CN110684781B

CN110684781B - Type 3 duck hepatitis A virus mutant gene ISA-A117C-T1142A and construction method thereof

Info

Publication number: CN110684781B
Application number: CN201910551112.9A
Authority: CN
Inventors: 汪铭书; 文兴建; 程安春; 吴莉萍
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2021-05-28
Anticipated expiration: 2039-06-24
Also published as: CN110684781A

Abstract

The invention discloses a 3-type duck hepatitis A virus mutant gene ISA-A117C-T1142A and a construction method, wherein the mutant gene ISA-A117C-T1142A is characterized in that the 117 th nucleotide of 5' UTR of a 3-type duck hepatitis A virus virulent strain genome is mutated from A to C; the 1142 th nucleotide is mutated from T to A, so that the 164 th amino acid of the virus VP0 protein is mutated from tyrosine of parent strain to asparagine. The mutant gene virus strain has the same antigenicity as the parent strain, has higher proliferation efficiency and virus titer on duck embryos than the parent strain, can be proliferated on chicken embryos, and has good immunogenicity and genetic stability. The pathogenicity of the mutant gene virus strain to the ducklings is obviously reduced, and the mutant gene virus strain can be replicated in the ducklings but does not cause diseases to the ducklings, so that an important foundation is laid for the virus pathogenic mechanism and vaccine development. In conclusion, the 3-type duck hepatitis A virus mutant gene ISA-A117C-T1142A virus strain is an ideal vaccine candidate strain and can be used for researching the tropism and weakening mechanism of a 3-type duck hepatitis A virus host, and the application prospect is wide.

Description

Type 3 duck hepatitis A virus mutant gene ISA-A117C-T1142A and construction method thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a 3-type duck hepatitis A virus mutant gene ISA-A117C-T1142A and a construction method thereof.

Background

Duck Viral Hepatitis (DVH) is an acute, highly contagious disease caused by Duck Hepatitis Virus (DHV) infection of ducklings. At present, the disease exists in main duck breeding areas in the world, has the characteristics of intermittent outbreak, local epidemic and the like, and is one of the main diseases harming the duck breeding industry. The disease mainly attacks ducklings within four-week age, and has the characteristics of acute disease onset, rapid spread, short disease course, high mortality and the like; clinically, the duck is mainly characterized in that the young duck is cramped before death, turns back with the head facing the back and is in the form of 'arcus retroflexus', and pathological changes mainly include hepatomegaly, inflammation and a large amount of hemorrhagic spots seen in a caesarean section. The disease is mainly caused by Duck Hepatitis A Virus (DHAV) belonging to the genus avian hepatis of the family picornaviridae. DHAV has three serotypes, type 1, type 2 and type 3. In recent years, the DHAV which is popular in China is mainly duck hepatitis A virus type 1 (DHAV-1) and duck hepatitis A virus type 3 (DHAV-3).

The reverse genetic operation technology is an important platform for developing virus molecular biology research, can perform artificial operations such as gene knockout, site-directed mutagenesis and the like on RNA virus genomes in vitro, can play an important role in explaining virus pathogenic mechanisms and vaccine development, and has the advantage of being shorter than a natural mutagenesis period. The key of the traditional RNA virus infectious cloning method is to obtain full-length cDNA fragment clone of virus genome, and the virus genome needs to be cloned into a proper vector after being converted into cDNA, in order to avoid the problem of instability of virus sequences in bacteria, researchers usually adopt a sectional cloning method, connect small fragments into large fragments, and finally obtain the full-length cDNA clone by the enzyme digestion connection method of the large fragments. However, the restriction of enzyme cutting site selection is more, the efficiency of connecting a plurality of large fragments in vitro is lower, and the cDNA cloning of other replicase genes has instability in bacteria, so the whole process of obtaining the virus genome full-length cDNA is not only troublesome in operation steps, but also long in time consumption and low in success rate, and meanwhile, the full-length cDNA of some viruses cannot be cloned or can be cloned into a vector but is easy to have variation in host bacteria so as to cause the virus to be unsuccessfully rescued. Currently, a technique called "Infectious Subgenomic replicons" (Infectious Subgenomic replicons) has been demonstrated to enable artificial rescue of single-stranded positive-strand RNA viruses in mammalian or mosquito cells, and has been applied in reverse genetics studies of viruses such as Japanese encephalitis virus, West Nile virus, Zika virus, yellow fever virus, dengue virus, and human saxiki virus. The technology is a novel 'bacteria-free' reverse genetics method, does not need to obtain a virus full-length cDNA plasmid and obtain a virus RNA transcript in vitro, and can directly save the virus by transfecting a DNA fragment with a homologous region. Specifically, the technical route adopted by the "infectious subgenomic replicon" is as follows: firstly, generating overlapped non-infectious subgenomic DNA fragments containing the whole virus genome by a PCR method, wherein the number of the overlapped subgenomic replicons can be 3 to 10, adjacent replicons have an overlapping region of about 100bp, and simultaneously, the 5 'end of the first fragment and the 3' end of the last fragment are respectively connected with a Cytomegalovirus early promoter (pCMV) sequence, a Hepatitis Delta Virus Ribozyme (HDVR) sequence and a simian vacuolating virus early polyadenylation signal (SV40 early mRNA polyadenylation signal, SV40pA) sequence which can help the subgenomic replicons to start transcription after being mixedly transfected into susceptible cells and spontaneously recombine by utilizing a homologous recombination mechanism of host cells to form an infectious complete virus transcript, which then leads to the replication and proliferation of the virus, finally obtaining the infectious rescue virus.

The prevention and control of DHAV in the current market mainly uses DHAV-1 attenuated vaccine, but lacks high-efficiency DHAV-3 live vaccine, and the research and development of novel molecular marker vaccine suitable for the DHAV-3 epidemic situation in China is imminent; meanwhile, basic researches such as virus genes and key sites of DHAV host tropism and virulence change can provide theoretical basis for preventing and treating duck hepatitis, and virus strains with host tropism and virulence change are urgently needed to be obtained.

Disclosure of Invention

The technical problem to be solved by the invention is that a molecular marker type 3 duck hepatitis A virus vaccine candidate strain is obtained by utilizing a gene modification platform; meanwhile, the obtained virus strain with virulence change can provide basic materials for researching virus genes, key sites and the like of DHAV virulence change.

In order to achieve the technical purpose, the invention is specifically realized by the following technical scheme:

a3-type duck hepatitis A virus mutant gene ISA-A117C-T1142A, the mutant gene ISA-A117C-T1142A mutates A to C from A to 117 th nucleotide of 5' UTR of 3-type duck hepatitis A virus virulent strain genome; the 1142 th nucleotide is mutated from T to A, so that the 164 th amino acid of the virus VP0 protein is mutated from tyrosine of parent strain to asparagine.

The type 3 Duck Hepatitis A Virus type 3 (DHAV-3) is preserved in China center for type culture Collection with the preservation number of CCTCC NO: v201305, deposit address: in the Wuhan university school of Wuhan 299 in the Wuchang area of Wuhan city, Hubei province, the preservation date is as follows: year 2013, month 3, and day 25.

G of 3403 th nucleotide of the mutant gene genome is mutated into T, and the T is used as a genetic marker of infectious clone, and the mutant gene can be distinguished from parent strains and wild strains by combining a PCR method and DNA sequencing.

The mutant gene virus strain containing the genetic marker can be stably propagated and passaged on 9-day-old duck embryos like parent viruses, the virus titer is higher, the mutant gene virus strain can be stably propagated and passaged on 9-day-old chick embryos, the genetic stability is good, and no mutation occurs after 10 continuous passages.

Compared with the parental virus, the mutant gene virus strain containing the genetic marker has the advantages that the mutant gene keeps the original immunogenicity but obviously reduces the pathogenicity to the ducklings, and the mutant gene is successfully copied in the ducklings but does not cause diseases to the ducklings.

In another aspect of the invention, the application of the mutant gene in preparing a duck hepatitis virus type 3 vaccine is provided.

Meanwhile, the mutant gene can also be used in basic researches on virus genes and key sites of duck hepatitis virus host tropism change, virulence attenuation mechanisms and the like.

In another aspect of the present invention, there is provided a method for constructing the mutant genovirus strain described above, comprising the steps of:

(1) uses the technology of infectious subgenomic replicon to construct the molecular marker type 3 duck hepatitis A virus mutant gene, it is characterized in that the whole genome of the parental virus is divided into three fragments with similar sizes (2.6kb, 2.6kb and 2.7kb) for PCR amplification, adding a Cytomegalovirus early promoter (pCMV) sequence at the 5 'end of the first fragment of the viral genome and introducing a mutation site in the 5' UTR and VP0 genes, introducing a nonsense mutation as a genetic marker site in the 2A gene of the second fragment, adding a Hepatitis Delta Virus Ribozyme (HDVR) sequence and a simian vacuolating virus early mRNA polyadenylation signal (SV40 early mRNA polyadenylation signal, SV40pA) sequence at the 3' end of a third fragment of a virus genome to obtain three DNA fragments to form an infectious subgenomic replicon of a type 3 duck Hepatitis A virus mutant gene ISA-A117C-T1142A;

(2) the constructed 3-type duck hepatitis A virus mutant gene ISA-A117C-T1142A 'infectious subgenomic replicon' is mixed with a transfection reagent to transfect duck embryo fibroblasts, the replicon is transcribed in a host cell and spontaneously recombines by utilizing a homologous recombination mechanism of the cell to form a complete virus transcript with infectivity, so that the virus is replicated and proliferated, and finally, the 3-type duck hepatitis A virus mutant gene ISA-A117C-T1142A virus strain containing genetic markers is obtained.

Further, the first fragment and the third fragment contain 74 and 83 base pair overlapping regions with the second fragment, respectively.

The invention has the beneficial effects that:

1. compared with the time period of mutagenesis by a natural method and the traditional reverse genetic operation technology, the time period of obtaining the 3-type duck hepatitis A virus mutant gene by using the method is shorter, and the process of cultivating the 3-type duck hepatitis A virus attenuated vaccine strain and researching the pathogenic mechanism of the virus can be accelerated.

2. The mutant gene ISA-A117C-T1142A virus strain obtained in the invention has antigenicity similar to that of parent strain and can keep stable genetic characteristics in the process of continuous passage, so the mutant gene virus strain can be used as a candidate vaccine strain for immunizing type 3 duck hepatitis A virus; meanwhile, the strain has higher proliferation efficiency and virus titer on duck embryos than a parent strain, and can improve the yield and reduce the cost if being used for vaccine production; in addition, the mutant gene virus strain can be propagated in the chick embryo, and the source of the cultured virus material is wider. If the method is used in vaccine production, the yield can be improved, the cost can be reduced, and the safety and the application range are higher.

3. The mutant gene ISA-A117C-T1142A virus strain obtained in the invention has obviously reduced pathogenicity to ducklings, can be replicated in bodies of the ducklings but does not cause diseases to the ducklings, so the mutant gene virus strain can be used as a candidate vaccine strain for 3-type duck hepatitis A virus immunization, and has better safety.

4. Compared with the parental strain, the mutant gene ISA-A117C-T1142A virus strain obtained in the invention has weakened virulence and can be propagated and changed in chick embryos, and can be used for researching virus genes and key sites of duck hepatitis virus host tropism and virulence change.

Drawings

FIG. 1 is a schematic diagram of construction of a molecular marker mutant gene ISA-A117C-T1142A "infectious subgenomic replicon" based on type 3 duck hepatitis A virus as a framework;

FIG. 2 is a diagram showing the result of duck embryo fibroblast transfected by type 3 duck hepatitis A virus mutant gene ISA-A117C-T1142A "infectious subgenomic replicon";

FIG. 3 shows the sequencing result of the 3403 rd nucleotide molecular genetic marker locus of the mutant gene virus genome;

FIG. 4 shows the result of nucleotide sequencing of the 117 th nucleotide target mutation site of the mutant gene virus genome;

FIG. 5 shows the result of nucleotide sequencing of the target mutation site at nucleotide 1142 of the viral genome of the mutant gene.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.

The materials and reagents used in the following examples are specifically as follows:

virus strain:

type 3 duck hepatitis a virus virulent strain: the laboratory is separated and preserved in China center for type culture Collection of Wuhan university in China, and the preservation numbers are as follows: CCTCC NO: v201305, category naming: avian Hepatitis virus genus picornaviridae type 3 Duck Hepatitis a virus (Duck Hepatitis virus type 3, DHAV-3).

Reagents and instruments:

TaKaRa MiniBEST Universal RNA Extraction Kit, PrimeSTAR Max DNA Polymerase, DNA Marker and the like from Takara bioengineering (Dalian) Co., Ltd; gel recovery kit, plasmid extraction kit and the like were purchased from Omega company of the United states; lipofectamine 3000 Lipofectamine was purchased from Invitrogen; other reagents are all domestic analytical purifiers.

Nucleic acid protein detector (Bio Rad, Smartspec3000), gradient PCR instrument (Biometra, Tgradient), electrophoresis instrument (Bio Rad, Powerpac 300) and gel imaging system (Bio Rad Versa Doc Model 2000).

Example 13 construction of Duck hepatitis A Virus mutant Gene ISA-A117C-T1142A "infectious subgenomic replicon" and Virus rescue

1.1. Design and Synthesis of primers

According to the complete genome sequence of the duck hepatitis A virus type 3 in GenBank, 8 pairs of primers are designed for amplifying the complete genome sequence of the virus, the pCMV and SV40pA sequences, the specific sequence information is shown in Table 1, and the primers are synthesized by Shanghai biological engineering Co.

TABLE 1 construction of type 3 Duck hepatitis A Virus mutant Gene ISA-A117C-T1142A infectious subgenomic replicon primers

1.2. Extraction of viruses

The virus whole genome RNA of the duck hepatitis A virus type 3 isolate was extracted from the allantoic fluid of duck embryos according to the procedure of the TaKaRa MiniBEST Universal RNA Extraction kit, and the nucleic acid concentration and purity thereof were measured by a nucleic acid protein detector (Bio Rad, Smartspec3000), and then stored at-70 ℃ for later use.

1.3. Cloning by amplification of Gene fragments

(1) The extracted total RNA is reversely transcribed into a cDNA template by using a PrimeScript II 1st Strand cDNA Synthesis kit, then primers F1-F, A117C-R, A117C-F, T1142A-R, T1142A-F and F1-R are respectively used for amplifying by using a DNA high fidelity PCR enzyme PrimeSTAR Max DNA Polymerase and taking a reverse transcription product of the total RNA of the parent strain as a template to obtain DHAV-3-F1-A117C, DHAV-3-F1-A117C-T1142A and DHAV-3-F1-T1142A fragments, primers F2-1-F, F2-1-R, F2-2-F and F2-2-R are respectively used for amplifying by using a reverse transcription product of the total RNA of the parent strain as a template to obtain a DHAV-3-F2-1 fragment and a DHAV 3-2-F2-R, using primers F3-HDVR-F and F3-HDVR-R to amplify by taking a reverse transcription product of the parent strain virus total RNA as a template to obtain a DHAV-3-F3-HDVR fragment; the eukaryotic expression plasmid pEGFP-C1 is used as a template, primers pCMV-F and pCMV-R are used for amplification to obtain a pCMV fragment, and primers HDVR-SV40pA-F and HDVR-SV40pA-R are used for amplification to obtain an HDVR-SV40pA fragment.

(2) By the fusion PCR technology, as shown in FIG. 1, DHAV-3-F1-A117C, DHAV-3-F1-A117C-T1142A and DHAV-3-F1-T1142A fragments are fused into F1-A117C-T1142A, and then pCMV and F1-A117C-T1142A fragments are fused into pCMV-F1; fusing the DHAV-3-F2-1 and DHAV-3-F2-2 fragments into an F2 fragment; the DHAV-3-F3-HDVR and HDVR-SV40pA fragments are fused into F3-Hdvrz/SV40pA fragments, and three finally obtained DNA fragments form a mutant gene ISA-A117C-T1142A 'infectious subgenomic replicon'. After the amplified fragments were separated by 1% agarose gel electrophoresis, the target fragments were recovered by gel cutting using a gel recovery kit (Omega). The DNA fragments were sent to Shanghai Bioengineering, Inc. for sequencing.

1.4. Transfection rescue of the mutant Gene ISA-A117C-T1142A "infectious subgenomic replicon

Primary duck embryo fibroblasts were prepared using 9-day-old duck embryos and equal amounts (1.5. mu.g) of pCMV-F1, F2 and F3-Hdvrz-SV40pA gene fragments were mixed with Lipofectamine 3000(Invitrogen) when cells in a 3.5cm dish were grown to 90% confluencyDuck embryo fibroblasts growing in an amount of 90% were transfected after the combination, and the control group was transfected only with Lipofectamine 3000 (Invitrogen). Cells were placed at 37 ℃ in 5% CO₂The culture observation is carried out in an incubator, the culture medium is replaced after 16 hours, cells of a transfection group have cell fragmentation phenomenon after 72 hours of transfection, and the cells of a control group have good growth condition. After 120 hours of transfection of duck embryo fibroblasts, the cell growth condition is shown in figure 2, the cells are repeatedly frozen and thawed 3 times after photographing and recording, 5 9-day-old duck embryos, 0.2 mL/egg, are inoculated by a cell culture solution through an allantoic cavity, are placed into an incubator after paraffin sealing for continuous incubation, the embryos are irradiated for 1 time every 8 hours, the death condition of the duck embryos after inoculation is observed, and the duck embryos which die within 24 hours are discarded. The result shows that the duck embryo dies between 24 hours and 48 hours after inoculation, the dead embryo body of the duck embryo bleeds seriously, and allantoic fluid is collected to be used as a first generation reverse genetic virus strain for preservation.

Example identification and characterization of the type 23 Duck hepatitis A Virus mutant Gene ISA-A117C-T1142A Virus Strain

2.1. Identification of genetic markers in rescued viruses

In order to eliminate the possibility that the rescued virus is possibly from the pollution of parent virus or wild strain in the transfection or passage process, the 3403 th base of the mutant gene genome is mutated from mutation G to T by using a reverse genetics method, the mutation does not change the composition of corresponding amino acid of 2A protein, the site is used as a molecular genetic marker site, and the mutant gene virus strain can be distinguished from the parent strain and the wild strain by combining a PCR method and DNA sequencing. Rescued virus was passaged and purified 5 times on duck embryos by limiting dilution. Extracting total RNA from allantoic fluid, performing PCR amplification on a DNA fragment containing a mutation site by using F2-F and F2-R primers after reverse transcription, separating the amplified fragment by 1% agarose gel electrophoresis, then cutting and recovering the gel by using a gel recovery kit (Omega), sending the DNA fragment to Shanghai Bioengineering limited company for sequencing, and displaying that the amplified product contains introduced silent mutation (G3403T) according to a sequencing result, wherein the sequencing result shows that the amplified product contains the correct rescued virus instead of the pollution of a parent strain or a wild-type strain as shown in figure 3.

2.2. Detection of genetic stability of rescued viruses and mutant sites thereof

In order to observe whether the rescued reverse genetic virus can be propagated and passaged in duck embryos and chicken embryos, the 1st generation of rescued virus is diluted by sterilized normal saline at a ratio of 1:100, and 5 9-day-old duck embryos or chicken embryos are inoculated. The results show that the death time of the duck embryo/chicken embryo is concentrated between 24 hours and 48 hours after inoculation, the embryo body is obviously diseased and continuously passes for 10 times, and virus liquid of each generation is collected during the period, and is stored in a refrigerator at minus 80 ℃. After RNA is extracted using the virus solutions of generations 1, 5 and 10 and reverse-transcribed into cDNA, a DNA fragment containing the mutation site is PCR-amplified, and the mutation site and the genetic marker site are detected, as shown in FIGS. 4 and 5. Sequencing results show that the 1st, 5 th and 10 th generation viruses have no mutation, which indicates that the virus has good genetic stability.

2.3. Virus propagation and content determination

Sterile normal saline solution is used for diluting type 3 duck hepatitis A virus in a multiple ratio to save virus and parent strain, and 10 percent of virus and parent strain is selected^-3、10^-4、10^-5、10^-6、10^-7、10^-8The 6 dilutions were inoculated with 5 9-day-old duck embryos in an allantoic cavity at a volume of 0.2mL per embryo, respectively, and then 5 sterilized normal saline controls were added, and after inoculation, the duck embryos were incubated in a 37 ℃ incubator at a constant temperature, and the dead duck embryos were counted within 24 hours, and the death and survival of the inoculated duck embryos within 7 days were observed and recorded, and ELD50 was calculated according to the Reed-Muech method, and the results showed that the rescued viruses and the parent strain viruses had different proliferation capacities, and the virus content in each 0.2mL allantoic fluid was 10^- ^7.55ELDs 50 and 10^-4.50The proliferation capacity of ELD50, mutant gene ISA-A117C-T1142A virus strain is obviously stronger than that of parent strain. For example, the production of virus antigen in vaccine can increase the yield and reduce the cost.

In addition, the same experimental operation is carried out on 9-day-old chick embryos, and the result shows that the 3-type duck hepatitis A virus mutant gene ISA-A117C-T1142A virus strain can be propagated in the chick embryos and cause the death of the chick embryos, and the virus titer is 10^-5.33ELD 50; while the parent strain is unable to kill the chick embryo. Mutant gene virus strains such as those used for antigen production in vaccines, provide a wider source of material for virus culture and reduce costs; and host preference of mutant gene virus strainsThe sex is changed, and the obtained mutant virus can be used as a basic material for researches on virus genes, key sites and the like of duck hepatitis virus host tropism and virulence change.

2.4. Seroneutralization assays to detect antigenicity of viruses

To test whether the reverse genetic strain generates mature progeny virions and whether the virus antigenicity is changed during duck embryo passage. The neutralizing titer of rabbit anti-type 3 duck hepatitis A virus serum to mutant gene virus strain and parent strain is determined by fixed virus dilution serum method, firstly, rabbit anti-type 3 duck hepatitis A virus standard serum (titer is more than or equal to 1:128) prepared in the early stage of laboratory of inventor is diluted by sterilized normal saline at 10 times ratio and 2 times^-1To 2^-9These 9 dilutions were made simultaneously with virus dilution to 200ELD50 per unit dose (0.2mL), then both were mixed in equal amounts and 5% of a cyan, streptomycin diabody were added to a water bath at 37 ℃ for 1 hour, and then inoculated into the allantoic cavity of 9 day old healthy duck embryos in a dose of 0.2mL per embryo, with 5 duck embryos inoculated at each dilution. And additionally arranging a negative serum control group (mixing healthy rabbit serum with the virus) and a blank control group (mixing sterilized normal saline with the virus), discarding duck embryos dead within 24 hours, observing and recording the death and survival conditions of the duck embryos within 7 days, and then calculating the neutralizing titer of the rabbit anti-type 3 duck hepatitis A virus standard serum to the virus.

The result shows that the rescued virus and the parental strain virus have the same antigenicity, and the duck embryos of the negative serum control group (the mixture of the serum of healthy rabbits and the virus) and the blank control group (the mixture of the sterilized normal saline and the virus) all die within 24 hours to 48 hours; when the serum dilution is 2^-1To 2^-6When the dilution is 2, the duck embryos of the mutant gene virus strain and the parent strain neutralization group are all healthy and alive^-7The duck embryo is not protected, and the duck embryo protection rate is lower along with the higher dilution degree till the dilution degree is 2^-9The duck embryo is completely unprotected. It is shown that if the mutant gene virus strain is used for preparing vaccine antigen, the type 3 duck hepatitis A virus infection can also be protected.

2.5. Toxicity and safety test for susceptible ducklings

Use of parent strainsAnd mutant gene virus strain, and collecting liver tissue homogenate of dead duck embryo in example 2.3 according to volume ratio of 1: adding sterilized phosphate buffer solution at a ratio of 100, grinding, repeatedly freezing and thawing for 3 times, centrifuging at 12000g for 10min, filtering the supernatant with 0.22 μm filter, sterilizing, inoculating 9-day-old duck embryo via allantoic cavity, measuring its ELD50, and diluting virus solution to 10^3.0ELD50/0.4 mL. In addition, 30 healthy ducklings of 1 day old are randomly divided into 3 groups, the ducklings of the test group are inoculated with 0.4mL parent strains or mutant gene virus strains through an intramuscular injection way, the ducklings of the control group are inoculated with equal-volume sterilized normal saline, the ducklings are separately fed in different animal rooms, water is freely drunk for ingestion, the morbidity and the mortality of the ducklings are recorded after the inoculation, the dead ducklings are immediately inspected, the living ducklings are inspected 7 days later, and the pathological changes of organs such as livers, kidneys and the like of the ducklings are recorded.

The morbidity and mortality of the ducklings after parent strain inoculation are 80% and 60%; the pathogenicity of the mutant gene virus strain to the ducklings is obviously reduced, the ducklings inoculated with the mutant gene virus strain have no clinical symptoms, the eating and drinking conditions are normal, and viruses are detected in the cloaca swab, which shows that the mutant gene virus strain is successfully copied in the ducklings but has no pathogenicity to the ducklings, has the potential of being used as a candidate strain of a low-virulent vaccine, and can distinguish the mutant gene virus strain from a parent strain by combining a PCR (polymerase chain reaction) method and DNA (deoxyribonucleic acid) sequencing.

Example 33 application of mutant Gene ISA-A117C-T1142A Virus Strain of Duck hepatitis A Virus in preparation of inactivated vaccine

Mutant gene virus strains tested according to example 2 had higher proliferation efficiency and virus titer on duck embryos than the parent strain; can also be propagated on chick embryos; the immunogenicity and the genetic stability are good; the pathogenicity of the duckling is obviously reduced, and the mutant gene virus strain can be replicated in the duckling body but does not cause diseases to the duckling, so that the mutant gene ISA-A117C-T1142A virus strain is an ideal vaccine candidate strain and can be used for developing a type 3 duck hepatitis A virus vaccine.

3.1 preparation of inactivated vaccines

Diluting a 3-type duck hepatitis A virus mutant gene ISA-T1142A virus strain serving as a seed virus by 100 times with sterilized normal saline, inoculating 20 9-day-old duck embryos into an allantoic cavity, continuously incubating the embryo in a constant-temperature incubator at 37 ℃, illuminating eggs for 1 time after 24 hours after inoculation, discarding dead embryos, illuminating eggs for 1 time every 8 hours later, taking out the dead duck embryos at any time till 48 hours, completely dying the duck embryos, standing the collected duck embryo air chambers upwards, cooling the duck embryos for 8 hours at 4 ℃, collecting the cooled aseptic duck embryo allantoic fluid, and storing the duck embryo allantoic fluid at-20 ℃ for later use.

Treating virus solution with 0.1% formaldehyde solution, inactivating at 37 deg.C for 24 hr, and diluting with sterilized normal saline to 10% virus content³And (3) emulsifying and uniformly mixing ELD50/0.1mL and Freund's incomplete adjuvant in the same volume to prepare the inactivated vaccine of the mutant gene virus strain of the duck hepatitis A virus type 3.

3.2 sterility and Mycoplasma detection

The inactivated vaccine is subjected to sterility and mycoplasma inspection according to the appendix of the current pharmacopoeia of the people's republic of China, and the detection results are negative.

3.3 detection of exogenous viruses

The inactivated vaccine is subjected to exogenous virus detection according to the appendix of the current pharmacopoeia of the people's republic of China, and the detection results are negative.

3.4 safety testing of inactivated vaccines

In order to test the safety of the inactivated vaccine, 10 ducklings of 1 day old are immunized with 10 times of immunization dose, each ducklings is inoculated with 0.2mL through a leg muscle inoculation way, meanwhile, 10 ducklings are taken to inject sterilized normal saline as a control group, isolated feeding is carried out, free drinking water is taken for feeding, the health condition of the ducklings is observed and recorded every day, the ducklings are dissected after 7 days of observation, and as a result, the ducklings of the immunization group and the control group have no disease within 7 days of inoculation, and the dissected result shows that no pathological change occurs in each immune organ and virus tropism tissue of the ducklings, and no virus is detected in a cloacal swab, which indicates that the inactivated vaccine has no pathogenicity on the ducklings of 1 day.

3.5 Immunity of inactivated vaccine

20 ducklings of 1 day old are randomly divided into 2 groups, 10 ducklings of an inactivated vaccine inoculation group are inoculated with a feather through a leg muscle injection way to obtain vaccine immunization, the other group is injected with equivalent sterilized normal saline as a contrast, two groups of ducklings are separately fed, free drinking water is taken, the condition of the ducklings is observed and recorded every day after inoculation, the parent strain virus with 10 times LD50 dosage is used for counteracting the virus after 7 days, the morbidity and the mortality of the ducklings are observed and recorded every day after counteracting the virus, the death ducklings are immediately inspected by skimming, the observation is continued for 1 week, the survival ducklings are inspected by skimming, and the pathological changes of organs such as liver, kidney and the like are recorded.

Within 7 days after immunization, the ducklings of the experimental group and the negative control group have no clinical symptoms, and the feeding and drinking conditions of the ducklings are normal. After the challenge, the negative control group has ducklings with lassitude and nervous symptoms on the 2 nd day, 6 ducklings die after the 7 th day, the surviving ducklings have pathological changes such as liver bleeding with different degrees, the morbidity is 80 percent (8/10), the mortality is 60 percent (6/10), and the ducklings of the inactivated vaccine immune group have no morbidity and death, and the inactivated vaccine prepared by the mutant gene virus strain is taken by normal drinking water, so that the inactivated vaccine is safe and effective, and can protect the homologous virulent challenge.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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<213> Artificial Sequence (Artificial Sequence)

<400> 10

caacctaggt aagtgagcac gat 23

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gtgctcactt acctaggttg gtt 23

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tggcaacttc ctgtctaacc tg 22

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ccttgaacac tggaacccaa 20

<210> 14

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aagtagccca ggtcggaccg cgaggaggtg gagatgccat gccgaccctt tttttttttt 60

ttagggtgg 69

<210> 15

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cggtccgacc tgggctactt cggtaggcta agggagaaga acttgtttat tgcagctta 59

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

taagatacat tgatgagttt gga 23

<210> 17

<211> 583

<212> DNA

<213> cytomegalovirus early promoter (pCMV)

<400> 17

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540

acggtgggag gtctatataa gcagagctgg tttagtgaac cgt 583

<210> 18

<211> 67

<212> DNA

<213> hepatitis delta virus ribozyme sequence (HDVR)

<400> 18

gggtcggcat ggcatctcca cctcctcgcg gtccgacctg ggctacttcg gtaggctaag 60

ggagaag 67

<210> 19

<211> 122

<212> DNA

<213> Simian vacuolating Virus early mRNA polyadenylation Signal (SV40pA)

<400> 19

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120

ta 122

Claims

1. A3-type duck hepatitis A virus mutant gene ISA-A117C-T1142A is characterized in that the mutant gene ISA-A117C-T1142A changes the 117 th nucleotide of 5' UTR of a 3-type duck hepatitis A virus virulent strain genome from A to C; the 1142 th nucleotide is mutated from T to A, so that the 164 th amino acid of the VP0 protein is mutated from tyrosine of parent strain to asparagine, the virulent strain of the type 3 duck hepatitis A virus is preserved in China center for type culture Collection with the preservation number of CCTCC NO: and V201305.

2. The type 3 duck hepatitis A virus mutant gene ISA-A117C-T1142A of claim 1, wherein the G of 3403 th nucleotide of the mutant gene ISA-A117C-T1142A is mutated to T, which is used as a genetic marker of infectious clone.

3. The use of the type 3 duck hepatitis A virus mutant gene ISA-A117C-T1142A of claim 1 in the preparation of a type 3 duck hepatitis virus vaccine.