CN110283835B

CN110283835B - Type-3 duck hepatitis A virus mutant gene ISA-T1142A-C4334A and construction method thereof

Info

Publication number: CN110283835B
Application number: CN201910551575.5A
Authority: CN
Inventors: 程安春; 文兴建; 汪铭书
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2022-10-14
Anticipated expiration: 2039-06-24
Also published as: CN110283835A

Abstract

The invention discloses a 3-type duck hepatitis A virus mutant gene ISA-T1142A-C4334A and a construction method, wherein the mutant gene ISA-T1142A-C4334A mutates the 1142 th nucleotide of a 3-type duck hepatitis A virus virulent strain genome from T to A, so that the 164 th amino acid of virus VP0 protein mutates from tyrosine of a parent strain to asparagine; the 4334 th nucleotide is mutated from C to A, so that the 71 th amino acid of the virus 2C protein is mutated from leucine of parent strain to isoleucine. A mutant strain of a 3-type duck hepatitis A virus mutant gene ISA-T1142A-C4334A is an ideal vaccine candidate strain; and can also be used in basic research such as 3-type duck hepatitis A virus attenuated molecular mechanism and the like, and has wide application prospect.

Description

Type-3 duck hepatitis A virus mutant gene ISA-T1142A-C4334A 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-T1142A-C4334A 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; the clinical manifestations are spasm of ducklings before death, head bending back to back and "arcus atelectasis", and pathological changes are mainly hepatomegaly, inflammation and massive hemorrhagic spots seen in the autopsy. 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 other replicase gene cDNA clones have 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 that the viruses cannot be successfully rescued. Currently, a technique called "Infectious Subgenomic replicons" (Infectious Subgenomic replicons) has been demonstrated to achieve 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, overlapping non-infectious subgenomic DNA fragments containing the whole virus genome are generated by a PCR method, the number of the overlapping subgenomic replicons can be 3 to 10, an overlapping region of about 100bp is formed between adjacent replicons, and meanwhile, 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 mRNA polyadenylation signal (SV 40 early mRNA polyadenylation replicon) 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, and finally obtain an 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 the 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-T1142A-C4334A, the said mutant gene ISA-T1142A-C4334A mutates from T to A with the 1142 bit nucleotide of the virulent strain genome of 3-type duck hepatitis A virus, thus make the 164 th amino acid of virus VP0 protein mutate to asparagine from tyrosine of the parent strain; the 4334 th nucleotide is mutated from C to A, so that the 71 th amino acid of the virus 2C protein is mutated from leucine of parent strain to isoleucine.

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.

The G of the 3403 th nucleotide of the mutant gene genome is mutated into T which is used as a genetic marker of infectious clone, and the mutant gene can be distinguished from a parent strain and a wild strain 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 parental viruses, the virus titer is higher, 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 is replicated in the duckling body but does not cause diseases to the duckling, has good safety and can be used as a candidate strain of a type 3 duck hepatitis A virus vaccine.

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 the research of duck hepatitis virus virulence attenuated molecular mechanism.

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

(1) Dividing the whole genome of a parent virus into three fragments (2.6 kb, 2.6kb and 2.7 kb) with similar sizes, carrying out PCR amplification, adding a Cytomegalovirus early promoter (pCMV) at the 5' end of a first fragment of a virus genome, introducing a mutation site in a VP0 gene, introducing a mutation site in a 2C gene of a second fragment and nonsense mutation of the 2A gene as a genetic marker site, adding a Hepatitis Delta Virus Ribozyme (HDVR) sequence and a simian vacuolation virus early mRNA polyadenylation signal (SV 40 early mRNA amplification signal SV, 40 pA) sequence at the 3' end of a third fragment of the virus genome, and obtaining three DNA fragments to form a 3 type A duck virus mutant gene ISA-T1142A-C4334A ' infectious subgenomic replicon;

(2) The constructed 3-type duck hepatitis A virus mutant gene ISA-T1142A-C4334A 'infectious subgenomic replicon' is mixed with a transfection reagent and then transfects duck embryonic 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 further caused to replicate and proliferate, and finally, the 3-type duck hepatitis A virus mutant gene ISA-T1142A-C4334A mutant 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 obtained low virulent strain ISA-T1142A-C4334A has antigenicity similar to that of parent strain and can keep stable genetic characteristic in the continuous passage process, so that the mutant gene mutant strain can be used as a candidate vaccine strain for immunizing 3-type duck hepatitis A virus; meanwhile, the strain has higher propagation efficiency and virus titer in duck embryos than parent strains, and can improve yield and reduce cost when being used for vaccine production.

3. The mutant gene mutant strain can be used as a candidate strain of a 3-type duck hepatitis A virus attenuated vaccine and has good safety.

4. Compared with the parental strain, the mutant gene ISA-T1142A-C4334A mutant strain obtained in the invention has reduced toxicity, so the mutant gene ISA-T1142A-C4334A mutant strain can be applied to basic research on viral genes, key sites and the like of duck hepatitis virus with changed toxicity.

Drawings

FIG. 1 is a schematic diagram of construction of a molecular marker mutant gene ISA-T1142A-C4334A 'infectious subgenomic replicon' based on type 3 duck hepatitis A virus as a framework;

FIG. 2 is the result of transfection of duck embryo fibroblast with type 3 duck hepatitis A virus mutant gene ISA-T1142A-C4334A "infectious subgenomic replicon";

FIG. 3 shows the sequencing result of 3403 nucleotide molecular genetic marker locus of mutant gene ISA-T1142A-C4334A virus genome;

FIG. 4 shows the result of nucleotide sequencing of the target mutation site at nucleotide 1142 of the viral genome of the mutant gene ISA-T1142A-C4334A;

FIG. 5 shows the result of nucleotide sequencing of target mutation site at nucleotide 4334 of virus genome of mutant gene ISA-T1142A-C4334A.

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, classification name: avian Hepatitis virus genus picornaviridae type 3 Duck Hepatitis A virus (Duck Hepatitis AVirus 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 are purchased from Omega company of America; lipofectamine3000 Lipofectamine was purchased from Invitrogen; other reagents are all domestic analytical purifiers.

Nucleic acid protein detector (Bio Rad, smartspec 3000), 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-T1142A-C4334A "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-T1142A-C4334A 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, smartspec 3000), 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 DHAV-3-F1-T1142A-R fragment is obtained by using DNA high fidelity PCR enzyme PrimeSTAR Max DNA Polymerase and primers F1-F and T1142A-R and using a reverse transcription product of the total RNA of the parent strain virus as a template, and DHAV-3-F1-T1142A-F fragment is obtained by using T1142A-F and F1-R and using a reverse transcription product of the total RNA of the parent strain virus as a template, respectively amplifying DHAV-3-F2-1 fragment, DHAV-3-F2-1 fragment and DHAV-3-F2-C4334A-2 fragment by using primers F2-1-F and F2-1-R, F-2-F and C4334A-R, C A-F and F2-2-R and taking a reverse transcription product of the total RNA of the parent strain virus as a template, and amplifying DHAV-3-F3-HDVR fragment by using primers F3-HDVR-F and F3-HDVR-R and taking a reverse transcription product of the total RNA of the parent strain virus as a template; eukaryotic expression plasmid pEGFP-C1 is taken 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) Through fusion PCR technology, as shown in FIG. 1, DHAV-3-F1-T1142A-F and DHAV-3-F1-T1142A-R fragments are fused into DHAV-3-F1-T1142A, and then pCMV and DHAV-3-F1-T1142A fragments are fused into pCMV-F1; fusing the DHAV-3-F2-1 segment, the DHAV-3-F2-C4334A-1 segment and the DHAV-3-F2-C4334A-2 segment into an F2 segment; the DHAV-3-F3-HDVR and HDVR-SV40pA fragments are fused into an F3-Hdvrz/SV40pA fragment, and as shown in figure 1, three finally obtained DNA fragments form a mutant gene ISA-T1142A-C4334A '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 mutant Gene ISA-T1142A-C4334A "infectious subgenomic replicon

Primary duck embryo fibroblasts were prepared using 9-day-old duck embryos, and when cells in a 3.5cm dish were grown to 90% confluency, equal amounts (1.5. Mu.g) of pCMV-F1, F2 and F3-HdvRz-SV40pA gene fragments were mixed with Lipofectamine3000 (Invitrogen) and transfected to reach 90% confluency, while control groups were transfected using Lipofectamine3000 (Invitrogen) alone. Cell lysis at 37 ℃ 5% CO ₂ The 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, after photographing and recording, the cells are repeatedly frozen and thawed for 3 times, 5 9-day-old duck embryos are inoculated with cell culture fluid through an allantoic cavity, each duck embryo is 0.2mL, the duck embryos are placed into an incubator for continuous incubation after paraffin is sealed, the embryos are irradiated for 1 time every 8 hours, and observation is carried outAnd (4) observing the death condition of the duck embryos after inoculation, and discarding the dead duck embryos within 24 hours. The result shows that the duck embryo dies between 24 hours and 72 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 type 23 Duck hepatitis A Virus mutant Gene ISA-T1142A-C4334A

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, a reverse genetics method is utilized, 3403 th base of a mutant gene genome is mutated from mutation G to T, 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 a PCR method and DNA sequencing can be combined to distinguish mutant gene mutant strains from parent strains and wild strains. 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 genetic marker site by using F2-F and F2-R primers after reverse transcription, separating the amplified fragment by 1% agarose gel electrophoresis, then cutting gel and recovering 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 an 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 a parent strain or a wild-type polluted 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 the duck embryo, the rescued virus of the 1st generation is diluted by sterilized normal saline solution to be 1. The results show that the death time of the duck embryo is concentrated between 24 hours and 72 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 by using virus liquid of 1st, 5 th and 10 th generations and is reversely transcribed into cDNA, a DNA fragment containing a genetic marker locus is amplified by PCR and the genetic marker locus is detected, as shown in figure 4 and figure 5, sequencing results show that the virus of 1st, 5 th and 10 th generations has no mutation, and 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 ^-8 The 6 dilutions were prepared by inoculating 5 9-day-old duck embryos in an amount of 0.2mL per embryo via allantoic cavity, inoculating with 5 sterilized normal saline as control, incubating in a 37 deg.C constant-temperature incubator for 24 hr without counting dead duck embryos, observing and recording the death and survival conditions of the inoculated duck embryos within 7 days, calculating ELD50 by Reed-Muech method, and finding that the rescued virus and the parent virus have different proliferation capacities, and the virus content in allantoic fluid of 0.2mL is 10 respectively ^-8.37 ELDs 50 and 10 ^-4.50 ELD50 shows that the proliferation capacity of the mutant strain of the 3-type duck hepatitis A virus mutant gene is obviously higher than that of a parent strain, and if the mutant strain is used for producing antigen in a vaccine, the yield can be increased, and the cost can be reduced.

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 in the duck embryo passage. The neutralizing titer of rabbit anti-type 3 duck hepatitis A virus serum on mutant gene mutant strains and parent strains is determined by adopting a fixed virus dilution serum method, firstly, the rabbit anti-type 3 duck hepatitis A virus standard serum (the titer is more than or equal to 1 128) prepared in the early stage of a laboratory where an inventor is located is diluted by 10 times with sterilized normal saline and then diluted from 2 times ^-1 To 2 ^-9 The virus was diluted to 200ELD50 per unit dose (0.2 mL) at these 9 dilutions, then mixed in equal amounts and added to a 5% solution of a bis-antibiotic for penicillin and streptomycin in a water bath at 37 ℃ for 1 hour, and then inoculated into the allantoic cavity of a 9-day-old healthy duck embryo in a dose of 0.2mL per embryo, and 5 duck embryos were inoculated at each dilution. Additionally arranging a negative serum control group (mixing healthy rabbit serum and virus) and a blank control group (mixing sterilized normal saline and virus), discarding duck embryos dying within 24 hours, observing and recording the death and survival conditions of the duck embryos within 7 days, and then calculating a rabbit anti-type 3 duck hepatitis A virus standardNeutralizing titer of serum against 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 ^-1 To 2 ^-6 In the meantime, the duck embryos of the mutant gene mutant strain and the parent strain neutralization group are all healthy and alive, and when the dilution is 2 ^-7 The 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 ^-9 The duck embryo is completely unprotected. It is shown that if the mutant gene mutant 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

The parent strain and mutant gene mutant strain are used for safety test, and the liver tissue homogenate treatment of the dead duck embryo in example 2.3 is collected according to the 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 ^3.0 ELD50/0.4mL. In addition, 30 healthy ducklings of 1 day old are randomly divided into 3 groups, and the ducklings of the test group are inoculated with 0.4mL parent strain or mutant gene mutant strain through intramuscular injection, each is inoculated with 10 ^3.0 ELD is 50/0.4mL, the ducklings of a control group are inoculated with equal volume of sterilized normal saline, separated and fed in different animal rooms, freely drunk by drinking water, observed every day after inoculation, the morbidity and the death condition of the ducklings are recorded, the dead ducklings are subjected to real-time analysis, the living ducklings are subjected to real-time analysis after 7 days of observation, and the pathological changes of organs such as livers, kidneys and the like of the ducklings are recorded.

Within 7 days, the ducklings of the control group have no clinical symptoms, the feeding and drinking conditions of the ducklings are normal, the morbidity of the ducklings after the inoculation of the parent strain is 80 percent, the mortality rate is 60 percent, and the typical duck hepatitis lesion of organs such as livers, kidneys and the like of the ducklings is observed through the autopsy of dead ducks; the duckling inoculated with the mutant gene mutant strain has no clinical symptoms, the eating and drinking conditions are normal, and the virus is detected in the cloaca swab, which shows that the pathogenicity of the mutant gene mutant strain to the duckling is obviously reduced, the duckling is replicated in vivo but has no pathogenicity to the duckling, the duckling mutant strain has the potential of being used as a duck hepatitis virus attenuated vaccine strain, and the mutant gene mutant strain and a parent strain can be distinguished by combining a PCR method and DNA sequencing. Moreover, the virulence of the mutant gene mutant strain is changed, and the mutant gene mutant strain can be used as a basic material for researching viral genes with the virulence change of the duck hepatitis virus, key sites thereof and the like.

Example 33 application of mutant Gene ISA-T1142A-C4334A of Duck hepatitis A Virus in preparation of inactivated vaccine

Mutant gene mutants tested according to example 2 had higher proliferation efficiency and virus titer on duck embryos than the parent strain; the immunogenicity and the genetic stability are good; the pathogenicity of the duckling is obviously reduced, and the mutant strain of the mutant gene ISA-T1142A-C4334A can be used as a vaccine candidate strain for preparing a type 3 duck hepatitis A virus vaccine.

3.1 preparation of inactivated vaccines

Diluting a mutant strain of a 3-type duck hepatitis A virus mutant gene ISA-T1142A-C4334A by 100 times by using sterilized normal saline as a seed virus, inoculating 20 9-day-old duck embryos into an allantoic cavity, wherein each embryo is 0.2mL, continuously incubating in a constant-temperature incubator at 37 ℃, irradiating eggs for 1 time after 24 hours after inoculation, discarding dead embryos, irradiating eggs for 1 time every 8 hours, taking out the dead duck embryos at any time till 48 hours, completely dying the duck embryos, vertically arranging a collected duck embryo air chamber upwards, cooling for 8 hours at 4 ℃, aseptically collecting duck embryo allantoic fluid after cooling, and storing at-20 ℃ for later use.

Treating the virus solution with 0.1% final concentration formaldehyde solution, inactivating at 37 deg.C for 24 hr, and diluting with sterilized normal saline to 10% virus content ³ And emulsifying and mixing ELD50/0.1mL and Freund incomplete adjuvant in equal volume to obtain the inactivated vaccine of the mutant gene mutant strain of the type 3 duck hepatitis A virus.

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 old.

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 with 10 times LD50 dosage is used for counteracting the virus after 7 days, the morbidity and the death condition of the ducklings are observed and recorded every day after counteracting the virus, the death ducklings are immediately necropsied and continuously observed for 1 week, the survival ducklings are necropsied, 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 in the 2 nd day has the ducklings with lassitude and nervous symptoms, and the ducklings die in total at the 7 th day, the surviving ducklings have pathological changes such as liver bleeding with different degrees, the morbidity is 80% (8/10), the mortality is 60% (6/10), while the ducklings of the inactivated vaccine immune group have no morbidity and death, and the normal drinking water is adopted, which shows that the inactivated vaccine prepared by the mutant gene mutant strain 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 modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. 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.

Sequence listing

<110> Sichuan university of agriculture

<120> type-3 duck hepatitis A virus mutant gene ISA-T1142A-C4334A and construction method

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tagttattaa tagtaatcaa ttacggggtc a 31

<210> 2

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

acaccacagc cgctttcaaa cggttcacta aaccagctct 40

<210> 3

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agagctggtt tagtgaaccg tttgaaagcg gctgtggtgt 40

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caacctgcca aaagtcaaac ca 22

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcactggatc taacaatgtg gatgc 25

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gcatccacat tgttagatcc agtga 25

<210> 7

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

attctgttac acctttacgc cccaca 26

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caacctaggt aagtgagcac gat 23

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gtgctcactt acctaggttg gtt 23

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tggcaacttc ctgtctaacc tg 22

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

acttgtgcat gatccggact gataa 25

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttatcagtcc ggatcatgca caagt 25

<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> monkey vacuolating virus early mRNA polyadenylation Signal (SV 40 pA)

<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-T1142A-C4334A, characterized by that, the said mutant gene ISA-T1142A-C4334A is to regard gene group reverse transcription product and eukaryotic expression plasmid pEGFP-C1 of the 3-type duck hepatitis A virus virulent strain as the template separately, adopt the primer shown in the following table to amplify and fuse and get, the said 3-type duck hepatitis A virus virulent strain is preserved in China center for type culture collection, the preserving number is CCTCC NO: v201305;

wherein, the genome reverse transcription product is taken as a template, primers F1-F, T1142A-R, T1142A-F and F1-R are used for respectively amplifying to obtain DHAV-3-F1-T1142A-R and DHAV-3-F1-T1142A-F fragments, primers F2-1-F, F2-1-R, F2-F, C4334A-R, C4334A-F and F2-2-R are used for respectively amplifying to obtain DHAV-3-F2-1 fragments, DHAV-3-F2-C4334A-1 fragments and DHAV-3-F2-C4334A-2 fragments, and primers F3-HDVR-F and F3-HDVR-R are used for amplifying to obtain DHAV-3-F3-HDVR fragments; the eukaryotic expression plasmid pEGFP-C1 is taken 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; then fusing the DHAV-3-F1-T1142A-F and the DHAV-3-F1-T1142A-R fragments into a fragment DHAV-3-F1-T1142A and fusing the pCMV and the DHAV-3-F1-T1142A fragments into a fragment pCMV-F1 by a fusion PCR technology; fusing the DHAV-3-F2-1 segment, the DHAV-3-F2-C4334A-1 segment and the DHAV-3-F2-C4334A-2 segment into a segment F2; the DHAV-3-F3-HDVR and the HDVR-SV40pA fragment are fused into a fragment F3-HdvRz/SV40pA, and the obtained fragment pCMV-F1, fragment F2 and fragment F3-HdvRz/SV40pA are sequentially connected to form the mutant gene ISA-T1142A-C4334A.

2. The use of the duck hepatitis A virus type 3 mutant gene ISA-T1142A-C4334A of claim 1 in the preparation of duck hepatitis virus type 3 vaccine.