CN111647610B - H9N2 subtype avian influenza virus with exchanged HA and NS1 deletion gene packaging signals and construction method and application thereof - Google Patents
H9N2 subtype avian influenza virus with exchanged HA and NS1 deletion gene packaging signals and construction method and application thereof Download PDFInfo
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Abstract
The invention provides an H9N2 subtype avian influenza virus with exchanged HA and NS1 deletion gene packaging signals and a construction method and application thereof, belonging to the technical field of vaccines. An H9N2 subtype avian influenza virus with exchanged HA and NS1 deleted gene packaging signals comprises recombinant NS-HAmut-NS and HA-NS1-128mut-HA genes. The nucleotide sequences of the recombinant NS-HAmut-NS and HA-NS1-128mut-HA genes are shown as SEQ ID NO.1 and SEQ ID NO.2 in sequence. The recombinant H9N2 subtype avian influenza virus constructed by the gene can effectively avoid the reassortment of HA and NS segments and wild strains, and HAs the characteristics of attenuation and good virus attack protection efficacy.
Description
Technical Field
The invention belongs to the technical field of vaccines, and particularly relates to an H9N2 subtype avian influenza virus exchanging HA and NS1 deletion gene packaging signals, and a construction method and application thereof.
Background
Avian influenza virus subtype H9N2 (AIV) infects a variety of poultry and wild birds. Although AIV strains of subtype H9N2 are less pathogenic to poultry, they cause significant economic losses to the poultry industry, mainly resulting in respiratory symptoms, immunosuppression and decreased egg production. H9N2 AIV has the property of binding to mammalian receptors and has been shown to be transmitted by aerosol between ferrets. In addition, H9N2 AIV can provide partial or all internal genes for H5N1, H7N9, H10N8 and H5N6 subtype AIV to be recombined to generate recombined virus, for example, in 2015-2016, H5N6 subtype AIV containing H9N2 subtype AIV internal gene cassettes is separated from live bird markets in eastern China; 6 internal gene segments separated in 2013 are all derived from a novel H7N9 influenza virus of H9N2 AIV, so that the prevention and control of H9N2 avian influenza also has important public health significance.
China uses H9N2 subtype avian influenza inactivated vaccine (Li CJ, et al, 2005) since 1998, and plays a key role in controlling the epidemic of H9N2 avian influenza. However, H9N2 AIV in different ages has virulence difference and antigen drift, and inactivated vaccines have the disadvantages of only inducing humoral immunity, high adjuvant price, long effective antibody production time, and the capability of producing high-level antibodies by immune chicken flocks, but not effectively inhibiting the detoxification of chicken flocks, so it is necessary to develop novel broad-spectrum vaccines.
Compared with inactivated vaccines, the live vaccine has the immune effect of more effectively generating humoral immunity, cellular immunity and mucosal immune response, reduces the infection rate and the toxin expelling rate of H9N2 AIV clinically, can adopt more convenient inoculation modes such as nasal drip, eye drip, drinking water, spraying and the like, and reduces the labor cost of immunization. The live vaccines are various in types, and can be designed by a gene modification method, for example, attenuated live vaccines can be obtained by a method of rescuing viruses through gene deletion and gene rearrangement reverse genetics or a method of obtaining cold-adapted strains through continuous low-temperature passage of chicken embryos. Live attenuated vaccines have been initially successful in preventing seasonal influenza in humans and have been successfully marketed. The successful case of the human influenza attenuated vaccine provides a development direction for the research of poultry vaccines. A series of live attenuated vaccine candidates against H9N2 subtype AIV have been developed, such as live cold-adapted attenuated vaccine strains grown by gradient cooling. The attenuated live vaccines can better induce mucosal immunity and cellular immunity, and can induce effective immune response in a short time with a smaller dose.
In recent years, an NS1 gene truncated H9N2 attenuated live vaccine strain (publication number CN 104830811, published Japanese 2015.8.12) is constructed and obtained by the research team, and animal experiments prove that the strain can provide good protection for SPF (specific pathogen free) chickens and laying hens and cannot expel toxin, so that the strain is an ideal attenuated live vaccine candidate strain. The research of avoiding recombination after the application of the attenuated live influenza virus vaccine is gradually the focus of the research of the attenuated live influenza virus vaccine. However, live attenuated vaccines have certain defects, such as the exchange of internal gene segments between vaccine strains and wild-type AIV in the environment, the appearance of new epidemic strains and the return of strong toxicity of live attenuated vaccines.
Disclosure of Invention
In view of the above, the invention aims to provide an H9N2 recombinant NS-HAmut-NS and HA-NS1-128mut-HA gene which exchange HA and NS deletion gene packaging signals and a recombinant virus constructed by the same, which can effectively prevent the reassortment of HA and NS fragments of an H9N2 subtype AIV attenuated live vaccine and ensure the safety of the vaccine.
The invention provides H9N2 recombinant NS-HAmut-NS and HA-NS1-128mut-HA genes which interchange HA and NS deletion gene packaging signals, wherein the nucleotide sequence of the NS-HAmut-NS is shown as SEQ ID No. 1; the nucleotide sequence of the HA-NS1-128mut-HA is shown in SEQ ID No. 2.
The invention provides a construction method of a recombinant H9N2 subtype avian influenza virus, which comprises the following steps:
(1) respectively constructing the recombinant NS-HAmut-NS and HA-NS1-128mut-HA genes on a pHW2000 plasmid vector to obtain pHW-NS-HAmut-NS and pHW-HA-NS1-128 mut-HA;
(2) taking an H9N2 subtype avian influenza virus TX strain as a parent strain, and co-transfecting the pHW-NS-HAmut-NS and pHW-HA-NS1-128mut-HA in the step 1) and other 6 fragment plasmids of the parent strain to obtain a recombinant H9N2 subtype avian influenza virus rTX-NS1-128 (mut).
Preferably, the eukaryotic cells in step 2) comprise MDCK cells and 293T cells.
Preferably, the co-transfection method in step 2) comprises liposome co-transfection.
Preferably, the remaining 6 fragments of the parental strain are pHW291-PB2, pHW292-PB1, pHW293-PA, pHW295-NP, pHW296-NA and pHW 297-M.
The invention provides a recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) constructed by the construction method, and the titer of HA is 6log 2.
Preferably, no independent reassortment occurs with the HA and NS fragments of the wild strain.
Preferably, the infection amount of half of SPF chick embryos is 7.48-8.18 log10EID50/0.1ml。
The invention provides an H9N2 attenuated live vaccine exchanging HA and NS deletion gene packaging signals, which is prepared from the recombinant H9N2 subtype avian influenza virus rTX-NS1-128 (mut).
The invention provides application of the recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) in an H9N2 attenuated live vaccine.
The invention provides H9N2 recombinant NS-HAmut-NS and HA-NS1-128mut-HA genes which interchange HA and NS deletion gene packaging signals, wherein the nucleotide sequence of the NS-HAmut-NS is shown as SEQ ID No. 1; the nucleotide sequence of the HA-NS1-128mut-HA is shown in SEQ ID No. 2. The invention successfully exchanges HA and NS1 deletion gene packaging signals by using a molecular cloning method, and specifically, ATG in a HA and NS1-128 fragment packaging signal region is mutated into TTG and NS1-128 fragment G57C shearing site mutation; performing synonymous mutation on the self-packaging signal sequences of the HA and the fragment in the NS1-128 open reading frame; and (3) recombining the mutant sequences by using a seamless cloning method to construct an H9N2 recombinant genome with exchanged HA and NS deletion gene packaging signals.
The invention provides the construction methodThe recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) is constructed. The double-exchange of pHW-NS-HAmut-NS and pHW-HA-NS1-128mut-HA into the maternal viral backbone successfully obtained the rescued virus rTX-NS1-128(mut), while the single-exchange of pHW-NS-HAmut-NS or pHW-HA-NS1-128mut-HA failed to obtain the rescued virus. The measurement result of the hemagglutination titer of the virus shows that the HA titer of the NS1 gene deletion recombinant virus with the exchanged packaging signals is 6log2, and is respectively reduced by 3 titers and 1 titer compared with the HA titers of the parental virus and the NS1 gene deletion virus. Simultaneously, the replication capacity of the virus was verified, and rTX, NS gene-deleted recombinant virus and packaging signal-exchanged virus were inoculated onto MDCK cells at the same infection dose with MOI of 0.001, and the half infection amount (TCID) of the virus tissue was measured by cell culture50) And the proliferation curves of the viruses were plotted, and the replication levels of rTX, rTX-NS1-128 and rTX-NS1-128(mut) were significantly different at different time points. The replication of the three viruses reaches the peak at 60 hours, and the propagation titer of the parental virus TX is obviously higher than that of NS1 gene-deleted virus (P) at all time points after infection<0.05), NS1 gene-deleted virus is significantly higher than packaging signal interchange virus rTX-NS1-128(mut) (P)<0.05). Therefore, compared with the parental virus rTX and NS1 gene deletion virus (rTX-NS1-128), the recombinant H9N2 subtype attenuated live virus constructed by exchanging HA and NS1 deletion gene packaging signals HAs slightly reduced replication capacity on SPF chick embryos and MDCK cells and half infection amount on SPF chick embryos.
The invention further defines that the recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) does not independently reassort with HA and NS segments of the wild strain. The rTX wild strain and rTX-NS1-128(mut) were inoculated onto MDCK cells simultaneously at an infection dose of MOI ═ 10, and cell culture and PCR identification indicated that independent reassortment of HA and NS fragments could not occur with rTX-NS1-128(mut), and all HA and NS fragments were detected from the parental viral rTX.
The invention provides an H9N2 attenuated live vaccine exchanging HA and NS deletion gene packaging signals, which is prepared from the recombinant H9N2 subtype avian influenza virus rTX-NS1-128 (mut). The results of animal experiments show that the dosage is 106EID50Dose of each medicine for nasal drop and eye drop immunization for 28 daysSPF chickens, compared with live attenuated viruses with H9N2 NS1 gene deletion, have the same characteristics of reduced pathogenicity, loss of contact transmission capacity, induction of good immune response and good protective efficacy on homologous TX strains and heterologous F98 strains.
Drawings
FIG. 1 is a scheme for constructing recombinant NS-HAmut-NS and HA-NS1-128mut-HA genes according to the present invention;
FIG. 2 shows the PCR amplification result of rTX-NS1-128(mut) virus cDNA provided by the present invention;
FIG. 3 shows the results of the growth curves of rTX-NS1-128(mut) virus provided by the present invention on MDCK cells;
FIG. 4 shows the result of the testing of rTX-NS1-128(mut) virus reassortment with HA and NS fragments of parental virus;
FIG. 5 shows the result of rTX-NS1-128(mut) virus provided by the present invention inducing animal immune response.
Detailed Description
The invention provides H9N2 recombinant NS-HAmut-NS and HA-NS1-128mut-HA genes which interchange HA and NS deletion gene packaging signals, wherein the nucleotide sequence of the NS-HAmut-NS is shown as SEQ ID No. 1; the nucleotide sequence of the HA-NS1-128mut-HA is shown in SEQ ID No. 2.
The construction strategy of the H9N2 recombinant NS-HAmut-NS and HA-NS1-128mut-HA gene of the invention is shown in figure 1. Specifically, ATG site mutation of HA and NS1-128 fragment packaging signal region into TTG and NS1-128 fragment G57C cutting site mutation; performing synonymous mutation on the self-packaging signal sequences of the HA and the fragment in the NS1-128 open reading frame; the above mutant sequences were recombined by a seamless cloning method to construct two gene fragments of NS-HAmut-NS and HA-NS1-128mut-HA, which were cloned into pHW2000 plasmid vector. The method comprises the following specific steps: the gene site-directed mutation is completed by a single point for multiple times, pHW294-HA and pHW298-NS1-128 are respectively used as templates, and the primers are shown as SEQ ID No. 3-SEQ ID No. 22. The final mutation-completed plasmids were designated pHW294-HAmut and pHW298-NS1-128 mut. The synonymous mutant HA ORF frame was amplified in two steps: in the first step, pHW294-HA is used as a template, an amplification product is obtained by using a primer pair HAOFRF/HAORF R1, and then the amplification product obtained in the first step is used as a template, and a final product HAmut ORF gene is obtained by using a primer pair HA ORF F/HAORF R2. The synonymous mutation NS1-128 ORF frame is obtained by using pHW298-NS1-128 as template and NS1-128mut ORF gene amplified by NS ORF F/NS ORF R primer pair. The primers are shown as SEQ ID No. 23-SEQ ID No. 27. The HAmut ORF and NS1-128mut ORF of the target gene obtained by synonymous mutation were cloned into a commercial Blunt3 vector as intermediate plasmids, and named as Blunt3-HAmut ORF and Blunt3-NS1-128mut ORF. Recombinant plasmids pHW-NS-HAmut-NS and pHW-HA-NS1-128mut-HA were constructed by seamless cloning using pHW294-HAmut, pHW298-NS1-128mut, Blunt3-HAmut ORF, Blunt3-NS1-128mut ORF, and pHW2000 empty plasmid as templates. The fragment amplification primers are designed according to the seamless cloning requirement, and the primers for seamless cloning are shown as SEQ ID No. 28-SEQ ID No. 40. All fragment amplification PCR reaction procedures were: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 15s, Tm annealing for 15s, extension at 72 ℃ for 30s-60s/kb, and 30 cycles; extending for 5min at 72 ℃, and storing at 4 ℃.
The invention provides a construction method of a recombinant H9N2 subtype avian influenza virus, which comprises the following steps:
(1) respectively constructing the recombinant NS-HAmut-NS and HA-NS1-128mut-HA genes on a pHW2000 plasmid vector to obtain pHW-NS-HAmut-NS and pHW-HA-NS1-128 mut-HA;
(2) taking the H9N2 subtype avian influenza virus TX strain as a parent strain, and co-transfecting the pHW-NS-HAmut-NS and pHW-HA-NS1-128mut-HA in the step 1) and the rest 6 fragments of the parent strain to obtain the recombinant H9N2 subtype avian influenza virus rTX-NS1-128 (mut).
The recombinant NS-HAmut-NS and HA-NS1-128mut-HA genes are respectively constructed on pHW2000 plasmid vectors to obtain pHW-NS-HAmut-NS and pHW-HA-NS1-128 mut-HA.
In the present invention, the pHW2000 plasmid vector is referred to reports in the prior art (Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G., and Webster, R. G. (2000). A DNA transfer system for generation of flubenza A viruses from light plasmids, Proc. Natl.Acad.Sci.U.S.A.97, 6108-6113. doi: 10.1073/pnas.100133697).
In the present invention, pHW2000 plasmid vectors into which recombinant NS-HAmut-NS and HA-NS1-128mut-HA genes are inserted, respectively, are inserted into the multiple cloning site BsmBI I by a seamless cloning method. The method for constructing the recombinant vector is not particularly limited in the present invention, and a method for constructing a recombinant vector known in the art may be used.
After recombinant vectors pHW-NS-HAmut-NS and pHW-HA-NS1-128mut-HA are obtained, the invention takes an H9N2 subtype avian influenza virus TX strain as a parent strain, and the pHW-NS-HAmut-NS and pHW-HA-NS1-128mut-HA and the rest 6 segments of the parent strain are co-transfected into eukaryotic cells to obtain the recombinant H9N2 subtype avian influenza virus rTX-NS1-128 (mut).
In the present invention, the H9N2 subtype avian influenza virus TX strain is the A/chicken/Taixing/10/2010(TX) strain, which has been published (see Zhu Y, Yang Y, Liu W, et al, Complex of biological characteristics of H9N2 avian influenza from different hosts, Archives of virology,2015,160: 917-. The Genebank sequence numbers corresponding to the 8-segment of the strain are as follows: PB 2: JN 653572; PB 1: JN 653588; PA: JN 653604; HA: JN 653620; NP: JN 653636; NA: JN 653652; m: JN 653668; and NS: JN 653684.
The present invention is not particularly limited in kind of the eukaryotic cell, and the eukaryotic cell may be infected with a conventional virus well known in the art. The eukaryotic cells preferably include MDCK cells and 293T cells.
The method of co-transfection is not particularly limited in the present invention, and a co-transfection method well known in the art may be used. In embodiments of the invention, the method of co-transfection preferably comprises lipofection. The remaining 6 fragments of the parental strain are pHW291-PB2, pHW292-PB1, pHW293-PA, pHW295-NP, pHW296-NA and pHW 297-M. The 6 fragments were constructed as described in the prior art (Chen S, ZhuY, Yang D, Yang Y, Shi S, Qin T, et al. efficiency of live-attached H9N2 infection Vaccine NS1 viral antigens H9N2 Avian Influenza viruses [ J ]. FrontMicrobiol.2017; 8: 1086.).
After the co-transfection, cell culture, cell enzymolysis and freeze thawing are also included. The condition of the cell culture is preferably that the cell is cultured for 8-10 h in a carbon dioxide incubator at 37 ℃. The method of cell enzymolysis is preferably as follows: blood-free DMEM 1.5ml containing TPCK pancreatin at a final concentration of 2. mu.g/ml, was incubated at 37 ℃ for a further 60 h. And (3) collecting cell supernatant after freeze thawing to obtain the recombinant H9N2 subtype avian influenza virus rTX-NS1-128 (mut).
The invention provides a recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) constructed by the construction method, and the titer of HA is 6log 2. Experiments demonstrated that the rTX-NS1-128(mut) did not reassort independently with the HA and NS fragments of the wild strain. The titer of the rTX-NS1-128(mut) is preferably 7.48-8.18 log10EID500.1 ml. Compared with the parental virus rTX and NS1 gene deletion virus rTX-NS1-128, the replication capacity of the rTX-NS1-128(mut) on SPF chick embryos and MDCK cells and the half infection amount of the SPF chick embryos are slightly reduced; and co-infecting MDCK cells with parent virus rTX, performing plaque purification on supernatant of the co-infected cells, extracting virus RNA, and performing PCR identification, wherein the result shows that the recombinant virus cannot independently reassort HA and NS fragments with wild viruses.
The invention provides an H9N2 attenuated live vaccine exchanging HA and NS deletion gene packaging signals, which is prepared from the recombinant H9N2 subtype avian influenza virus rTX-NS1-128 (mut).
The invention provides application of the recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) in an H9N2 attenuated live vaccine.
In the invention, when the rTX-NS1-128(mut) is used as a H9N2 attenuated live vaccine, animal test results show that 10 is used6EID50The single dose nasal drop eye immunization 28-day-old SPF chicken has the same pathogenicity reduction compared with the H9N2 NS1 gene deletion attenuated live virus, loses the capability of contact transmission, can induce and generate good immune response and provides good protective efficacy for homologous TX strains and heterologous F98 strains.
The present invention provides an avian influenza virus subtype H9N2 with the deletion of HA and NS1 gene packaging signals, and the construction method and application thereof, which are described in detail in the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Vector construction
8 fragments of the H9N2 TX strain used in the invention are cloned to pHW2000 plasmid, and NS1 gene deletion plasmid is constructed (see Chen S, ZhuY, Yang D, Yang Y, Shi S, Qin T, et al. effective of Live-extended H9N2 Influenza Vaccine H9N 1 derived from natural sources, H9N2 Avian Influenza Viruses [ J ]. Front Microbiol.2017; 8: 1086) and named pHW291-PB2, pHW292-PB1, pHW293-PA, pHW294-HA, pHW295-NP, pHW296-NA, pHW297-M, pHW298-NS, pHW-NS 1-298. The nucleotide sequences of NS-HAmut-NS and HA-NS1-128mut-HA genes which interchange HA and NS1 to delete the gene packaging signals are sequentially shown as SEQ ID NO.1 and SEQ ID NO. 2. The two recombinant genes were artificially synthesized and ligated to the pHW2000 plasmid vector. Site-directed mutagenesis and primers are shown in Table 1, head-to-tail synonymous mutagenesis primers for HA and NS1-128 ORF frames are shown in Table 2, and seamless cloning fragment amplification primers are designed according to the requirement of seamless cloning and are shown in Table 3.
TABLE 1 HA and NS1-128 Point mutation primers
TABLE 2 HA and NS1-128 ORF synonymous mutant primers
TABLE 3 seamless splicing primers
The amplification system (25. mu.L) is shown in Table 4:
TABLE 4 Gene amplification System
After the system is mixed evenly and separated instantly, the PCR amplification is preferably carried out according to the following steps: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 15s, Tm annealing for 15s, extension at 72 ℃ for 30s-60s/kb for 30 cycles; extending for 5min at 72 ℃, and storing at 4 ℃. At the end of the reaction, the present invention is preferably identified by electrophoresis on a 1% agarose gel, which recovers the band of interest. The recovered target fragment was ligated to pHW2000, preferably in the system shown in Table 5, and the plasmid was extracted after the ligation transformation. Preferred transformation steps are: dissolving 50 μ l Trans T1 competent cells on ice, adding 4 μ l ligation product, standing on ice for 30min, heat-shocking at 42 deg.C for 45s, standing on ice for 2min, adding 300 μ l LB culture solution, culturing at 37 deg.C with shaking table (220rpm) for 60min, collecting 100 μ l LB-coated cellsA+The plates were cultured in 37 ℃ incubator for 12 h. The gene plasmid with the correct band size is identified by PCR and sent to Nanjing Kingsry company for sequencing. The positive recombinant plasmids were designated pHW-NS-HAmut-NS and pHW-HA-NS1-128 mut-HA.
TABLE 5 Gene fragment ligation System
The invention preferably proves the correctness of the obtained gene sequence mutation by sequencing.
(2) Virus rescue
A method for constructing an H9N2 attenuated virus with a deleted packaging signal of HA and NS1 genes interchanged, comprising the following steps:
after pHW-NS-HAmut-NS and pHW-HA-NS1-128mut-HA were obtained, the parental viral TX backbone was single-and double-interchanged, respectively. Specifically, 600 ng/well of each of three fragments pHW291-PB2, pHW292-PB1 and pHW293-PA, 300 ul/well of each of pHW-NS-HAmut-NS, pHW295-NP, pHW296-NA and pHW297-M, pHW-HA-NS1-128mut-HA were added to 100 ul/well of DMEM without antibiotic and serum, and the DMEM was gently blown to mix the plasmid well. Thereafter, 3. mu.l/well of polyjet transfection reagent was added and mixed again and allowed to act at room temperature for 13 min. During the liposome action, co-cultured MDCK and 293T cells (number ratio 1:3, fusion 60-80%) cultured in 35mm diameter dish were washed 2 times with antibiotic-free, bloodless DMEM, followed by 1ml of antibiotic-and serum-free DMEM. After the liposome reaction was completed, the above prepared 8 fragment recombinant plasmid and transfection reagent mixture was added dropwise to dish. Culturing the culture dish in a carbon dioxide incubator at 37 ℃ for 8-10 h, changing the culture solution to 1.5ml of nonreactive bloodless DMEM containing TPCK pancreatin with the final concentration of 2 mu g/ml, continuously culturing for about 60h at 37 ℃, sealing the whole culture dish by using a sealing film, and repeatedly freezing and thawing for three times in a refrigerator at-70 ℃. Cell supernatants subjected to freeze-thaw treatment were collected, inoculated with 9-day-old SPF chick embryos (0.3 ml/embryo), and cultured in an incubator at 37 ℃. After culturing for 72h, the HA titer of the chick embryo is measured by a conventional method, and the allantoic fluid with the highest titer is collected.
The rescue results show that double-exchange of pHW-NS-HAmut-NS and pHW-HA-NS1-128mut-HA into the maternal viral backbone successfully obtained the rescued virus rTX-NS1-128(mut), and single-exchange of pHW-NS-HAmut-NS or pHW-HA-NS1-128mut-HA failed to obtain the rescued virus. Then inoculating allantoic fluid obtained by inoculating chick embryos of 9 days old to chick embryos of 9 days old for continuous passage, finally obtaining virus fluid for 5 times of continuous passage, extracting virus RNA, carrying out reverse transcription cDNA for PCR identification, wherein HA segment identification primers are SEQ ID No. 41-SEQ ID No.42, NS segment identification primers are SEQ ID No. 43-SEQ ID No.44, and specifically shown in Table 6, an amplification system is shown in Table 7, and PCR reaction procedures for all segment amplification are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, Tm annealing for 15s, extension at 72 ℃ for 15s/kb, 35 cycles; extending for 5min at 72 ℃, and storing at 4 ℃.
TABLE 6 rTX-NS1-128(mut) HA and NS fragment amplification primers
The amplification system (25. mu.L) is shown in Table 7:
TABLE 7 Gene amplification System
The results are shown in FIG. 2, where M: DL2000 marker; lane 1: NS-HAmut-NS fragment size (1835 bp); lane 2: HA-NS1-128mut-HA fragment size (990 bp). As can be seen from FIG. 2, the successful rescue of the H9N2 NS1 gene-deleted virus rTX-NS1-128(mut) with the interchanged packaging signal.
Example 2
Hemagglutination titer of virus, determination of half infection amount of SPF chick embryo and determination of growth curve on MDCK cell
Adding 25 μ l chick embryo allantoic fluid into 25 μ l PBS to obtain 21-211After dilution in multiple proportion, 25. mu.l of 1% chicken erythrocyte suspension was added, negative control wells were made simultaneously, the chamber was left at 37 ℃ for 10min, the hemagglutination titer was read, and the results are shown in Table 8, in which the HA titers of the NS1 gene-deleted recombinant virus, whose packaging signals were interchanged, were reduced by 3 and 1 titers, respectively, compared to the parental virus and the NS1 gene-deleted virus.
TABLE 8 Virus HA Titers
Using antibiotic-containing PBS as raw material of allantoic fluid of virus as 105～1010The solution was diluted in gradient, inoculated with 5 SPF embryos of 9 days old, 0.1 ml/egg, and incubated at 37 ℃ for 72h in an incubator. Allantoic fluid was collected, HA titer was measured, and half of the infected amount was calculated, and the results are shown in Table 9.
TABLE 9 infection of chick embryos with recombinant viruses
Compared with maternal virus and NS1 gene deletion virus, the rTX-NS1-128(mut) recombinant virus has the advantage that the infection amount of half of SPF chick embryos by the rTX-NS1-128(mut) recombinant virus is reduced to different degrees.
Will be 1 × 106MDCK cells of (a) were cultured in 6-well plates, and rTX, NS gene-deleted recombinant virus, and packaging signal-exchanged virus were inoculated at an infectious dose with MOI of 0.001. After 1h of adsorption, the cells were washed 3 times with PBS and cultured in anti-blood-free DMEM containing pancreatin at a final concentration of 2. mu.g/ml TPCK. Cell supernatants were collected at 12, 24, 36, 48, 60 and 72h post-infection, respectively, and the median infectious load (TCID) of viral tissues was determined50) And the proliferation curve of the virus is plotted.
The results are shown in FIG. 3. On MDCK cells, there were significant differences in the replication levels of rTX, rTX-NS1-128 and rTX-NS1-128(mut) at different time points. The replication of all three strains of virus peaked at 60 hours, and at all time points after infection, the propagation titer of the parental virus TX was significantly higher than that of the NS1 gene-deleted virus (P <0.05), and that of the NS1 gene-deleted virus was significantly higher than that of the packaging signal-exchanged virus (P < 0.05).
Example 3
HA and NS fragment recombination assays
Will be 1 × 106The MDCK cells of (a) were cultured in a 6-well plate, and inoculated with both rTX wild strain and NS1-128(mut) packaging signal-exchanged NS truncated attenuated virus at an infection dose of MOI ═ 10, adsorbed for 1 hour, washed 3 times with PBS, and then cultured with anti-anemic DMEM 1ml containing TPCK pancreatin at a final concentration of 2 μ g/ml. Supernatants were collected by three freeze-thaw cycles after 12 hours post infection and centrifuged for use.
MDCK cells grown full of monolayers were cultured in 12-well plates, and the plates were washed three times with PBS after media was discarded. Gradient dilution of virus to 10% in non-anti-haemophilus MEM medium-5Add 200. mu.l of virus dilution along the well wall, shake the plate and mix well. The 12-well plate was incubated at 37 ℃ for 1h, during which time the plate was shaken and mixed once every 15 minutes. The inoculum was then removed, the plate washed three times with PBS, and 1 mL/well of a 1:1 mixture of 2 × EMEM medium containing TPCK pancreatin at a final concentration of 1 μ g/mL and 1.6% agarose water was added and allowed to solidify at room temperature. Pouring at 37 deg.CIncubate for 72h, during which time plaque formation was observed daily. After 72h, the cells were washed with PBS 1: diluting 0.33% neutral red solution by 20 times, mixing with 1.6% agarose solution at a ratio of 1:1, adding into 12-well plate, incubating at 1 mL/well, standing at 37 deg.C under dark condition for 24h, and placing single plaque into finger-shaped tube containing 400 μ l MEM. After the plaque sample is frozen and thawed for three times, 200 mu l of supernatant is inoculated into a 24-well plate paved with MDCK cells, and after incubation for 1h, the solution is changed into MEM culture medium with the final concentration of 2 mu g/ml TPCK pancreatin. And (3) incubating at 37 ℃ for 72h, measuring the HA titer of the supernatant, collecting the supernatant with the titer, extracting virus RNA, performing reverse transcription to obtain cDNA, and designing a primer for PCR identification.
TABLE 10 HA and NS fragment identification primers
The HAJD F and HA/NS JD R primer pair is used for identifying HA segment sources, the amplification size of the parent strain HA segment is 1217bp, and the amplification size of the recombinant virus HA segment is 1321 bp. The primer pair of NS JD F and HA/NS JD R is used for identifying the source of NS segment, the amplification size of the parent strain NS segment is 408bp, and the amplification size of the recombinant virus NS segment is 532 bp. The co-infected samples were plaque purified to obtain a total of 48 effective samples. PCR detection was performed, and as a result, as shown in FIG. 3, independent reassortment of HA and NS fragments did not occur in all plaque samples, and all HA and NS fragments were detected to be from the maternal virus rTX.
Example 4
To determine the pathogenicity of the packaging signal interchange virus to chickens. At 106EID50Virus dose of 200 μ l 4 week old SPF chickens, 11 chickens per group, were inoculated in a nasal drop-and-eye manner. After challenge, 21d of continuous observation was carried out.
To determine the replication capacity of the virus in the tissues, 3 chickens were anesthetized 3d and 5d post challenge to harvest tracheal and lung tissues, respectively. A portion of the collected tissue was collected, PBS was added at a ratio of 1g/0.3 ml, and the tissue was homogenized electrically, centrifuged at 8,000 r.times.10 min, and the supernatant was collected. The supernatant was inoculated with SPF chick embryos for EID determination50The results are shown in Table 11. Two chickens in the NS truncated attenuated virus rTX-NS1-128 group can feelThe titers detected in the trachea at day 3 and day 5 after virus infection were 2.05. + -. 0.28log10EID, respectively500.1ml and 0.81. + -. 0.2 log10EID500.1 ml. Only one chicken in the package signal-interchanged NS truncated attenuated virus rTX-NS1-128(mut) group could be detected in the trachea at day 3 and day 5 post-virus infection, with titers of 2.77log10EID500.1ml and 2.33 log10EID500.1 ml. Virus could not be detected in lung tissue on both day 3 and day 5 post-infection. The female parent virus group rTX can detect viruses with higher replication titer in trachea and lung, and can respectively reach 2.41 +/-1.08 log on day 310EID500.1ml and 1.67log10EID500.1 ml. The rTX-NS1-128 was not significantly different from the rTX-NS1-128(mut) group (p)>0.05)。
TABLE 11 replication titers of viruses in tracheal and pulmonary tissues of chickens
Note: in the tableaPositive swab/total swab;bno virus was isolated.
Example 5
In order to detect the detoxification of the virus in the chickens, throat swabs and cloaca cotton swabs were collected at 3d, 5d and 7d after challenge, respectively, and the chicken embryos were inoculated after the cotton swabs were treated by a conventional method, and the results are shown in table 12. The rTX-NS1-128 group can expel toxin from larynx, the toxin expelling rate on day 3 is 10/10, the toxin expelling rate on day 5 is 8/10, and the toxin expelling rate on day 7 is reduced to 3/10; the strain can not expel toxin from cloaca after infecting chickens; the rTX-NS1-128(mut) group can also expel toxin from larynx, the expelling rate of 3 days is 8/10, which is lower than that of the control group, the expelling rate of 5 days is 8/10, and the expelling rate of 7 days is reduced to 2/10; the strain can not be detoxified from cloaca after infecting chickens; female parent toxin rTX group laryngeal detoxification was similar to rTX-NS1-128 groups, but this group of chickens could be cleared by cloaca on day 3 (3/10). rTX-NS1-128(mut) was not significantly different from rTX-NS1-128 (p > 0.05).
TABLE 12 detoxification of chicken infected with virus
Note: in the tableaThroat swab;bcloacal swabs.
Example 6
Test for viral Transmission in chickens
In the spread test, each group contained 15 SPF chickens, 10 were inoculated with 10 SPF chickens6EID500.2ml of virus/200. mu.l was used as donor group; 5 chickens were placed in the same cage 24h after inoculation as a contact group. And 3d, 5d and 7d after inoculation and exposure are respectively used for collecting throat swabs and cloaca swabs of chickens in the inoculation group and the contact group, treating the cotton swabs, and then inoculating chick embryos to determine the detoxification condition. At 14d post inoculation or exposure, chicken blood was collected, sera separated and HI measured to observe the positive shift in sera.
The virus isolation results are shown in Table 13. Detoxification was detected from throat swabs only on days 3 and 5 post-inoculation in the rTX-NS1-128 group, with 80% and 50% detoxification rates, respectively. The corresponding detoxification rates of the rTX-NS1-128(mut) groups were 60% and 40%, respectively, and the differences between the two groups were not significant (p > 0.05). On day 14, all vaccinated breeders developed a seroconversion to positive. None of the chickens in the contact group detected virus and had developed a seroconversion positive phenomenon.
TABLE 13 test for Transmission Properties of viruses
Note: in the tableaThroat swab;bcloacal swabs.
Example 7
Recombinant virus induced antibody levels and duration
To check antibody levels and duration, packaging signals were exchanged for virus to106 EID50After 4-week-old SPF chickens were inoculated nasally at a dose of 200. mu.l, sera were separated at 7d intervals and HI values were determined for 11 weeks. HI antibody levels approaching 4log2 were achieved one week post immunization, antibody levels began to rise in the second week, peaks at an average of 7.89 ± 0.87log2 three weeks post immunization, and then began to decline slowly, reaching 3.85 ± 0.25 log2 11 weeks post immunization (fig. 5).
Example 8
Challenge protection test
At 106EID50A dose of 200. mu.l was used to immunize 4-week-old SPF chickens by nasal drop and eye drop, and PBS inoculated chickens were used as controls. After 21d immunization, the dose was changed to 106EID50A200. mu.l dose of homologous TX and heterologous F98 (see Liu JH, Okazaki K, Shi WM, Wu QM, Mweene AS, Kida H (2003) Photogenic analysis of neuroamidinate gene of H9N2 inflenza viruses preservation in chicken in China drug 1995-2002.) the chickens were challenged by nasal drip. And collecting throat swabs and cloaca cotton swabs after challenge at 3d, 5d and 7d respectively to determine detoxification conditions. The challenge results of the immune chicken flock show (table 14), and the vaccine strain can provide 100% protection for the female parent virus rTX; for heterologous strain F98, 80% protection was provided and detoxification could be detected in throat swabs on day 3 post challenge (2/10). For the homologous virus TX and heterologous virus F98 challenge control group, larynx detoxification can be detected on the 3 rd day after challenge, the detoxification rate is 100%, the cloaca detoxification rate is 40% and 30%, and the peak of detoxification is formed; the detoxification rate began to decrease 5 days after challenge.
TABLE 14 SPF Chicken vaccine candidate immunoprotection experiments
Note: in the tableaThroat swab;bcloacal swabs.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Yangzhou university
Qingdao Yibang Bioengineering Co.,Ltd.
<120> H9N2 subtype avian influenza virus with exchanged HA and NS1 deletion gene packaging signals and construction method and application thereof
<160> 47
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1917
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agcaaaagca gggtgacaaa aacatattgg attccaacac tgtgtcaagc ttccagctag 60
actgctttct ttggcttgtc cgcaaacgat ttgcagacca agaaatggaa accatctccc 120
tcatcaccat cctgctggtc gtcacggtct cgaatgcaga caaaatctgc atcggctatc 180
aatcaacaaa ctccacagaa actgtagaca cactaacaga aaacaatgtc cctgtgacac 240
atgccaaaga attgctccac acagagcata atgggatgct gtgtgcaaca ggcttgggac 300
atcctcttat tctagacacc tgtaccattg aaggactaat ctatggcaat ccttcttgtg 360
atctactgct gggaggaaga gaatggtcct atatcgtcga gagaccatca gctgttaacg 420
gactgtgtta ccccgggaat gtagagaatc tagaagagct aaggtcactt tttagttctg 480
ctagttctta tcaaaggatc cagatttttc cagacacaat ctggaatgtg acttacagtg 540
ggacaagcaa agcatgttca gattcattct acagaagcat gagatggttg acccaaaaga 600
acaacgctta ccctattcaa gacgcccaat acacaaataa tcaagaaaag aacattcttt 660
tcatgtgggg cataaatcat ccccccaccg atactgtgca gacaaatctg tacacaagaa 720
ccgacacaac aacgagtgtg gcaacagaag aaataaatag gaccttcaaa ccattgatag 780
gaccaaggcc tcttgttaat ggtatgcagg gaagaatcga ctattattgg tcggtattga 840
aaccgggtca aacgctgcga ataagatcca atggaaatct aatagctcca tggtatggac 900
acattctttc tggagagagc cacggaagaa tcctgaagac tgatttaaaa aggggtagct 960
gcacagtgca atgtcagacc gaaaaaggtg gcttaaacac aacattgcca ttccaaaacg 1020
taagtaagta tgcatttgga aactgctcga aatacattgg aataaagagt ctcaaactcg 1080
cagttggtct gaggaatgtg ccttctagat ctagtagagg actattcggg gccatagcag 1140
gattcataga gggaggctgg tcaggactag ttgctggttg gtatggattc cagcattcaa 1200
atgaccaagg ggttggtatg gcagctgata gagactcaac ccaaaaggca attggcaaaa 1260
taacatccaa agtgaataat atagtcaaca aaatgaacaa gcagtatgaa attattgatc 1320
atgaattcag cgaggttgaa gctagactta acatgatcaa taataagatt gatgatcaaa 1380
tccaagacat atgggcatat aatgcagaat tactagttct gcttgaaaac cagaaaacac 1440
tagatgagca tgatgcaaat gtaaacaatc tatataataa agtgaagagg gcattgggtt 1500
ccaatgcaat ggaagatggg aaaggatgtt tcgagctata ccacaaatgt gataaccagt 1560
gcatggagac gattcggaac gggacctaca acaggaggaa atatcaagag gaatcaaaat 1620
tagaaagaca gaaaatagag ggggtcaagc tggaatctga aggaacttac aaaatcctca 1680
ccatttattc gactgtcgca agcagcctcg taatcgccat gggattcgcc gcttttctat 1740
tttgggcgat gagcaacggc agctgtaggt gtaatatctg catctgaaaa ttacggaaaa 1800
tagctttgag caaataactt ttatgcaagc cttacaacta ttgcttgaag tggaacaaga 1860
gataagaact ttctcgtttc agcttattta atgataaaaa acacccttgt ttctact 1917
<210> 2
<211> 1019
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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agcgaaagca ggggaatttc acaacactca agttggagac aatatcacta ataactatac 60
tactagtagt aacagtaagc attgcagaca aaatctgcat atggacagca ataccgtcag 120
ttctttccag gtagattgct ttctttggca tgtccgcaaa cgatttgcag accaagaaat 180
gggtgatgcc ccatttctag accggcttcg ccgagatcag aagtccctga gaggaagaag 240
cagcactctt ggtctggaca tcagaactgc tactcgtgaa ggaaagcata tagtggaacg 300
gattttagag gacgagtcag atgaagcatt taaaatgagt attgcttcag tgccagcccc 360
acgctaccta actgacatga ctcttgaaga aatgtcaaga gattggttaa tgctcattcc 420
caaacagaaa gtaacagggt ccctttgcat tagaatggac caagcaatag tggacaaaaa 480
catctaggaa ggagcaattg tgggcgaaat ctcaccatta ccttctcttc caggacatac 540
tgacaaggat gtcaaaaatg caattgaggt cctcatcgga ggatttgaat ggaatgataa 600
cacagttcga gtctctgaaa ctctacagag attcgcttgg agaagcagcg atgaggatgg 660
gagacctcca ctctctacaa agtagaaacg gtaaatggag agaacagcta agccagaagt 720
tcgaagaaat aagatggttg attgaagaag tacgacatag attaaaaatt acggaaaata 780
gctttgagca aataactttt atgcaagcct tacaactatt gcttgaagtg gagcaggaaa 840
tccggacctt tagcttccaa ttgatctaaa acttacaaaa tcctcaccat ttattcgact 900
gtcgcctcat cccttgtgat tgcattgggg tttgctgcct tcttgttctg ggccttgtcc 960
attgggtctt gcagttgcaa catttgtatt tgattggcaa aaacaccctt gtttctact 1019
<210> 3
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctcaagttgg agacaatatc actaataact atactactag tag 43
<210> 4
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gatattgtct ccaacttgag tgttgtgaaa ttcccctgct tt 42
<210> 5
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acagtaagca ttgcagacaa aatctgcatc ggct 34
<210> 6
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tttgtctgca atgcttactg ttactactag tagtatag 38
<210> 7
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tgtgattgca ttggggtttg ctgccttctt gtt 33
<210> 8
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gcaaacccca atgcaatcac aagggatgag gcg 33
<210> 9
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gttctgggcc ttgtccattg ggtcttgc 28
<210> 10
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
acccaatgga caaggcccag aacaagaag 29
<210> 11
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ggtcttgcag ttgcaacatt tgtatttgat tggcaaaaac 40
<210> 12
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
caaatgttgc aactgcaaga cccaatggac aagg 34
<210> 13
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
catattggat tccaacactg tgtcaagctt c 31
<210> 14
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cagtgttgga atccaatatg tttttgtcac cctgcttttg ct 42
<210> 15
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aagcttccag ctagactgct ttctttggct tgtccg 36
<210> 16
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
aagcagtcta gctggaagct tgacacagtg ttgg 34
<210> 17
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tttctttggc ttgtccgcaa acgatttgca g 31
<210> 18
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tttgcggaca agccaaagaa agcagtctac ctgg 34
<210> 19
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aataactttt ttgcaagcct tacaactatt gcttgaag 38
<210> 20
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gcttgcaaaa aagttatttg ctcaaagcta ttttccgtaa ttt 43
<210> 21
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
agcttattta ttgataaaaa acacccttgt ttctact 37
<210> 22
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ttttttatca ataaataagc tgaaacgaga aagttc 36
<210> 23
<211> 68
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
atggaaacca tctccctcat caccatcctg ctggtcgtca cggtctcgaa tgcagacaaa 60
atctgcat 68
<210> 24
<211> 67
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ccaaaataga aaagcggcga atcccatggc gattacgagg ctgcttgcga cagtcgaata 60
aatggtg 67
<210> 25
<211> 65
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tcagatgcag atattacacc tacagctgcc gttgctcatc gcccaaaata gaaaagcggc 60
gaatc 65
<210> 26
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
atggacagca ataccgtcag ttctttccag gtagattgct ttctttggca tgtcc 55
<210> 27
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ttagatcaat tggaagctaa aggtccggat ttcctgctcc acttcaagc 49
<210> 28
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
aataacccgg cggcccaa 18
<210> 29
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
cccccccaac ttcggag 17
<210> 30
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ctccgaagtt gggggggagc aaaagcaggg tg 32
<210> 31
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ggagatggtt tccatttctt ggtctgcaaa 30
<210> 32
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tttgcagacc aagaaatgga aaccatctcc 30
<210> 33
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
attttccgta attttcagat gcagatat 28
<210> 34
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
atatctgcat ctgaaaatta cggaaaat 28
<210> 35
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
ctccgaagtt gggggggagc gaaagcaggg g 31
<210> 36
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gtattgctgt ccatatgcag attttgtc 28
<210> 37
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gacaaaatct gcatatggac agcaatac 28
<210> 38
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ggattttgta agttttagat caattgga 28
<210> 39
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tccaattgat ctaaaactta caaaatcc 28
<210> 40
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
ttgggccgcc gggttattag tagaaacaag ggt 33
<210> 41
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
tccagctaga ctgctttc 18
<210> 42
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
taagctgaaa cgagaaag 18
<210> 43
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gggaatttca caacactc 18
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tttgccaatc aaatacaaat 20
<210> 45
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
aagaacaacg cttaccct 18
<210> 46
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gaaggagcaa ttgtgggcga 20
<210> 47
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
agtagaaaca agggtgtttt t 21
Claims (5)
1. An H9N2 recombinant NS-HAmut-NS and HA-NS1-128mut-HA gene which exchange HA and NS deletion gene packaging signals, wherein the nucleotide sequence of the NS-HAmut-NS is shown as SEQ ID No. 1; the nucleotide sequence of the HA-NS1-128mut-HA is shown in SEQ ID No. 2.
2. A construction method of a recombinant H9N2 subtype avian influenza virus is characterized by comprising the following steps:
(1) the recombinant NS-HAmut-NS and HA-NS1-128mut-HA genes of claim 1 are constructed on pHW2000 plasmid vector to obtain pHW-NS-HAmut-NS and pHW-HA-NS1-128 mut-HA;
(2) taking an H9N2 subtype avian influenza virus TX strain as a parent strain, and co-transfecting the pHW-NS-HAmut-NS and pHW-HA-NS1-128mut-HA in the step (1) and the rest 6 fragments of the parent strain to obtain a eukaryotic cell, so as to obtain a recombinant H9N2 subtype avian influenza virus rTX-NS1-128 (mut);
the Genebank sequence numbers corresponding to the remaining 6 segments of the parental strain are as follows: PB 2: JN 653572; PB 1: JN 653588; PA: JN 653604; NP: JN 653636; NA: JN 653652; m: JN 653668; cloning the 6 fragments into pHW2000 plasmid vector to obtain pHW291-PB2, pHW292-PB1, pHW293-PA, pHW295-NP, pHW296-NA and pHW297-M fragments;
the co-transfection method includes a liposome co-transfection method;
the eukaryotic cells are MDCK and 293T cells; the number ratio of the MDCK to the 293T cells is 1:3, and the fusion degree of the MDCK and the 293T cells is 60-80%;
after the co-transfection, cell culture is also included; the cell culture condition is characterized in that the cell culture condition is carried out in a DMEM (DMEM) without antibiotics and serum at 37 ℃ for 8-10 h, the solution is changed into a non-resistant blood-free DMEM containing TPCK pancreatin with the final concentration of 2 mu g/ml, the cell culture condition is carried out at 37 ℃ for 60h, and cell supernatant is collected after freezing and thawing at-70 ℃.
3. The recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) constructed by the construction method of claim 2.
4. An H9N2 attenuated live vaccine exchanging HA and NS deletion gene packaging signals, which is prepared from the recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) of claim 3.
5. The use of the recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) as claimed in claim 3 in a H9N2 attenuated live vaccine.
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