEF cells infected with the rCEA- Δ5a-NDV-F strain was detected by indirect immunofluorescence with monoclonal antibodies to newcastle disease virus F protein.

2. Immune protection evaluation of rCEA-delta 5a-NDV-F strain

(1) Immune efficacy evaluation test: the 40 SPF chickens of 5 days are randomly divided into 4 groups (10 chickens in each group) which are respectively fed into a negative pressure isolator, and the chickens can eat and drink water freely. Wherein group 1 and group 2 chicks used rCEA-. DELTA.5a-NDV-F strain (10 ^5.5 EID ₅₀ ) Immunization was performed with 100 μl of each nasal drop; groups 3 and 4 served as control groups, and 100. Mu.L of SPF chick embryo allantoic fluid was dropped on each nose.

(2) Clinical protection evaluation: 20 days after immunization, 1,3 groups of chicks were treated with the homologous parental virulent LDT3/03 (10 ^5.5 EID ₅₀ 0.1 ml) were subjected to nasal drip attacks, 100 μl each; group 2,4 chicks were treated with Newcastle disease virus virulent HLJ/1/06 strain (10 ^5.0 EID ₅₀ 0.1 ml) were subjected to nasal drip attacks, 100 μl each; after the toxicity is removed, the disease condition and death condition of the chicken flock are observed and recorded daily. The results are shown in Table 2, and the chickens in rCEA-delta 5a-NDV-F immune group 1 do not have morbidity and mortality after the virulent LDT3/03 strain of avian infectious bronchitis attacks; 1 chicken in immune group 2 shows lassitude after being challenged by Newcastle disease virus virulent HLJ/1/06 strain, and does not die; the control group 3 is totally ill and dies 2 after LDT3/03 strain attack; and chickens of control group 4 all developed and died after HLJ/1/06 challenge. The result shows that rCEA-delta 5a-NDV-F strain has good immunogenicity and has good protective effect on the virulent of chicken infectious bronchitis parent and newcastle disease virus.

TABLE 2 evaluation of immunopotency of rCEA-. DELTA.5a-NDV-F strains

Example 9: preparation and evaluation of chicken infectious bronchitis virus chimeric newcastle disease virus F gene recombinant vector vaccine strain

1. Preparation of chicken infectious bronchitis virus chimeric newcastle disease virus F gene recombinant vector vaccine

(1) VaccinePreparing a stock solution: taking rCEA-delta 5a-NDV-F strain constructed in example 8, diluting with sterile physiological saline 1000 times, inoculating 9-day-old SPF chick embryo allantoic cavity, incubating at 37deg.C for 60-120 h, discarding 24h dead chick embryo, harvesting allantoic fluid of 48h dead chick embryo or living embryo, preserving at 2-8deg.C, and performing sterile test to obtain the final product with virus content not less than 10 ^6.5 EID ₅₀ /0.1ml。

(2) Preparation of live vaccine: when the vaccine is prepared, the virus culture solution which is qualified by sterile inspection and virus content measurement is mixed and then is mixed with a sucrose gelatin protective agent according to the volume ratio of 8.5:1 (virus liquid: protective agent) is prepared into seedlings, the freeze-drying protective agent is preferably 40-50 ℃ (8% gelatin and 40% sucrose protective agent) and is sterilized at 115 ℃ for 40 minutes under high pressure, and the seedlings are preserved at 4 ℃ and used up within 72 hours. The virus liquid is continuously shaken in the adding process, and the vaccine stock solution is obtained after the virus liquid is fully and uniformly mixed. And (3) quantitatively subpackaging the vaccine stock solution in an aseptic mode, quickly freezing, drying in vacuum, capping and sealing to obtain the live vaccine, and preserving at the temperature below-15 ℃.

2. Safety verification of chicken infectious bronchitis virus chimeric newcastle disease virus F gene recombinant vector vaccine:

Vaccine for test: the live vaccines prepared by the preparation method have the following batch numbers: 202301, 202302. 1000 parts per bottle. Vaccine preservation conditions and expiration date: preserving at below-15 ℃ and keeping the effective period for 12 months.

And (3) verifying the safety of the live vaccine: 1-2 groups (10 groups/group) of SPF chicks with the age of 1-2 days are respectively inoculated with 202301 and 202302 batches of live vaccine, and 10 using doses (about 0.03-0.05 ml) are inoculated on each nasal drip. Group 3 SPF chickens 1-2 days old were each given a drop of sterile saline. The clinical manifestations of each group of chickens are continuously observed after immunization, and the morbidity and mortality are recorded. After the observation is finished, the chickens of each group are sectioned and examined to see whether specific lesions of the infectious bronchitis of the chickens appear, such as: part of the sick chickens have serous or catarrhal secretions in the trachea and sinuses, or the kidneys swell, and typical changes of 'plaque kidneys' appear.

The results show that after the 2 live vaccine immune groups are immunized for 20 days at the dosage of 10 times, the clinical symptoms of infectious bronchitis such as head throwing, listlessness, feather loosening and the like do not appear in the same way as the blank control group, 0/10 of the clinical symptoms occur in the observation period, kidney swelling, specific lesions of 'spotted kidneys' are not found in the dissection, and each group is normal in performance (see table 3). The live vaccine prepared by the chicken infectious bronchitis virus chimeric newcastle disease virus F gene recombinant vector vaccine strain is safe.

TABLE 3 safety test data for avian infectious bronchitis Virus chimeric newcastle disease Virus F Gene recombinant vector vaccine

3. Immune efficacy verification of chicken infectious bronchitis virus chimeric newcastle disease virus F gene recombinant vector vaccine

Vaccine for test: 202301 and 202302 batches of vaccine of the chicken infectious bronchitis virus chimeric newcastle disease virus F gene recombinant vector vaccine. 1000 parts per bottle. Vaccine preservation conditions and expiration date: preserving at below-15 ℃ and keeping the effective period for 12 months.

Usage and dosage: the live vaccine is diluted by normal saline according to the labeling of the bottle label, the vaccine is sucked by a dropper, and each chicken is dropped into one drop (about 0.03-0.05 ml).

Live vaccine efficacy validation: groups 1 to 2 (10/group) and 3 to 4 (10/group) 5 day old SPF chicks were vaccinated with 202301 and 202302 batches of live vaccine, respectively, with 1 dose (about 0.03-0.05 ml) per nasal drip. 5 th to 6 th groups (10/group) of SPF chickens at 5 days of age were used as a control group, and one drop of sterilized normal saline was dropped into each nose. 20 days after immunization, group 1, group 3 and group 5 chickens were challenged with LDT3/03 strain virulence by nasal drip (10 ^6.0 EID ₅₀ ) After the toxicity attack, the clinical manifestations of the chickens of each group are continuously observed, and the morbidity and mortality are recorded. Meanwhile, the 2 nd, 4 th and 6 th chickens were respectively intramuscular injected to attack Newcastle disease virus virulent HLJ1/06 strain (10) ^4.0 ELD ₅₀ ) The clinical manifestations of each group of chickens were continuously observed, the morbidity and mortality were recorded, and the chickens were observed for 14 days.

The results show that after 20 days of immunization of 2 live vaccine immune groups, IBV virulent attack is carried out, wherein all physiological saline control groups are ill, all the clinical symptoms of infectious bronchitis such as head throwing, listlessness, feather loosening and the like appear, 10/10 of the clinical symptoms occur in the observation period, 2/10 of the clinical symptoms die, kidney swelling, specific lesions of 'spotted kidneys' are found in the anatomy, and the vaccine immune groups are normal. The newcastle disease virus virulent attack is carried out, wherein all physiological saline control groups are ill and dead, and the clinical protection rate of vaccine immune groups reaches 8/10-9/10, which shows that after the live vaccine prepared by rCEA-delta 5a-NDV-F strain is used for immunizing chickens, good immune protection response can be generated for 2 virulent. The detailed results are shown in Table 4.

TABLE 4 evaluation of immunization efficacy of avian infectious bronchitis Virus chimeric Newcastle disease Virus F Gene recombinant vector vaccine

The result shows that the active immune protection rate of the survival vaccine prepared by the constructed avian infectious bronchitis virus chimeric newcastle disease virus F gene recombinant vector vaccine strain reaches more than 80%, which shows that the method can be used for researching and developing avian coronavirus vector vaccine.

Comparative example 1: alignment of transcription efficiency of chimeric transcription regulatory sequences

Taking the rescue method of CEA strain in example 2 as an example, the present invention compares the rescue efficiencies corresponding to CEA strain rescue using different chimeric transcription regulatory sequences (TRS 1, TRS2 … … TRS 9). TRS1 consists of a hepatitis delta virus ribozyme (Hdvrz) sequence, a monkey vacuolated virus 40 (SV 40) polyA signal sequence and a CMV promoter sequence, and is shown in SEQ ID NO.1; TRS2 is the TRS selected in the embodiment of the invention; TRS3 consists of a hepatitis delta virus ribozyme (Hdvrz) sequence, a Bovine Growth Hormone (BGH) polyA signal sequence and a CMV promoter sequence, and is shown in SEQ ID NO.3; TRS4 consists of a hepatitis delta virus ribozyme (Hdvrz) sequence, a Bovine Growth Hormone (BGH) polyA signal sequence, a Cytomegalovirus (CMV) enhancer sequence and a CMV promoter sequence, and is shown in SEQ ID NO.4; TRS5 consists of a hepatitis delta virus ribozyme (Hdvrz) sequence, a monkey vacuolated virus 40 (SV 40) polyA signal sequence and a chicken beta-actin promoter sequence, and is shown in SEQ ID NO.5; TRS6 consists of a hepatitis delta virus ribozyme (Hdvrz) sequence, a monkey vacuole virus 40 (SV 40) polyA signal sequence, a Cytomegalovirus (CMV) enhancer sequence and a chicken beta-actin promoter sequence, and is shown in SEQ ID NO.6; TRS7 consists of a hepatitis delta virus ribozyme (Hdvrz) sequence, a Bovine Growth Hormone (BGH) polyA signal sequence and a chicken beta-actin promoter sequence, and is shown in SEQ ID NO.7; TRS8 comprises a hepatitis delta virus ribozyme (Hdvrz) sequence, a Bovine Growth Hormone (BGH) polyA signal sequence, a Cytomegalovirus (CMV) enhancer sequence and a chicken beta-actin promoter sequence, and is shown in SEQ ID NO.8; TRS9 consists of a hepatitis delta virus ribozyme (Hdvrz) sequence, a T7RNA polymerase terminator sequence, and a T7RNA polymerase promoter sequence, as shown in SEQ ID NO.9.

(1) 9 chimeric transcription regulatory sequence combinations TRS1-TRS9 containing elements such as a ribozyme sequence, a transcription termination signal sequence, a promoter sequence and the like are synthesized, cloned into pUC57 vectors respectively for sequencing identification to obtain plasmids pUC-TRS 1-pUC-TRS 9, primers are respectively designed at two ends of the TRS, pUC-TRS 1-pUC-TRS 9 are respectively used as templates, and the designed and synthesized TRS sequence upstream and downstream primers are utilized to obtain TRS fragments TRS1-TRS9 by amplification of high-fidelity DNA polymerase.

(2) The 9 genomic fragments of CEA obtained in step (2) of example 2 and the TRS fragment obtained by amplifying in step (1) were used as templates, CPER was performed with high-fidelity DNA polymerase PrimeSTAR GXLDNAPolymerase to obtain circular products pTRS1-CEA, pTRS2-CEA … … pTRS9-CEA containing the genomic sequences of TRS1-TRS9 and CEA strain, respectively, and the reaction system and procedure were the same as in example 2.

(3) Co-transfecting chicken embryo fibroblasts (DF-1) with 25. Mu.l of the circular products pTRS1-CEA to pTRS8-CEA obtained in (2) and 2. Mu.g of the expression vector pCAGGS-N obtained in step (3) of example 2, respectively, using a TransIT LT-1 transfection reagent; mu.l of the circular product pTRS9-CEA obtained in (2) and 2. Mu.g of the expression vector pCAGGS-N obtained in step (3) of example 2 were additionally co-transfected with 2. Mu.g of the plasmid pCAG-T7pol expressing T7 polymerase using TransIT LT-1 transfection reagent (DF-1); cells and supernatant were collected 4 days after transfection.

(4) Repeating freezing and thawing for 3 times on the cells and the supernatant obtained in the step (3), inoculating 30 SPF chick embryo allantoic cavities of 8 days old respectively, continuously culturing for 96 hours, and collecting allantoic fluid.

(5) RNA was extracted from allantoic fluid collected from each embryo in step (4), RT-PCR detection was performed using primers AVCov8-F and AVCov8-R, and a 3680bp target band was found in the electrophoresis of positive samples, which showed a large difference in the efficiency of 9 chimeric transcription regulatory sequences to rescue viruses, wherein the positive rate of the transcript rescue viruses containing TRS2 (i.e., TRS described in the examples) was the highest, and the positive rate was 20/30 (Table 5).

TABLE 5 Effect of different transcription regulatory sequences on viral rescue efficiency validation results

Comparative example 2: comparison of viral genome cDNA cloning efficiency

The invention takes CEA strain in rescue example 2 as an example, and compares rescue efficiencies corresponding to the rescue CEA strain obtained by using different virus genome cDNA cloning methods.

(1) In vitro enzyme digestion ligation cloning method: the 9-piece genome described in example 2 was cloned into pBR322 plasmid one by one using the CEA strain as the target virus using in vitro fusion ligation and restriction enzyme ligation according to the methods described in the literature (Dan Shan, shougxi Fang, zongxi Han, huiAi, wenjun Zhao, yuqiu Chen, lei Jiang, shingang Liu, effects of hypervariable regions in spike protein on pathogenicity, troprism, and serotypes of infectious bronchitis virus. Virus Research 2018, 250:104-113) and the invention patent (bulletin No. CN106119207B, bulletin No. CN110079541A, a method for constructing IBVVEro cell line adapted based on reverse genetics technology, bulletin No. CN110079541A, a method for constructing coronavirus infectious clones and applications thereof), construction of a full-length viral cDNA plasmid carrying a T7 promoter at the 5' -end, taking the full-length genomic cDNA constructed as described above and the nucleoprotein expression vector pCAGGS-N constructed in example 2, performing in vitro transcription using in vitro Transcription T in vitro transcription kit (Dalianbao bioengineering Co., ltd.) while adding 2.5 mM/. Mu.L of RNA cap structure (NEB Co.) to the transcription system, constructing 50. Mu.L of reaction system according to the in vitro transcription kit product specification, adding 2. Mu.L of DNaseI after transcription for 3h at 37℃for 20min, and obtaining transcripts Transfection of BHK21 cells, CO at 37 ℃ ₂ After culturing in the incubator for 12 hours, the supernatant was discarded, and 3mL of DMEM high-sugar medium containing 10% fetal bovine serum was added for further culturing. After 48h, the culture solution and the cell mixture are frozen and thawed 3 times, 9-day-old SPF chick embryos are inoculated, and after 48h of incubation, chick embryo allantoic fluid is harvested for RT-PCR identification. Results full-length cDNA cloning of the virus was completed and in vitro transcription and rescue of the virus were completed using in vitro enzyme ligation cloning, all taking 90 days, with a rescue success rate of 5/30 (see Table 6).

(2) Bacterial Artificial Chromosome (BAC) system cloning: according to the methods described in the literature (Na Xing, zhishengWang, jichunWang, marianaNascimento, anan Jongkaewwattana, jakob trim, nikolaus Osterrieder, dusan Kunec, engineering and Characterization of Avian Coronavirus Mutants Expressing Fluorescent Reporter Proteins from the Replicase Gene. Journal of virology,2022,96 (14), e 0065322) and the invention (bulletin No. CN107190022B, entitled "method for rapidly constructing reverse genetics strain of avian infectious bronchitis Virus"), a full-length cDNA clone of the CEA genome of avian infectious bronchitis Virus was constructed by using a BAC vector as a backbone and by using in vitro homologous recombination technique, and CMV promoter was added at 5', HDVR sequence was added at 3', and transcription was performed in the cells after transfection to obtain infectious transcripts, and virus packaging was completed, and a mixture of cells and culture medium was inoculated with SPF chickembryos and passaged to obtain reverse genetics strain of avian infectious bronchitis Virus CEA. All take 60 days, the rescue success rate is 12/30 (see Table 6).

(3) Yeast Artificial Chromosome (YAC) system cloning: according to the literature (Thi Nhu Thao, T., labroussaa, F., ebert, N., V ' Kovski, P., stalder, H., portmann, J., kelly, J., steiner, S., holwerda, M., kratzel, A, gultom, M., schmied, K., laloli, L., husser, L., wider, M., pmaender, S., hirt, D., cippa, V., crespo-Pomar, S., schroder, S., muth, d., niemeier, d., corman, v.m., muller, m.a., drosten, c., dijkman, r., jores, j., thiel, v.,2020.Rapid reconstruction of SARS-CoV-2using a synthetic genomics platform.Nature582,561-565) and methods of the invention (publication No. CN115896173a, entitled a method of rapidly constructing infectious clones of avian infectious bronchitis viruses, and products and applications thereof), taking CEA strain as a target virus, adding a CMV promoter to the 5' end of the 1 st segment of the 9 th segment gene fragment described in the example 2, adding Ploy (A) to the 3' end of the 9 th segment, introducing HB sequence, transforming 9 DNA fragments and linearized shuttle plasmids into Saccharomyces cerevisiae for recombination, screening plasmids containing CEA full-length genes, co-transfecting BHK-21 cells with the N gene expression plasmid pCAGGS-N constructed in the expression example 2, collecting supernatant and cell mixture after transfection, inoculating SPF chicken embryo after repeated freeze thawing, and screening to obtain the chicken infectious bronchitis virus CEA reverse genetic strain. All take 60 days, the rescue success rate is 15/30 (see Table 6).

(4) Poxvirus vector system cloning: recombinant poxviruses were constructed by adding the T7 promoter to the 5' end of segment 1 and Ploy (A) to the 3' end of segment 9 of the 9 gene fragment and introducing HB sequences and T7 stop codons according to the methods described in the literature (Ye Zhao, jinlong Cheng, gang Xu, volker Thielb, guozhong Zhang. Successfull establishment of a reverse genetic system for QX-type infectious bronchitis virus and technical improvement of the rescue Process virus Research,2019, 272:197726) and (R.Casais, V.Thiel, S.G.Siddell, D.Cavanagh, P.Britton.Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus. J Virol.2001Dec;75 (24): 12359-69), using the CEA strain as the target virus, inserting fragments comprising the T7 promoter, full-length cDNA and 3' end Ploy (A), HB sequences and T7 stop codons into poxvirus vectors by homologous recombination methods; meanwhile, constructing a T7RNA polymerase gene recombinant poxvirus, allowing the T7RNA polymerase gene recombinant poxvirus to act for 60 minutes after infecting chicken embryo kidney cells, transfecting the chicken embryo kidney cells with a linearized gene fragment containing CEA full-length genes and transcription initiation and termination elements, which is obtained by enzyme digestion of the recombinant poxvirus, incubating for 16 hours, replacing a maintenance medium, harvesting the cells after 3 days, centrifuging after freeze thawing for three times, filtering the supernatant through a 0.22 mu m needle filter, inoculating the chicken embryo kidney cells, incubating and passaging, observing cytopathy, and identifying the rescued viruses by RT-PCR by using the identification primers shown in the example 2. All take 6 months, the rescue success rate is 16/30 (see Table 6).

(5) Circular Polymerase Extension Reaction (CPER) method: the invention simplifies the steps of constructing genome full-length clone without sectioning clone gene fragment, complex time-consuming enzyme cutting or connecting process, omits the in vitro transcription process, synthesizes annular products by one-time annular polymerase extension reaction of the optimized chimeric transcription element group and avian coronavirus genome, and transforms the annular products with virus N gene expression plasmid I to cells, can efficiently transcribe in the cells to obtain transcripts, and assembles virus particles. The time from amplifying the gene fragment to obtaining the rescuing virus is about 10 days, wherein the time for amplifying the gene fragment and sequencing is 1 day, the time for obtaining a ring product containing TRS sequence and whole genome cDNA through CPER is 1 day, the time from cell transfection to chick embryo inoculation is 8 days, and the rescuing success rate is 20/30 (see table 6).

TABLE 6 time consuming and rescue efficiency for obtaining rescue CEA strains by different virus genome cDNA cloning methods

Comparative example 3 comparison of cloning efficiency for different segments of avian coronavirus genome

Taking CEA strain in rescue example 2 as an example, the invention compares rescue efficiencies corresponding to the rescue CEA strain obtained by dividing the CEA strain genome into different numbers of segments.

(1) The CEA strain genome sequence was randomly divided into 3, 5, 8, 10, 12 fragments of similar size, primers were designed at both ends of each fragment according to example 2, wherein the 5 'end primer of fragment 1 comprises 50-60 nt homologous to the 3' end of the TRS sequence, the 3 'end primer of the last fragment comprises 50-60 nt homologous to the 5' end of the TRS sequence, the sequence of the head and tail portions of each fragment was repeated approximately 50nt with the adjacent fragments, and the annealing temperature (tm) values of the primer pairs between fragments were similar.

(2) Extracting CEA RNA, transcribing the RNA into cDNA by using random primers, and amplifying 3, 5, 8, 10 and 12 DNA fragments respectively by using high-fidelity DNA polymerase according to 3, 5, 8, 10 and 12 sets of primers respectively designed in the step (1);

(3) CPER was performed with high-fidelity DNA polymerase PrimeSTAR GXLDNAPolymerase using 3, 5, 8, 10 and 12 DNA fragments obtained in (2) and the TRS fragment obtained in step (4) of example 1, respectively, as templates, to obtain a circular product pTRS-CEA containing the genomic sequences of the TRS and CEA strains, respectively, according to the method described in example 1;

(4) Co-transfecting chicken embryo fibroblasts (DF-1) with 25. Mu.l of the circular product pTRS-CEA obtained in (3) and 2. Mu.g of the expression vector pCAGGS-N obtained in example 1 using TransIT LT-1 transfection reagent; cells and supernatant were collected 4 days after transfection;

(5) The cells obtained in (4) and the supernatant were inoculated with chick embryos and rCEA strain identified as described in example 2, and the success rate of rescuing the virus was compared.

The result shows that when the genome sequence of the CEA strain is randomly divided into 3 fragments with approximate sizes, 3 complete fragments are difficult to obtain by high-fidelity DNA polymerase amplification, and the success rate is 1/10 after multiple attempts; the success rate of the genome sequence is 3/10 when the genome sequence is randomly divided into 5 fragments; the cloning success rate can reach 10/10 when the genome sequence is randomly divided into 8, 10 and 12 DNA fragments, and the shorter the fragments are, the higher the success rate is, but when CPER is carried out, the more fragments are unfavorable for fusion to form a circular genome structure, and accordingly, the virus rescue efficiency is also reduced (see Table 7). Therefore, the genome fragment is divided into 8-10 fragments, and the amplification efficiency and virus rescue efficiency are highest.

TABLE 7 comparison of Gene amplification and Virus rescue efficiency for different segmented amplification protocols of avian coronavirus genome

Comparative example 5 comparison of rescue efficiency of different cell lines transfected with viral genomic cDNA

The method comprises the steps of co-transfecting 25. Mu.l of the annular product pTRS-CEA obtained in the step (4) of the example 2 and 2. Mu.g of the expression vector pCAGGS-N obtained in the step (3) of the example 2 with a TransIT LT-1 transfection reagent respectively to obtain BHK-21 cells, VERO cells and chick embryo fibroblasts (DF-1) cells, culturing the transfected chick embryo fibroblasts for 4 days, collecting cells and supernatant, repeatedly freezing and thawing 3 times, inoculating 8-9-day-old SPF chick embryo allantoic cavities, culturing for 96-120 hours, collecting chick embryo allantoic fluids, and identifying the rescued viruses by using RT-PCR by using the identification primers described in the example 2. As a result, the rCEA virus was obtained by transfecting three cells with the circular product containing the transcription regulatory sequence and the cDNA genome sequence of CEA virus strain. It is demonstrated that all three cells can be used in the rescue system of avian coronavirus.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method of rescuing avian coronavirus comprising the steps of:

s2, respectively designing primers at two ends of the TRS sequence by taking the TRS sequence in the S1 as a template;

s3, randomly dividing the genome sequence of the avian coronavirus into N fragments with approximate sizes, respectively designing primers at two ends of each fragment, wherein the 5 '-end primer of the fragment 1 comprises 50-60 nt homologous to the 3' -end of the TRS sequence in S1, the 3 '-end primer of the fragment N comprises 50-60 nt homologous to the 5' -end of the TRS sequence in S1, and in addition, the sequence of the head and tail parts of each fragment is repeated with the adjacent fragments by about 40-60 nt, and the annealing temperature of primer pairs among the fragments is similar;

S5, extracting avian coronavirus RNA, transcribing the RNA into cDNA, and respectively amplifying the cDNA by using high-fidelity DNA polymerase by adopting the primer designed in the S3 to obtain N genome fragments;

2. The method of claim 1, wherein the chimeric transcription regulatory sequence of S1 consists of a ribozyme sequence, a transcription termination signal sequence, an enhancer sequence, and a promoter sequence as set forth in SEQ ID No. 2.

3. The method of claim 1, wherein the cell line of S8 is hamster kidney cells, african green monkey kidney cells, or chick embryo fibroblasts.

4. A reverse genetics rescue strain of avian coronavirus obtained by rescue according to the method of any one of claims 1-3.

5. Use of the avian coronavirus reverse genetics rescue strain obtained by the method of any one of claims 1-3 for studying biological functions and effects of avian coronavirus genes, bases and amino acids.

6. A method of rescuing a recombinant avian coronavirus expressing a foreign gene comprising the steps of:

S1-S5, S1-S5 as in claim 1;

s7 is the same as S6 in claim 1;

7. The method of claim 6, wherein the exogenous gene of S6 is an EGFP gene or an NDV-F gene, the nucleotide sequence of the EGFP gene is shown in SEQ ID NO.11, the nucleotide sequence of the NDV-F gene is shown in SEQ ID NO.12, and the nucleotide sequence of the 5a gene is shown in SEQ ID NO. 10.

8. The recombinant avian coronavirus reverse genetics rescue strain expressing the exogenous gene obtained by rescue according to the method of any one of claims 6 or 7.

9. Use of the recombinant avian coronavirus reverse genetics rescue strain expressing the exogenous gene obtained by rescue according to any one of the methods of claim 6 or 7 in antiviral drug screening of avian coronavirus reporter viruses.

10. Use of the recombinant avian coronavirus reverse genetics rescue strain expressing the exogenous gene obtained by the method of any one of claims 6 or 7 for preparing infectious bronchitis virus vector vaccine.