阅读说明：本技术 一种互换ha和ns1缺失基因包装信号的h9n2亚型禽流感病毒及其构建方法和应用 (H9N2 subtype avian influenza virus with exchanged HA and NS1 deletion gene packaging signals and construction method and application thereof ) 是由 陈素娟 王辉 彭大新 秦涛 杜元钊 楚电峰 于 2020-06-02 设计创作，主要内容包括：本发明提供了一种互换HA和NS1缺失基因包装信号的H9N2亚型禽流感病毒及其构建方法和应用,属于疫苗技术领域。一种互换HA和NS1缺失基因包装信号的H9N2亚型禽流感病毒,包括重组NS-HAmut-NS和HA-NS1-128mut-HA基因。所述重组NS-HAmut-NS和HA-NS1-128mut-HA基因的核苷酸序列依次如SEQ ID NO.1和SEQ ID NO.2所示。利用本发明所述基因构建得到的重组H9N2亚型禽流感病毒能有效避免HA和NS片段与野生毒株发生重配,且具有减毒特性和良好的攻毒保护效力。(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.)

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 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).

3. The method according to claim 2, wherein the eukaryotic cells in step 2) comprise MDCK cells and 293T cells.

4. The method of claim 2, wherein the co-transfection method in step 2) comprises lipofection.

5. The method of any one of claims 2 to 4, wherein the remaining 6 fragments of the parental strain in step 2) are pHW291-PB2, pHW292-PB1, pHW293-PA, pHW295-NP, pHW296-NA and pHW 297-M.

6. The recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) constructed by the construction method of any one of claims 2 to 5, wherein the titer of HA is 6log 2.

7. The recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) according to claim 6, characterized in that no independent reassortment with HA and NS fragments of the wild strain occurs.

8. Root of herbaceous plantThe recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) according to claim 6, wherein the titer is 7.48-8.18 log₁₀EID₅₀/0.1ml。

9. 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) as claimed in any one of claims 6 to 8.

10. The use of the recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) as claimed in any one of claims 6 to 8 in a H9N2 attenuated live vaccine.

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 No. CN104830811, 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 does not 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 a recombinant H9N2 subtype avian influenza virus rTX-NS1-128(mut) constructed by the construction method. 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 10⁶EID₅₀The 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.

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 NS1-128ORF 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 sciences of H9N2 avian influenza viruses isolated from different strains, 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. effective of live-attached H9N2 infection Vaccine NS1 clones against H9N2 Avian Influ 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 used⁶EID₅₀Dosage ofThe 28-day-old SPF chickens immunized by nasal drop and eye injection have the same pathogenicity reduction compared with the live attenuated virus with H9N2 NS1 gene deletion, lose the capability of contact transmission, can induce to generate good immune response and provide 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.