CN110295180B - Type 3 duck hepatitis A virus mutant gene ISA-A117C-C4334A and construction method thereof - Google Patents
Type 3 duck hepatitis A virus mutant gene ISA-A117C-C4334A and construction method thereof Download PDFInfo
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Abstract
The invention discloses a type-3 duck hepatitis A virus mutant gene ISA-A117C-C4334A and a construction method, wherein the mutant gene ISA-A117C-C4334A is obtained by mutating the 117 th nucleotide of 5' UTR of genome of a type-3 duck hepatitis A virus virulent strain to C; 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 a parent strain to isoleucine. The pathogenicity of the mutant gene virus strain to the ducklings is obviously reduced, the mutant gene virus strain can be replicated in the ducklings but does not cause diseases to the ducklings, and the mutant gene virus strain has good safety, so that an important foundation is laid for the virus pathogenic mechanism and vaccine development. The 3-type duck hepatitis A virus mutant gene ISA-A117C-C4334A virus strain is an ideal vaccine candidate strain; meanwhile, a basic material is provided for researching virus genes with changed tropism and virulence of duck hepatitis virus hosts and key sites thereof, and the application prospect is wide.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a type 3 duck hepatitis A virus mutant gene ISA-A117C-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 duckling. 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 weeks, and has the characteristics of acute disease onset, rapid spread, short disease course, high mortality rate and the like; clinically, the disease is mainly characterized in that the young duck is cramped before death, turns back with head facing to 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 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 amplics) 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 saxivirus. The technology is a novel 'bacteria-free' reverse genetics method, does not need to obtain a virus full-length cDNA plasmid or obtain a virus RNA transcript in vitro, and can directly save the virus by transfecting a DNA fragment with a homologous region. In particular, the technical route adopted by "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 meanwhile, the 5 'end of the first fragment and the 3' end of the last fragment are respectively flanked by a Cytomegalovirus early promoter (pCMV) sequence, a Hepatitis Delta Virus Ribozyme (HDVR) sequence and a simian vacuolating virus early mRNA polyadenylation signal (SV 40early mRNA polyadenylation signal, SV40 pA) sequence, which can help the subgenomic replicons to start transcription after being mixed and 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 replication and proliferation of the virus, and finally obtains the infectious rescue virus.
The existing market mainly uses DHAV-1 attenuated vaccine for preventing and controlling DHAV, lacks high-efficiency DHAV-3 live vaccine, and is in need of developing novel molecular marker vaccine suitable for the DHAV-3 epidemic situation in China; 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:
a type 3 duck hepatitis A virus mutant gene ISA-A117C-C4334A, wherein the mutant gene ISA-A117C-C4334A is characterized in that the 117 th nucleotide of 5' UTR of a type 3 duck hepatitis A virus virulent strain genome is mutated from A to C; 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 Wuchang zone of Wuhan city, hubei province, the preservation period 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 as well as 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 obviously reduced pathogenicity of the mutant gene to the ducklings, and the mutant gene is successfully replicated 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 virus genes with changed tropism and virulence of duck hepatitis virus hosts and basic research of key sites.
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) Dividing the whole genome of the parental virus into three segments with similar sizes for PCR amplification, wherein the first segment and the third segment respectively contain 74 and 83 base pairs of overlapping regions with the second segment; adding a Cytomegalovirus early promoter (pCMV) at the 5 'end of a first fragment of a virus genome, introducing a mutation site in 5 UTR, introducing a mutation site in 2C gene of a second fragment, taking 2A gene nonsense mutation as a genetic marker site, and adding a Hepatitis Delta Virus Ribozyme (HDVR) sequence and a simian vacuolating virus early mRNA polyadenylation signal (SV 40early mRNA polyadenylation signal, SV40 pA) sequence at the 3' end of a third fragment of the virus genome to obtain three DNA fragments which form a duck Hepatitis A virus mutation gene ISA-A117C-C4334A infectious subgenomic replicon type 3;
(2) The constructed 3-type duck hepatitis A virus mutant gene ISA-A117C-C4334A 'infectious subgenomic replicon' is mixed with a transfection reagent and then transfects duck embryo fibroblasts, a 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, a 3-type duck hepatitis A virus mutant gene ISA-A117C-C4334A virus strain containing a genetic marker is obtained.
Further, the first fragment and the third fragment contain 74 and 83 base pair overlapping regions with the second fragment, respectively.
The beneficial effects of the invention are as follows:
1. compared with the time period of natural method mutagenesis and the traditional reverse genetic operation technology, the time period of the mutant gene of the type 3 duck hepatitis A virus obtained by the method is shorter, and the process of cultivating the attenuated vaccine strain of the type 3 duck hepatitis A virus and researching the pathogenic mechanism of the virus can be accelerated.
2. The mutant gene ISA-A117C-C4334A obtained in the invention has antigenicity similar to that of a parent virus strain thereof and can keep stable genetic characteristics, so that the mutant gene virus strain can be used as a candidate vaccine strain for 3-type duck hepatitis A virus immunization and can be used as a production strain for preparing 3-type duck hepatitis vaccine; meanwhile, compared with a parent strain, the mutant strain has higher propagation efficiency and virus titer on duck embryos, and can keep stable genetic characteristics in the continuous passage process. When the method is used in vaccine production, the yield can be improved, and the cost can be reduced.
3. The mutant gene ISA-A117C-C4334A virus strain obtained in the invention has obviously reduced pathogenicity on ducklings, can be replicated in the bodies of the ducklings but does not cause diseases on the ducklings, so the mutant gene virus strain can be used as a candidate vaccine strain for immunizing 3-type duck hepatitis A virus and has better safety.
4. Compared with the parental strain, the mutant gene ISA-A117C-C4334A virus strain obtained in the invention has reduced toxicity and can be propagated in chick embryos, so that the mutant gene ISA-A117C-C4334A virus strain can be used for basic research applications such as virus genes and key sites of duck hepatitis virus host tropism and toxicity change.
Drawings
FIG. 1 is a schematic diagram of construction of a molecular marker mutant gene ISA-A117C-C4334A 'infectious subgenomic replicon' based on type 3 duck hepatitis A virus as a framework;
FIG. 2 is the result chart of transfection of duck embryo fibroblast by type 3 duck hepatitis A virus mutant gene ISA-A117C-C4334A "infectious subunit genomic replicon";
FIG. 3 shows the sequencing result of 3403 nucleotide molecular genetic marker locus of the mutant gene ISA-A117C-C4334A virus genome;
FIG. 4 shows the result of nucleotide sequencing of 117 th nucleotide target mutation site of mutant gene ISA-A117C-C4334A virus genome;
FIG. 5 shows the result of nucleotide sequencing of target mutation site at nucleotide 4334 of viral genome of mutant gene ISA-A117C-C4334A.
Detailed Description
The following examples are intended to facilitate a better understanding of the invention, but are not intended to limit the invention thereto. The experimental procedures in the following examples are all conventional ones 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:
the 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 number is as follows: CCTCC NO: v201305, sort 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 kits, plasmid extraction kits, and the like were purchased from Omega, usa; lipofectamine 3000 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 1 construction of type 3 Duck hepatitis A Virus mutant Gene ISA-A117C-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-A117C-C4334A infectious subgenomic replicon primers
1.2. Extraction of viruses
The virus whole genome RNA of duck hepatitis A virus type 3 isolate was extracted from the allantoic fluid of duck embryo by the procedure of 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 future use.
1.3. Cloning by amplification of Gene fragments
(1) The extracted total RNA is reversely transcribed into cDNA template by using a PrimeScript II 1st Strand cDNA Synthesis kit, then DHAV-3-F1-A117C-F and DHAV-3-F1-A117C-R fragments are obtained by using primers F1-F and A117C-R, A117C-F and F1-R respectively and amplifying by using a reverse transcription product of the parent strain virus total RNA as a template by using a DNA high fidelity PCR enzyme PrimeSTAR Max DNA Polymerase, respectively amplifying DHAV-3-F2-1 fragments, DHAV-3-F2-C4334A-1 fragments and DHAV-3-F2-C4334A-2 fragments by using primers F2-1-F and F2-1-R, F2-2-F and C4334A-R, C4334A-F and F2-2-R and reverse transcription products of the total RNA of the parental strain virus as templates, and amplifying DHAV-3-F3-HDVR fragments by using primers F3-HDVR-F and F3-HDVR-R and reverse transcription products of the total RNA of the parental strain virus as templates; the eukaryotic expression plasmid pEGFP-C1 is taken as a template, a pCMV fragment is obtained by using primers pCMV-F and pCMV-R for amplification, and an HDVR-SV40pA fragment is obtained by using primers HDVR-SV40pA-F and HDVR-SV40pA-R for amplification.
(2) Through a fusion PCR technology, as shown in figure 1, DHAV-3-F1-A117C-F and DHAV-3-F1-A117C-R fragments are fused into F1-A117C, and then pCMV and DHAV-3-F1-A117C 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 the HDVR-SV40pA fragment are fused into an F3-Hdvrz/SV40pA fragment, and three finally obtained DNA fragments form a mutant gene ISA-A117C-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 the mutant Gene ISA-A117C-C4334A "infectious subgenomic replicon
Preparation of Primary Duck embryo fibroblasts Using 9 day old Duck embryos, equal amounts (1.5. Mu.g) of pCMV-F1, F2 and F3-HdvRz-SV40pA gene fragments were mixed with Lipofectamine 3000 (Invitrogen) and transfected to reach 90% confluency when cells grown in a 3.5cm dish, and control groups were used to transfect only 90% of the grown Duck embryo fibroblastsTransfection was performed with Lipofectamine 3000 (Invitrogen). Cell lysis at 37 ℃ 5% CO 2 The observation of culture 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 growth condition of cells of a control group is good. After transfecting the duck embryo fibroblasts for 120 hours, the growth condition of the cells is shown in figure 2, the cells are repeatedly frozen and thawed for 3 times after photographing and recording, 5 9-day-old duck embryos, each 0.2mL, are inoculated into cell culture solution through an allantoic cavity, the cells are placed into an incubator for continuous incubation after sealing a paraffin, 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 bleeding of the dead duck embryo body is serious, and allantoic fluid is collected to be used as a first generation of reverse genetic virus strain for preservation.
Example 2 identification of mutant Gene ISA-A117C-C4334A Virus Strain of type 3 Duck hepatitis A Virus
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, the parent strain and the wild strain can be distinguished 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 reverse transcription, performing PCR amplification on a DNA fragment containing a mutation site by using F2-F and F2-R primers, performing electrophoresis separation on the amplified fragment by using 1% agarose gel, then performing gel cutting recovery by using a gel recovery kit (Omega), sending the DNA fragment to Soyazao bioengineering limited company for sequencing, and displaying that an amplified product contains an introduced silent mutation (G3403T) according to a sequencing result, wherein as shown in figure 3, the result shows that the correct rescued virus is obtained instead of contamination of a parent strain or a wild strain.
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 chick embryos, the rescued virus of the 1st generation is diluted by sterilized normal saline solution to be 1. The result shows 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 extracting RNA using the virus solution of generations 1, 5 and 10, and reverse transcribing it 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 parental strains, and 10 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, preparing 5 sterilized normal saline controls, incubating in a 37 deg.C incubator at constant temperature after inoculation, counting dead duck embryos within 24 hours, observing and recording the death and survival conditions of the inoculated duck embryos within 7 days, and calculating ELD50 by Reed-Muech method, which shows that rescued virus and parent strain of virus have different proliferation capacities, and the virus content in allantoic fluid of 0.2mL is 10 respectively -7.55 ELDs 50 and 10 -4.50 The ELD50, mutant gene ISA-A117C-C4334A virus strain has obviously stronger multiplication capacity than the parent strain, and can increase the yield and reduce the cost when being used for the production of antigen in vaccine.
In addition, the same experimental operation is carried out on 9-day-old chick embryos, and the result shows that the virus strain of the type-3 duck hepatitis A virus mutant gene ISA-A117C-C4334A can be propagated in the chick embryos and cause death of the chick embryos, and the virus titer in allantoic fluid of the chick embryos is 10 -5.33 ELD50; while the parent strain was unable to proliferate in and kill the chick embryos. 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 tropism development of mutant gene virus strainsThe 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. Serum neutralization assay for detecting 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 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 and 128) prepared in the early stage of a laboratory where a inventor is located is diluted by 10 times with sterilized normal saline, and the ratio of the diluted serum is 2 times -1 To 2 -9 The virus was diluted to 200ELD50 per unit dose (0.2 mL) at these 9 dilutions, then the two were mixed in equal amounts and 5% of a bis-antibiotic to penicillin and streptomycin was added in a 37 ℃ water bath 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 per dilution. And additionally arranging a negative serum control group (mixing the serum of healthy rabbits with the virus) and a blank control group (mixing the 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 standard serum of the rabbit anti-type 3 duck hepatitis A virus on 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 serum of the healthy rabbit is mixed with the virus) and the blank control group (the sterilized normal saline is mixed with the virus) all die within 24 hours to 48 hours; when the serum dilution is 2 -1 To 2 -6 In the middle, the duck embryos of the mutant virus and the parent strain virus neutralization group are all healthy and alive, and when the dilution is 2 -7 The duck embryo is not protected until the dilution is 2 -9 The duck embryo is completely unprotected. It is shown that the type 3 duck hepatitis A virus infection can be protected by using mutant gene virus strain to prepare vaccine antigen.
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 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, 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 timely inspected by a dissecting way, the living ducklings are inspected by a dissecting way after 7 days of the observation, and the pathological changes of organs such as livers, kidneys and the like of the ducklings are recorded.
The ducklings of the control group have no clinical symptoms within 7 days, the ducklings have normal feeding and drinking conditions, the incidence rate of the ducklings after the inoculation of the parent strains is 80 percent, the mortality rate is 60 percent, the pathogenicity of the mutant gene virus strains to the ducklings is obviously reduced, the ducklings inoculated with the mutant gene virus strains have no clinical symptoms, the feeding and drinking conditions are normal, viruses are detected in a cloacal swab, the result shows that the mutant gene virus strains are successfully replicated in the ducklings but do not cause diseases to the ducklings, the potential of serving as candidate strains of attenuated vaccines is realized, and the mutant gene virus strains and the parent strains can be distinguished by combining a PCR method and DNA sequencing.
Example 3 application of mutant Gene ISA-A117C-C4334A Virus Strain of type 3 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 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-C4334A virus strain is an ideal vaccine candidate strain and can be used for preparing a type 3 duck hepatitis A virus vaccine.
3.1 preparation of inactivated vaccines
Diluting a virus strain of a 3-type duck hepatitis A virus mutant gene ISA-A117C-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 the embryo in a constant-temperature incubator at 37 ℃, irradiating eggs for 1 time after 24 hours after inoculation, removing 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, cooling for 8 hours at 4 ℃, aseptically collecting duck embryo allantoic fluid after cooling, 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 content 3 And emulsifying and mixing ELD50/0.1mL and Freund's incomplete adjuvant in equal volume to obtain the inactivated vaccine of the mutant gene virus strain of the type 3 duck hepatitis A virus.
3.2 sterility and Mycoplasma detection
And (3) carrying out sterility and mycoplasma inspection on the inactivated vaccine according to the appendix of the existing pharmacopoeia of the people's republic of China, wherein the detection results are negative.
3.3 detection of exogenous viruses
And (3) detecting the exogenous viruses of the inactivated vaccine according to the appendix of the current pharmacopoeia of the people's republic of China, wherein the detection results are negative.
3.4 safety testing of inactivated vaccines
In order to test the safety of the inactivated vaccine, 10 young ducks of 1 day old are immunized with 10 times of immunization dose, each young duck is inoculated with 0.2mL through a leg muscle inoculation way, meanwhile, 10 young ducks are injected with sterilized normal saline to serve as a control group and are fed in an isolated mode, free drinking water is taken for feeding, the health conditions of the young ducks are observed and recorded every day, the young ducks are dissected after 7 days of observation, and as a result, the young ducks of the immunization group and the control group are free of diseases 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 young ducks, and no virus is detected in cloacal swabs, which indicates that the inactivated vaccine is not pathogenic to the young ducks 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 for immunization, the other group is injected with equivalent sterilized normal saline as a contrast, the two groups of ducklings are separately fed, free drinking water is adopted for feeding, the condition of the ducklings is observed and recorded every day after inoculation, the parent strain virus with 10-time 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 inspected, the observation is continuously carried out for 1 week, the living ducklings are inspected, 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 day 2, 6 ducklings die after day 7, the surviving ducklings have pathological changes such as liver hemorrhage with different degrees, the morbidity is 80% (8/10), and the mortality is 60% (6/10), while the ducklings of the inactivated vaccine immunization group have no morbidity death, and normal drinking water intake shows that the inactivated vaccine prepared by the mutant gene virus strains is safe and effective, and can protect the challenge of homologous virulent viruses.
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 without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Sequence listing
<110> Sichuan university of agriculture
<120> type-3 duck hepatitis A virus mutant gene ISA-A117C-C4334A and construction method
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Claims (2)
1. A3-type duck hepatitis A virus mutant gene ISA-A117C-C4334A is characterized in that the mutant gene ISA-A117C-C4334A is obtained by respectively taking a genome reverse transcription product of a 3-type duck hepatitis A virus virulent strain and a eukaryotic expression plasmid pEGFP-C1 as templates and adopting primers shown in the following table for amplification and fusion, wherein the 3-type duck hepatitis A virus virulent strain is preserved in a China center for type culture collection with the preservation number of CCTCC NO: v201305;
wherein, the genome reverse transcription product is taken as a template, primers F1-F and A117C-R, A117C-F and F1-R are used for respectively amplifying to obtain DHAV-3-F1-A117C-F and DHAV-3-F1-A117C-R fragments, primers F2-1-F and F2-1-R, F2-2-F and 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-A117C-F and the DHAV-3-F1-A117C-R fragment into a fragment F1-A117C and fusing the pCMV and the F1-A117C fragment 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-A117C-C4334A.
2. The use of the duck hepatitis A virus type 3 mutant gene ISA-A117C-C4334A of claim 1 in the preparation of a duck hepatitis virus type 3 vaccine.
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| CN105754959A (en) * | 2016-03-13 | 2016-07-13 | 山东省农业科学院家禽研究所（山东省无特定病原鸡研究中心） | NDV (Newcastle disease virus) recombinant virus expressing DHAV-1 and DHAV-3 VP1 genes and application thereof |
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