QX type avian infectious bronchitis virus recombinant attenuated strain as well as preparation method and application thereof

Abstract

The invention provides a QX-type avian infectious bronchitis virus recombinant attenuated strain as well as a preparation method and application thereof, belongs to the technical field of veterinary biological products, and can solve the problems of long period and poor adaptability in acquisition of attenuated strains through multiple passage of chick embryos. According to the QX type avian infectious bronchitis virus recombinant attenuated strain, cDNA of a QX type avian infectious bronchitis virus serves as a template, after PCR amplification is conducted, infectious clone plasmids containing a QX type avian infectious bronchitis virus whole genome are constructed through a yeast homologous recombination technology, and the infectious clone plasmids are subjected to a PCR amplification to obtain the QX type avian infectious bronchitis virus recombinant attenuated strain. And co-transfecting a host cell with a recombinant plasmid carrying the QX type avian infectious bronchitis virus N protein gene and a 3'untranslated region, and then carrying out genetic rescue. The QX type avian infectious bronchitis virus recombinant attenuated strain disclosed by the invention is good in in-vitro proliferation characteristic and stable in genome heredity; the feed additive is good in safety and has no pathogenic risk to SPF chicken; the immune protection efficacy is prominent, and chicken flocks can obtain 100% protection against QX virulent virus attack after being immunized for 14 days; the method can be used for preparing vaccines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of veterinary biological products, and particularly relates to a QX type avian infectious bronchitis virus recombinant attenuated strain and a preparation method and application thereof. BACKGROUND

[0002] Avian infectious bronchitis (IB) is a major viral infectious disease faced by the global poultry industry, which has the characteristics of high infectivity, fast transmission speed and wide host range, and continuously threatens the economic benefits of the industry. The susceptible host of IB is mainly domestic chicken, and chickens of all ages are susceptible to infection, with young chicks being the most sensitive; waterfowl such as ducks and geese are mostly subclinical infections with no obvious clinical symptoms, but they can carry the virus for a long time and become a transmission medium, in addition, quail, turkey and other birds can also be infected, further expanding the transmission range of the disease. In a chicken house with high density, the virus can spread rapidly through respiratory secretions, and usually within 1-2 days, the whole flock can be infected, with an incidence rate of almost 100%. In the actual breeding scene, IB often occurs mixed infection or secondary infection with Escherichia coli, Mycoplasma gallisepticum, Newcastle disease virus (NDV) and other pathogens, which can significantly aggravate the disease and cause various complications, and the mortality rate can rise to more than 30%. In addition, IB has specific damage to the reproductive system of laying hens and breeding hens: adult laying hens after infection will have a 10%-50% decline in egg production, and the egg quality will be poor; if the chicks are infected at 1-3 weeks of age, the virus will permanently damage the development of the oviduct, and even if they recover and grow into adult chickens, they will also be unable to lay eggs normally, causing more serious long-term losses to the breeding industry.

[0003] Infectious bronchitis virus (IBV) belongs to the coronavirus family of the coronavirus genus, and is a single-stranded positive-sense RNA virus with a capsid, with a genome of about 27600 nt. At present, multiple genotypes of IBV have been detected in China, including GI-1, GI-19, GI-22, GI-23, GII-2, GIV-1, GV-1, GVI-1, etc., among which the detection rate of GI-19 (QX type) has been consistently high, becoming the dominant epidemic genotype of IBV in China and the most common strain type causing IB outbreaks in chicken flocks. QX type IBV can infect chickens of all ages: clinically, chickens show listlessness, disheveled feathers, and respiratory symptoms such as coughing, nasal discharge, and panting; the typical lesion is kidney enlargement and hemorrhage, showing characteristic "spotted kidney", and severe cases can cause mass mortality of chicks.

[0004] Vaccination is the most core and effective means for the prevention and control of IBV. The vaccines currently used for the prevention and control of IBV are divided into inactivated vaccines and attenuated live vaccines. The routine immunization of broilers and laying hens mainly uses attenuated live vaccines, and the booster immunization of laying hens and breeding hens generally uses inactivated vaccines. Due to the poor adaptability of the immune mechanism, the inactivated vaccine mainly stimulates humoral immunity and is difficult to induce strong mucosal immunity; while the attenuated live vaccine can replicate locally in the mucosa of the bird body to simulate the natural infection process, can more efficiently induce the production of mucosal antibodies and the activation of cellular immunity, and form a targeted mucosal protective barrier. The QX type IBV mainly invades the bird body through the respiratory and digestive mucosa, and mucosal immunity is the first key defense line against its infection. At present, the QX type inactivated vaccine is less used, and the QX type attenuated live vaccine is widely used in the market. However, the QXL87 strain and the LDT3-A strain, which are the most widely used QX type attenuated live vaccines, are attenuated strains obtained by the traditional method of passing through chicken embryos for multiple times. This process is time-consuming and long (usually months to years), and not only is difficult to match the characteristics of the subtypes of IBV, which are multiple and change rapidly, but also due to the lagging behind of the research and development efficiency, the vaccine strain cannot be updated in time to synchronize with the epidemic strain, which is easy to cause the antigenic difference between the two and further lead to the risk of immune escape.

[0005] The Chinese invention patent CN118620850A discloses a preparation of an avian infectious bronchitis virus attenuated strain and its application. The virulence of the QX type IBV is attenuated based on the reverse genetics of codon pair deoptimization. However, the above technical solution has obvious limitations: although the reverse genetics system of the QX strain itself is used as the basis, the gene fragments of the exogenous H120 strain are introduced for replacement, which not only increases the complexity of the technical operation, but also may cause unpredictable interactions due to the integration of the heterologous genes. At the same time, the deoptimization of this method is directed to the non-structural proteins Nsp14, Nsp15 and Nsp16, and the modification region is wide. Since these proteins are all key molecules in the replication cycle of the virus, and there is a functional synergy between them (such as the repair function of Nsp14 depends on the methylation modification of Nsp16), a large-scale modification may destroy their natural interaction network, and further lead to the risk of virulence reversion of the attenuated strain in the later stage. In addition, the base modification length of the attenuated strain in this scheme is as high as 2918 nt. The long modification fragment not only increases the economic and time costs of gene synthesis before the experiment, but also increases the difficulty of virus genome assembly, and may affect the stability of the virus. The damage to the functions of Nsp14, Nsp15 and Nsp16 will directly lead to a significant decrease in the virus replication efficiency, which is not conducive to the subsequent large-scale production of the vaccine.

[0006] In view of this, the present application provides a QX type avian infectious bronchitis virus recombinant attenuated strain and a preparation method and application thereof. SUMMARY

[0007] The present application is directed to the technical problems of long cycle and poor adaptability existing in the process of obtaining attenuated strains through multiple passages of chicken embryos, and provides a recombinant attenuated QX type avian infectious bronchitis virus and a preparation method and application thereof. The recombinant attenuated QX type avian infectious bronchitis virus obtained by the present application has good in vitro proliferation characteristics and stable genome; the safety is good, and the pathogenic risk to specific pathogen free (SPF) chickens is low; the immunoprotective efficacy is outstanding, and the chicken population can be protected by 100% after QX virulent challenge 14 days after immunization; the key fragments of the QX type IBV genome are directedly modified through the reverse genetic manipulation system and codon pair deoptimization strategy, the virulence attenuation can be accurately realized at the molecular level, the cycle from genetic modification to successful rescue of the vaccine strain is shortened to several weeks (more than 90% shorter than the traditional method), and the research and development efficiency is greatly improved.

[0008] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: a recombinant attenuated QX type avian infectious bronchitis virus, wherein the cDNA of a QX type avian infectious bronchitis virus parent strain is used as a template, amplified by PCR, and then an infectious clone plasmid is constructed by using yeast homologous recombination technology, and the recombinant plasmid carrying the N protein gene and the 3' untranslated region of the QX type avian infectious bronchitis virus is co-transfected into host cells to obtain the recombinant attenuated QX type avian infectious bronchitis virus.

[0009] In one embodiment, the nucleotide sequence of the N protein of the QX type avian infectious bronchitis virus parent strain is shown in SEQ ID NO. 1, and the nucleotide sequence of the N protein of the recombinant attenuated QX type avian infectious bronchitis virus is shown in SEQ ID NO. 2.

[0010] The present application also provides a preparation method of the recombinant attenuated QX type avian infectious bronchitis virus, which comprises the following steps: using the cDNA of the QX type avian infectious bronchitis virus as a template, amplifying by PCR to obtain an assembly template of the reverse genetic vector of the QX type avian infectious bronchitis virus, re-encoding the 637th-1008th position of the N protein coding gene by codon pair deoptimization and replacing it to the original position, and obtaining an infectious clone plasmid by homologous recombination with a recombinant backbone; using the N-3'UTR gene sequence as a template, using the specific primers shown in SEQ ID NO. 61-62 to obtain a gene fragment by PCR amplification, and performing homologous recombination with a pcDNA3.1 vector to construct a recombinant plasmid; co-transfecting the infectious clone plasmid and the recombinant plasmid into BHK-21 cells for expression to rescue the recombinant virus, and obtaining the recombinant attenuated QX type avian infectious bronchitis virus.

[0011] In an embodiment, the nucleotide sequence of the N protein coding gene re-encoded from 637th to 1008th by codon pair deoptimization is shown as SEQ ID NO. 3.

[0012] In an embodiment, the construction of the recombinant backbone comprises: sequentially connecting the linearized pYES1L vector with CMV promoter, 5' end hammerhead ribozyme sequence, double enzyme digestion site, 3' end hepatitis D virus ribozyme sequence and bGH polyA sequence to construct the recombinant vector backbone.

[0013] In an embodiment, the nucleotide sequence of the CMV promoter is shown as SEQ ID NO. 4, the hammerhead ribozyme sequence is shown as SEQ ID NO. 5, the hepatitis D virus ribozyme sequence is shown as SEQ ID NO. 6, and the bGH polyA sequence is shown as SEQ ID NO. 7.

[0014] In an embodiment, the double enzyme digestion site is BamHI and PmeI.

[0015] In an embodiment, the N-3'UTR gene sequence is shown as SEQ ID NO. 8.

[0016] Another aspect of the present application also provides the use of the QX type avian infectious bronchitis virus recombinant attenuated strain in the preparation of vaccines.

[0017] Another aspect of the present application also provides a vaccine containing the QX type avian infectious bronchitis virus recombinant attenuated strain.

[0018] Compared with the prior art, the present application has the following advantages and positive effects: (1) The present application directly takes the QX type IBV epidemic strain as the research object, and constructs a QX type specific attenuated vaccine strain through directional modification, which not only fills the research gap of QX type IBV special vaccine, but also enriches the product system of IBV vaccine, provides more targeted prevention and control options for the market, and promotes the upgrade of IBV vaccine research and development from traditional strain coverage to precise matching of epidemic strains. Compared with conventional vaccines, the recombinant vaccine strain screened in the present application is only modified to the QX type IBV genome itself, without introducing gene fragments of other strains, which maximally reduces the adverse effects such as virulence return and stability decline of the vaccine strain in the subsequent passage process, fills the research gap of QX type IBV special vaccine, enriches the product system of IBV vaccine, and provides more targeted prevention and control options for the market; the recombinant vaccine carrier is used, which can accurately distinguish between wild strains and vaccine strains, providing convenience for disease monitoring; the recombinant vaccine strain obtained by construction can provide 100% protection for chicken flocks after 14 days of immunization; the present application can realize precise modification at the viral gene level, and the operation is more efficient and fast; the cellular immunity and humoral immunity of the body can be activated at the same time, effectively avoiding the immune defects of inactivated vaccines, and the immune activation speed is fast, which can avoid the protection window period; the attenuated live vaccine does not need to be prepared through long-term passage, which avoids the problem of long time consumption of traditional methods, shortens the time from vaccine research and development to market, and improves the vaccine research and development efficiency.

[0019] (2) In view of the problem of single immune efficacy and short duration of the existing QX type IBV inactivated vaccine, the QX type attenuated live vaccine strain constructed in the present application can moderately replicate in the avian body, and simultaneously activate the humoral immunity (produce specific neutralizing antibodies) and the cellular immunity (activate T cells and other immune cells), forming a double immune barrier; such comprehensive immune response not only can more effectively resist the invasion and replication of QX type IBV, but also can prolong the immune memory time, significantly prolong the protection period compared with inactivated vaccines, reduce the frequency of supplementary immunization, and reduce the breeding operation cost and chicken flock stress.

[0020] (3) In view of the problem of long time consumption and low efficiency of traditional passage attenuation method for developing QX type IBV vaccine, the present application combines reverse genetic manipulation system and codon pair deoptimization strategy, and through directional modification of key fragments of QX type IBV genome (without relying on blind transmission), the virulence attenuation can be accurately realized at the molecular level, the period from genetic modification to successful rescue of the vaccine strain is shortened to several weeks (more than 90% shorter than the traditional method), which greatly improves the research and development efficiency; at the same time, the present application can quickly adapt to the genome sequence of new variant strains, providing technical support for emergency vaccine research and development, and solving the core pain point of traditional method in response to variation lag.

[0021] (4) For the problem of IBV live vaccine passage attenuation and virulence return, the recombinant attenuated vaccine strain constructed by the application directly realizes controllable attenuation of virus virulence at the molecular level, without relying on repeated in vitro passage screening in traditional technology, can maximize the reduction of the opportunity of random mutation of virus genome in the passage process while ensuring the immunogenicity of the vaccine strain, fundamentally cuts off the molecular basis of virulence return caused by random mutation, and further effectively guarantees the genetic stability and biological safety of the recombinant vaccine strain in production and clinical application.

[0022] (5) For the problem that the existing IBV vaccine cannot distinguish between immune and wild virus infection, when the codon pair of the N protein fragment is de-optimized, the base sequence of the same segment of the wild strain can be effectively distinguished; in clinical application, only nucleic acid needs to be collected from the pharyngeal swab or anal swab of the chicken flock, and the RT-qPCR detection or gene sequencing for the marker can quickly distinguish whether the chicken flock is vaccinated or infected with wild virus, providing precise technical tools for the epidemiological monitoring, epidemic tracing and breeding end purification of QX type IBV.

[0023] (6) For the problem that the existing IBV vaccine modification technology relies on exogenous genes and has low modification efficiency, the application modifies the QX type IBV strain genome accurately, only de-optimizes part of the fragments of the structural protein (N protein), does not introduce any exogenous gene fragments, can retain the original immunogenicity and moderate replication ability of the strain, and further shortens the virus rescue time, improves the rescue efficiency, and maximizes the risk of virulence return and stability decrease in the passage process of the strain, which lays a more reliable foundation for the safety and large-scale production of the vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The agarose gel electrophoresis map of the QX parent strain genomic DNA fragment provided by the embodiment of the application, M represents Trans2K Plus II DNA Marker, QXF1 represents the first fragment PCR product with QX parent strain cDNA as a template, QXF2 represents the second fragment PCR product with QX parent strain cDNA as a template, QXF3 represents the third fragment PCR product with QX parent strain cDNA as a template, QXF4 represents the fourth fragment PCR product with QX parent strain cDNA as a template, QXF5 represents the fifth fragment PCR product with QX parent strain cDNA as a template, QXF6 represents the sixth fragment PCR product with QX parent strain cDNA as a template, and QXF7 represents the seventh fragment PCR product with QX parent strain cDNA as a template; Figure 2PCR identification result of pcDNA3.1-CMV-N-3'UTR recombinant plasmid provided by the embodiment of the application, M represents Trans2K Plus II DNA Marker, and N-3'UTR represents PCR identification of pcDNA3.1-CMV-N-3'UTR recombinant plasmid; Figure 3 Recombinant strain modification region RT-PCR agarose gel electrophoresis chart, M represents Trans2K Plus II DNA Marker, 1 represents RT-PCR identification of S-1 target sequence, 2 represents RT-PCR identification of S-2 target sequence, 3 represents RT-PCR identification of N-1 target sequence, and 4 represents RT-PCR identification of N-2 target sequence; Figure 4 Deoptimization site PCR agarose gel electrophoresis chart, M represents Trans2K Plus II DNA Marker, S-1 from left to right is respectively PCR identification of 6th-10th generation QX-S-1-deopt recombinant strain, S-2 from left to right is respectively PCR identification of 6th-10th generation QX-S-2-deopt recombinant strain, N-1 from left to right is respectively PCR identification of 6th-10th generation QX-N-1-deopt recombinant strain, and N-2 from left to right is respectively PCR identification of 6th-10th generation QX-N-2-deopt recombinant strain; Figure 5 Proliferation curve of different strains in SPF embryos in the embodiment of the application; Figure 6 Dissection chart of SPF chicken inoculated with QX strain and died in the embodiment of the application; Figure 7 Dissection chart of SPF chicken inoculated with QX-N-1-deopt strain and died in the embodiment of the application; Figure 8 Dissection chart of SPF chicken inoculated with QX-N-2-deopt strain and died in the embodiment of the application; Figure 9 Dissection chart of SPF chicken inoculated with PBS and died in the embodiment of the application; Figure 10 Survival curve of safety evaluation of different strains in the embodiment of the application; Figure 11 Survival curve of efficacy evaluation of different strains in the embodiment of the application. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0026] Yeast homologous assembly technology (TAR) can realize precise assembly of large fragment DNA by virtue of the high-efficiency homologous recombination in Saccharomyces cerevisiae to repair double-stranded DNA break, and the highest assembly efficiency can reach megabase (Mb) level. Codon optimization, as one of the key technologies for gene expression regulation, has important application potential in the field of viral vaccine research and development. The core principle is that different species have inherent preference for the usage frequency of synonymous codons (i.e. different codons encoding the same amino acid), and when the synonymous codon usage pattern of an exogenous gene is highly matched with the preference of a host cell, the transcription and translation efficiency of the exogenous gene will be significantly improved, and finally the protein expression level is improved. If this principle is applied reversely to IBV modification: by adjusting the synonymous codon of the key gene of the virus in a targeted manner, the matching degree of the codon preference between the virus and the host (chicken cell) is reduced, and the expression level of the viral protein is moderately reduced, and then the biological activity (such as the replication ability) of the virus is weakened - this process is expected to weaken the virulence of the virus while retaining its immunogenicity (i.e. the core antigen structure that stimulates the host immune response). Compared with the blindness and inefficiency of the traditional “chick embryo multiple passage attenuation”, the present application combines the virus rescue technology of reverse genetics system and the codon pair deoptimization strategy, and can precisely and controllably modify IBV at the molecular level, providing an innovative technical path for developing safer and more efficient new IBV vaccines.

[0027] The present application provides a preparation method and application of a QX type avian infectious bronchitis virus recombinant attenuated strain. The present application uses the cDNA of the QX type IBV parent strain as a template to amplify and synthesize a genomic assembly template by PCR technology, uses yeast homologous recombination (TAR) technology to construct an infectious clone (i.e. reverse genetic vector) containing a complete QX type IBV genome, co-transfects the infectious clone and a eukaryotic expression plasmid (named pcDNA3.1-N-3'UTR) carrying the IBV nucleocapsid protein (N protein) gene and 3' untranslated region (3'UTR) into BHK-21 cells, inoculates the cell culture into chicken embryos for passage after transfection, and finally realizes the rescue of the recombinant virus, and selects the QX type avian infectious bronchitis virus recombinant attenuated strain QX-N-2-deopt.

[0028] The present application is directed to precise genetic modification research of the QX strain itself genome, according to the codon usage bias of the original chicken and other factors, five gene fragments corresponding to the two key structural proteins S protein and N protein of the virus are respectively subjected to codon pair deoptimization modification, which can not only avoid the potential risks brought by the introduction of foreign genes, but also can maintain the replication ability of the virus while ensuring the attenuation effect by limiting the modification region and shortening the modification length, which has important theoretical significance and practical application value. The reverse genetics technology constructed based on TAR technology can quickly and accurately modify IBV at the gene level to achieve specific research goals, effectively solving the pain point of the traditional chicken embryo passage method that the long cycle hinders the development of vaccines, at the same time, the risk of random variation caused by high-frequency mutation of RNA virus can be reduced through directional modification, which can greatly shorten the vaccine development cycle and significantly improve the efficiency of scientific research and industrialization.

[0029] The specific technical solutions of the present application will be described in detail below in combination with examples.

[0030] Example 1: Parental strain genome sequencing and determination of codon pair deoptimization sites The isolation method of the QX type IBV strain used in the present application includes: using MagaBio plus virus DNA / RNA purification kit (Hangzhou Boyi) to extract the viral RNA genome of the strain, then using RevertAid Reverse Transcriptase (Thermofisher, America) with random primers to reverse transcribe the extracted viral RNA genome into cDNA; using the obtained cDNA as a template, the designed primers shown in Table 1 are used to amplify the full-length genome fragment of the virus by PCR; the PCR product is purified by Gel Extraction Kit (Omega, China), and then T-Vector pMD TM 19 kit (Takara, China) is used for T vector directional cloning; finally, positive clones are selected and sent to Shengong Bioengineering (Shanghai) Co., Ltd. for Sanger sequencing.

[0031] Table 1: PCR amplification primer sequence

[0032] According to the Sanger sequencing results, five gene sites of S-1 (142-1296 bp), S-2 (1297-2400 bp), S-3 (2401-3441 bp) on the key structural protein spike protein (S protein) and N-1 (112-636 bp), N-2 (637-1008 bp) on the nucleocapsid protein (N protein) are codon pair de-optimized, avoiding key sites (such as S protein cleavage site and protein two-end conserved sequence). The gene sequence is synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd. Among them, the DNA sequence of the S-1 coding region after codon pair de-optimization is as shown in SEQ ID NO. 9, the DNA sequence of the S-2 coding region after codon pair de-optimization is as shown in SEQ ID NO. 10, the DNA sequence of the S-3 coding region after codon pair de-optimization is as shown in SEQ ID NO. 11, the DNA sequence of the N-1 coding region after codon pair de-optimization is as shown in SEQ ID NO. 12, and the DNA sequence of the N-2 coding region after codon pair de-optimization is as shown in SEQ ID NO. 3.

[0033] Example 2 Construction of infectious clone and recombinant plasmid Based on the QX parent strain genome sequencing results, the segmented amplification primers shown in Table 2 were designed and synthesized, and the whole genome sequence was amplified by PCR to obtain 7 QX parent strain genomic DNA fragments, as shown in Table 2, named QXF1~QXF7, respectively. At the same time, the five codon pair de-optimized sequences were replaced into the original site by overlap PCR to replace the original sequence, as the core template for subsequent experiments. Figure 1

[0034] ​The reverse genetic vector construction method comprises the following steps: taking a linearized pYES1L vector as a basic framework, sequentially connecting a CMV promoter, a 5' end hammerhead ribozyme sequence, a double enzyme digestion site, a 3' end hepatitis D virus (HDV) ribozyme sequence and a bGH polyA sequence to form a recombinant vector skeleton which can be directly obtained by double enzyme digestion, wherein the double enzyme digestion site is BamHI and PmeI; then mixing the corresponding IBV genomic DNA fragment with the linearized vector obtained by enzyme digestion, and transforming into MaV203 yeast competent cells according to the reagent kit instructions; culturing the yeast for 2-3 days after transformation, picking suspected positive colonies for expansion culture; using the E.Z.N.A. Yeast Plasmid Mini Kit of China Omega Company to extract the yeast plasmid, and verifying the plasmid recombination correctness by PCR; electrically transforming the yeast colonies positive in PCR verification into E. coli TOP10 competent cells according to the reagent kit standard process, picking positive clones for expansion culture again, and saving the plasmid glycerol bacteria after sub-PCR sequencing is correct, so as to obtain five reverse genetic vectors, namely infectious clone plasmids, which are named as pYES1L-QX-S-1-deopt, pYES1L-QX-S-2-deopt, pYES1L-QX-S-3-deopt, pYES1L-QX-N-1-deopt and pYES1L-QX-N-2-deopt.

[0035] Table 2 Sub-fragment amplification primer sequences

[0036] Taking the N-3'UTR gene sequence as a template, the specific primers shown in Table 3 are designed, and the N-3'UTR gene sequence is amplified by PCR. The amplified gene fragment is subjected to homologous recombination with a pcDNA3.1 vector to construct a pcDNA3.1-CMV-N-3'UTR recombinant plasmid. The plasmid PCR identification result is shown in Table 4, and the pcDNA3.1-CMV-N-3'UTR recombinant plasmid is co-expressed with the infectious clone in BHK-21 cells to assist the rescue of IBV virus. Figure 2

[0037] Table 3 Specific primer sequences

[0038] Example 3 Rescue of recombinant virus ​The plasmids pYES1L-QX-S-1-deopt, pYES1L-QX-S-2-deopt, pYES1L-QX-S-3-deopt, pYES1L-QX-N-1-deopt, pYES1L-QX-N-2-deopt, and pcDNA3.1-CMV-N-3'UTR were extracted using an Omega endotoxin-free plasmid extraction kit (D6922-04), and the DNA concentrations of the plasmids were calibrated after completion. The BHK-21 cells were digested and inoculated into a 6-well plate, and the culture medium was replaced before cell transfection. The above five infectious clone plasmids were co-transfected with the pcDNA3.1-CMV-N-3'UTR plasmid using Lipofectamine 3000 transfection reagent. The culture medium was replaced 8 hours after transfection, and the cells were cultured. After 48 hours of BHK-21 cell transfection, the cell samples were stored in a -80°C freezer, repeatedly frozen and thawed three times, and then centrifuged to collect the supernatant. 2 mL of supernatant was taken using a disposable syringe and inoculated into the allantoic cavities of five 10-day-old SPF chicken embryos (note: chicken embryos that died within 24 hours were determined to be caused by improper operation and were excluded). After blind transmission of the chicken embryos for three generations, the recombinant virus liquid was collected. The nucleic acid of the obtained recombinant virus liquid was extracted, and positive results were obtained by RT-qPCR detection and verification. The experimental results showed that, except for the pYES1L-QX-S-3-deopt infectious clone corresponding to the recombinant virus rescue failure, the other four infectious clones successfully obtained recombinant viruses, and the failure reasons were as follows: after codon optimization of the S-3 region, the gene expression level of the fragment was significantly decreased, and the S-3 region contained the key functional region of the S protein (directly affecting virus adsorption and invasion of host cells), and the insufficient expression amount caused fatal inhibition to the virus replication process, ultimately leading to virus rescue failure. The four recombinant viruses successfully rescued were named QX-S-1-deopt, QX-S-2-deopt, QX-N-1-deopt, and QX-N-2-deopt. Using the PrimeScript One Step RT-PCR Kit Ver.2 kit, the gene modification regions of the four recombinant viruses were amplified and sequenced using the positive virus nucleic acid as a template. The agarose gel electrophoresis results are shown in Figure 3 The sequencing results showed that the amplified fragments were the base sequences after codon optimization, and therefore it was confirmed that the rescued strains were the target optimized strains (non-wild strains), and the possibility of cross contamination was excluded.

[0039] Example 4: Genetic stability identification of recombinant vaccine strains The four recombinant vaccine strains (QX-S-1-deopt, QX-S-2-deopt, QX-N-1-deopt, and QX-N-2-deopt) obtained in Example 3 were respectively continuously passaged on 10-day-old SPF chicken embryos, and were passed to the 10th generation; the RNA genomes of the 6th, 7th, 8th, 9th, and 10th generation virus liquids of each recombinant virus were extracted, and cDNA was synthesized through reverse transcription, wherein the QX-S-1-deopt, QX-S-2-deopt, QX-N-1-deopt, and QX-N-2-deopt strains were respectively subjected to PCR identification and sequencing using the primer pairs S-1-F / R, S-2-F / R, N-1-F / R, and N-2-F / R described in Table 4, and the detection results are shown in FIG. 6, wherein the 6th-10th generation cDNA was used as a template from left to right, and the expected positive bands were amplified for each passage of the four recombinant viruses, and the sequencing results were completely consistent with the target sequence, and no genetic deletion, insertion, or recombination variation was detected, indicating that the four recombinant vaccine strains constructed in the application can be stably inherited during continuous passage on SPF chicken embryos. Figure 4

[0040] Table 4: Detection primer sequences

[0041] Example 5: Titer test and growth curve determination of recombinant vaccine strains 5.1 Titer test of recombinant vaccine strains The EID 50 (50% chicken embryo infectious dose) method was used to quantitatively determine the titer of the four recombinant vaccine strains (QX-S-1-deopt, QX-S-2-deopt, QX-N-1-deopt, and QX-N-2-deopt), and the specific method was as follows: healthy 10-day-old SPF chicken embryos were selected, and QX-S-1-deopt vaccine strain was taken as an operation example, 25 chicken embryos were randomly divided into 5 groups for parallel experiments, 5 chicken embryos in each group; the QX-S-1-deopt virus liquid was diluted by 10 times in gradient with sterile saline, and 10 -5 , 10 -6 , 10 -7 , 10 -8 four dilutions were prepared; four chicken embryos in each group were inoculated with 0.1 mL of virus liquid of the corresponding dilution in the allantoic cavity, and the remaining one chicken embryo was inoculated with 0.1 mL of sterile saline as a negative control group; after inoculation, the chicken embryos were incubated in an incubator for 6 days, and the EID 50 ​Potency: Calculated, the potency of the four recombinant vaccine strains QX-S-1-deopt, QX-S-2-deopt, QX-N-1-deopt, and QX-N-2-deopt were 10. -6.625 / 0.1mL, 10 -7.5 / 0.1mL, 10 -7.5 / 0.1mL, 10 -7.625 / 0.1mL.

[0042] 5.2 Determination of growth curve of recombinant vaccine strain With a titer of 10 2 EID 50 QX-S-1-deopt, QX-S-2-deopt, QX-N-1-deopt, and QX-N-2-deopt recombinant virus dilutions, as well as laboratory-preserved QX parental strain virus dilutions, were inoculated into the allantoic cavity of 18 10-day-old SPF chicken embryos. Allantoic fluid was collected from 3 chicken embryos at six time points: 12h, 24h, 36h, 48h, 60h, and 72h. RNA was extracted from the allantoic fluid and then amplified by RT-qPCR using primers IBV-F and IBV-R as shown in Table 4. The standard curve was y = -3.23x + 41.21 (R). 2 =0.999), calculate the copy number of each viral RNA, and plot the growth curve based on the copy number, such as Figure 5 As shown in the figure, the replication rates of all four recombinant vaccine strains were lower than those of the QX parent strain, indicating that the codon pair deoptimization strategy can act on the viral replication system and slow down viral replication. Specifically, the replication rates of QX-N-1-deopt and QX-N-2-deopt were slightly lower than those of the parent strain, while the replication rates of QX-S-1-deopt and QX-S-2-deopt were significantly lower than those of the parent strain. Considering subsequent production costs, the QX-N-1-deopt and QX-N-2-deopt recombinant vaccine strains were ultimately selected for subsequent safety and efficacy evaluations.

[0043] Example 6 Safety assessment of recombinant vaccine strain Select 40 1-day-old SPF chickens, randomly divided into 4 groups, 10 in each group, fed in isolator, given sufficient feed and drinking water; four groups of chickens were inoculated by eye drops route PBS, QX parent strain, recombinant vaccine strain QX-N-1-deopt, QX-N-2-deopt, respectively, and observed for 14 days after immunization, among them, PBS factory is shanghai shenguo biological engineering co., ltd., and the item number is E607008-0500; the virus inoculation group was inoculated at 10 times the recommended inoculation dose of IBV live vaccine, and the observation indexes included the state of mind of the chickens, the fecal characteristics, the respiratory symptoms, etc., and if any dead chickens appeared during the period, necropsy should be performed in time. The specific safety evaluation scheme is shown in Table 5, the safety evaluation results are shown in Table 6, and the chicken dissection diagram is shown in Figures 6-9

[0044] Table 5 Safety evaluation scheme of recombinant vaccine strain

[0045] Table 6 Safety evaluation results of recombinant vaccine strain

[0046] Referring to Table 6 and Figures 6-9 During the 14-day observation period, the SPF chickens in each group performed as follows: in the PBS inoculation group, all the chickens survived, and there were no any symptoms of disease throughout the whole process, and the health status was good; in the QX parent strain inoculation group, 100% of the chickens were sick, and there were obvious respiratory symptoms, and all the chickens died within 14 days, and the necropsy of the dead chickens showed that the kidneys were swollen, indicating that the QX parent strain had very strong virulence; in the QX-N-1-deopt inoculation group, 7 / 10 chickens were sick, and there were different degrees of respiratory rales, and finally 4 / 10 survived, and the necropsy of the dead chickens showed that the kidneys were swollen, indicating that the QX-N-1-deopt vaccine strain had certain pathogenic risk; in the QX-N-2-deopt inoculation group, 10 / 10 chickens all survived, and all the chickens were in normal state during the observation period, and there were no any clinical symptoms, and the safety reached 100%, and the necropsy after 14 days showed that the kidneys were not swollen. The survival curves of chickens inoculated with different strains are shown in Figure 10 As can be seen from the figure, the virulence of the two recombinant vaccine strains (QX-N-1-deopt and QX-N-2-deopt) was significantly weaker than that of the parent strain QX, indicating that the codon-optimized strategy could effectively achieve virulence attenuation, and the QX-N-2-deopt vaccine strain had the best safety, so it was determined to select this vaccine strain for subsequent efficacy evaluation.

[0047] Example 7 Efficacy evaluation of recombinant vaccine strain ​Select 20 1-day-old SPF chickens, randomly divided into 2 groups, 10 each, reared in isolators, given sufficient feed and water; two groups of chickens are immunized by eye drops, one group is inoculated with 1 feather QX-N-2-deopt strain (dose 0.1 mL, containing 10 3.5 EID 50 ), the other group is inoculated with the same volume of PBS as a control; 14 days after immunization, both groups of chickens are challenged with 10 4.0 EID 50 QX parent strain, the observation indicators include the mental state of chickens, fecal characteristics, respiratory symptoms, etc., if there is chicken death during the period, it needs to be dissected immediately. The specific efficacy evaluation scheme is shown in Table 7, and the efficacy evaluation results are shown in Table 8, and the survival curves of different strains are shown in Figure 11 .

[0048] Table 7 Efficacy evaluation scheme of recombinant vaccine strain

[0049] Table 8 Efficacy evaluation results of recombinant vaccine strain

[0050] Referring to Table 8 and Figure 11 , the SPF chickens in each group showed the following performance within the 14-day observation period after QX parent strain challenge: in the negative control group immunized with PBS, the morbidity of the chicken population reached 100%, the chickens had different degrees of respiratory rales, occasionally had poor spirits, and even died, with a mortality rate of 80%, fully demonstrating the strong pathogenicity of the parent strain; in the group pre-immunized with QX-N-2-deopt vaccine strain, the chickens had no clinical symptoms throughout the challenge, and all individuals remained normal and healthy, with an immune protection effect of 100%.

[0051] In summary, the QX-N-2-deopt vaccine strain screened by the application has good in vitro proliferation characteristics and stable genome; it is safe and has no pathogenic risk to SPF chickens; it has outstanding immune protection efficacy, and can provide 100% protection against QX virulent challenge to the chicken population 14 days after immunization.

[0052] The above-described embodiments only describe the preferred embodiments of the application and do not limit the scope of the application. Without departing from the design spirit of the application, various modifications, modifications, evolution, improvements of the technical solutions of the application made by those skilled in the art shall fall within the protection scope of the application.

Claims

1. A recombinant attenuated strain of QX-type avian infectious bronchitis virus, characterized in that, The recombinant attenuated strain of QX-type avian infectious bronchitis virus was obtained by using the cDNA of the parent strain of QX-type avian infectious bronchitis virus as a template, amplifying it by PCR, and constructing an infectious cloning plasmid containing the complete genome of QX-type avian infectious bronchitis virus using yeast homologous recombination technology. After co-transfection of host cells with the recombinant plasmid carrying the N protein gene and 3' untranslated region of the QX-type avian infectious bronchitis virus, genetic rescue was performed.

2. The recombinant attenuated strain of QX-type avian infectious bronchitis virus according to claim 1, characterized in that, The nucleotide sequence of the N protein of the parent strain of QX-type avian infectious bronchitis virus is shown in SEQ ID NO.1, and the nucleotide sequence of the N protein of the recombinant attenuated strain of QX-type avian infectious bronchitis virus is shown in SEQ ID NO.

2.

3. The method for preparing the recombinant attenuated strain of QX-type avian infectious bronchitis virus according to claim 1, characterized in that, Includes the following steps: Using the cDNA of QX-type avian infectious bronchitis virus as a template, the assembly template of the QX-type avian infectious bronchitis virus reverse genetic vector was obtained by PCR amplification. The N protein coding gene was re-encoded from position 637 to position 1008 by codon pair optimization and then replaced in place. After homologous recombination with the recombinant backbone, an infectious cloning plasmid was obtained. Using the N-3'UTR gene sequence as a template, the gene fragment obtained by PCR amplification with the specific primers shown in SEQ ID NO.61~62 was homologously recombinated with the pcDNA3.1 vector to construct a recombinant plasmid; The infectious cloning plasmid and the recombinant plasmid were co-transfected into BHK-21 cells for expression, thereby rescuing the recombinant virus and obtaining the recombinant attenuated strain of QX-type avian infectious bronchitis virus.

4. The method for preparing a recombinant attenuated strain of QX-type avian infectious bronchitis virus according to claim 3, characterized in that, The nucleotide sequence after recoding positions 637 to 1008 of the N protein coding gene by codon pair optimization is shown in SEQ ID NO.

3.

5. The method for preparing a recombinant attenuated strain of QX-type avian infectious bronchitis virus according to claim 3, characterized in that, The construction of the recombinant backbone includes: sequentially linking the linearized pYES1L vector to the CMV promoter, the 5' hammerhead ribozyme sequence, the double restriction site, the 3' hepatitis D virus ribozyme sequence, and the bGH polyA sequence to construct the recombinant vector backbone.

6. The method for preparing a recombinant attenuated strain of QX-type avian infectious bronchitis virus according to claim 5, characterized in that, The nucleotide sequence of the CMV promoter is shown in SEQ ID NO.4, the hammerhead ribozyme sequence is shown in SEQ ID NO.5, the hepatitis D virus ribozyme sequence is shown in SEQ ID NO.6, and the bGH polyA sequence is shown in SEQ ID NO.

7.

7. The method for preparing a recombinant attenuated strain of QX-type avian infectious bronchitis virus according to claim 5, characterized in that, The double restriction sites are BamHI and PmeI.

8. The method for preparing a recombinant attenuated strain of QX-type avian infectious bronchitis virus according to claim 3, characterized in that, The N-3'UTR gene sequence is shown in SEQ ID NO.

8.

9. The use of the recombinant attenuated strain of QX type avian infectious bronchitis virus according to claim 1 or 2 in the preparation of vaccines.

10. A vaccine, characterized in that, The vaccine contains a recombinant attenuated strain of QX type avian infectious bronchitis virus as described in claim 1 or 2.

Images