CN117568288B - Method for efficiently rescuing avian coronavirus and application thereof - Google Patents

Method for efficiently rescuing avian coronavirus and application thereof Download PDF

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CN117568288B
CN117568288B CN202311330504.5A CN202311330504A CN117568288B CN 117568288 B CN117568288 B CN 117568288B CN 202311330504 A CN202311330504 A CN 202311330504A CN 117568288 B CN117568288 B CN 117568288B
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刘胜旺
孙军峰
韩宗玺
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Harbin Veterinary Research Institute of CAAS
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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 adopts CPER technology to connect the chimeric transcription element group sequence and the avian coronavirus genome sequence from head to tail according to the 5 'to 3' direction by optimizing and constructing the chimeric transcription element group, 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 the 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 alpha, beta, delta and gamma coronaviruses and comprise 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 (Infectiousbronchitis, IB) which is widely prevalent in chickens raised worldwide, mainly damaging the respiratory, kidney and reproductive systems, and can infect chickens of all ages and different types, causing serious economic losses. The pheasant coronavirus (PhCoV) in avian coronavirus 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 chicks 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 manner, 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 full-length IBV cDNA, then enzyme-cleave the full-length IBV cDNA from poxvirus genome by restriction enzyme cleavage, transcribe in vitro to obtain 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 AVCov1 and AVCov-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 rLDT/03 strain obtained by rescue in example 1 after negative staining with allantoic fluid
FIG. 3 is a graph showing the result of AVCov7 fragment sequencing of the allantoic fluid of strain rLDT/03 obtained by rescue in example 1;
FIG. 4 is a graph showing the result of AVCov fragment sequencing of each generation of allantoic fluid obtained after 5 successive passages of rLDT/03 strains obtained by rescue in example 1;
FIG. 5 is a graph showing the results of Western blot detection of N protein in allantoic fluid of strain rLDT/03 obtained by rescue of example 1; wherein M is Marker,1 is negative allantoic fluid, 2 is rLDT/03 allantoic fluid;
FIG. 6 is an immunofluorescence of rCEA- Δ5a-EGFP strain after infection of CEF cells for various times;
FIG. 7 is an immunofluorescence of CEF cells infected with rCEA- Δ5a-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 is 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 cristatus)and teal(Anas).Journal ofGeneral Virology,2005,86(3):719-725, genBank accession number is 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 infectious bronchitis virus suitable for cell replication and proliferation and the application thereof are in patent document, and the Genbank accession number is ON036185.
The pheasant coronavirus strain I0623/17 is described in literature Zongxi Han,Liwen Xu,Mengting Ren,Jie Sheng,Tianxin Ma,Junfeng Sun,Yan Zhao,Shengwang Liu.*Genetic,antigenic and pathogenic characterization ofavian coronaviruses isolated from pheasants(Phasianus colchicus)in China.Veterinary Microbiology,2020,240:108513 under Genbank accession number MK423877.
Monoclonal antibody 4F10 is disclosed in the literature ：Zongxi Han,Fei Zhao,Yuhao Shao,Xiaoli Liu,Xiangang 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 nucleocapsidprotein.Virus 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 immune effect thereof, 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 LDT3/03 strain genome sequence is randomly divided into 8 fragments (respectively named AVCov1, AVCov2 and … … AVCov 8) with approximate sizes, primers (AVCov, upstream primer AVCov-F and AVCov1-R are respectively shown as SEQ ID NO.17 and SEQ ID NO.18, AVCov, upstream primer AVCov-F and AVCov-R are respectively shown as SEQ ID NO.19 and SEQ ID NO.20, and … … AVCov, upstream primer AVCov-F and AVCov-R are respectively shown as SEQ ID NO.31 and SEQ ID NO. 32) are designed at two ends of each fragment, wherein primer AVCov-F at the 5 'end of fragment 1 comprises 50-60 nt homologous to the 3' end of TRS sequence in (1), primer AVCov-R at the 3 'end of fragment 8 comprises 50-60 nt homologous to the 5' end of TRS sequence in (1), and the head-tail portion of each fragment is further annealed to the adjacent repeated fragments at about 50nt (tmd) and the temperature of each fragment is similar to that of the primers (tmd).
AVCov1-F base set was 1-44nt in the TRS 3 'end 60nt+LDT3/03 genome, AVCov2-F base set was 2841-2888nt in the LDT3/03 genome, AVCov3-F base set was 6989-7039nt in the LDT3/03 genome, AVCov4-F base set was 10428-104 nt in the LDT3/03 genome, AVCov5-F base set was 13399-13447nt in the LDT3/03 genome, AVCov6-F base set was 16585-16636nt in the LDT3/03 genome, AVCov7-F base set was 19726-19774nt in the LDT3/03 genome, AVCov8-F base set was 23358-238 nt in the LDT3/03 genome, and AVCov8-R base set was 27667-2799+5' end of the TRS complementary sequence in the LDT3/03 genome.
(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) by using 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 AVCov fragment 1 to AVCov fragment) each in an amount of 0.1pmol; 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 TRANS IT LT-1 transfection reagent, and the cells and supernatant were collected 4 days after transfection.
(9) After repeating freeze thawing for 3 times, inoculating 8-day-old SPF chick embryo allantoic cavity, continuously culturing for 96 hours, collecting allantoic fluid, wherein chick embryo can see typical symptoms of IBV infection, slow development and contracture, and the obtained LDT3/03 reverse genetics rescue strain is named rLDT/03 strain.
Identification and passaging of rLDT/03 strain:
The resulting rLDT/03 strain was negatively stained with allantoic fluid and observed by electron microscopy, and spherical coronavirus particles having fibrils were observed (see FIG. 2). RNA of rLDT strain 3/03 allantoic fluid was extracted, RT-PCR detection was performed using primers AVCov-F and AVCov-7-R, a 3683bp target band was found by electrophoresis, and sequencing results showed that the sequence was consistent with the theoretical sequence of AVCov fragment of rLDT/03 strain AVCov (see FIG. 3). The rLDT/03 strain was serially passaged 5 times (F1-F5) in chick embryos, RNA of allantoic fluid of each generation was extracted, RT-PCR amplification was performed by using primers AVCov-F and AVCov-R, and sequencing was performed, and the results showed that the F1-F5 generation rLDT/03 strain could be amplified to AVCov7 fragment and matched with wild strain sequence, indicating that the rLDT3/03 strain has good genetic stability (see FIG. 4). N protein in the allantoic fluid of strain rLDT/03 was detected by Western blot using mAb 4F10 to IBV N protein, and the result showed that N protein could be detected in the allantoic fluid of strain rLDT3/03 (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 of similar size (designated AVCov1', AVCov2', … … AVCov '), designing primers at both ends of each fragment (AVCov 1' upstream and downstream primers AVCov '-F and AVCov1' -R respectively as shown in SEQ ID NO.33 and SEQ ID NO.34, AVCov 'upstream and downstream primers AVCov' -F and AVCov '-R respectively as shown in SEQ ID NO.35 and SEQ ID NO.36, … … AVCov' upstream and downstream primers AVCov '-F and AVCov' -R respectively as shown in SEQ ID NO.49 and SEQ ID NO. 50), wherein the 5 'primer AVCov' -F of fragment 1 comprises 50-60 nt homologous to the 3 'end of the TRS sequence in step (1) of example 1, the 3' primer AVCov '-R of fragment 9 comprises 50-60 nt homologous to the 5' end of the TRS sequence in step (1) of example 1, and the sequence of each fragment is annealed at a temperature similar to the adjacent pair of primers (tmd) between the respective pairs of primers.
AVCov1'-F base composition is 1-30nt in the 3' end 60nt+CEA genome of TRS, AVCov2'-F base composition is 2473-2526nt in CEA genome, AVCov3' -F base composition is 5258-5317nt in CEA genome, AVCov4'-F base composition is 8066-8121nt in CEA genome, AVCov5' -F base composition is 10686-10735nt in CEA genome, AVCov6'-F base composition is 13727-13781nt in CEA genome, AVCov7' -F base composition is 17005-17054nt in CEA genome, AVCov8'-F base composition is 20132-20178nt in CEA genome, AVCov9' -F base composition is 23762-231 nt in CEA genome, AVCov9'-R base composition is 27457-27486+TRS reverse complement of 5' end 60 in CEA genome.
(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 'to AVCov').
(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) Using the 9 genome fragments obtained in (2) and the TRS fragment obtained in step (4) of example 1 as templates, CPER was performed with high-fidelity DNA polymerase PRIMESTAR GXL DNAPOLYMERASE 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 the 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 TRANS IT 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; typical symptoms of IBV infection can be seen in chick embryos, development is delayed and the chick embryos are contracted, and the obtained CEA reverse genetics rescue strain is named rCEA strain.
RCEA strains were identified and passaged as in example 1:
The obtained rCEA strains were negatively stained with allantoic fluid and observed by electron microscopy, and spherical coronavirus particles having fibers were observed. RNA of rCEA allantoic fluid is extracted, RT-PCR detection is carried out by using the primer pair AVCov '-F/R in the invention, a target band of 3679bp is found by electrophoresis, and sequencing results show that the target band accords with AVCov' fragment theoretical sequence of rCEA strain. The rCEA strains are continuously passaged in chick embryos for 5 times, RNA of allantoic fluid of each generation is extracted, RT-PCR amplification is carried out by using the primer pair AVCov '-F/R and sequencing is carried out, and the result shows that AVCov' fragments can be amplified in 5 generations rCEA strains and are consistent with wild strain sequences, thus indicating that the rCEA strain has good genetic stability. N protein in the allantoic fluid of rCEA strains was detected by Westernblot using monoclonal antibody 4F10 of IBV N protein, and the result shows that N protein can be detected in the allantoic fluid of rCEA strains.
Example 3: rescue of recombinant rCEA-LS virus of avian infectious bronchitis virulent LDT3/03 strain S gene chimeric avian infectious bronchitis virus attenuated 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 AVCov '-F and S-R of example 2, using the attenuated CEA strain AVCov' fragment of example 2 as a template, amplifying by PCR with high fidelity DNA polymerase KOD-Plus-Neo to obtain fragment 1 of 293 nt; using the primer S-F and AVCov' R in 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 1as a template.
② And (3) carrying out fusion PCR on the fragment 1 and the fragment 2 obtained in ① by using a low-toxicity CEA strain primer AVCov' -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 LS fragment was obtained by PCR amplification using primers AVCov '-F and AVCov' -R and the constructed pGEM-LS plasmid as template and the high fidelity DNA polymerase KOD-Plus-Neo.
(3) The circular product pCMV-CEA-LS containing the transcription element and CEA strain genomic sequence of chimeric LDT3/03 strain S gene was obtained by using the TRS fragment obtained in example 1, the AVCov ' to AVCov ' and AVCov ' fragments of CEA obtained in example 2, and the LS fragment in step (2) of this example as templates and performing CPER with high-fidelity DNA polymerase PRIMESTAR GXLDNAPOLYMERASE. The reaction system and the reaction procedure were carried out 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-CEA-LS obtained in (3) and 2. Mu.g of the expression vector pCAGGS-N using TRANS IT 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 CEA strain of the S gene of the chimeric LDT3/03 strain, which is named rCEA-LS strain.
RCEA-LS strains were identified as described in example 1:
The spherical virus particles with fibers exist in allantoic fluid of the rCEA-LS strain obtained through rescue can be observed by an electron microscope; extracting RNA of rCEA-LS allantoic fluid, carrying out RT-PCR detection by using a primer pair AVCov' -F/R, 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 in allantoic fluid of rCEA-LS strain was detected by Westernblot using IBVN protein mab 4F10, which showed that N protein could be detected in 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 AVCov' -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 CS fragment was obtained by PCR amplification using the primer pair AVCov' -F/R of example 2 and the constructed pGEM-CS plasmid as a template, using the high-fidelity DNA polymerase KOD-Plus-Neo.
(3) Using the TRS fragment obtained in example 1, the AVCov.about. AVCov6 and AVCov fragments of LDT3/03 and the CS fragment of this example as templates, CPER was performed with high-fidelity DNA polymerase PRIMESTAR GXL DNAPOLYMERASE to obtain the circular product pCMV-LDT3/03-CS containing the transcription element and the genomic sequence of LDT3/03 strain of chimeric CEA S gene, the reaction system and procedure were carried out 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 TRANS IT 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 further culturing for 96 hours, the allantoic fluid of the chick embryo is collected to obtain a recombinant LDT3/03 strain of the S gene of the chimeric CES strain, which is named rLDT3/03-CS strain.
RLDT3/03-CS strain was identified as in example 3:
The spherical virus particles with fibers exist in allantoic fluid of the rLDT/03-CS strain obtained by rescue can be observed by an electron microscope; extracting RNA of rLDT3/03-CS strain allantoic fluid, carrying out RT-PCR detection by using a primer pair AVCov-F/R 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 rLDT/03-CS strain was detected by Westernblot using IBVN protein monoclonal antibody, which showed that N protein could 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 AVCov1", AVCov2", … … AVCov ", respectively) and primers were designed at both ends of each fragment, wherein the 5 'end primer AVCov" -F of fragment 1 contained 50-60 nt homologous to the 3' end of the TRS sequence in step (1) of example 1, the 3 'end primer AVCov "-R of fragment 10 contained 50-60 nt homologous to the 5' end of the TRS sequence in step (1) of example 1, and the sequence of the head-to-tail portion 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 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) The 10 genomic fragments obtained in (2) and the TRS fragment obtained in step (4) of example 1 were used as templates, and CPER was carried out using a 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 TRANS IT 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 AVCov ' -F and AVCov ' -R, a 3680bp target band is found by electrophoresis, and a sequencing result shows that the sequence accords with a AVCov ' fragment theoretical sequence of rI0623/17 strain. The rI0623/17 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 by using primers AVCov ' -F and AVCov ' -R, and sequencing is carried out, so that the result shows that the rI0623/17 strain of 5 generations can be amplified to AVCov ' fragment and is consistent with wild strain sequence, and 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 infectious bronchitis virulent rLDT/03 strain of chicken obtained in example 1 has genome consistent with that of the isolated wild strain, and has strong pathotype, morbidity of 100% and mortality of 40% for 1 day-old SPF chicken; the infectious bronchitis attenuated rCEA strain obtained in example 2 is consistent with the CEA strain genome of the LDT3/03 strain cell adaptation attenuated strain, and has no pathogenicity to SPF chicken of 1 day old, 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 gene chimeric chicken infectious bronchitis virus attenuated CEA obtained in example 3 and recombinant rLDT/03-CS virus of chicken infectious bronchitis attenuated CEA strain S gene chimeric chicken infectious bronchitis virulent LDT3/03 obtained in example 4 are constructed, and rLDT/03 strain as described in example 1 and rCEA strain as pathogenic virulent strain and nonpathogenic attenuated strain contrast is constructed. 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, and the chicks were free to eat and drink water. Group 1 chicks were vaccinated with rLDT strain 3/03 (10 5.5EID50), 100 μl per nasal drip; group 2 chicks were vaccinated with rCEA strains (10 5.5EID50), 100 μl per nasal drip; group 3 chicks were vaccinated with rLDT/03-CS strain (10 5.5EID50), 100 μl per nasal drip; group 4 chicks were vaccinated with rCEA-LS strain (10 5.5EID50), 100 μl per nasal drip; 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. As a result, rLDT/03-CS strain, rCEA strain and blank control group have no obvious morbidity and mortality, and the specific pathological changes such as tracheal bleeding, kidney enlargement and the like are not seen after the observation and the finishing of the section examination; the morbidity of the strain rLDT/03 and the strain rCEA-LS on SPF chicks 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 primer is designed by taking AVCov' fragment of the attenuated CEA strain in the example 2 as a template, replacing a coding region of a 5a gene with an EGFP gene by fusion PCR with high-fidelity DNA polymerase KOD-Plus-Neo, cloning the EGFP gene into a pGEM-T Easy vector, and carrying out sequencing identification to obtain a plasmid pGEM-delta 5a-EGFP containing the 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) The TRS fragment obtained in example 1, the AVCov 'to AVCov' fragment of CEA obtained in example 2, and the Δ5a-EGFP fragment in this example (2) were used as templates, CPER was performed with high-fidelity DNA polymerase PRIMESTAR GXLDNAPOLYMERASE, and a circular product pCMV-CEA-. DELTA.5a-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 that the templates were different.
(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 TRANS IT 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; 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;
the rCEA- Δ5a-EGFP strain was identified as in example 1:
The electron microscope can observe that spherical virus particles with fibers exist in allantoic fluid of the rCEA-delta 5a-EGFP strain obtained by rescue; extracting RNA of allantoic fluid of rCEA-delta 5a-EGFP strain, carrying out RT-PCR detection by using a primer pair AVCov' -F/R 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 plated in 6-well plates at a cell number of 1X 10 6/well. 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: the CEF cells were treated with monoclonal antibody 3A11 strain, diltiazem, paxilline reagent respectively 30min after rCEA- Δ5a-EGFP strain infection, the positive control was positive for the positive serum treatment group of avian infectious bronchitis virus, the negative control was untreated, and the number of green fluorescent lesions plaques was observed 48h after 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 Paxilline agent treated group was not significantly reduced (see figure 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 TRS fragment obtained in example 1, the AVCov 'to AVCov' fragment of CEA obtained in example 2, and the Δ5a-NDV-F fragment in this example (2) were used as templates, CPER was performed with high-fidelity DNA polymerase PRIMESTAR GXLDNAPOLYMERASE, and a circular product pCMV-CEA-. DELTA.5a-NDV-F containing a transcription element and the genomic sequence of the CEA strain of the chimeric Δ5a-NDV-F fragment gene was obtained, and the reaction system and the 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 TRANS IT 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; and (3) 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 a newcastle disease virus F gene, wherein the recombinant CEA strain is named rCEA-delta 5a-NDV-F.
The rCEA-. DELTA.5a-NDV-F strain was identified as in example 1:
The spherical virus particles with fibers exist in allantoic fluid of the rCEA-LS strain obtained through rescue can be observed by an electron microscope; extracting RNA of rCEA-delta 5a-NDV-F strain allantoic fluid, carrying out RT-PCR detection by using a primer pair AVCov' -F/R 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 a newcastle disease virus F gene; the presence of newcastle disease virus F protein in CEF cells infected with rCEA- Δ5a-NDV-F strain was detected by indirect immunofluorescence with monoclonal antibodies to newcastle disease virus F protein.
2. Evaluation of immune protection of rCEA- Δ5a-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 were vaccinated with rCEA- Δ5a-NDV-F strain (10 5.5EID50), 100 μl per nasal drip; 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: at 20 days after immunization, 1,3 groups of chicks were subjected to nasal drip challenge with homologous parental virulent LDT3/03 (10 5.5EID50/0.1 ml), 100 μl each; the chicks of the 2,4 groups are subjected to nose drip attack by using Newcastle disease virus virulent HLJ/1/06 strain (10 5.0EID50/0.1 ml), and each chick is 100 mu L; 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 rCEA-delta 5a-NDV-F immunized group 1 chicken does not have morbidity and mortality after the infectious bronchitis virulent LDT3/03 strain is challenged; 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 the parent infectious bronchitis and the virulent of newcastle disease virus.
TABLE 2 evaluation of the immunopotency of rCEA- Δ5a-NDV-F Strain
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) Preparing vaccine stock solution: taking rCEA-delta 5a-NDV-F strain constructed in example 8, diluting with sterile physiological saline 1000 times, respectively inoculating 9-day-old SPF chick embryo allantoic cavity, incubating at 37 ℃ for 60-120 h, discarding 24h dead chick embryo, harvesting allantoic fluid of 48h dead chick embryo or living embryo, preserving at 2-8 ℃ and performing sterile test at the same time, and measuring that the virus content is not less than 10 6.5EID50/0.1 ml.
(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 202301-202302 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 groups 3 to 4 (10/group) of 5-day-old SPF chicks were vaccinated with 202301 and 202302 batches of live vaccine, respectively, and each nasal drip was vaccinated with 1 dose (about 0.03-0.05 ml). 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. The method for dripping nose of chicken in 1,3 and 5 groups attacks LDT3/03 strain virulent strain (10 6.0EID50) 20 days after immunization, and clinical manifestations of chicken in each group are continuously observed after the virulent strain is attacked, and morbidity and mortality are recorded. Meanwhile, the 2 nd, 4 th and 6 th chickens are respectively challenged with the Newcastle disease virus virulent HLJ1/06 strain (10 4.0ELD50) by an intramuscular injection method, the clinical manifestations of the chickens of each group are continuously observed, the morbidity and mortality conditions are recorded, and the chickens are 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 immunity groups reaches 8/10-9/10, which shows that the live vaccine prepared by rCEA-delta 5a-NDV-F strain can generate good immune protection response to 2 virulent after being used for immunizing chickens. 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 invention compares the corresponding rescue efficiencies of CEA strains by using different chimeric transcription regulatory sequences (TRS 1, TRS2 … … TRS 9). TRS1 consists of a hepatitis delta virus ribozyme (HdvRz) sequence, a monkey vacuole 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 comprises 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 vacuole 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, see SEQ ID NO.9.
(1) 9 Chimeric transcription regulatory sequence combinations TRS 1-TRS 9 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 TRS 1-TRS 9 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) above were used as templates, and CPER was carried out using high-fidelity DNA polymerase PRIMESTAR GXLDNAPOLYMERASE to obtain circular products pTRS1-CEA, pTRS2-CEA … … pTRS9-CEA containing genomic sequences of TRS1-TRS9 and CEA strains, 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 TRANS IT 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 TRANS IT LT-1 of a transfection reagent into chick embryo fibroblasts (DF-1) with 2. Mu.g of the plasmid pCAG-T7pol expressing T7 polymerase; 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), and RT-PCR was performed using primers AVCov-F and AVCov-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: according to the method described in document (Dan Shan,Shouguo Fang,Zongxi Han,HuiAi,Wenjun Zhao,Yuqiu Chen,Lei Jiang,Shengwang Liu,Effects of hypervariable regions in spike protein on pathogenicity,tropism,and serotypes of infectious bronchitis virus.Virus Research 2018,250:104-113) and patent of invention (bulletin No. CN106119207B, bulletin No. CN110079541A, bulletin No. CN 32 cell adapted strain constructed based on reverse genetics technology, a method for constructing coronavirus infectious clone and application thereof), CEA strain is used as target virus, 9 segments of genome described in example 2 are constructed into pBR322 plasmid one by cloning through in vitro fusion connection and enzyme cutting connection, full-length virus cDNA plasmid with T7 promoter at 5' end is constructed, full-length genome cDNA constructed as described above and nucleoprotein expression vector pCAGGS-N constructed in example 2 are taken, in vitro transcription is carried out by adopting in vitro Transcription T in vitro transcription kit (Dalianbao bioengineering Co., ltd.) and simultaneously, 2.5 mM/. Mu.L of RNA cap structure (NEB Co.) is added into transcription system, 50. Mu.L of reaction system is constructed according to the specification of in vitro transcription kit, DNaseI is added after 3h of transcription at 37 ℃, the obtained transcripts are transfected BHK21 cells are placed into a culture tank at 37 ℃ for 12h, and serum culture medium containing 10% of high bovine serum is further removed. 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 method disclosed in the document (Na Xing,ZhishengWang,JichunWang,MarianaNascimento,Anan Jongkaewwattana,Jakob Trimpert,Nikolaus Osterrieder,Dusan Kunec,Engineering and Characterization of Avian Coronavirus Mutants Expressing Fluorescent Reporter Proteins from the Replicase Gene.Journal ofVirology,2022,96(14),e0065322) and the invention patent (publication No. CN107190022B, the invention name is a method for quickly constructing avian infectious bronchitis virus reverse genetics strain), a BAC vector is taken as a framework, an in vitro homologous recombination technology is applied to construct full-length cDNA clone of avian infectious bronchitis virus CEA genome, a CMV promoter is added at 5', an HDVR sequence is added at 3', transfected cells are transcribed in the cells to obtain infectious transcripts, virus packaging is completed, and a mixed solution of the cells and a culture medium is inoculated with SPF chickembryos and passaged to obtain the avian infectious bronchitis virus CEA reverse genetics strain. All take 60 days, the rescue success rate is 12/30 (see Table 6).
(3) Yeast Artificial Chromosome (YAC) system cloning: according to the method disclosed in the document (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.,Pfaender,S.,Hirt,D.,Cippa,V.,Crespo-Pomar,S.,Schroder,S.,Muth,D.,Niemeyer,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 the invention patent (publication No. CN115896173A, the invention name is a method for quickly constructing infectious clone of avian infectious bronchitis virus, and a product and application thereof), CEA strain is taken as a target virus, CMV promoter is added to the 5 'end of the 1 st segment of the 9 segment of gene fragment described in the example 2, ploy (A) is added to the 3' end of the 9 segment and HB sequence is introduced, 9 DNA fragments and linearized shuttle plasmid are transformed into Saccharomyces cerevisiae for recombination, plasmid containing CEA full-length gene is screened and transfected into BHK-21 cells together with N gene expression plasmid pCAGGS-N constructed in the expression example 2, supernatant and cell mixture are collected after transfection, SPF chickembryo is inoculated after repeated freeze thawing, and chicken infectious bronchitis virus CEA reverse genetic strain is obtained by screening. 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 of the 9-segment gene fragment described in example 2, adding Ploy (a) to the 3' -end of segment 9 and introducing HB sequence and T7 stop codon, ligating by in vitro fusion, and inserting fragments comprising T7 promoter, full-length cDNA of the virus and Ploy (a) at 3' -end, HB sequence and T7 stop codon into poxvirus vector by homologous recombination method according to the methods described in documents (Ye Zhao,Jinlong Cheng,Gang Xu,Volker Thielb,Guozhong Zhang.Successful establishment of a reverse genetic system for QX-type infectious bronchitis virus and technical improvement of the rescue procedure.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); 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) The loop 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 circular product containing a 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) Using the 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, CPER was performed using high-fidelity DNA polymerase PRIMESTAR GXLDNAPOLYMERASE, respectively, according to the method described in example 1, to obtain a circular product pTRS-CEA containing the genomic sequences of the TRS and CEA strains;
(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 TRANS IT 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 strains were 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 the more fragments are in favor of fusion to form a circular genome structure when CPER is carried out, 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
Mu.l of the circular product pTRS-CEA obtained in the step (4) of example 2 and 2. Mu.g of the expression vector pCAGGS-N obtained in the step (3) of example 2 were co-transfected with TRANS IT LT-1 transfection reagent respectively into BHK-21 cells, VERO cells, chick embryo fibroblasts (DF-1) cells, after 4 days of culture after transfection, the cells and supernatant were collected, freeze-thawed repeatedly for 3 times, 8-9 day old SPF chick embryo allantoic cavities were inoculated, and after further culture for 96-120 hours, chick embryo allantoic fluid was collected, and the rescued virus was identified by RT-PCR using the identification primers described in 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 (9)

1. A method of rescuing avian coronavirus comprising the steps of:
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;
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 40-60 nt with the adjacent fragments, and the annealing temperature of primer pairs 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 high-fidelity DNA polymerase by adopting the primer designed in the S3 to obtain N genome fragments;
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;
And S9, repeatedly freezing and thawing the cells and the supernatant in the step 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.
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. A method of rescuing a recombinant avian coronavirus expressing a foreign gene comprising the steps of:
S1-S5, S1-S5 as in claim 1;
S6, replacing the 5a gene in the genome segment of the cDNA in S5 with an exogenous gene; the nucleotide sequence of the 5a gene is shown as SEQ ID NO. 10;
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 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.
6. The method of claim 5, 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, and the nucleotide sequence of the NDV-F gene is shown in SEQ ID NO. 12.
7. The recombinant avian coronavirus reverse genetics rescue strain expressing the exogenous gene obtained by rescue according to the method of any one of claims 5 or 6.
8. 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 5 or 6 in antiviral drug screening of avian coronavirus reporter viruses.
9. Use of the recombinant avian coronavirus reverse genetics rescue strain expressing the exogenous gene obtained by the method of any one of claims 5 or 6 for preparing infectious bronchitis virus vector vaccine.
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CN102382812A (en) * 2010-12-15 2012-03-21 江苏省家禽科学研究所 Reverse genetics operating system of infectious bronchitis virus H120 vaccine strain and operating method thereof
CN107190022A (en) * 2017-06-28 2017-09-22 浙江大学 A kind of method of rapid build avian infectious bronchitis virus reverse genetic strain
CN107435041A (en) * 2017-09-01 2017-12-05 扬州大学 Chimeric the newcastle Disease poisonous carrier H9 live vaccines Candidate Strain and its construction method for overcoming chicken Newcastle disease maternal antibody to influence
CN110079541A (en) * 2019-05-05 2019-08-02 华南农业大学 A kind of method and its application of building coronavirus infectivity clone

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