CN102876638A - Recombinant influenza virus capable of realizing high-efficiency expression of HA protein, and preparation method and application of recombinant influenza virus - Google Patents

Abstract

The invention discloses a PR8 recombinant influenza virus. The recombinant influenza virus comprises HA and/or NA genes of H3 subtype influenza virus and six internal genes (PB1, PB2, PA, NP, M and NS genes) of PR8 virus, wherein the HA and/or NA genes comprise the following mutation sites: the NS gene coded NS2 protein comprises E67S point mutation or E74S point mutation, or E67S/E74S point mutation; and the NP gene coded NP protein comprises G132A point mutation. The invention further discloses a preparation method and application of the PR8 recombinant influenza virus. The PR8 recombinant influenza virus prepared by construction of recombinant plasmids, cotransfection of cells and amplification chicken embryo can realize high-efficiency of the HA protein and/or the NA gene and can be applied to large-scale preparation of influenza vaccines.

Description

Efficiently express recombinant influenza of HA albumen and its preparation method and application

The application is to be on September 8th, 2011 applying date, and application number is 201110266082.0, and denomination of invention is divided an application for the patent application of " efficiently expressing recombinant influenza of HA albumen and its preparation method and application ".

Technical field

The invention belongs to biological technical field, relate to the production field of vaccine, particularly, relate to recombinant influenza of a kind of HA of efficiently expressing albumen and its preparation method and application.

Technical field

Influenza is a kind of acute, the height contagious disease that is caused by orthomyxoviridae family Influenzavirus A influenza virus, and high pathogenic avian influenza is defined as the category-A epidemic disease by the International Animal Health tissue.Influenza virus can be divided into A, B, C three types according to the difference of stromatin (Matrix protein, M).According to influenza virus hemagglutinin (Hemagglutinin, HA) and the antigenic difference of neuraminidase (Neuraminidase, NA), influenza virus can be divided into different hypotypes again, A type influenza virus is divided into 16 hypotypes according to HA, is divided into 9 hypotypes according to NA.Influenza virus has hyperinfection, can be by droplet transmission, so it can occur suddenly in a short time, and rapid spread cause in various degree popular, even is very popular in the world.1983-1984, the high pathogenic avian influenza in U.S. Binzhou is broken out, and causes 1,700 ten thousand poultry dead, loses nearly 6,500 ten thousand dollars.The end of the year in 2003, highly pathogenic bird flu has been broken out in a plurality of countries and regions, Asia.Breaking out of bird flu makes more than 100,000,000 poultry dead or slaughtered, and Countries poultry output and sales volume sharply descend, price drops, and poultry and goods thereof are imported and exported and are supspended; In addition, poultry farming, feedstuff industry and tourism also all are adversely affected.Estimate that according to food and agricultural organization the loss that suffers thus is at least 500,000,000 dollars.In addition, the avian influenza virus of many hypotypes has the host of striding and is transmitted to Man's Power, thereby breaking out of this disease also endangering social public security simultaneously.Twentieth century has broken out being very popular of three influenzas, is respectively spanish influenza (H1N1), nineteen fifty-seven Asia influenza (H2N2) and nineteen sixty-eight Mao flu (H3N2), wherein the Flow Behavior spanish influenza of maximum-norm in 1918.This influenza epidemic disease causes global 200d 00,000 people's death, surpasses World War I death toll, is listed as first of all Death of Infectious Diseases of the whole world.

The genome of influenza virus is by sub-thread, and negative adopted RNA fragment forms..A type influenza virus is divided into 8 fragments, the 11 kinds of functional proteins of encoding, fragment 1,2,3 encode respectively three polymerase protein PB2, PB1 and PA; Fragment 4 coding hemagglutinin HA; Fragment 5 coding Nucleocapsid protein P; Fragment 6 coding neuraminidase NA; Fragment 7 coding matrix prote m1 and ionic channel M2; Fragment 8 coding non-structural protein NS 1 and NS2.Wherein HA and NA are the topmost two kinds of surface glycoproteins of influenza virus, and HA albumen is the most important protective antigen of influenza virus.

At present, various countries have taked different measures to the prevention and control of animal and human's influenza, but vaccine inoculation remains the optimal selection of flu-prevention, therefore develop effective influenza vaccines and are extremely important for the control influenza pandemic.Inactivated virus vaccine is present most widely used vaccine, and this vaccine safety is good, virulence can not occur and return danger strong and variation, can stand the attack of subtype influenza virus of the same race.The influenza vaccines that gone on the market at present substantially all are to use chicken embryo culture preparation, use chicken embryo culture vaccine that 50 years history has been arranged both at home and abroad.Need to consume a large amount of chicken embryos because the chicken embryo is produced influenza vaccines, there is the potentially contaminated possibility in the chicken germ band, and culture cycle is long, is not easy to enlarge output, is unfavorable for tackling large-scale flu outbreak.For this reason, the World Health Organization, United States Government etc. all the encourage growth cell culture technology substitute present chick embryo technique runoff yield in next life influenza vaccine.For accelerating the exploitation of cell cultures influenza vaccines technology, United States Government determines 1,100,000,000 dollars of subsidies of investment GlaxoSmithKline PLC, edImmune, Novartis, DynPort, Solvay and 6 main influenza vaccines development new technologies of pasteur.In 2007, Novartis of one of biopharmaceutical company that the whole world is maximum announced its human influenza vaccines Optaflu listing, became the human cell cultures influenza vaccines of unique granted (European Union's approval), was one of the most great innovation of 50 years influenza vaccines production histories.

No matter adopt chick embryo method and mass cell preparation method to obtain virus, important influence factor is that vaccine kind poison itself is the virus strain of high yield.A/Puerto Rico/8/34 (PR8) is that a strain chicken embryo adapts to virus strain, it is present one of high yield strain on the chicken embryo, usually with 6 internal gene of PR8 and HA and the NA gene recombination (6+2 pattern) of epidemic isolates, recombinant virus is improved virus titer as vaccine strain in the vaccine development.In order further to improve the virus titer of PR8, satisfy when influenza is broken out greatly, huge vaccine demand, the scientific research personnel has carried out a large amount of research, improves virus strain output by optimizing virogene.Research finds that certain amino acid sites of these some albumen of influenza virus has important impact to this viral multiplication capacity, and upper 55 L-glutamic acid (Glu) of the tyrosine upper 360 such as PB2 (Tyr) and NS1 is also played a role.

Summary of the invention

One of purpose of the present invention is to provide a kind of PR8 sudden change recombinant influenza, and this recombinant influenza can efficiently express HA albumen, is applicable to producing influenza vaccine in large-scale.

Realize that the above-mentioned purpose technical scheme is as follows:

A kind of PR8 recombinant influenza, it contains HA and/or the NA gene of H1 subtype influenza virus, 6 internal gene (PB1 that contain PR8 virus, PB2, PA, NP, M, the NS gene), wherein one of NS and NP gene or the two contain following mutational site: have the E67S point mutation on the NS2 albumen of NS genes encoding, or E74S point mutation, or E67S/E74S point mutation, has the G132A point mutation on the NP albumen of NP genes encoding, described H1 subtype influenza virus is that (coding has the E67S point mutation for H1 subtype influenza virus except PR8 virus, or E74S point mutation, or the NS gene of the NS2 albumen of E67S/E74S point mutation, and coding has the NP gene of the NP albumen of G132A point mutation, its nucleotide sequence is the nucleotide sequence shown in SEQ ID.NO:20-23 respectively, and its aminoacid sequence is shown in SEQ ID.NO:24-27).

A kind of PR8 recombinant influenza, it contains HA and/or the NA gene of H3 subtype influenza virus, 6 internal gene (PB1 that contain PR8 virus, PB2, PA, NP, M, the NS gene), wherein one of NS and NP gene or the two contain following mutational site: have the E67S point mutation on the NS2 albumen of NS genes encoding, or E74S point mutation, or E67S/E74S point mutation, (coding has the E67S point mutation to have the G132A point mutation on the NP albumen of NP genes encoding, or E74S point mutation, or the NS gene of the NS2 albumen of E67S/E74S point mutation, and coding has the NP gene of the NP albumen of G132A point mutation, its nucleotide sequence is the nucleotide sequence shown in SEQ ID.NO:20-23 respectively, and its aminoacid sequence is shown in SEQ ID.NO:24-27).

A kind of PR8 recombinant influenza, it contains HA and/or the NA gene of H4 subtype influenza virus, 6 internal gene (PB1 that contain PR8 virus, PB2, PA, NP, M, the NS gene), wherein one of NS and NP gene or the two contain following mutational site: have the E67S point mutation on the NS2 albumen of NS genes encoding, or E74S point mutation, or E67S/E74S point mutation, (coding has the E67S point mutation to have the G132A point mutation on the NP albumen of NP genes encoding, or E74S point mutation, or the NS gene of the NS2 albumen of E67S/E74S point mutation, and coding has the NP gene of the NP albumen of G132A point mutation, its nucleotide sequence is the nucleotide sequence shown in SEQ ID.NO:20-23 respectively, and its aminoacid sequence is shown in SEQ ID.NO:24-27).

A kind of PR8 recombinant influenza, it contains HA and/or the NA gene of H5 subtype influenza virus, 6 internal gene (PB1 that contain PR8 virus, PB2, PA, NP, M, the NS gene), wherein one of NS and NP gene or the two contain following mutational site: have the E67S point mutation on the NS2 albumen of NS genes encoding, or E74S point mutation, or E67S/E74S point mutation, (coding has the E67S point mutation to have the G132A point mutation on the NP albumen of NP genes encoding, or E74S point mutation, or the NS gene of the NS2 albumen of E67S/E74S point mutation, and coding has the NP gene of the NP albumen of G132A point mutation, its nucleotide sequence is the nucleotide sequence shown in SEQ ID.NO:20-23 respectively, and its aminoacid sequence is shown in SEQ ID.NO:24-27).

A kind of PR8 recombinant influenza, it contains HA and/or the NA gene of H6 subtype influenza virus, 6 internal gene (PB1 that contain PR8 virus, PB2, PA, NP, M, the NS gene), wherein one of NS and NP gene or the two contain following mutational site: have the E67S point mutation on the NS2 albumen of NS genes encoding, or E74S point mutation, or E67S/E74S point mutation, (coding has the E67S point mutation to have the G132A point mutation on the NP albumen of NP genes encoding, or E74S point mutation, or the NS gene of the NS2 albumen of E67S/E74S point mutation, and coding has the NP gene of the NP albumen of G132A point mutation, its nucleotide sequence is the nucleotide sequence shown in SEQ ID.NO:20-23 respectively, and its aminoacid sequence is shown in SEQ ID.NO:24-27).

A kind of PR8 recombinant influenza, it contains HA and/or the NA gene of H7 subtype influenza virus, 6 internal gene (PB1 that contain PR8 virus, PB2, PA, NP, M, the NS gene), wherein one of NS and NP gene or the two contain following mutational site: have the E67S point mutation on the NS2 albumen of NS genes encoding, or E74S point mutation, or E67S/E74S point mutation, (coding has the E67S point mutation to have the G132A point mutation on the NP albumen of NP genes encoding, or E74S point mutation, or the NS gene of the NS2 albumen of E67S/E74S point mutation, and coding has the NP gene of the NP albumen of G132A point mutation, its nucleotide sequence is the nucleotide sequence shown in SEQ ID.NO:20-23 respectively, and its aminoacid sequence is shown in SEQ ID.NO:24-27).

A kind of PR8 recombinant influenza, it contains HA and/or the NA gene of H9 subtype influenza virus, 6 internal gene (PB1 that contain PR8 virus, PB2, PA, NP, M, the NS gene), wherein one of NS and NP gene or the two contain following mutational site: have the E67S point mutation on the NS2 albumen of NS genes encoding, or E74S point mutation, or E67S/E74S point mutation, (coding has the E67S point mutation to have the G132A point mutation on the NP albumen of NP genes encoding, or E74S point mutation, or the NS gene of the NS2 albumen of E67S/E74S point mutation, and coding has the NP gene of the NP albumen of G132A point mutation, its nucleotide sequence is the nucleotide sequence shown in SEQ ID.NO:20-23 respectively, and its aminoacid sequence is shown in SEQ ID.NO:24-27).

A kind of PR8 recombinant influenza, it contains HA and/or the NA gene of H10 subtype influenza virus, 6 internal gene (PB1 that contain PR8 virus, PB2, PA, NP, M, the NS gene), wherein one of NS and NP gene or the two contain following mutational site: have the E67S point mutation on the NS2 albumen of NS genes encoding, or E74S point mutation, or E67S/E74S point mutation, (coding has the E67S point mutation to have the G132A point mutation on the NP albumen of NP genes encoding, or E74S point mutation, or the NS gene of the NS2 albumen of E67S/E74S point mutation, and coding has the NP gene of the NP albumen of G132A point mutation, its nucleotide sequence is the nucleotide sequence shown in SEQ ID.NO:20-23 respectively, and its aminoacid sequence is shown in SEQ ID.NO:24-27).

Another object of the present invention provides the method for the above-mentioned PR8 recombinant influenza of preparation.

The technical scheme that realizes this purpose is as follows:

A kind of method for preparing above-mentioned PR8 recombinant influenza may further comprise the steps:

Make up and comprise respectively the HA of H1, H3, H4, H5, H6, H7, H9, H10 subtype influenza virus and the recombinant plasmid of NA gene;

Structure comprises the recombinant plasmid of PR8 virus mutation gene fragment, this PR8 virus mutation gene fragment is selected from NS or the NP gene fragment of following sudden change: coding contains the PR8 virus N S gene fragment of the NS2 albumen of E67S or E74S or NS2E67/74S point mutation, and coding contains the PR8 virus NP gene fragment of the NP albumen of G132A point mutation;

With the recombinant plasmid of the HA gene of above-mentioned each subtype influenza virus and the recombinant plasmid of NA gene, by respective combination transfection 293T cell, cultivate the cell after the transfection with the recombinant plasmid of the above-mentioned PR8 of comprising virus mutation gene fragment and the plasmid that comprises respectively PR8 virus PA, PB1, PB2, M, NP or NS internal gene;

The cultured cells supernatant is inoculated in the chicken embryo, in brooder, after the suitable time of cultivation, gathers in the crops chick embryo allantoic liquid, detect the blood clotting of this allantoic fluid, if hemagglutination activity is arranged, and through after sequential analysis determines not have unexpected sudden change, namely obtain the PR8 recombinant influenza.

In specific embodiments of the invention, prepare the method for above-mentioned PR8 recombinant influenza, may further comprise the steps:

(1), construction recombination plasmid:

A, acquisition H1, H3, H4, H5, HA and the NA gene of H6, H7, H9, H10 subtype influenza virus;

Obtaining H1, H3, H4, H5, H6, H7, H9, the HA of H10 subtype influenza virus and the method for NA gene is:

Total RNA of difference extracting H1, H3, H4, H5, H6, H9 and H10 subtype influenza virus;

Take total RNA as template, the cDNA of H1, H3, H4, H5, H6, H9, H10 subtype influenza virus is synthesized in reverse transcription respectively;

Take the cDNA that obtains as template, be the upstream and downstream primer with SEQ ID.NO:13 and SEQ ID.NO:14 and SEQ ID.NO:11 and SEQ ID.NO:12 respectively, amplify respectively the NA gene of the HA gene of H1, H3, H4, H6, H9, H10 subtype influenza virus and H1, H3, H4, H5, H6, H9, H10 subtype influenza virus;

Take the cDNA of H5 subtype influenza virus as template, with the primer SEQ ID.NO:15 in sudden change H5HA alkaline bleach liquor cleavage site and SEQ ID.NO:13, and primer SEQ ID.NO:16 and SEQ ID.NO:14 increase respectively, carry out PCR with SEQ ID.NO:13 and SEQ ID.NO:14 primer again and merge amplification, acquisition contains the HA gene of the H5N1 subtype influenza virus in low pathogenicity bird flu strain alkaline bleach liquor cleavage site;

Being prepared as of the HA of described H7 subtype influenza virus and NA gene: the NA gene of synthetic H7 subtype influenza virus and the HA gene that contains low pathogenicity bird flu strain alkaline bleach liquor cleavage site, and be the upstream and downstream primer with SEQ ID.NO:13 and SEQ ID.NO:14 and SEQ ID.NO:11 and SEQ ID.NO:12, increase respectively, amplify respectively HA gene and the NA gene of H7 subtype influenza virus.

B, obtain coding respectively and contain the PR8 virus NP gene fragment of NP albumen of G132A point mutation and the PR8 virus N S gene fragment of the NS2 albumen that coding contains E67S or E74S or NS2E67/74S point mutation;

Preferably, describedly obtain respectively being prepared as of PR8 virus N S gene fragment that coding contains the PR8 virus NP gene fragment of NP albumen of G132A point mutation and the NS2 albumen that coding contains E67S or E74S or NS2E67/74S point mutation:

Take the recombinant plasmid that contains the PR8 virus NP gene as template, under the effect of Pfx archaeal dna polymerase, carry out respectively pcr amplification with primer SEQ ID.NO:7 and SEQ ID.NO:6 and primer SEQ ID.NO:5 and SEQ ID.NO:8 respectively; Take two sections PCR products as template, take SEQ ID.NO:7 and SEQ ID.NO:8 as primer, carry out second time pcr amplification and merge, obtain the PR8 virus NP gene fragment of point mutation G132A;

Take the PBD-PR8NS recombinant plasmid as template, under the effect of Pfx archaeal dna polymerase, carry out respectively pcr amplification with primer SEQ ID.NO:9 and SEQ ID.NO:2 and primer SEQ ID.NO:1 and SEQ ID.NO:10 respectively; Take two sections PCR products as template, take SEQ ID.NO:9 and SEQ ID.NO:10 as primer, carry out second time PCR and merge, obtain the NS gene fragment that coding contains E67S rite-directed mutagenesis NS2 albumen;

Take the PBD-PR8NS recombinant plasmid as template, under the effect of Pfx archaeal dna polymerase, carry out respectively pcr amplification with primer SEQ ID.NO:9 and SEQ ID.NO:4 and SEQ ID.NO:3 and primer SEQ ID.NO:10 respectively; Take two sections PCR products as template, take SEQ ID.NO:9 and SEQ ID.NO:10 as primer, carry out second time PCR and merge, obtain the NS gene fragment that coding contains E74S rite-directed mutagenesis NS2 albumen;

The NS gene fragment that contains E67S rite-directed mutagenesis NS2 albumen take above-mentioned coding is as template, carries out respectively pcr amplification with primer SEQ ID.NO:9 and SEQ ID.NO:4 and primer SEQ ID.NO:3 and SEQ ID.NO:10 under the effect of Pfx archaeal dna polymerase respectively; Take two sections PCR products as template, take SEQ ID.NO:9 and SEQ ID.NO:10 as primer, carry out second time PCR and merge, obtain the NS gene fragment that coding contains E74S and E74S rite-directed mutagenesis NS2 albumen simultaneously; C, preparation recombinant plasmid: the PR8 virus N S gene fragment that the coding that will obtain respectively contains the PR8 virus NP gene fragment of NP albumen of G132A point mutation and the NS2 albumen that contains E67S or E74S or NS2E67/74S point mutation of being connected be connected the HA of acquisition and be connected enzyme with the NA gene and cut, connect and transform, obtain corresponding recombinant plasmid; Described recombinant plasmid is with lower more than one: PBD-(H1) HA, PBD-(H1) NA; PBD-(H3) HA, PBD-(H3) NA; PBD-(H4) HA, PBD-(H4N2) NA; PBD-(H5) HA, PBD-(H5) NA; PBD-(H6) HA, PBD-(H6) NA; PBD-(H7) HA, PBD-(H7) NA; PBD-(H9) HA, PBD-(H9) NA; PBD-(H10) HA, PBD-(H10) NA; PBD-PR8NS-NS2E67/74S, PBD-PR8NS-NS2E67S, PBD-PR8NS-NS2E74S, PBD-PR8NP-G132A, PBD-PR8PB1, PBD-PR8PB2, PBD-PR8PA, PBD-PR8NP, PBD-PR8M, PBD PR8NS (Zejun Li, et al.JVI, 2005,79 (18): 12058-12064).

(2), preparation PR8 recombinant influenza: according to respective combination, transfection is in the 293T cell with the recombinant plasmid of above-mentioned acquisition; After cell conditioned medium after the transfection was processed with TPCK-Trypsin, inoculation SPF chicken embryo was cultivated; The results chick embryo allantoic liquid obtains above-mentioned PR8 recombinant influenza.

Another object of the present invention is the application of above-mentioned PR8 recombinant influenza.

Concrete technical scheme is as follows:

The application of above-mentioned arbitrary described PR8 recombinant influenza in the preparation influenza vaccines.

The present inventor finds to sport S (E67S point mutation) when NS2 albumen the 67th amino acids of PR8 virus strain by E, or NS2 albumen the 74th amino acids is mutated into S (E74S point mutation) by E, or NS2 albumen the 64th and 74 amino acids are mutated into S (E67S/E74S point mutation) by E simultaneously, and the multiplication capacity of virus mutation strain on the chicken embryo obviously improves.In addition, when the 132nd amino acids of NP albumen sports A by G (G132A point mutation), the multiplication capacity of the virus strain of mutant on cell obviously improves.Utilize internal gene and different subtype (H1, H3, H4, H5, H6, H7, H9, the H10) influenza virus of these high proliferation ability thumping viruses to carry out artificial recombination, obtain the recombinant virus that efficiently expresses HA antigen of the present invention, these recombinant viruses can be used for preparing on a large scale influenza vaccines.

Description of drawings

The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.

Fig. 1 recombinate PR8 mutated viruses and the restructuring hemagglutination activity of PR8 virus on the chicken embryo.

Fig. 2 recombinate PR8 mutated viruses and the restructuring hemagglutination activity of PR8 virus on cell.

Embodiment

The Construction and identification of embodiment 1 recombinant plasmid

1, design of primers

The NS of design flow Influenza Virus PR8 and the mutant primer of NP; The universal primer of influenza virus 12bp reverse transcription primer, A type influenza, H5 and H7HA cracking site mutant primer are by this lab design.The concrete sequence of above-mentioned primer sees Table 1 (used primer sequence among the present invention specifically sees Table 1), and is synthetic by Shanghai Invitrogen company.

2,2 rite-directed mutagenesises

Adopt the two-step pcr method to expecting that the Nucleotide in mutating acid site suddenlys change.At first take PBD-PR8NP as template, be the upstream and downstream primer with BSPQI-NP-forward and PR8-NP-400R and PR8-NP-387F and BSPQI-NP-reverse respectively, under the effect of Pfx archaeal dna polymerase (Invitrogen), carry out respectively pcr amplification.Two fragments that PCR obtains reclaim test kit by glue and reclaim.Take two sections PCR products reclaiming as template, take BSPQI-NP-forward and BSPQI-NP-reverse as primer, carry out PCR fusion second time.So obtain the NP gene fragment of the NP albumen of coding G132A point mutation.The pcr amplification program is 94 ℃ of denaturation 5min, enters following circulation, 94 ℃ of sex change 45s, and 53 ℃ of annealing 45s, 72 ℃ are extended 1min-1min45s, moves 30 circulations, at last 72 ℃ of extension 10min again.After reaction finished, the PCR product carried out electrophoresis experiment at 1% sepharose.

Use the same method, utilize NS2 mutant primer in the table 1: PR8-NS2-193F, PR8-NS2-204R, PR8-NS2-215F, PR8-NS2-224R and NS gene amplification primer: BSPQI-NS-forward and BSPQI-NS-reverse obtain respectively to contain the NS gene PCR amplified production of NS2E67S, E74S and NS2E67/74S point mutation.

Specific as follows: as take the PBD-PR8NS recombinant plasmid as template, under the effect of Pfx archaeal dna polymerase, to carry out respectively pcr amplification with primer SEQ ID.NO:9 and SEQ ID.NO:2 and primer SEQ ID.NO:1 and SEQ ID.NO:10 respectively; Take two sections PCR products as template, take SEQ ID.NO:9 and SEQ ID.NO:10 as primer, carry out second time PCR and merge, obtain the NS gene fragment that coding contains E67S rite-directed mutagenesis NS2 albumen;

Take the PBD-PR8NS recombinant plasmid as template, under the effect of Pfx archaeal dna polymerase, carry out respectively pcr amplification with primer SEQ ID.NO:9 and SEQ ID.NO:4 and SEQ ID.NO:3 and primer SEQ ID.NO:10 respectively; Take two sections PCR products as template, take SEQ ID.NO:9 and SEQ ID.NO:10 as primer, carry out second time PCR and merge, obtain the NS gene fragment that coding contains E74S rite-directed mutagenesis NS2 albumen;

Contain the NS gene fragment template of E67S rite-directed mutagenesis NS2 albumen with above-mentioned coding, under the effect of Pfx archaeal dna polymerase, carry out respectively pcr amplification with primer SEQ ID.NO:9 and SEQ ID.NO:4 and primer SEQ ID.NO:3 and SEQ ID.NO:10 respectively; Take two sections PCR products as template, take SEQ ID.NO:9 and SEQ ID.NO:10 as primer, carry out second time PCR and merge, obtain the NS gene fragment that coding contains E74S and E74S rite-directed mutagenesis NS2 albumen simultaneously.

3, the pcr amplification of HA and NA gene

HA and NA gene source are in the influenza virus (HxNy represents H1N1, H3N2, H4N2, H5N1, H6N4, H7N7, H9N2, H10N8 subtype influenza virus) of different subtype.Except the H7N7 subtype influenza, other virus Trizol (Invitrogen) extracted total RNA.

With reverse transcription test kit (TakaRa), according to its specification sheets, usefulness 12bp primer 5 '-AGCAAAAGCAGG-3 ' (table 1) is Auele Specific Primer, synthetic cDNA the first chain.Take the first chain of the cDNA that obtains as template, be that the upstream and downstream primer (contains the BspQI restriction enzyme site with BSPQI-HA-forward, BSPQI-HA-reverse and BSPQI-NA-forward, BSPQI-NA-reverse, such as table 1), amplify respectively the NA gene of the HA of H1N1, H3N2, H4N2, H6N4, H9N2, H10N8 subtype influenza virus of fragment and H1N1, H3N2, H4N2, H5N1, H6N4, H9N2, H10N8 subtype influenza virus.The pcr amplification program is 94 ℃ of denaturation 5min, enters following circulation, 94 ℃ of sex change 45s, and 53 ℃ of annealing 45s, 72 ℃ are extended 1min45s, moves 30 circulations, at last 72 ℃ of extension 10min again.After reaction finished, the PCR product carried out the electrophoresis checking at 1.0% sepharose.

After extracting obtains H5N1 subtype influenza virus HA gene, upstream primer H5-forward and BSPQI-HA-reverse with the downstream primer H5-reverse in sudden change H5HA alkaline bleach liquor cleavage site and BSPQI-HA-forward, sudden change alkaline bleach liquor cleavage site carry out respectively pcr amplification, merge PCR with BSPQI-HA-forward, BSPQI-HA-reverse primer again.The pcr amplification program is 94 ℃ of denaturation 5min, enters following circulation, 94 ℃ of sex change 45s, and 53 ℃ of annealing 45s, 72 ℃ are extended 1min45s, moves 30 circulations, at last 72 ℃ of extension 10min again.After reaction finished, the PCR product carried out the electrophoresis checking at 1.0% sepharose.Acquisition contains the HA gene of the H5 in low pathogenicity bird flu strain alkaline bleach liquor cleavage site.

In this research synthetic the H7N7 influenza virus the NA gene and contain the HA gene in low pathogenicity bird flu strain alkaline bleach liquor cleavage site, and be that the upstream and downstream primer (contains the BspQI restriction enzyme site with BSPQI-HA-forward, BSPQI-HA-reverse and BSPQI-NA-forward, BSPQI-NA-reverse, such as table 1), carried out respectively pcr amplification.The pcr amplification program is 94 ℃ of denaturation 5min, enters following circulation, 94 ℃ of sex change 45s, and 53 ℃ of annealing 45s, 72 ℃ are extended 1min45s, moves 30 circulations, at last 72 ℃ of extension 10min again.After reaction finished, the PCR product carried out the electrophoresis checking at 1.0% sepharose.

4, the rubber tapping of PCR product is reclaimed

Under UV-light, downcut the sepharose of target DNA fragment after electrophoresis finishes from gel, reclaim fast test kit with DNA and reclaim DNA.Concrete grammar is as follows: downcut the sepharose that contains target DNA under ultraviolet lamp, put into the EP pipe of an aseptic 1.5ml, the Buffer DE-A (coagulant liquid) that adds 3 times of gel volumes (100mg=100ul volume), mix rear in 75 ℃ of heating, be interrupted and mix (2-3min), until gel piece melts (about 6-8min) fully.Add the Buffer DE-B (in conjunction with liquid) of 0.5 Buffer DE-A volume, mix; When the dna fragmentation that reclaims during less than 400bp, add the Virahol of 1 gel volume.Mixed solution is transferred to DNA prepare in the pipe, the centrifugal 1min of 12000 * g outwells the waste liquid in the collection tube.To prepare pipe and put in the recovery collector, and add 500ul Buffer W1 (washings), the centrifugal 30s of 12000 * g discards the waste liquid in the collection tube.To prepare pipe and put in the recovery collector, and add 700ul Buffer W2 (liquid desalts), the centrifugal 1min of 12000 * g discards the waste liquid in the collection tube, washes once with same method again.To prepare pipe and put in the recovery collector, 12000 * g sky is from 1min.To prepare at last pipe and place clean 1.5ml EP pipe, at the deionized water of the adding 30ul of preparation film central authorities, room temperature leaves standstill 1min, and the centrifugal 1min eluted dna of 12000 * g places-20 ℃ to save backup.

5, enzyme is cut, is connected and transforms

Above-mentioned PCR purified product and PBD carrier (Zejun Li, et al.JVI, 2005,79 (18): 12058-12064) digest with the BSPQI restriction enzyme respectively, reclaim the enzyme of test kit recovery purpose fragment and PBD plasmid with glue and cut product, the PCR product after then with the T4 ligase enzyme enzyme being cut is connected with the PBD carrier that enzyme is cut processing.Connect product and transform in competent cell JM109 (Shanghai Suo Lai bio tech ltd), coat under the aseptic condition on the LB solid medium that contains Amp, cultivate 8-20h for 37 ℃.

6, the evaluation of recombinant plasmid

Single bacterium colony on the picking LB solid medium is put into and is added the test tube that the 3ml that has an appointment contains the LB liquid nutrient medium of Amp, 37 ℃ of shaking culture 10h.With the plasmid of bacterium liquid with alkaline extraction process extracting, verify with PCR method.Be accredited as positive plasmid and carry out sequencing, carry out sequential analysis with the DNAstar sequence analysis software, determine that sequence is correct.The respectively nucleotide sequence shown in SEQ ID.NO:20-23, or its aminoacid sequence is SEQ ID.NO:24-27.In addition, the sequence of the HA of each hypotype and NA gene also is correct through examining.

Rite-directed mutagenesis primer and the A type influenza virus universal primer of table 1NS2, NP gene

The rescue of embodiment 2 restructuring PR8 mutated viruses

1, the transfection plasmid is prepared

Extract the recombinant plasmid that aforesaid method makes up with ultrapure extraction agent box (OMEGA), comprising: PBD-(H1) HA, PBD-(H1) NA; PBD-(H3) HA, PBD-(H3) NA; PBD-(H4) HA, PBD-(H4) NA; PBD-(H5) HA, PBD-(H5) NA; PBD-(H6) HA, PBD-(H6) NA; PBD-(H7) HA, PBD-(H7) NA; PBD-(H9) HA, PBD-(H9) NA; PBD-(H10) HA, PBD-(H10) NA; PBD-PR8NS-NS2E67/74S, PBD-PR8NS-NS2E67S, PBD-PR8NS-NS2E74S, PBD-PR8NP-G132A, PBD-PR8PB1, PBD-PR8PB2, PBD-PR8PA, PBD-PR8NP, PBD-PR8M, PBD PR8NS, and measure plasmid concentration.

2, rescue obtains restructuring PR8 mutated viruses

According to the combination that designs, utilize liposome 2000 cotransfections in the 293T cell above-mentioned plasmid.6h after the transfection discards cell conditioned medium, adds 2ml OPTI-MEM, places 37 ℃ CO ₂Cultivate 72h in the incubator.After cell conditioned medium after the transfection is processed with TPCK-Trypsin, be inoculated in 9-11 age in days SPF chicken embryo (the logical laboratory animal technology company limited of Beijing Cimmeria dimension), be placed on paraffin sealing and continue hatching in 37 ℃ of brooders.Put into 4 ℃ behind the 48-72h and spend the night, take out, the results chick embryo allantoic liquid.Allantoic fluid is measured with hemagglutination test and is had or not agglutination activity.

The present invention saves and has obtained to contain the HA of H1 subtype influenza virus and PR8 recombinant virus and PR8 sudden change recombinant virus: the H1N1-PR8 (being called for short 1-PR8) of NA gene, H1N1-PR8-NS2E67S (being called for short 1-67), H1N1-PR8-NS2E74S (being called for short 1-74), H1N1-PR8-NS2E67S/E74S (being called for short 1-67/74), H1N1-PR8-NP-G132A (being called for short 1-132) and H1N1-PR8-NPG132A-NS2E67S/E74S (being called for short 1-132/67/74).

The present invention saves and has obtained to contain the HA of H3 subtype influenza virus and PR8 recombinant virus and PR8 sudden change recombinant virus: the H3N2-PR8 (being called for short 3-PR8) of NA gene, H3N2-PR8-NS2E67S (being called for short 3-67), H3N2-PR8-NS2E74S (being called for short 3-74), H3N2-PR8-NS2E67S/E74S (being called for short 3-67/74), H3N2-PR8-NPG132A (being called for short 3-132) and H3N2-PR8-NPG132A-NS2E67S/E74S (being called for short 3-132/67/74).

The present invention saves and has obtained to contain the HA of H4 subtype influenza virus and PR8 recombinant virus and PR8 sudden change recombinant virus: the H4N2-PR8 (being called for short 4-PR8) of NA gene, H4N2-PR8-NS2E67S (being called for short 4-67), H4N2-PR8-NS2E74S (being called for short 4-74), H4N2-PR8-NS2E67S/E74S (being called for short 4-67/74), H4N2-PR8-NPG132A (being called for short 4-132) and H4N2-PR8-NPG132A-NS2E67S/E74S (being called for short 4-132/67/74).

The present invention saves and has obtained to contain the HA of H5 subtype influenza virus and PR8 recombinant virus and PR8 sudden change recombinant virus: the H5N1-PR8 (being called for short 5-PR8) of NA gene, H5N1-PR8-NS2E67S (being called for short 5-67), H5N1-PR8-NS2E74S (being called for short 5-74), H5N1-PR8-NS2E67S/E74S (being called for short 5-67/74), H5N1-PR8-NPG132A (being called for short 5-132) and H5N1-PR8-NPG132A-NS2E67S/E74S (being called for short 5-132/67/74).

The present invention saves and has obtained to contain the HA of H6 subtype influenza virus and PR8 recombinant virus and PR8 sudden change recombinant virus: the H6N4-PR8 (being called for short 6-PR8) of NA gene, H6N4-PR8-NS2E67S (being called for short 6-67), H6N4-PR8-NS2E74S (being called for short 6-74), H6N4-PR8-NS2E67S/E74S (being called for short 6-67/74), H6N4-PR8-NPG132A (being called for short 6-132) and H6N4-PR8-NPG132A-NS2E67S/E74S (being called for short 6-132/67/74).

The present invention saves and has obtained to contain the HA of H7 subtype influenza virus and PR8 recombinant virus and PR8 sudden change recombinant virus: the H7N7-PR8 (being called for short 7-PR8) of NA gene, H7N7-PR8-NS2E67S (being called for short 7-67), H7N7-PR8-NS2E74S (being called for short 7-74), H7N7-PR8-NS2E67S/E74S (being called for short 7-67/74), H7N7-PR8-NPG132A (being called for short 7-132) and H7N7-PR8-NPG132A-NS2E67S/E74S (being called for short 7-132/67/74).

The present invention saves and has obtained to contain the HA of H9 subtype influenza virus and PR8 recombinant virus and PR8 sudden change recombinant virus: the H9N2-PR8 (being called for short 9-PR8) of NA gene, H9N2-PR8-NS2E67S (being called for short 9-67), H9N2-PR8-NS2E74S (being called for short 9-74), H9N2-PR8-NS2E67S/E74S (being called for short 9-67/74), H9N2-PR8-NP132A (being called for short 9-132) and H9N2-PR8-NPG132A-NS2E67S/E74S (being called for short 9-132/67/74).

The present invention saves and has obtained to contain the HA of H10 subtype influenza virus and PR8 recombinant virus and PR8 sudden change recombinant virus: the H10N8-PR8 (being called for short 10-PR8) of NA gene, H10N8-PR8-NS2E67S (being called for short 10-67), H10N8-PR8-NS2E74S (being called for short 10-74), H10N8-PR8-NS2E67S/E74S (being called for short 10-67/74), H10N8-PR8NP-G132A (being called for short 10-132) and H10N8-PR8-NPG132A-NS2E67S/E74S (being called for short 10-132/67/74).

3, the evaluation of recombinant virus

With the total RNA of allantoic fluid of Trizol extracting recombinant virus, and with the reverse transcription of 12bp primer, obtain cDNA the first chain.Take cDNA the first chain as template, be the upstream and downstream primer with BSPQI-HA-forward and BSPQI-HA-reverse, BSPQI-NA-forward and BSPQI-NA-reverse, BSPQI-NP-forward and BSPQI-NP-reverse, BSPQI-NS-forward, BSPQI-NS-reverse, with the method for PCR increase respectively HA, NA, NP and NS fragment, to check order behind these PCR product purifications, the contained fragment of sequencing result confirmation restructuring PR8 mutated viruses is all expection, does not find unexpected sudden change.

Embodiment 3 rescues the recombinant virus growth characteristics that obtain and identifies

1, rescues the recombinant virus EID that obtains ₅₀Measure

Contain the chick embryo allantoic liquid of virus according to 10 times of doubling dilutions, with 10 ^-5～10 ^-9Each extent of dilution is inoculated into respectively in the SPF chicken embryo of 3 pieces of 9-11 ages in days, and 37 ℃ are continued hatching 48h.Judge by measuring the hemagglutination activity that infects embryo allantoic liquid whether it infects, utilize the Reed-Muench method to calculate EID ₅₀(chicken embryo median infective dose).Recombinant virus EID ₅₀Measurement result (wherein, viral dilution liquid is long-pending is 100ul) as shown in table 2.

The EID of table 2 recombinant virus ₅₀

2, rescue the recombinant virus TCID that obtains ₅₀Measure

From 1: 10 ^-2 Begin 10 times of dilutions, different dilution recombinant viruses are connect poison in 48 orifice plates that cover with the individual layer mdck cell, and the process that connects poison is as follows: clean mdck cell twice with PBS first, then add 100ul virus in each hole, each extent of dilution is done 3 repetitions, and 48 orifice plates are put into 37 ℃ of CO ₂Incubator to cell, rocks one time culture plate every 20min by viruses adsorption, behind the 1.5h-2h liquid in the Tissue Culture Plate is discarded, and cleans cell twice with PBS, then adds the serum free medium 300ul that contains TPCK-Trypsin, and cell is at CO ₂Incubator continues to cultivate 72h, then measures the hemagglutination activity in each hole, utilizes the Reed-Muench method to calculate TCID ₅₀(histocyte median infective dose).Recombinant virus TCID ₅₀Measurement result (wherein, viral dilution liquid is long-pending is 100ul) as shown in table 3.

The TCID of table 3 recombinant virus ₅₀

3, rescuing the recombinant virus growth characteristics on the chicken embryo that obtain compares

To contain 100EID ₅₀The SPF chicken embryo of recombinant virus diluent 100ul inoculation 9-11 age in days, 6h after connecing poison, 12h, 24h, 36h during 48h, takes out respectively 3 pieces, collects allantoic fluid and measures their hemagglutinative titer.The blood clotting titre of different subtype recombinant virus after chicken embryo propagation presents similar result, connect rear 12 hours allantoic fluids with interior virus inoculation chicken embryo of poison and all do not have blood clotting, 24-48h, recombinant virus (the HxNy-PR8-NS2-E67S/E74S that contains six internal gene of mutated viruses PR8-NS2-E67/74S, abbreviation x-67/74) hemagglutinative titer is the highest, other recombinant virus hemagglutinative titers are from high to low successively: the recombinant virus (HxNy-PR8-NS2-E67S that contains six internal gene of mutated viruses PR8-NS2-E67S, be called for short x-67), recombinant virus (the HxNy-PR8-NS2-E74S that contains six internal gene of PR8-NS2-E74S, be called for short x-74), the recombinant virus (HxNy-PR8 is called for short x-PR8) that contains six internal gene of PR8 virus, recombinant virus (the HxNy-PR8-NP-G132A that contains six internal gene of PR8-NP-G132A, abbreviation x-132), the recombinant virus (HxNy-PR8-NP-G132A-NS2-E67S/E74S is called for short x-132/67/74) that contains six internal gene of PR8-NP-G132A-NS2-E67S/E74S.As shown in Figure 1, with H1N1, H3N2, H4N2, H5N1, H6N4, H7N7, H9N2 and H10N8 hypotype recombinant virus, showed intuitively the above results.

4, rescue the recombinant virus that obtains and the comparison of PR8 growth characteristics on mdck cell

The recombinant virus diluent 200ul that will contain 100TCID50, the mdck cell in the inoculation T25 Tissue Culture Flask.6h after connecing poison, 12h, 24h, 36h during 48h, collects respectively its cell conditioned medium and measures hemagglutinative titer, the growing state of heavier papova on mdck cell.The blood clotting titre of different subtype recombinant virus after cell propagation presents similar result, connects in the rear 12h of poison, and the recombinant virus-infected cell supernatant is all without hemagglutination activity.24h after connecing poison, 36h and 48h, recombinant virus (x-132) the blood clotting valency of six internal gene that contains PR8-NP-G132A is the highest, and other recombinant virus hemagglutinative titers are from high to low successively: the recombinant virus (x-132/67/74) that contains six internal gene of PR8-NP-G132A-NS2-E67S/E74S, the recombinant virus (x-PR8) that contains six internal gene of PR8 virus, the recombinant virus (x-67/74) that contains six internal gene of mutated viruses PR8-NS2-E67/74S, contain mutated viruses PR8-NS2-E67S six internal gene recombinant virus (x-67) and contain the recombinant virus (x-74) of six internal gene of PR8-NS2-E74S.As shown in Figure 2, with H1N1, H3N2, H4N2, H5N1, H6N4, H7N7, H9N2 and H10N8 hypotype recombinant virus, showed intuitively the above results.

The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims (4)

1. PR8 recombinant influenza, it is characterized in that, its contain H3 subtype influenza virus HA and/or NA gene, contain 6 internal gene (PB1 of PR8 virus, PB2, PA, NP, M, the NS gene), wherein one of NS and NP gene or the two contain following mutational site: have E67S point mutation or E74S point mutation or E67S/E74S point mutation on the NS2 albumen of NS genes encoding, have the G132A point mutation on the NP albumen of NP genes encoding.

2. a method for preparing PR8 recombinant influenza claimed in claim 1 is characterized in that, may further comprise the steps:

Make up and comprise respectively the HA of H3 subtype influenza virus and the recombinant plasmid of NA gene;

Structure comprises the recombinant plasmid of PR8 virus mutation gene fragment, this PR8 virus mutation gene fragment is selected from NS or the NP gene fragment of following sudden change: coding contains the PR8 virus N S gene fragment of the NS2 albumen of E67S or E74S or NS2E67/74S point mutation, and coding contains the PR8 virus NP gene fragment of the NP albumen of G132A point mutation;

With the recombinant plasmid of the HA gene of above-mentioned H3 subtype influenza virus and the recombinant plasmid of NA gene, by respective combination transfection 293T cell, cultivate the cell after the transfection with the recombinant plasmid of the above-mentioned PR8 of comprising virus mutation gene fragment and the plasmid that comprises respectively PR8 virus PA, PB1, PB2, M, NP or NS internal gene;

The cultured cells supernatant is inoculated in the chicken embryo, in brooder, after the suitable time of cultivation, gathers in the crops chick embryo allantoic liquid, detect the blood clotting of this allantoic fluid, if hemagglutination activity is arranged, and through after sequential analysis determines not have unexpected sudden change, namely obtain the PR8 recombinant influenza.

3. the application of PR8 recombinant influenza claimed in claim 1 in the preparation influenza vaccines.

4. a vaccine is characterized in that, comprises PR8 recombinant influenza claimed in claim 1.

Images