CN101886084B - Codon-optimized H3HA/XJ3-07 gene and nucleic acid vaccine thereof - Google Patents

Abstract

The invention belongs to the technical field of medical biology, and relates to a codon-optimized H3HA/XJ3-07 gene and a nucleic acid vaccine thereof. In the invention, according to an HA protein sequence of an equine A-type influenza virus A/Equine/Xinjiang/3/07 (H3N8), an H3HA/XJ3-07 gene segment which can express a corresponding protein is chemically synthesized after codon optimization, and the segment is cloned into a nucleic acid vaccine vector pJW4303 to construct an equine influenza H3HA nucleic acid vaccine; and further, HA genes are modified, an H3-XJ HA natural signal peptide is replaced by a human tPA signal peptide or the gene segments of part of the HA genes are removed to make the HA genes only express an extracellular domain of the HA protein. The experimental result shows that three H3HA nucleic acid vaccines can be expressed in a 293T cell with high efficiency, and can induce the generation of specific anti-H3HA antibodies in New Zealand white rabbits.

Description

A kind of codon optimized H3HA/XJ3-07 gene and nucleic acid vaccine thereof

Technical field

The invention belongs to the medical biotechnology field, relate to a kind of codon optimized H3HA/XJ3-07 gene and nucleic acid vaccine thereof.

Background technology

In recent years, A type influenza virus is widely current in different species, like high pathogenic avian influenza H5N1, novel pig source H1N1 influenza or the like.The frequently outburst in equus equally of A type influenza.(hemagglutinin, HA) (neuraminidase, the NA) difference of gene have been divided into 16 hypotypes with HA, and NA is divided into 9 hypotypes for gene and neuraminidase according to the hemagglutinin of influenza virus in the world.Wherein equine influenza virus comprises H7N7 and H3N8 hypotype.Over nearly 30 years, H7N7 type equine influenza virus is not separated to again, and H3N8 type equine influenza worldwide often has outburst, and the existing vaccine of prompting can not effectively be controlled the popular of this virus.

Existing research shows that H3N8 type equine influenza appears strides species and propagates trend, can infect dog, pig etc.The influenza virus of other H3 hypotypes can infect multiple host, also comprises the people, thereby control H3N8 type equine influenza can reduce the potential threat of this influenza virus to people and other species.Most countries adopts the equine influenza virus deactivation vaccine in the world at present, and a small amount of use attenuated live vaccine and carrier bacterin etc. are also arranged.The equine influenza vaccine research work of China is sluggish relatively, and commercialized vaccine comprises inactivated vaccine vest one type, vest two types and the mixed vaccine that the seventies is succeeded in developing.Use Superlysoform inactivation of viruses immunity horse, the immanoprotection action that is produced only can be kept about three months.The present domestic development research that novel vaccine is arranged of not seeing.The 2008 Beijing Olympic Games, owing to lack the equine influenza vaccine of protection definite effect, China is from French import H3N8 poxvirus live vector vaccine immunity horse.Thereby China's independent research H3N8 type equine influenza vaccine novel, that immunogenicity is good is imperative.

Nucleic acid vaccine (nucleic vaccine) has another name called gene vaccine (gene vaccine) or dna vaccination (DNAvaccine); Its essence is the carrier for expression of eukaryon that contains the pathogen antigen gene; After it is imported into animal body; The antigen protein that can be absorbed and express pathogenic agent by the animal cell, thus induce body to this proteic immunoreation.At present, all verified in a lot of species, the immunity of utilization dna vaccination can be induced stronger specific immunity protective reaction.Dna vaccination is superior to traditional inactivated vaccine because of body fluid and the cellular immunization that it can evoke body simultaneously, but adds its low production cost scale prodn, be easy to preserve and have an extensive market prospects.Up to the present; There have been four kinds of dna vaccinations that apply to animal to obtain the listing permission, comprised the west Nile virus dna vaccination, the sick dna vaccination of infectivity hematopoietic tissue necrosis that applies to salmon that apply to horse, apply to the melanoma dna vaccination of dog and reduce the growth hormone releasing hormone dna vaccination of young pig pathogenicity rate and lethality rate.

As emerging in recent years a kind of vaccine, it is attracting people with its unique advantage: (1) antigen is synthetic similar with the natural infection of cause of disease with the submission process, through MHC I class and the direct submission immunity system of II quasi-molecule.Specific C D particularly ⁸⁺The immunoreation of lymphocyte (CTL), this is that inactivated vaccine and subunit vaccine can not be compared; (2) immunogenic unicity has only the required antigen gene transfered cell of coding to obtain expressing, and itself does not have antigenicity carrier.And the virus live vector vaccine of reorganization also has huge and complex immunological albumen except destination gene expression; (3) be easy to make up and prepare, good stability, with low cost, be suitable for large-scale production.But the limitation of dna vaccination is lower, the less immunogenic of antigenic expression.How to optimize antigen gene sequences, promote the expression and the secretion of vaccine, improve immunogenicity, become the key point of structure and design dna vaccine.

Antigenic expression amount is to influence the immunogenic important factor of dna vaccination.Dna vaccination utilizes transcribing of host and translation system; Because there is the preference property of codon in the nature biotechnology body; In the heterologous host body, possibly be difficult to effective expression from the gene clone in pathogenic agent source; Therefore the effective immunity system of stimulation of host just makes it to produce immanoprotection action preferably, and this is to cause the lower major cause of present nucleic acid vaccine immunity originality.

Summary of the invention

The objective of the invention is to overcome the deficiency of prior art; Provide a kind of, separate the hemagglutinin gene of the susceptible poison that effluents when this sequence is Fuyun County, Altay Prefecture, Xinjiang Uygur Autonomous Regions's in October, 2007 outburst H3N8 hypotype equine influenza through codon optimized H3HA/XJ3-07 gene.

Another object of the present invention is to make up three kinds of nucleic acid vaccines that contain this H3HA/XJ3-07 gene.

Technical scheme of the present invention is:

A kind of codon optimized H3N8 hypotype equine influenza H3HA/XJ3-07 gene, sequence is SEQ ID NO.1.

A kind of equine influenza H3HA nucleic acid vaccine, this vaccine are that H3HA/XJ3-07 gene and the carrier for expression of eukaryon of SEQ ID NO.1 formed by sequence.

Wherein, described carrier for expression of eukaryon is for cutting the carrier segments that pJW4303 obtains through restriction endonuclease pstI and BamHI.

A kind of equine influenza H3HA-tPA nucleic acid vaccine, this vaccine is made up of natural signals peptide gene H3HA/XJ3-07 gene fragment that obtains and the carrier for expression of eukaryon that contains tPA (people tissue plasminogen activator) signal peptide gene that the described H3HA/XJ3-07 gene excision of claim 1 carries.

Wherein, The H3HA/XJ3-07 gene fragment that the natural signals peptide gene that described excision carries obtains is, and to be template with SEQ IDNO.1 insert the NheI restriction enzyme site through what pcr amplification obtained at 5 ' end; Hold the H3HA gene fragment that comprises the BamHI site 3 ', sequence is SEQ ID NO.2.

Described carrier for expression of eukaryon is for cutting the big fragment of carrier that pJW4303 obtains through restriction endonuclease NheI and BamHI, and wherein the sequence between NheI and pstI restriction enzyme site is the tPA signal peptide gene.

A kind of H3HA/XJ3-07-dTM nucleic acid vaccine, this vaccine is made up of with the sequence of striding film district part only encoding influenza virus hemagglutinin H3HA/XJ3-07 extracellular domain gene fragment partly that obtains and the carrier for expression of eukaryon that contains the tPA signal peptide gene natural signals peptide gene and encoding influenza virus hemagglutinin H3HA/XJ3-07 born of the same parents internal area that described H3HA/XJ3-07 gene excision carries.

Wherein, The gene fragment of described influenza virus hemagglutinin H3HA/XJ3-07 extracellular domain of encoding is to be that template is inserted the NheI restriction enzyme site through what pcr amplification obtained at 5 ' end with SEQ ID NO.1; Hold the H3HA gene fragment that comprises the BamHI site 3 ', sequence is SEQ ID NO.3.

Described carrier for expression of eukaryon is for cutting the big fragment of carrier that pJW4303 obtains through restriction endonuclease NheI and BamHI; Sequence behaviour tissue plasminogen activator (human tissueplasminogen activator, the tPA) signal peptide gene between NheI and pstI restriction enzyme site wherein.

The plasmid physical map of above-mentioned three kinds of vaccines is seen Fig. 2, and three kinds of expressed HA proteose intentions of vaccine are seen Fig. 3.

The design of codon optimized H3HA gene and synthetic:

(1) at first uses gene software MacVector 7.2 analysis of encoding wild-type H3HA/XJ3-07 gene orders; Find out its codon and use preference, and find out in the gene order of wild-type H3HA-XJ3-07 and use preference and use the sub-site of preference different ciphers with e. coli codon with the Mammals codon.Use the identical codon of preference for Mammals with intestinal bacteria; With Mammals and intestinal bacteria all the codon of preference substitute and use the sub-site of preference different ciphers in the wild-type H3HA/XJ3-07 gene, design codon optimized H3HA gene order SEQ ID NO.1 then.

(2) the H3HA gene optimized of numeral is synthetic by German Geneart company, and the carrier pGA15 that packs into is built into recombinant plasmid pGA15/H3HA.Confirm that through order-checking the synthetic sequence is correct.With DNAman software comparison wild-type H3HA gene with through the difference of codon optimized H3HA gene at Mammals and expression in escherichia coli.See Fig. 1.

Beneficial effect of the present invention:

1, compares with wild type gene; The codon frequency of occurrences of the codon of mammalian cell preference and intestinal bacteria preference increases in the codon optimized gene; But the H3HA Argine Monohydrochloride sequence of its coding is constant, thereby makes it be more suitable for the protein expression in mammalian cell and intestinal bacteria.

Through gene sequencing to wild-type H3HA/XJ3-07; The contriver thinks because there is the preference property of codon in the nature biotechnology body; In the heterologous host body, be difficult to effective expression from the H3HA/XJ3-07 gene clone in pathogenic agent source; Therefore the effective immunity system of stimulation of host just makes it to produce immanoprotection action preferably.In order to improve the expression efficiency of heterologous gene in Mammals and intestinal bacteria, often need be optimized nucleotide coding sequence.Because the optimization to nucleotide sequence does not still have unified standard or principle at present.Therefore to aminoacid sequence, different researchists can design expression and the manufacturing that different nucleotide sequences is used for target polypeptides fully, and correspondingly expression efficiency also possibly there are differences.Nucleotide coding sequence according to we optimize has overcome above-mentioned defective, has improved the H3HA protein expression level, and by the immunogenicity of this gene constructed nucleic acid vaccine

2, the contriver directly is cloned into carrier for expression of eukaryon with the H3HA gene of codon optimization, has made up equine influenza H3HA nucleic acid vaccine, this vaccine can be in eukaryotic cell 293T cell effective expression, immune animal can stimulate and produces specific antibody.

3, the contriver has designed the equine influenza H3HA-tPA nucleic acid vaccine that has tPA (human tissueplasminogen activator, people tissue plasminogen activator) signal peptide on the basis that makes up equine influenza H3HA nucleic acid vaccine.Under the guiding of tPA signal peptide, antigen protein can be secreted to the extracellular more can effectively stimulate body immune system.Confirm that through cell expressing and animal immune the pJW4303/H3HA-tPA nucleic acid vaccine is expressed better, has better immunogenicity.

4, the contriver has designed on the basis of structure equine influenza H3HA nucleic acid vaccine and has had tPA (human tissue plasminogenactivator, people tissue plasminogen activator) signal peptide and remove its membrane-spanning domain and the H3HA nucleic acid vaccine H3HA/XJ3-07-dTM nucleic acid vaccine of born of the same parents' internal area.More effectively stimulate body immune system to the extracellular thereby make antigen protein to secrete.

Description of drawings

Fig. 1 wild-type and codon optimized H3HA/XJ3-07 gene are in the comparison (index＞1 is a mammalian cell institute preference) of the preference of mammalian cell expression, and ordinate zou is represented the preference index, and X-coordinate is represented the nucleotides sequence column position.Left side figure is a wild-type H3HA/XJ3-07 gene, and right figure is codon optimized H3HA/XJ3-07 gene.

Three kinds of nucleic vaccine plasmid physical maps of Fig. 2.

A H3HA nucleic vaccine plasmid physical map.Sequence between PstI and BamHI restriction enzyme site is the H3HA gene.

B H3HA-tPA nucleic vaccine plasmid physical map.Sequence between NheI and BamHI restriction enzyme site is to remove the H3HA gene of natural signals peptide gene, joins with tPA signal peptide on the eukaryon expression plasmid pJW4303, constitutes the H3HA nucleic acid vaccine that has the tPA signal peptide.

C H3HA-dTM nucleic acid vaccine is further made amendment, and that removes the H3HA gene strides film district and born of the same parents' internal area, only keeps the extracellular domain part.

Fig. 3 H3HA-wt, H3HA-tPA, the expressed HA proteose intention of H3HA-dTM nucleic acid vaccine

The evaluation of Fig. 4 pJW4303/H3HA, pJW4303/H3HA-tPA, pJW4303/H3HA-dTM nucleic acid vaccine.1:1kb DNA marker, 2:pJW4303/H3HA, 3:pJW4303/H3HA is through the BamHI single endonuclease digestion; 4:pJW4303/H3HA is through the PstI+BamHI double digestion, 5:pJW4303/H3HA-tPA, and 6:pJW4303/H3HA-tPA is through the BamHI single endonuclease digestion; 7:pJW4303/H3HA-tPA is through the PstI+BamHI double digestion; 8:pJW4303/H3HA-dTM, 9:pJW4303/H3HA-dTM are through the BamHI single endonuclease digestion, and 10:pJW4303/H3HA-dTM is through the PstI+BamHI double digestion.

Fig. 5 western blot method detects pJW4303/H3HA, pJW4303/H3HA-tPA, pJW4303/H3HA-dTM nucleic acid vaccine and the expression of empty carrier pJW4303 in eukaryotic cell 293T cell.(antigen is above-mentioned four kinds of plasmid transfection 293T cell conditioned medium liquid S and lysate L, and used antiserum(antisera) is a pJW4303/H3HA-tPA immunize rabbit serum, dilution in 1: 500)

Fig. 6 nucleic acid vaccine immunity NZw situation.

Fig. 7 pJW4303/H3HA, pJW4303/H3HA-tPA, pJW4303/H3HA-dTM nucleic acid vaccine immunity rabbit anteserum specific antibody answering time curve (antigen pJW4303/H3HA-dTM transfection 293T cell conditioned medium, dilution in 1: 5; Used antiserum(antisera) is the dilution in 1: 500 of each group rabbit immune serum, and arrow is represented the immunity time).

Fig. 8 pJW4303/H3HA, pJW4303/H3HA-tPA, pJW4303/H3HA-dTM nucleic acid vaccine immunity rabbit anteserum antibody titers (antigen pJW4303/H3HA-dTM transfection 293T cell conditioned medium, dilution in 1: 5; Used antiserum(antisera) is between each group rabbit immune serum).

Embodiment

Structure and the evaluation of embodiment 1. nucleic acid vaccine pJW4303/H3HA

1.1 preparation competence intestinal bacteria HB101

1.1.1 the intestinal bacteria HB101 that glycerine is preserved melts on ice, gets 5ul and is inoculated in the 5ml LB liquid, places 37 ℃ of constant incubators, 180rpm shakes bacterium and spends the night.

1.1.2 under the aseptic condition, get incubated overnight liquid 1ml, be inoculated in the fresh 200ml LB liquid, place 37 ℃ of constant incubators, 180rpm shook bacterium 2 hours.

1.1.3 will go up the step bacterium put cooled on ice 15min.

1.1.4 it is aseptic subpackaged to 50ml centrifuge tube (autoclaving), 4 ℃, 2500rpm, centrifugal 15min.

1.1.5 add cold 0.1M Cacl ₂100ml (Cacl ₂Autoclaving), resuspended bacterium hatches 30min on ice.

1.1.6 it is aseptic subpackaged to 50ml centrifuge tube (autoclaving), 4 ℃, 2500rpm, centrifugal 15min.

1.1.7 add 0.1M Cacl cold, that contain 20% glycerine ₂2ml, resuspended bacterium.

1.1.8100ul/ pipe divides to be filled to 1.5ml sterilization EP centrifuge tube ,-70 ℃ frozen subsequent use.

1.2 the recombinant vectors pGA15/H3HA transformed competence colibacillus intestinal bacteria HB101 that contains target sequence that provides with genome company; The picking mono-clonal extracts plasmid in a small amount and identifies; Step is carried out (TaKaRa miniBESTPlasmid Purification Kit ver 2.0, Dalian is precious biological) by the test kit specification sheets.Plasmid is with PstI and BamHI double digestion, and the enzyme system of cutting is:

10x?buffer(with?BSA) 5.0ul

Plasmid (0.5ug/ul) 20.0ul

PstI(10u/ul) 1.5ul

BamHI(10u/ul) 1.5ul

ddH ₂O 22ul

TV 40ul was hatched 2 hours for 37 ℃

1.3pJW4303 carrier is used PstI and BamHI double digestion equally, it is the same that enzyme is cut system.

1.4 1.2 and 1.3 enzymes are cut product and are all used 1% gel electrophoresis, reclaim test kit (E.Z.N.A. with dna gel ^TMGelExtraction Kit (50), OMEGA BIO-Tek company) reclaims pJW4303 carrier segments about purifying 1707bp size H3HA gene fragment SEQ IDNO.1 and 5.0Kb.It is following that glue reclaims concrete steps:

1.4.1 sepharose behind the electrophoresis is placed under the Ultraviolet Detector, downcut the gel that contains target DNA, put into the 1.5ml EP pipe of weighing.

1.4.2 analytical balance claims to contain the quality of the EP pipe of blob of viscose, deducts the quality of EP pipe itself, the quality of acquisition blob of viscose.Press the volume that 1g=1ml calculates blob of viscose.Add isopyknic Binding buffer, place 55～65 ℃ of water-bath 7min, the heating and melting blob of viscose, every separated 2min mixing once melts until blob of viscose fully.

1.4.3 the liquid that melts is all taken out, adds (each 700ul takes out in the adding pipe after redundance is centrifugal, continues centrifugal) among the HinbindingDNA Mini Spin Colomn again.10000g, centrifugal 1min.

1.4.4 discard filtrating, add 300ul Binding buffer, 10000g, centrifugal 1min.

1.4.5 discard filtrating, add 700ul SPW wash buffer, room temperature leaves standstill 2-3min, 10000g, centrifugal 1min.

1.4.6 repeat the 1.2.5 step.

1.4.7 discard filtrating, 10000g, centrifugal 1min.This step is very crucial.

1.4.8 clean 1.5ml EP pipe is put in interior pipe taking-up, is added 20-50ul Elution buffer or aqua sterilisa or TE, 10000g, centrifugal 1min, eluted dna according to the experiment needs.The DNA of results can be used for doing the experiment in downstream.

1.5 H3HA gene fragment that step 1.4 is obtained and pJW4303 carrier segments be according to 3: 1 mixed,, connect with T4DNA ligase enzyme (Promega company) and to spend the night, reaction conditions is following:

Ligase?buffer 1ul

T4DNA?ligase 1ul

PJW4303 (purifying fragment behind PstI and the BamHI double digestion) 2ul

H3HA gene (purifying fragment behind PstI and the BamHI double digestion) 6ul

TV 10ul, 4 ℃ of connections are spent the night

1.6 connect product transformed competence colibacillus intestinal bacteria HB101, concrete steps are following:

1.6.1 competence intestinal bacteria HB101 is in melting on ice.

Spend the night and connect product and add competence HB101 1.6.2 get 5ul, the springing tube wall is with the mixing plasmid.Hatch 30min on ice.

1.6.3 reaction tubes moves to 42 ℃ of water tanks, heat-shocked 90s.Be transferred on ice immediately.

1.6.4 add 800ul sterilization LB nutrient solution, in 37 ℃ of constant incubators, 70-80rpm shakes bacterium 45min.

Add the bacterium ware central authorities that contain solid LB substratum 1.6.5 get the 100ul culture bacteria, bacterium liquid evenly is coated with aseptic spreading rod, is paved with whole petridish.Treat that bacterium liquid is dry a little, back-off places 37 ℃ of constant incubators, overnight cultures.

1.6.6 second day little upgrading grain of picking mono-clonal, step is carried out (TaKaRa miniBESTPlasmid Purification kit ver 2.0, Dalian is precious biological) by the test kit specification sheets.

1.6.7 cut the correct clone of evaluation through electrophoresis, enzyme, continuous three times strokes of plates carry out bacterial classification and preserve.

1.6.8 correct plasmid prepares in a large number, step is carried out (QIAGEN Plasmid MegaKit (5), QIAGEN company) by the test kit specification sheets.

PJW4303/H3HA nucleic vaccine plasmid physical map is seen Fig. 2 a.Sequence between PstI and BamHI restriction enzyme site is the H3HA gene.Enzyme is cut qualification result and is seen the Lane1 of Fig. 4, lane2, Lane3, lane4.Behind the PstI+BamHI double digestion, discharge target gene fragment H3HA, length is about 1700, conforms to expection, the pJW4303/H3HA nucleic acid vaccine makes up successfully.

Structure and the evaluation of embodiment 2. nucleic acid vaccine pJW4303/H3HA-tPA

2.1 design PCR primer is respectively: upstream primer SEQ ID N0.4, downstream primer SEQ ID N0.5; With recombinant plasmid pGA15/H3HA is template, and amplification contains GCTAGC(NheI) and GGATCC(BamHI) the H3HA gene fragment that does not contain natural signals peptide gene of restriction enzyme site (SEQ ID NO.2).Reaction conditions is: 94 ℃ of 4min, 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 1.5min totally 25 circulations, 72 ℃ of 7min.

2.2PCR product is got 2ul and is run the evaluation of 1% agarose gel electrophoresis.

Reclaim test kit (E.Z.N.A. 2.3 identify correct PCR product with dna gel ^TMGel Extraction Kit (50), OMEGABIO-Tek company) recovery purifying H3HA gene fragment.Glue reclaims concrete steps like 1.4 operating processes.

2.4 the PCR product of purifying and carrier pJW4303 are respectively through NheI and BamHI double digestion; Enzyme is cut system:

10x?buffer(with?BSA) 5.0ul

Plasmid/H3HA gene fragment (0.5ug/ul) 20.0ul

NheI(10u/ul) 1.5ul

BamHI(10u/ul) 1.5ul

ddH ₂O 22ul

TV 40ul was hatched 2 hours for 37 ℃

2.5pJW4303 carrier segments and H3HA PCR enzyme are cut product and be further purified (BioSpin PCRPurification Kit, BioFlux company) respectively, concrete steps are following:

2.5.1 enzyme is cut product all to be transferred in the 1.5ml EP pipe.

2.5.2 add the Binding buffer of 2 times of volumes of reactant, mixing.

2.5.3 mixture all is transferred among the Spin Colomn the centrifugal 1min of 6000g.

2.5.4 abandon filtrating, add 650ul wash buffer, room temperature leaves standstill 2-3min, the centrifugal 1min of 12000g.

2.5.5 repeat the step (very crucial).

2.5.612000g 1min centrifugal more once (very crucial).

2.5.7 Spin Colomn is transferred in the clean 1.5ml EP pipe, add 20-50ul Elutionbuffer or aqua sterilisa or TE as required, room temperature leaves standstill 1min.

2.5.8 the centrifugal 1min of 12000g collects DNA.

2.6 pJW4303 carrier segments and H3HA PCR enzyme that 2.5 steps obtained are cut product by 1: 3 mixed, connect with T4 dna ligase (Promega company) and spend the night, reaction conditions is following:

Ligase?buffer 1ul

T4?DNA?ligase 1ul

PJW4303 (the purifying fragment approximately behind NheI and the BamHI double digestion) 2ul

H3HA gene (purifying fragment behind NheI and the BamHI double digestion) 6ul

TV 10ul, 4 ℃ of connections are spent the night

2.7 connect product transformed competence colibacillus intestinal bacteria HB101, step is with 1.6.

2.8 the little upgrading grain of picking mono-clonal, step is carried out (TaKaRa miniBEST PlasmidPurification kit ver 2.0, Dalian is precious biological) by the test kit specification sheets.

2.9 cut the correct clone of evaluation through electrophoresis, enzyme, continuous three times strokes of plates carry out bacterial classification and preserve.

2.10 correct plasmid prepares in a large number, step is carried out (QIAGEN Plasmid MegaKit (5), QIAGEN company) by the test kit specification sheets.

PJW4303/H3HA-tPA nucleic vaccine plasmid physical map is seen Fig. 2 b.Sequence between NheI and BamHI restriction enzyme site is the H3HA gene.Enzyme is cut qualification result and is seen the Lane1 of Fig. 4, lane5, Lane6, lane7.Behind the Nhe+BamHI double digestion, discharge target gene fragment H3HA-tPA, length is more smaller than 1700bp, conforms to expection, the pJW4303/H3HA-tPA nucleic acid vaccine makes up successfully.The structure and the evaluation of embodiment 3.pJW4303/H3HA-dTM nucleic acid vaccine

3.1 design two pairs of PCR primers, be template all with recombinant plasmid Pga15/H3HA, with upstream primer SEQ ID NO.4, downstream primer SEQ ID NO.6, amplification contains GCTAGC(NheI) and GGATCC(BamHI) the H3HA gene fragment of restriction enzyme site (SEQ IDNO.3).The PCR reaction conditions is with 2.1 operation stepss.This gene fragment is the gene fragment of having excised the only encoding influenza virus hemagglutinin H3HA/XJ3-07 extracellular domain part that H3HA/XJ3-07 gene natural signals peptide gene and coding born of the same parents internal area and the sequence of striding film district part obtain.

3.2PCR product is got 2ul and is run the evaluation of 1% agarose gel electrophoresis.

Reclaim test kit (E.Z.N.A. 3.3 identify correct PCR product with dna gel ^TMGel Extraction Kit (50), OMEGA BIO-Tek company) recovery purifying H3HA gene fragment.Glue reclaims concrete steps like 1.4 operating processes.

3.4 the gene fragment of purifying is with NheI and BamHI double digestion, carrier pJW4303 is with NheI and BamHI double digestion.Enzyme is cut system and same 1.2 operation stepss of reaction conditions.

3.5 enzyme is cut product and is further purified (BioSpin PCR Purification Kit, BioFlux company) respectively, concrete steps are with 2.5.

3.6 enzyme is cut purified product by about 1: 3 mixed, the connection of T4 ligase enzyme is spent the night.Reaction system is following:

Ligase?buffer 1ul

T4?DNA?ligase 1ul

PJW4303 (purifying fragment behind NheI and the BamHI double digestion) 2ul

H3HA gene (purifying fragment behind KpnI and the BamHI double digestion) 6ul

TV 10ul, 4 ℃ of connections are spent the night

3.7 connect product transformed competence colibacillus intestinal bacteria HB101, step is with 1.6.

3.8 the little upgrading grain of picking mono-clonal, step is carried out (TaKaRa miniBEST PlasmidPurification kit ver 2.0, Dalian is precious biological) by the test kit specification sheets.

3.9 cut the correct clone of evaluation through electrophoresis, enzyme, continuous three times strokes of plates carry out bacterial classification and preserve.

3.10 correct plasmid prepares in a large number, step is carried out (QIAGEN Plasmid MegaKit (5), QIAGEN company) by the test kit specification sheets.

PJW4303/H3HA-dTM nucleic vaccine plasmid physical map is seen Fig. 2 c.Sequence between NheI and BamHI restriction enzyme site is the H3HA gene.Enzyme is cut qualification result and is seen the Lane1 of Fig. 4, lane8, Lane9, lane10.Behind the Nhe+BamHI double digestion, discharge target gene fragment H3HA-dTM, length is about 1500bp, conforms to expection, the pJW4303/H3HA-dTM nucleic acid vaccine makes up successfully.

The expression of embodiment 4. recombinant plasmids

4.1 the transfection of recombinant plasmid (adopting cationic polymers PEI infection protocol, Invitrogen company), step is following:

4.1.1 preceding 24 hours 293T cells of transfection (for preserving this chamber) are passaged to 100mm Tissue Culture Plate, 5x10 ⁶Individual/ml.37 ℃, 5%CO ₂Cultivate.Used substratum is for containing the DMEM (Hyclone) of 10% foetal calf serum (Hyclone) and penicillium mould (100U/ml), Streptomycin sulphate (0.1mg/ml).

4.1.2 cell density about 50%～70% in second day.Get 100ul PEI and add 1ml serum-free DMEM nutrient solution (containing green grass or young crops, Streptomycin sulphate), fully mixing adds 20ug plasmid (plasmid and PEI ratio are 1: 5, and this ratio is applicable to the petridish of virtually any size size).Abundant mixing, room temperature leaves standstill 15min.

4.1.3 the said mixture multiple spot is splashed into Tissue Culture Plate.Slightly rock with mixing.Continuing to put into incubator cultivates.

4.1.4 after 8-10 hour, go culture supernatant gently, add the DMEM (in 37 ℃ of preparatory temperature, adding the fashionable cell of carefully avoiding having dashed in advance) that 6ml does not have foetal calf serum, continue to be cultured to 48-72 hour.

4.1.5 transfectional cell results: the culture supernatant of absorption transfectional cell (supernatant, S) to the 10ml centrifuge tube, the centrifugal 10min of 2500rpm, packing, mark ,-70 ℃ are frozen subsequent use.Transfectional cell adds 5ml lxPBS (cell is used), washes culture plate gently, draws cell suspension to the 10ml centrifuge tube, the centrifugal 10min of 2500rpm; Supernatant discarded adds the 200ul lysate, fully inhales and beats mixing, hatches 15min on ice; 12000rpm, 4 ℃ centrifugal 1 hour, drawing supernatant is lysis (lysis; L), packing, mark ,-70 ℃ are frozen subsequent use.

4.2Western blot detects the proteic expression of H3HA.Step is following:

4.2.1 at first prepare 10% separation gel (5ml ddH ₂O, 5ml 30% acrylamide soln, 5.7ml 1MTris/ClpH8.8,150 μ l 10%SDS, 150 μ l, 10% ammonium persulfate, 6 μ l TEMED), encapsulating, the liquid level top adds entry, polymerization 20～30min under the room temperature.

4.2.2 abandon pouring, filter paper blots, the spacer gel of refabrication 5% (4.1ml ddH ₂O, lml 30% acrylamide soln, 0.75ml 1MTris/Cl pH6.8,60 μ l 10%SDS, 60 μ l, 10% ammonium persulfate, 6 μ l TEMED), on spacer gel, insert broach, treat that glue condenses fully after, pull up broach.

4.2.3 specimen preparation: each 20ul of the cracking of plasmid transfection cell and supernatant, add 5x albumen sample-loading buffer 5 μ l, 100 ℃, boil 5min.

4.2.4 the above-mentioned sample of handling well is carried out electrophoresis, and condition is 20mA l hour, 40mA, 1.5 hours.

4.2.5 commentaries on classics film.Adopt wet commentaries on classics method.Albumen on the glue is forwarded on the pvdf membrane to 100v, 65min.

4.2.6 the film that takes a turn for the better seals with 5% skim-milk, 4 ℃, spends the night.

4.2.7 wash film twice with 1xPBST.Film is immersed in the immune serum (is anti-, with 1% skim-milk PBST dilution) of dilution in 1: 500 room temperature, horizontal shaking table 25rpm, 1 hour.

4.2.8 1xPBST washes film, horizontal shaking table 80rpm, 6min/ time, 1 hour.

4.2.9 two anti-hatching are goat anti-rabbit igg (H+L)-HRP (couplet section is biological, the import packing), dilution in 1: 5000 (with 1% skim-milk PBST dilution).Room temperature, horizontal shaking table 25rpm, 1 hour.

4.2.10 1xPBST washes film, horizontal shaking table 80rpm, 6min/ time, 1 hour.

4.2.11 luminous agent (PierceECL Western Blotting KIT) is added on the film scotography.

The Westernblot detected result is seen Fig. 5, can be known by figure: the H3HA albumen in three kinds of vaccines has all obtained expressing in various degree (about 72kd).Wherein H3HA-dTM also can detect proteic expression (about 90kd) in supernatant; And behind the independent transfection pJW4303; All detect proteic expression in lysis and the supernatant less than H3HA; All can be behind prompting nucleic acid vaccine H3HA, H3HA-tPA, the H3HA-dTM transfection 293T cell at cell inner expression, H3HA-dTM can also be secreted into the extracellular with expression product.

Embodiment 5 animal immunes

5.1 a large amount of preparations of nucleic acid vaccine (the big extraction reagent kit QIAGEN of plasmid Plasmid Mega Kit (5), Qiagen)

Identify that correct bacterium preservation liquid 5 μ L are inoculated in 5ml and contain in the LB nutrient solution of penbritin 5.1.1 draw, 37 ℃, 200rpm, overnight growth.

Contain in the LB nutrient solution of penbritin 5.1.2 culture bacteria liquid in (1) is inoculated in 1000ml by 1: 500,37 ℃, 200rpm, overnight growth.

5.1.3 bacterium moved on in the 250ml centrifugal bottle in second day, 4 ℃ of centrifugal 15min of 6000g abandon supernatant, collect bacterium.

5.1.450ml the resuspended bacterial precipitation of damping fluid P1, jolting repeatedly, until can't see bacterial aggregate, bacterium all is resuspended in the solution.

5.1.5 50ml damping fluid P2 adds resuspended liquid in centrifugal bottle, gentleness is put upside down 5-6 time, and it is blue that solution is, and leaves standstill 5min.

5.1.6 50ml damping fluid P3 adds in the above-mentioned mixing liquid, gentleness is put upside down 5-6 time, and blue solution disappears, the solution layering, and the upper strata is blocky oyster white agglomerate, lower floor is limpid liquid, places 30min on ice.

5.1.7 4 ℃, 20000g, centrifugal 30min keeps supernatant, is transferred to another centrifugal bottle.Under this condition again with the centrifugal 10min of supernatant.

5.1.8 level pad QBT35ml wet balanced extraction column.

5.1.9 the supernatant that obtains in (7) is added extraction column, cross post naturally, discard filtered solution.

5.1.10 add lavation buffer solution QC200ml, cross post naturally, discard filtered solution.

5.1.11 add elution buffer QF35ml, cross post naturally, collect filtered solution.

5.1.12 in collecting liquid, add the 24.5ml Virahol, 4 ℃, 15000g, centrifugal 30min abandons supernatant.

5.1.13 with precipitating the centrifugal 10min of normal temperature 15000g, supernatant discarded in the resuspended centrifuge tube of 7ml 70% ethanol.

5.1.14 will have sedimentary centrifuge tube to dry naturally, the 1ml physiological saline solution in super clean bench.

5.1.15 (wavelength 260nm～280nm) is plasmid content quantitatively for ultraviolet spectrophotometry.

5.1.16 add elution buffer QF35ml, again with pillar wash-out 1 time.

5.1.17 for improving the utilization ratio of adsorption column, the second day solution with autogamy carries out 1 plasmid again by the step of 1-15 and extracts in a large number.

5.1.18 the plasmid of extracted twice is mixed, carries out digestion with restriction enzyme and identify that qualification result is seen Fig. 4.

5.2 animal immune: this institute uses animal to be 2.0kg left and right sides NZw.Immunize rabbit can produce a large amount of immune serums, enough carries out the research of antibody test and the effect of antibody passive protection.A large amount of recombinant plasmids that extract are diluted to 1.0ug/ul with saline water and are used for immunity, the 200ug/ rabbit.Immunization protocol is the 0th, 2,4,8 weeks of animal to be total to immunity 4 times, and immunization strategy is seen Fig. 6; Immunization method is that intramuscular injection powers up and transcribes, WJ-2002 live body gene introducing apparatus, technical parameter: voltage 100V, positive and negative each 3 times of pulse number, the wide 20ms of ripple; It is effective to be regarded as electrotransfection with rabbit leg muscle generation shake.Animal immune divides 3 of pJW4303 empty carrier control groups (Fig. 6 D group), vaccine experimental group pJW4303/H3HA (Fig. 6 A group), pJW4303/H3HA-tPA (Fig. 6 B group), pJW4303/H3HA-dTM (Fig. 6 C group), and each organizes 5 rabbits.

5.3 immune serum preparation: reach each immunity two weeks of back before the first immunisation, arteria auricularis is gathered animal blood, room temperature placement 1 hour, and the centrifugal 10min of 2500rpm draws supernatant and uses the centrifugal 10min of 8000rpm again.Separate obtaining immune serum, packing and mark are clear, and be frozen in-70 ℃, is used for the detection of specific antibody.

The detection of embodiment 6 immunne responses

6.1 EUSA (ELISA), step is following:

6.1.1 antigen coated: by dilution in 1: 5, the 100ul/ hole added elisa plate to the supernatant of transfectional cell (S) with 1xPBS (ELISA with), and 4 ℃ encapsulate and spend the night.Each antigen is two multiple holes and encapsulates.

6.1.2 taking-up elisa plate, 1xPBST are washed 5 times.(PBST constitutes 10mMPBS and 0.05%Tween-20).

6.1.35% every hole 200 μ l of skim-milk (joining) with 1xPBST.37 ℃, sealed 1 hour.

6.1.4 abandon confining liquid, wash plate 5 times with 1XPBST.

6.1.5 one anti-is immune serum to be checked, (PBS),, hatched 1 hour in the 100ul/ hole by 37 ℃ for anti-diluent a: 4%whey, 0.5%Tween-20 in dilution in 1: 500.

Resist 6.1.6 abandon one, wash plate 5 times with 1XPBST.

6.1.7 two is anti-: be goat anti-rabbit igg (H+L)-HRP (couplet section is biological, the import packing), dilution in 1: 5000 (two anti-diluent: 4%whey, 0.5%Tween-20, PBS)., hatched 1 hour in the 100ul/ hole by 37 ℃.

6.1.8TMB colour developing.(TMB solution formula: 1 in TMB tablet, 0.1M phosphoric acid citrate buffer 5ml, distilled water 5ml, 30% ydrogen peroxide 50,2 μ l), every hole adds 100 μ l, room temperature, 3.5min; Every hole adds the H of 50 μ l1M ₂SO ₄Color development stopping.

6.1.9 detect.Each hole A450 value is measured and write down to ELIASA, calculates multiple hole MV.

Experimentation on animals is the result show: in two weeks of back of immunity for the second time, all can detect specific anti H3HA antibody in the animal serum of three groups of nucleic acid vaccine immunities; In two weeks of back of immunity for the third time, specific anti H3HA antibody horizontal obviously raises.Along with the increase of immune time, it is fastest that H3-tPA immune serum specific antibody level rises, and is significantly higher than H3HA and H3HA-dTM immune group (p＜0.01).And two groups of immune serums of H3HA and H3HA-dTM, specific antibody level rising situation no significant difference.Negative control group does not have antibody response.See Fig. 7.

In the 4th two weeks of immunity back, the average titer of specific anti H3HA antibody is respectively in three groups of immune serums: the H3HA-tPA group: 56300, and the H3HA group: 13500, H3HA-dTM group: 6500.The negative control group antibody titers is less than 10.See Fig. 8.

H3HA gene, H3HA/XJ3-07 gene used in the present specification all refer to same sequence, interchangeable use.

Related reagent information such as following table among the embodiment:

Taq enzyme Promega company

T4DNA ligase enzyme Promega company

The precious biotech firm in DNAmarker (DL 3000) Dalian

Restriction enzyme BamH I, Pst I and Sac I Fermentas company

Dna gel reclaims the precious biotech firm in test kit Dalian

DMEM high glucose medium Hyclone company

RPM1640 cell culture fluid Hyclone company

Foetal calf serum Gibico company

Plasmid extracts test kit Qiagen company in a small amount

The a large amount of test kit Qiagen companies that extract of grain

PEI Invitrogen company

The sheep anti-mouse igg BD company of HRP mark

The streptavidin SouthernBiotech company of HRP mark

The goat anti-rabbit igg SouthernBiotech company of HRP mark

Biotin labeled sheep anti-mouse igg SouthernBiotech company

Biotin labeled goat anti-rabbit igg SouthernBiotech company

CTB protein Sigma company

TMB tablet Sigma company

Sequence table

< 110>Wang Shixia, Zhou Jianhua, Xu Ying, Huang Zuhu, Lu Shan

< 120>a kind of codon optimized H3HA-XJ3-07 gene and nucleic acid vaccine thereof

<160>6

 

<210>1

<211>1707

<212>DNA

< 213>artificial sequence

<220>

< 223>codon optimized H3HA-XJ3-07 gene order

<400>1

atgaaaacca?ccatcatcct?gaacttcatc?ctgctgaccc?actgggccta?cagccagaac?60

cccatcagca?acaacaacac?cgccaccctg?tgcctgggcc?accacgccgt?ggccaacggc?120

accctggtga?aaaccatcag?cgacgaccag?atcgaggtga?ccaacgccac?cgagctggtg?180

cagagcatca?ccatgggcaa?gatctgcaac?aacagctaca?gaatcctgga?cggccggaac?240

tgcaccctga?tcgacgccat?gctgggcgac?cctcactgcg?acgtgttcca?gtacgagaac?300

tgggacctgt?tcatcgagcg?gagcagcgcc?ttcagcaact?gctaccccta?cgacatcccc?360

gactacgcca?gcctgcggag?catcgtggcc?agcagcggca?cactggaatt?caccgccgag?420

ggcttcacct?ggaccggcgt?gacccagaac?ggcatcagcg?gcgcctgcaa?gaggggcagc?480

gccgacagct?tctttagcag?actgaactgg?ctgaccaaga?gcggcaactc?ctaccccacc?540

ctgaacgtga?ccacccccaa?caacaagaac?ttcgacaagc?tgtacatctg?gggcatccac?600

caccccagca?gcaaccagga?acagaccaag?ctgtatatcc?aggaaagcgg?cagggtcacc?660

gtgagcacca?agcggagcca?gcagaccatc?atccccaaca?tcggcagccg?gccctgggtg?720

cggggccaga?gcggccggat?cagcatctac?tggaccatcg?tgaagcccgg?cgacatcctg?780

atgatcaaca?gcaacggcaa?tctggtggcc?cccaggggct?acttcaagct?gaaaaccggc?840

aagagcagcg?tgatgcggag?cgacgtgccc?atcgacatct?gcgtgagcga?gtgcatcacc?900

cccaacggca?gcatctccaa?cgacaagccc?ttccagaacg?tgaacaaggt?gacctacggc?960

aagtgcccca?agtacatccg?gcagaacacc?ctgaagctgg?ccaccggcat?gcggaacgtg?1020

cccgagaagc?agatccgggg?catcttcggc?gccattgccg?gcttcatcga?gaacggctgg?1080

gagggcatgg?tggacgggtg?gtacggcttc?agataccaga?acagcgaggg?caccggccag?1140

gccgccgacc?tgaagagcac?ccagaccgcc?atcgaccaga?tcaacgagaa?gctgaaccgg?1200

gtgatcgagc?ggaccaacga?gaagttccac?cagatcgaaa?aagaattcag?cgaggtggag?1260

ggcagaatcc?aggacctgga?aaagtacgtg?gaggacacca?agatcgacct?gtggagctac?1320

aacgccgagc?tgctcgtggc?cctggaaaac?cagcacacca?tcgacctgac?cgacgccgag?1380

atgaacaagc?tgttcgagaa?aaccaggcgg?cagctgcggg?agaacgccga?ggacatgggc?1440

ggaggatgct?tcaagatcta?ccacaagtgc?gacaacgcct?gcatcggcag?catccggaac?1500

ggcacctacg?accactacat?ctaccgggac?gaggccctga?acaaccggtt?ccagatcaag?1560

ggcgtggagc?tgaagagcgg?ctacaaggac?tggattctgt?ggatcagctt?cgccatcagc?1620

tgctttctga?tctgcgtggt?gctgctgggc?ttcatcatgt?gggcctgcca?gaagggcaac?1680

atccgctgca?atttctgcat?ctgatga 1707

<210>2

<211>1674

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>2

gctagcgcct?acagccagaa?ccccatcagc?aacaacaaca?ccgccaccct?gtgcctgggc?60

caccacgccg?tggccaacgg?caccctggtg?aaaaccatca?gcgacgacca?gatcgaggtg?120

accaacgcca?ccgagctggt?gcagagcatc?accatgggca?agatctgcaa?caacagctac?180

agaatcctgg?acggccggaa?ctgcaccctg?atcgacgcca?tgctgggcga?ccctcactgc?240

gacgtgttcc?agtacgagaa?ctgggacctg?ttcatcgagc?ggagcagcgc?cttcagcaac?300

tgctacccct?acgacatccc?cgactacgcc?agcctgcgga?gcatcgtggc?cagcagcggc?360

acactggaat?tcaccgccga?gggcttcacc?tggaccggcg?tgacccagaa?cggcatcagc?420

ggcgcctgca?agaggggcag?cgccgacagc?ttctttagca?gactgaactg?gctgaccaag?480

agcggcaact?cctaccccac?cctgaacgtg?accaccccca?acaacaagaa?cttcgacaag?540

ctgtacatct?ggggcatcca?ccaccccagc?agcaaccagg?aacagaccaa?gctgtatatc?600

caggaaagcg?gcagggtcac?cgtgagcacc?aagcggagcc?agcagaccat?catccccaac?660

atcggcagcc?ggccctgggt?gcggggccag?agcggccgga?tcagcatcta?ctggaccatc?720

gtgaagcccg?gcgacatcct?gatgatcaac?agcaacggca?atctggtggc?ccccaggggc?780

tacttcaagc?tgaaaaccgg?caagagcagc?gtgatgcgga?gcgacgtgcc?catcgacatc?840

tgcgtgagcg?agtgcatcac?ccccaacggc?agcatctcca?acgacaagcc?cttccagaac?900

gtgaacaagg?tgacctacgg?caagtgcccc?aagtacatcc?ggcagaacac?cctgaagctg?960

gccaccggca?tgcggaacgt?gcccgagaag?cagatccggg?gcatcttcgg?cgccattgcc?1020

ggcttcatcg?agaacggctg?ggagggcatg?gtggacgggt?ggtacggctt?cagataccag?1080

aacagcgagg?gcaccggcca?ggccgccgac?ctgaagagca?cccagaccgc?catcgaccag?1140

atcaacgaga?agctgaaccg?ggtgatcgag?cggaccaacg?agaagttcca?ccagatcgaa?1200

aaagaattca?gcgaggtgga?gggcagaatc?caggacctgg?aaaagtacgt?ggaggacacc?1260

aagatcgacc?tgtggagcta?caacgccgag?ctgctcgtgg?ccctggaaaa?ccagcacacc?1320

atcgacctga?ccgacgccga?gatgaacaag?ctgttcgaga?aaaccaggcg?gcagctgcgg?1380

gagaacgccg?aggacatggg?cggaggatgc?ttcaagatct?accacaagtg?cgacaacgcc?1440

tgcatcggca?gcatccggaa?cggcacctac?gaccactaca?tctaccggga?cgaggccctg?1500

aacaaccggt?tccagatcaa?gggcgtggag?ctgaagagcg?gctacaagga?ctggattctg?1560

tggatcagct?tcgccatcag?ctgctttctg?atctgcgtgg?tgctgctggg?cttcatcatg?1620

tgggcctgcc?agaagggcaa?catccgctgc?aatttctgca?tctgatgagg?atcc 1674

 

<210>3

<211>1554

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>3

gctagcgcct?acagccagaa?ccccatcagc?aacaacaaca?ccgccaccct?gtgcctgggc?60

caccacgccg?tggccaacgg?caccctggtg?aaaaccatca?gcgacgacca?gatcgaggtg?120

accaacgcca?ccgagctggt?gcagagcatc?accatgggca?agatctgcaa?caacagctac?180

agaatcctgg?acggccggaa?ctgcaccctg?atcgacgcca?tgctgggcga?ccctcactgc?240

gacgtgttcc?agtacgagaa?ctgggacctg?ttcatcgagc?ggagcagcgc?cttcagcaac?300

tgctacccct?acgacatccc?cgactacgcc?agcctgcgga?gcatcgtggc?cagcagcggc?360

acactggaat?tcaccgccga?gggcttcacc?tggaccggcg?tgacccagaa?cggcatcagc?420

ggcgcctgca?agaggggcag?cgccgacagc?ttctttagca?gactgaactg?gctgaccaag?480

agcggcaact?cctaccccac?cctgaacgtg?accaccccca?acaacaagaa?cttcgacaag?540

ctgtacatct?ggggcatcca?ccaccccagc?agcaaccagg?aacagaccaa?gctgtatatc?600

caggaaagcg?gcagggtcac?cgtgagcacc?aagcggagcc?agcagaccat?catccccaac?660

atcggcagcc?ggccctgggt?gcggggccag?agcggccgga?tcagcatcta?ctggaccatc?720

gtgaagcccg?gcgacatcct?gatgatcaac?agcaacggca?atctggtggc?ccccaggggc?780

tacttcaagc?tgaaaaccgg?caagagcagc?gtgatgcgga?gcgacgtgcc?catcgacatc?840

tgcgtgagcg?agtgcatcac?ccccaacggc?agcatctcca?acgacaagcc?cttccagaac?900

gtgaacaagg?tgacctacgg?caagtgcccc?aagtacatcc?ggcagaacac?cctgaagctg?960

gccaccggca?tgcggaacgt?gcccgagaag?cagatccggg?gcatcttcgg?cgccattgcc?1020

ggcttcatcg?agaacggctg?ggagggcatg?gtggacgggt?ggtacggctt?cagataccag?1080

aacagcgagg?gcaccggcca?ggccgccgac?ctgaagagca?cccagaccgc?catcgaccag?1140

atcaacgaga?agctgaaccg?ggtgatcgag?cggaccaacg?agaagttcca?ccagatcgaa?1200

aaagaattca?gcgaggtgga?gggcagaatc?caggacctgg?aaaagtacgt?ggaggacacc?1260

aagatcgacc?tgtggagcta?caacgccgag?ctgctcgtgg?ccctggaaaa?ccagcacacc?1320

atcgacctga?ccgacgccga?gatgaacaag?ctgttcgaga?aaaccaggcg?gcagctgcgg?1380

gagaacgccg?aggacatggg?cggaggatgc?ttcaagatct?accacaagtg?cgacaacgcc?1440

tgcatcggca?gcatccggaa?cggcacctac?gaccactaca?tctaccggga?cgaggccctg?1500

aacaaccggt?tccagatcaa?gggcgtggag?ctgaagagcg?gctactgagg?atcc 1554

 

<210>4

<211>35

<212>DNA

< 213>artificial sequence

<220>

< 223>upstream primer

<400>4

gtcacttcgc?tagcgcctac?agccagaacc?ccatc 35

 

<210>5

<211>25

<212>DNA

< 213>artificial sequence

<220>

< 223>downstream primer

<400>5

gagctcggat?cctcatcaga?tgcag 25

 

<210>6

<211>36

<212>DNA

< 213>artificial sequence

<220>

< 223>downstream primer

<400>6

gagctcggat?cctcagtagc?cgctcttcag?ctccac 36

Claims (7)

1. codon optimized H3N8 hypotype equine influenza H3HA/XJ3-07 gene, sequence is SEQ ID NO.1.

2. equine influenza H3HA nucleic acid vaccine is characterized in that this vaccine is that H3HA/XJ3-07 gene and the carrier for expression of eukaryon of SEQ ID NO.1 formed by sequence.

3. nucleic acid vaccine according to claim 2 is characterized in that described carrier for expression of eukaryon is for cutting the carrier segments that pJW4303 obtains through restriction endonuclease pstI and BamHI.

4. equine influenza H3HA-tPA nucleic acid vaccine; It is characterized in that this vaccine excises the natural signals peptide gene H3HA/XJ3-07 gene fragment that obtains and the carrier for expression of eukaryon that contains the tPA signal peptide gene that carry by the described H3HA/XJ3-07 gene of claim 1 and forms, the H3HA/XJ3-07 gene fragment order that the natural signals peptide gene that described excision carries obtains is SEQ ID NO.2.

5. nucleic acid vaccine according to claim 4 is characterized in that described carrier for expression of eukaryon for cutting the big fragment of carrier that pJW4303 obtains through restriction endonuclease NheI and BamHI, and wherein the sequence between NheI and pstI restriction enzyme site is the tPA signal peptide gene.

6. H3HA/XJ3-07-dTM nucleic acid vaccine; It is characterized in that this vaccine excises the natural signals peptide gene and the encoding influenza virus hemagglutinin H3HA/XJ3-07 born of the same parents internal area that carry by the described H3HA/XJ3-07 gene of claim 1 and forms with the sequence of striding film district part only encoding influenza virus hemagglutinin H3HA/XJ3-07 extracellular domain gene fragment partly that obtains and the carrier for expression of eukaryon that contains the tPA signal peptide gene, the gene fragment order of described only encoding influenza virus hemagglutinin H3HA/XJ3-07 extracellular domain part is SEQID NO.3.

7. the described nucleic acid vaccine of root pick claim 6 is characterized in that described carrier for expression of eukaryon for cutting the big fragment of carrier that pJW4303 obtains through restriction endonuclease NheI and BamHI, and wherein the sequence between NheI and pstI restriction enzyme site is the tPA signal peptide gene.

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