CN101353375A - Method for producing influenza hemagglutinin multivalent vaccine - Google Patents

Abstract

The invention relates to a method for producing a polyvalent vaccine of an influenza hemagglutinin. The invention provides a method for preparing a recombined influenza vaccine by using the technology of DNA. The produced vaccine is polyvalent, preferably to be trivalent and is based on the influenza vaccine of a recombined hemagglutinin antigen compound cloning from an influenza virus with an epidemic potential. The recombined hemagglutinin antigen is whole-length glycosidoprotein which is not cut (HAO), generated from a rhabdovirus expression vector for culturing an insect cell and purified under a non-denatured condition. In the preferable implementing scheme, the cloned HA gene is decorated by cutting a natural hydrophobic signal peptide sequence and using a novel rhabdovirus of chitinase signal peptide to replace. The invention also discloses a common method used for effectively distilling and purifying the recombined HA protein generated from the insect cell from the rHA protein purified from the influenza viruses of A subtype and B type.

Description

Produce the method for influenza hemagglutinin polyvalent vaccine

The application is to be May 26 nineteen ninety-five the applying date, and application number is 95197928.0, and denomination of invention is divided an application for the application for a patent for invention of " produce influenza hemagglutinin polyvalent vaccine method ".

Background of invention

In general, the invention belongs to the recombinant flu vaccines field.

Influenza all takes place every year, and is significantly pathogenic, lethal reason in the world wide.Children have the highest infection rate, and influenza virus propagation is socially taken the main responsibility.The elderly and have the people of potential health problem that influenza infection complication and hospital care are had higher danger.Only in the U.S., seven influenzas in the period of 1956 and 1988 are in season, each infects season because pneumonia and influenza all have 10, the death of people more than 000, report 2 mid-season each all have greater than 40,000 people's death (latest information: influenza activity-U.S. and world wide, and 1992-1993 influenza vaccines data The weekly that causes death causes a disease, the U.S., healthy and human department, publilc health service centre, 41/No.18:315-323,1992) and influenza virus is by 2 surface glycoproteins, the many types of particle of height that hemagglutinin (HA) and neuraminidase (NA) are formed.The HA valency is led virus and is reached the fusion between virus-cytolemma in virus intrusion cell processes with combining of host cell.The influenza virus gene group is made up of 8 strand sense-rna fragments, and wherein length is the 4th fragment coding HA gene.Be divided into A, B and C type according to the antigenic difference influenza virus.Influenza A virus is described by comprising hypotype or type, geographic origin, strain number and separating the nomenclature in age, as A/Beijing 353/89.HA has 13 hypotype (H at least ₁-H ₁₃), NA has 9 hypotype (N at least ₁-N ₉).All hypotypes all are found in the bird, but have only H ₁-H ₃And N ₁-N ₂Be found in the mankind, pig and Malaysia and China (Murphy and Webster, " orthomyxovirus ", in " virusology ", editor: Fields, B.N., Knipe, D.M., Chanock, R.M., 1091-1152 (Raven press, New York, 1990)).

In the antibody of anti-HA and virus and form the infection that influenza causes autarcetic basis (Clements, " influenza vaccines " are at vaccine: the new exploration of immunological problem, editor RonaldW.Ellis, pp.129-150 (MA 1992 for Butterworth Heinemann, Stoneham)).The antigenicity of HA molecule changes the limitation that has caused the frequent outburst of influenza and utilized immune control to infect.

The three-dimensional structure of HA and be widely studied with the sialic interaction of its cell receptor (Wilson, etc., " structure of influenza virus hemagglutinin membrane glycoprotein under 3A ° of resolving power ", From So, 289:366-378 (1981); Weis, etc., " with the structure of its acceptor sialic acid compound influenza virus hemagglutinin ", Nature, 333:426-431 (1988); Murphy andWebster, 1990).The HA molecule exists with trimeric form in virus particle.Each monomer comprises two chains, and HA1 and HA2 are connected by a disulfide linkage.Infected host cell produces a molecular weight and is about 85,000 glycosylated polypeptide precursor (HA0), is cracked into HA1 and HA2 then.

Influenza HA special in the existence and its anti-infective relevant with disease (Clements, 1992) of IgG and IgA antibody.The intact virus of deactivation or partially purified (subunit of cutting apart) influenza vaccines are demarcated with the HA content of every strain.Influenza vaccines contain the HA that 7 to 25 micrograms obtain usually from each of three kinds of influenzas.

Another main surface glycoprotein NA is not also determining the protection antibody immunity of influenza and the effect in the T-cell response.Neuraminidase very unstable in purifying and storage process (Murphy and Webster, 1990), and the content of NA does not also have stdn in the present influenza vaccines.The HA of purifying rather than NA vaccine can prevent that the animal that attacked by influenza from avoiding (the Johansson that falls ill, Deng., " influenza virus hemagglutinin of purifying and neuraminidase are of equal value in stimulating antibody response but induce the immunity to infecting of opposite types ", Journal of Virology, 63:1239-1246 (1989)).A kind ofly in test, find no provide protection (Orga etc., transmissible disease magazine 135:499-506 (1997)) to the people based on the antigenic experimental vaccine of neuraminidase.

The influenza vaccines of approval are made up of the subunit goods complete or chemical cracking of the formalin deactivation that obtains from two kinds of influenza A hypotypes (H1N1 and H3N2) and a kind of influenza B subtype virus.Before season, U.S. food and bureau of drug's vaccine and associated biomolecule consultative committee are the composition of recommending a kind of trivalent influenza vaccines upcoming season in each influenza.The 1992-93 vaccine contains A/Texas/36/91-like (H1N1), A/Beijing/353/89-like (H3N2) and B/Panama/45/90 virus.FDA suggestion 1993-94 influenza vaccines should comprise identical Texas and Panama strain, and a kind of new influenza ABeijing strain (A/Beijing/32/92).

In annual influenza before season, be the effective measures that reduce the influenza influence to high risk population's vaccination.At present the limitation of the vaccine that uses comprise low rate of utilization, to the elderly and children's weak effect, originate from egg, antigenic change and side effect.

Centre for Disease Control (CDC) estimates to have inoculated vaccine (MMWR, 1992) less than 30% influenza susceptible individual every year.Present inactivated vaccine can produce disease-resistant efficiently protectiveness to normal normal adults under the antigen of the vaccine situation very close with the popular influenza virus.In the elderly, very different to the protection ratio of disease, particularly to the elderly in those nursing house (Clements, 1992).Powers and Belshe ( The transmissible disease magazine167:584-592 (1993)) in the nearest research, observes less than 30% among 65 years old or the above experimenter a kind of trivalent subvirral particle influenza vaccines are produced obvious antibody response.

The seed virus of influenza A and B vaccine is copied into the strain of generation naturally that height is tired at the egg allantoic cavity.In addition, the virus strain of influenza A composition is a kind of reprovision virus that contains correct surface antigen gene.Reprovision virus is meant the virus that contains each parental virus strain feature owing to virus genomic fragmentation.When more than a kind of strains of influenza viruses infected cell, these viral fragments mix, and produce to contain the progeny virus that the range gene from parents distributes.

Using the provide protection of present influenza vaccines complete or that isolate is short-term, and along with its protectiveness of antigen drift of popular strains of influenza viruses can fade away.It is the result who the virus that has the aminoacid sequence change in the hemagglutinin molecule is carried out immunoselection that influenza virus produces the antigenicity drift.Under the perfect condition, the vaccine virus strain conforms to pathogenic strains of influenza viruses.But the production process of influenza vaccines is restricted because of virus is bred in the egg of tool embryo at present.Not every strains of influenza viruses can both duplicate in egg well; Therefore virus must adaptation or is made up viral reprovision.With compare from the isolating influenza virus that grows in mammalian cell of infected individuals at first, the hemagglutinin that originates in the influenza virus of egg occur widely heterology (Wang, etc., virusology 171:275-279 (1989); Rajakumar, etc., Proc. Natl. Acad. Sci.USA 87:4154-4158 (1990)).In the selection and production process of influenza vaccines, the variation of HA can produce the mixture with the visibly different viral subgroup of antigenicity.Therefore the virus in the vaccine can be different from the mutation in the epidemic strain, causes being lower than the protection of optimum level.

There is serious egg people hypersensitive to produce anaphylaxis immediately to egg albumen residual in the vaccine.Porcine influenza vaccine in 1976 is accompanied by the increase of Guillain-Barre syndromes frequency.Find that also subsequently the vaccine from other strains of influenza viruses preparation causes the generation of this rare disease.

A kind of method that does not need the production influenza vaccines that breed in egg can produce purer and cause harmful littler product of immunoreactive possibility.In addition, pure vaccine production thing does not need organic extraction of inactivation of virus or viromembrane component, therefore avoids the sex change of antigenic surface and the safety worries that is caused by residual chemical in the vaccine.

In addition, do not need the influenza vaccines of egg propagation production can avoid the genetic heterogeneity that in egg, adapts to and go down to posterity and cause.Can obtain the vaccine that more mates with the influenza virus epidemic strain like this, produce better effect.

Therefore provide that a kind of not need the method for the production influenza vaccines of duplicating in egg be a target of the present invention.

Another object of the present invention provide a kind of rapidly, cost produces the methods of influenza vaccines effectively, to heavens, its allows to produce vaccine from the primary source of influenza.

The present invention's general introduction

The invention provides the method for utilizing baculovirus expression system in insect cell, to prepare the recombinant influenza hemagglutinin through expression.Resulting albumen is useful the multivalence influenza vaccines of preparation based on the reorganization hemagglutinin antigen mixture of cloning from the influenza virus with popular potentiality.The reorganization hemagglutinin is total length, uncracked (HA0) glycoprotein, be included in be purified under the non-sex change condition 95% or more than, be preferably the HA1 and the HA2 subunit (HAO) of 99% purity.

A kind of method of utilizing specially designed oligonucleotide probe and polymerase chain reaction (PCR) method from influenza A and B virus clone influenza hemagglutinin gene is also disclosed.In preferred embodiments, clone's HA gene is through the Nucleotide of the natural hydrophobic signal peptide sequence of deletion coding and replace a new baculovirus signal peptide and modify, and produces the sequence that a coding is close to the signal peptide of hemagglutinin.This mosaic gene is introduced in the rhabdovirus expression vector, makes baculovirus polyhedrin body protein promotor instruct the expressed in insect cells of the HA albumen of reorganization in infection.18 glycosylation pathway differ that amino acid whose baculovirus signal peptide instructs the translation of rHA to enter insect cell, and it is not present in the sophisticated rHA glycoprotein.In preferred embodiments, designed a kind of any amino acid whose carrier that interleaves of between signal peptide and hemagglutinin, not encoding.

This method can be generalized to the influenza virus of all models, includes but not limited to infect human popular A (H1N1) hypotype, A (H3N2) hypotype and Type B, and the influenza virus that infects other Mammalss and birds.

Disclose the proteic general method of reorganization HA that a kind of effective extraction and purifying originate in insect cell and be used for the rHA albumen that purifying derives from A hypotype and Type B influenza virus.Recombiant vaccine can form from the primary source of influenza, for example from the nasal discharge of infected individual, and need not be to form from the virus that adapts to and be incubated at egg.This makes can directly form vaccine from popular strains of influenza viruses fast, avoids adapting to egg by virus and cultivates and the problem of generation and the reaction that patient produces the egg pollutent in the vaccine that is produced.

Embodiment has confirmed that containing from FDA be the prescription and the clinical effectiveness of vaccine the antigenic immunizing dose form of rHA of three kinds of strains of influenza viruses purifying recommending for 1993/1994 and 1994/1995 influenza pandemic season.Utilize quantitatively in conjunction with the ability of influenza hemagglutinin, blocking-up influenza virus aggegation erythrocyte or in and the test of the antibody of influenza virus, measured the function immunity.In to the animal that is subject to influenza infection or in the mankind's attack experiment, measured the protective immune response of rHA vaccine.

Brief description of the drawings

Fig. 1 is from the viral RNA goods clone HA gene of influenza A strain by purifying, the diagram of the purifying of the rHA of expression and the biological characteristics of rHA.

Abbreviation: FDA, food and bureau of drug; MDCK, Madin Darby dog kidney; TPCK, tosylphenylalanine chloromethyl ketone; RNA, Yeast Nucleic Acid; CDNA, complementary DNA (cDNA); HA, hemagglutinin; FBS, foetal calf serum; PCR, the polymerase chain reaction; BV, baculovirus.

Fig. 2 is that the method with Fig. 1 is used to clone the more detailed diagram with the HA gene of expression of influenza A/Texas/36/91 strain.The influenza HA gene is from having infected the mdck cell purified RNA of influenza A/Texas/36/91, utilized reversed transcriptive enzyme and universal primer (SEQ ID NO.1), takes turns through 2 then that pcr amplification and clone obtain.As shown in the figure, in first round PCR reaction, used the terminal primer SEQ ID NO.3 of 5 ' terminal primer SEQ ID NO.2 and 3 '.Take turns in the PCR reaction second, used the terminal primer SEQ ID NO.5 of 5 ' terminal primer SEQ ID NO.4 and 3 '.Made up a kind of baculovirus recombinant vectors, this carrier contains the polyhedrin promotor, and (molecular weight is about 61 with deriving from baculovirus 61K gene, the baculovirus gene of 000 coded signal peptide) signal peptide sequence is thereafter the complete proteic encoding sequence of ripe HA.This recombinant vectors is used to make up the rhabdovirus expression vector that produces HA from this virus strain then.

Fig. 3 is the diagram of mouse the 42nd day anti-HA immunne response, and n=5 has shown pure rHA0-

Vaccine, rHA0 aluminium is 0.5 μ g (secret note band) at dosage, 0.1 μ g (shade band), 0.02 μ g (some band), the antibody titers during 0.004 μ g (dead zone).

Fig. 4 a, 4b and 4c are the trivalent vaccines with rHA or approval, the immunne response of the anti-HA of 1994-1995 prescription mice immunized, (Fig. 4 a) to HAI A/Texas/36/91 for number of weeks after the vaccination, HAI A/Shangdong/9/93 (Fig. 4 b), HAI B/Panama/45/90 (Fig. 4 c), rHA (rhombus) and be incubated at egg

The HIA titre of attenuated vaccine (square).

Detailed description of the present invention

The method for preparing recombinant flu vaccines is described. (HA0) total length, that be not cut hemagglutinin antigen that rhabdovirus expression vector production in the insect cell of utilize cultivating obtains from influenza virus, and under non-Denaturing purifying. To mix from two or more hemagglutinin antigens that influenza A and/or influenza B strain purifying obtain, produce the multivalence influenza vaccines. Recombinant antigen can make up to strengthen effectiveness with a kind of assistant carrier.

Utilize recombinant DNA technology to produce influenza vaccines and have several advantages: the recombinant DNA influenza vaccines can produce under the condition of safer and stricter control; Not needing to infect in egg influenza breeds; Restructuring HA albumen can by more highly purified, in fact have been eliminated the side effect that is caused by contaminating protein; Therefore the purge process of restructuring HA needn't comprise deactivation or organic extraction viromembrane component of virus, has avoided other safety worries that residual chemical causes in the sex change of antigen and the vaccine; Utilize recombinant DNA technology production HA to avoid the hereditary heterologous that adapts to and go down to posterity and produce because of in egg, this can make vaccine be complementary with epidemic strain better, causes it to render a service and strengthens; Recombination method also allows to carry out Strain in the after a while period of this year and selects, so that select based on more reliable epidemic data if having time.

Baculovirus expression system

Baculoviral is the dna virus of Baculoviridae section. Known this viroid has narrower host range, mainly is confined to the insect (butterfly and moth) of Lepidopteran kind. Baculoviral autographa california nuclear polyhedrosis virus (AcNPV) has become the prototype of baculoviral, can be in the sensitive insect cell of cultivating efficient replication. AcNPV has a double-stranded closed hoop DNA gene, and is about 130, and 000bp, its host range, molecular biology and genetics characteristic are more clearly.

The many baculovirals that comprise AcNPV form large albumin crystal occlusion body in the nuclear of infection cell. A kind of polypeptide that is called polyhedrin accounts for 95% of these occlusion body albumen qualities. Polyhedron gene exists with single copy in the AcNPV viral genome. Because polyhedrin gene pairs virus copying in cultured cell is optional, it can be at an easy rate be used for expression alien gene by modification. Exogenous gene sequence inserts polyhedrin promoter sequence 3 ' end in the AcNPV gene, makes it be subject to the control of transcribing of polyhedrin promoter.

Utilize baculovirus DNA and contain the recombinant baculovirus of the homologous recombination construction expression alien gene between the chimeric plasmid of target gene sequences.Can become the feature detection of homogeneity to go out recombinant virus with plaque purification by they special plaque forms.

Baculovirus is specially adapted to as eukaryotic cell clone and expression vector.Host range by them is narrow, and only limitation is formed on arthropodan characteristic, therefore normally safe.Three kinds of baculovirus Pest Control have been agreed to use by U.S. environment protection mechanism (EPA).Under the EPA experiment usage license, AcNPV was used to crop many years.

AcNPV wild-type and recombinant virus comprise from mythimna separata Spodoptera frugiperda (Lepidoptera in autumn at various insect cells; Noctuidae) duplicate in the continuous cell line of Chan Shenging.The population doubling time of S.frugiperda cell is 18 to 24 hours, can breed in individual layer or free suspending nutrient solution.

Reorganization HA albumen can be produced in the cell from Lepidopteran kind Spodoptera frugiperda, but is not limited thereto kind of a cell.Other can as the cell that obtains from Bombix mori, Galleria mellanoma, Trichplusia ni or Lamanthriadispar kind, also be can be used as and produce the proteic suitable substrate of reorganization HA by the insect cell of baculovirus infection.

It is most preferred that to utilize recombinant baculovirus to produce proteic host cell system be Sf900+.Another preferably utilizes recombinant baculovirus to produce proteic host cell is to be Sf9.Sf900+ and Sf9 are from mythimna separata Spodoptera frugiperda (Lepidoptera in autumn; Noctuidae) the non-conversion of Chan Shenging, nononcogenic continuous cell line.Sf900+ and Sf9 cell are at 28 ± 2 ℃, and supplying carbon dioxide is not bred down.The substratum that is used for the Sf9 cell is TNMFH, a kind of salt, VITAMIN, sugar and amino acid whose simple mixtures, and other adds 10% foetal calf serum.Except foetal calf serum, there are not other animal derived products (being trypsinase etc.) to be used for cell proliferation.The substratum of serum-free (as the Sf900 substratum, Gibco BRL, Gaithersburg MD) also can be used for cultivating the Sf9 cell, and is preferred for the breeding of Sf900+ cell.

The population doubling time of Sf9 cell is 18 to 24 hours, can breed in individual layer or suspending nutrient solution.Also report demonstration S.frugiperda cell is not supported duplicating of any known mammalian virus.

It will be appreciated by those skilled in the art that this expression vector is not confined to baculovirus expression system.Reorganization HA albumen also can be other expression vectors such as entomopoxvirus (poxvirus of insect), cytoplasmic polyhedrosis virus (CPV) and with the expressed in insect cells of recombinate HA gene or constitutive expression gene transformation.

The separation of strains of influenza viruses

Can be from being isolated one or more strains of influenza viruses the individuality of disease infection.Preferably, strains of influenza viruses is had the virus strain of popular potentiality by what food and medicine office (FDA) or CDC identified in influenza subsequently in season.The advantage of method described herein is clinical sample can be used as virus as the nasal discharge that is subjected to the influenza infection patient a direct material.In addition, these also can obtain from FDA or CDC.

The breeding of strains of influenza viruses

Then with the high virus titer of virus strain propagation production in cell, as Madin Darby dog kidney (MDCK) cell (can obtain from American type culture collection, preserving number is ATCCCCL34).For example, the trypsinase of tosylphenylalanine chloromethyl ketone (TPCK) part deactivation and can produce Gao Shoudai go down to posterity virus titer best foetal calf serum concentration in the presence of, infect mdck cell.To analyze (Rosen according to standard HA, " hemagglutinin and animal virus " be editor: K.Habel and N.P.Salzman in " virusology basic technology ", pp.276-28 (academic press, New York 1969), this paper quotes its instruction) the low infection multiplicity (0.1 to 0.5) measured infects mdck cell with strains of influenza viruses.Infected cell was cultivated 48 hours at 33 ℃, utilized the viral yield in the hemagglutination activity analyzing and testing substratum.Can produce the active condition of the highest HA and be used for preparing influenza virus in enormous quantities later on.

The purifying of virus

Use known purification process, as sucrose density gradient centrifugation, the virion that purifying produces from the first-generation from substratum.For example, after infecting 24 to 48 hours, the centrifugal substratum results virus of the mdck cell of centrifugal influenza infection.The virus precipitation of gained is resuspended in the damping fluid, and centrifugal by the saccharose gradient damping fluid.At the 40-45% of gradient sucrose district results influenza virus band,, pass through 100 centrifugation under the 000xg with the damping fluid dilution.With the virus precipitation of the resuspended purifying of damping fluid ,-70 ℃ of preservations.

The clone of influenza hemagglutinin gene

Fig. 1 has shown the summary of clone HA genetic method.Basically, with the strains of influenza viruses cells infected that will clone.Results virus is isolated viral RNA (to influenza A strain) or mRNA (to influenza B strain) from cell culture medium.Extracting viral RNA from the virion of purifying (RNA), analyzes on the formaldehyde agarose gel with standard program.To influenza A strain virus RNA universal primer system, or to influenza B strain mRNA with the synthetic cDNA of random primer.With to all influenza A and B viral hemagglutinin RNA fragment all be the synthetic positive standard complementary DNA (cDNA) of the general oligonucleotide primer of homologous (5 '-AGCAAAAGCAGG-3 ' (SEQ ID NO.1)) (Davis etc., " contain the structure and the evaluation of the bacterial clone of influenza virus WSN strain hemagglutinin gene (HON1) " gene, 10:205-218 (1980)).Design and influenza hemagglutinin gene 5 ' and 3 ' end conserved regions homologous primer.5 ' and 3 ' primer end all contain undiscovered restriction enzyme site in the hemagglutinin gene.

With suitable influenza A or B primer and influenza cDNA mixing, with Standard PC R program amplification hemagglutinin gene fragment, the double chain DNA fragment of gained contains the sequence of complete encoding mature hemagglutinin.With the total HA gene of polymerase chain reaction (PCR) amplification, be cloned into then in the suitable host bacterium such as E.coli.Measure 5 ' terminal sequence to identify the signal peptide of HA gene, do not contain the HA gene of signal peptide then with pcr amplification.Thereafter with its subclone in the plasmid transfer vector that contains AcNPV polyhedrin promotor.That the transfer vector of gained contains is following 5 '-＞3 ' sequence, polyhedrin promotor from baculovirus autographa california NPV, the ATG translation initiation codon, the baculovirus signal peptide of 61K, the encoding sequence of ripe hemagglutinin, natural hemagglutinin translation stop codon, polyhedrin RNA polyadenylation signal, and flank baculovirus DNA.

The chimeric transferring plasmid DNA of purifying that will contain clone's hemagglutinin gene then mixes with AcNPV wild-type DNA, and with the calcium co-precipitation, the S.frugiperda cell is advanced in transfection.Select recombinant baculovirus according to the plaque form, be further purified with additional several plaque purifying of taking turns.Clone's recombinant baculovirus carries out the hemagglutinin expression screening, and one of them baculovirus hemagglutinin expression vector is selected produces main virus base.

The influenza virus A strain:

From the viral RNA goods of purifying, clone HA gene from the influenza virus A strain.From containing 1,000-2 extracts viral RNA in the influenza A virus particle of the 100-200 microlitre purifying of 000 hemagglutinin unit (HAU).1HAU is the virus quantity (Rosen of energy aggegation 50% erythrocyte in the standard aggegation is analyzed, 1969), handle virion with digestible protein with protein kinase K, use equal-volume phenol and chloroform extracting viral RNA then, in the presence of the tRNA carrier, use ethanol sedimentation.Resuspended viral RNA is removed contaminating dna with the dnase digestion that does not contain the RNA enzyme in damping fluid, repeat then to extract and settling step.As Maniatis, etc., molecular cloning: laboratory manual .pp.86-96 and 366-367 described (cold spring harbor laboratory, cold spring port, New York 1982), with formaldehyde agarose gel analysis viral RNA (vRNA).

The influenza virus B strain:

Clone HA gene in the total messenger RNA(mRNA) (mRNA) that from the cell that is infected by the influenza virus B strain, extracts from the influenza virus B strain.From cells infected, extract total RNA then.Cracking harvested cell in the presence of guanidine thiocyanate, the total RNA of purifying cells is as using PharmaciaBiotech Inc. (Piscataway, RNA NJ) ExtractTest kit.From cell RNA, extract total mRNA with oligomerization (dT)-Mierocrystalline cellulose column spinner, as using Pharmacia Biotech Inc. (Piscataway, mRNA NJ) PurifyingTest kit.

The expression and the processing of reorganization hemagglutinin in the insect cell

Reorganization hemagglutinin antigen is expressed in the S.frugiperda cell that is infected by AcNPV-hemagglutinin carrier high-levelly.Initial gene product is unprocessed, total length hemagglutinin (rHA0), and it is not secreted, just on the periphery film attached to cells infected.Reorganization HA0 is that molecular weight is 68,000 albumen, the glycosylation that tool N-connects, and its glycan is a high mannose type, is different from the glycan that viral protein expression produces in Mammals and birds cell.Evidence show that rHA0 forms tripolymer after translation, focus on the cytoplasmic membrane.

HAO and other proteic expression vectors

HAO is an improved vaccine, because it is better than the stability of HAI/HA2 mixture, and can keep correct folding in the process of purifying and preservation.This advantages of excellent stability shows obviously especially in the Type B virus strain, and this makes its titre exceed about 5 times than the attenuation B virus strain that obtains from commerce.

Described in following embodiment, when with restriction enzyme site pMGS12 is arrived in the HA gene clone, HA is ripe, and signal peptide is removed, and replaces baculovirus chitinase signal peptide, i.e. the 61kD signal peptide.Because the HA gene is connected by cleavage site with the chitinase signal peptide, therefore, 3 to 5 amino acid are arranged between ripe HAO albumen and the 61kD signal peptide according to the difference of selected restriction site.Although no problem to the influenza virus A strain, the HAO of the several amino acid that has interpolation that the B strain is expressed can not correctly fold.

Existing two kinds of methods overcome this problem.First kind is to use another kind of novel vector pMGS3, its 61kD signal peptide of not encoding.The HAO that will have the original signal peptide is cloned on this carrier, expresses then.When identifying, demonstrate better glycosylation and processing when on pMGS12, expressing with the B strain HAO of this vector expression with SDS-PAGE.HAO is finely folding, can change into HA1/HA2 in quantity.Regrettably detect according to the Western trace, its output is not high.Second method utilizes the 61kD signal peptide of pMGS12 to instruct the HA expression of gene of inserting without Restriction Enzyme to increase output.This novel vector comprises 61kD signal peptide and HAO gene, does not contain the amino acid whose sequence of the external insertion of coding, is called as pMGS27.

PMGS27 can be used to clone and express any gene in baculovirus expression system.Goal gene is not to be cloned on the carrier with being connected by restriction enzyme digestion, but is cloned on the carrier by annealing.Reagent instructs clone's system at its PCR-from Clontech and obtains.PMGS27 is designed to and can adds dATP with the T4DNA polysaccharase and handle linearizing pMGS27 in the terminal linearizing of chitinase signal peptide coding region, forms two long strand afterbodys.

With a pair of be designed to produce with T4DNA polysaccharase and dTTP handle after the PCR fragment oligonucleotide with the afterbody complementary strand afterbody of the pMGS27 that handles, with polymerase chain reaction (" PCR ") or reverse transcription-PCR (" RT-PCR ") amplifying target genes.Simple then annealing can be combined into the cyclic plasmid that is suitable for transforming the host with two molecules.Except cut than the conventional limited enzyme-connection method with the HA gene clone to pMGS12 rapider and simple, pMGS27 also has important advantage, and it does not produce the unnecessary amino acid by the restriction enzyme site coding that produces between chitinase signal peptide and the ripe HA albumen.These unnecessary amino acid bring some difficulties sometimes, make signal peptidase can not excise signal peptide, or as in B strain HA, coded albumen can not correctly fold.

The purifying of reorganization HA0

Infect after several days, can be with non-sex change, nonionic washing agent or other well known by persons skilled in the art being used for from the method for insect cell purification of recombinant proteins, include but not limited to affinity or gel chromatography, antibodies, selective extraction rHA0 from the periphery film of the cell that infected by the AcNPV-hemagglutinin.Further use DEAE ion-exchange and LentilLectin affinity chromatography or other correlation method purifying well known by persons skilled in the art dissolve in the rHA0 of washing agent.

In preferred embodiments, use milder and from the influenza virus B strain, obtain the method purifying rHAO of the rHAO of higher output yield.This method is roughly as follows:

With the pH value is 9.5 to 10.5 relative viscid alkaline solution extracting cell, separates the HAO albumen that forms the insect cell membrane inner portion from soluble proteins, peripheral membrane protein and most of DNA and RNA.When containing concentration and be about the sucrose of 250mM, viscosity increases.With the concentration that prevents that effectively proteic disulfide linkage forms in the mixture.Add the disulfide bond reduction agent, as beta-mercaptoethanol with cell suspension in extracting damping fluid, homogenate, centrifugal then.(electric conductivity is generally less than 1mS, contains under pH10.5) in the low ionic strength buffer liquid of disulfide bond reduction agent through the homogenate washing precipitation, and is centrifugal then at alkaline pH.From containing 0.3 to 1.5% washing agent such as Triton, a certain amount ofly can effectively stop the decondensation agent that forms mixture because of coulombic interaction, as (preferred 9.5) under the alkaline pH 0.3 to the 1.0M trimethyl-glycine or PaurineThe precipitation of damping fluid in extract HAO.Use HAO in the cation-exchange chromatography purifying supernatant subsequently with anion-exchange chromatography then.Behind damping fluid balance columns, HAO is crossed anion column, as DEAE or Q-with washing agent that contains about 1/10 concentration and disulfide bond reduction agent

(a kind of sepharose 4B post with quaternary amine group) uses and extract phase damping fluid together, but will dilute at least 1: 2 with other damping fluid.Reduce the pH value then to being about 8.5, wash-out HAO.Use essentially identical damping fluid to cross cationic exchange coloum the HAO of wash-out, reduce pH to about 7.4, wash-out impurity.Increase salt concn to 0.15M NaCl, wash-out HAO.

This preferred purification process will be described in detail below.

Contain the preparation of the film fragment of the HA that recombinates.The cell (the 6.2g cell that comes from the 0.34L culture) of express recombinant HA is suspended in ice-cold 100mM trisodium phosphate with 100mg/mL, 100mM sodium-chlor, 250mM sucrose, 0.1% beta-mercaptoethanol is in the damping fluid of pH10.5.With (Brinkman Instruments Inc., Westbury is NY) 4 grades of smudge cellses 2 minutes for homogenizer.Need with alkaline homogenate medium to increase the purity of proteic solubility of impurity and membrane product.Homogenate under 9.200g centrifugal 30 minutes.Abandon supernatant, collecting precipitation.Subsequently with low ionic strength buffer liquid washing film fraction goods.Deposition is resuspended in the 0.1% ice-cold beta-mercaptoethanol of original volume, 10.5In, use

Homogenizer was 4 grades of homogenate 2 minutes.Homogenate under 9.200g centrifugal 30 minutes.Abandon supernatant, collecting precipitation.The redundance of peripheral membrane protein has been removed in current low ionic strength washing.Gained film fraction goods obvious enrichments reorganization HA, removed the nucleic acid that pollutes.

The extraction of reorganization HA.Under the condition that does not make the antigen sex change, selective extraction reorganization HA from the film precipitation.At the ice-cold 10mM thanomin pH9.5 of 41mL, 1%Triton N101,0.1% beta-mercaptoethanol, 25mM NaCl in the 40mM trimethyl-glycine, precipitates 2 minutes with the Polytron homogenizer at 4 grades of homogenate films.After 40 minutes, centrifugal mixture is 30 minutes under 9.200g 23 ℃ of cultivations.The supernatant decant that will contain the HA that recombinates is used 2 times of dilutions of same buffer then.

With sds polyacrylamide cohesion electrophoretic analysis albumen.Under the condition of 2% sodium laurylsulfonate (SDS) and the existence of 5% beta-mercaptoethanol, in boiling water bath, destroyed sample 10 minutes.With 11% the polyacrylamide gel electrophoresis that contains 0.1%SDS, use Coomassie blue stain then.

Chromatography purification.Simplify the chromatography purification of reorganization HA, use LentilThe affinity chromatography of the costliness of lectin sepharose is replaced by a kind of two chromatography purification processes that go on foot, and that this method can prepare is unmodified, be suitable for the high purity reorganization HA antigen as the influenza vaccines component that is used for the people.Employed chromatography gel matrix is Pharmacia Q-

Fast Flow and CM-SepharoseFast

Anion-exchange chromatography.All chromatographies all are at room temperature to carry out.Divided Pharmacia Q-Sepharose Fast with the extract that contains the HA that recombinates of preparation as mentioned above with 1mL/ Post (containing 5mL in the C10/10 Pharmacia post), post are earlier with 10mM thanomin pH9.5,0.1%

N101,0.01% beta-mercaptoethanol, 25mM NaCl, 400mM trimethyl-glycine balance.Reduce to baseline with level pad washing column to the UV absorption of effluent liquid then.Under this condition, reorganization HA is combined on the post, and impurity partly flows out.Use 30mM diethanolamine pH8.5 then, 0.1%

N101,0.01% beta-mercaptoethanol, 25mM NaCl, the partially purified reorganization of 400mM trimethyl-glycine wash-out HA.

Cation-exchange chromatography.With 30mM diethanolamine pH8.5,0.1% N101,0.01% beta-mercaptoethanol, 10mM NaCl, 400mM trimethyl-glycine twice dilution Q-Sepharose elutriant (23mL).Use 35mL 10mM sodium phosphate pH7.4 then, 0.1%

N101,0.01% beta-mercaptoethanol, 10mM NaCl, 400mM beet alkali cleaning post.This processing makes impurity wash-out from post, and reorganization HA still is combined on the CM agarose.Then through with 10mM sodium phosphate pH7.4,10mM NaCl washes post and absorbs to the UV of effluent liquid and reduce to baseline and remove washing agent.Reorganization HA with phosphate buffered saline buffer pH7.5 (PBS) wash-out purifying.

The rHA0 of purifying is resuspended in the isoosmotic buffered soln.After removing washing agent, the rHA0 of purifying is the aggegation erythrocyte effectively.

Structure and the biological nature of reorganization HA0

RHA0 is purified at least 95% purity, is preferably 99%.To be mainly single molecular weight be 68,000 polypeptide in its migration on the SDS-polyacrylamide gel.Detect the antigenic quaternary structure of reorganization HA0 of purifying with Electron Microscopy, trypsin-resistant, density sedimentation analysis and aggegation red corpuscle ability.These data presentation reorganization HA0 forms tripolymer, is assembled into bow structure.

The rHA0 of purifying can not the aggegation cell before removing washing agent, and this shows that for crosslinked chicken erythrocyte antigen must form mixture (bow structure structure).The quantitation capabilities of the rHA0 aggegation cell of purifying can be used to measure antigenic corresponding denseness.When 1 hemagglutinin unit is defined in and does the standard hemagglutinin and analyze with the chicken erythrocyte, required antigen quantity during aggegation 50%.Comparative data shows that the ability of the rHA0 antigen aggegation erythrocyte of purifying is with observed quite with the susceptible malicious particle of complete stream.

Reorganization HA0 can be cleaved at the disulfide linkage place, causes conformational change, forms two chain HA1 and HA2, as Carr, and C.M. and Kim, P.S., " the jump load mechanism of influenza hemagglutinin conformational change ", Cell73:823-832 (1993) is described, and this literary composition has enrolled in the reference of this paper.The cracking to reorganization HA0 has more detailed description among the embodiment 6 hereinafter.Can be sure of, after natural HA0 is cracked into HA1 and HA2, because the ability of acquisition and cytogamy, and have infectivity, thus the enhanced immunne response produced.The processing of antigen such as influenza hemagglutinin is carried out with main combining of histocompatibility (MHC) molecule through antigen peptide.Antigen/MHC mixture is started immunne response by the T cell recognition, as described in the summary of Harding and Geuze, and CurrentOpinion in Cell Biology 5:596-605 (1993), this literary composition has enrolled in the reference of this paper.Yet as complete molecule, the rHAO that produces in baculovirus is high stability and has immunogenic.Show that to comparing this glycan is different with the HAO that expresses in Mammals or birds cell at the glycan molecule on the HAO of expressed in insect cells.

The preparation of fusion rotein

Lower or when having the advantage that causes multiple antigenic immunne response when the second proteic antigenicity, can prepare the HAO fusion rotein that merges of second antigen protein therewith.A kind of preferred second antigenic example is the neuraminidase that influenza virus produces.This antigen can be by cell, virus or cell protein, or comprises that at least 5 to 8 amino acid whose its antigenic portions form.Other antigens comprise Australia antigen(AA), HIV antigen and carcinomebryonic antigen." immunne response " used herein is meant with the humoral response at antigenic antibody measurement that produces, or discharges the cell response of measuring with what mediate at antigenic T cell of replying.In some cases, can between HA and antigen, insert the amino acid " joint " of no antigen, compare the further antigenic antigenicity that increased with HA.This process comprises a kind of DNA plasmid of structure, is used to use oligonucleotide probe and polymerase chain reaction (PCR) method, merges purpose antigen gene and total length or part influenza virus HA gene.

Modify HA purpose antigen fusion gene it can correctly be expressed at insect cell through removing natural hydrophobic signal peptide sequence and replacing new baculovirus signal peptide.Fusion gene is inserted rhabdovirus expression vector, make baculovirus polyhedrin body promotor instruct fusion rotein transcribing in the insect cell that infects.18 amino acid whose baculovirus signal peptides instruct the translation of HA purpose antigen fusion polypeptide to enter the insect cell glycosylation pathway differ, but it is not present in sophisticated fusion rotein.

For example, plasmid pA9440 comprises the A/Beijing/32/92 strain HA gene that is present among the baculovirus transferring plasmid pMGS 12 as described below, with it as template, use supplier (Gene Amp PCR Cloning Kit, Perkin Elmer Cetus) scheme of Tui Jianing is with polymerase chain reaction (PCR) amplification HA gene.PCR reaction mixture (100 μ l) contains and is designed so that section H A gene annealed 20pmol primer.5 ' and 3 ' primer tip designs restriction endonuclease site of having HA gene inside not have, the segmental 5 ' PCR primer of HA0 and HA1 (O-567) starts from natural HA gene coded sequence 5 ' end downstream 52 base pairs.Deletion natural signals peptide sequence, 5 ' end is right after the HA encoding sequence and inserts a SmaI site.Segmental 5 ' PCR the primer of HA2 (O-651) starts from natural HA gene 11 08 Nucleotide place, is right after behind the codon of coding arginine residues, and when HA0 was cracked into HA1 and HA2, this residue was removed.Segmental 3 ' PCR the primer of HA0 and HA2 (O-680) is designed to add a kpnI site after being right after the HA encoding sequence, removes natural terminator codon.3 ' PCR primer (O-679) brachymemma of HA1 the gene before the arginine residues of removing when abutting against the HA0 cracking.30 circulations are carried out in the amplification of HA gene fragment, comprise 94 ℃ of following sex change 1 minute at every turn.55 ℃ of following primer annealings 2 minutes, and 72 ℃ prolong 2 minutes down.HA gene fragment after the amplification is carried out agarose gel electrophoresis, and (Bio 101, and Inc.) purifying from gel is connected to the plasmid (pCRII that is designed to accept pcr amplified fragment with the GeneClean test kit; Invitrogen) on.Plasmid pB 142, the pB144 and the pB330 that are so contained HA0, HA1 or HA2 gene fragment respectively.

Cut the HA gene fragment with SmaI and KpnI Restriction Enzyme from plasmid pB142, pB144 and pB330, use then the standard recombinant dna technology (Sambrook etc., 1989) subclone is to AcNPV transferring plasmid pMGS12.The pMGS12 plasmid from 5 ' to 3 ' comprise AcNPV polyhedron promotor, an ATG initiator codon, it is 61 that a coding derives from molecular weight, 000 baculovirus glycoprotein (61K) but the sequence of cleavable signal peptide, SmaI and KpnI Restriction Enzyme cloning site, and the general termination codon subsequence of TAA.These regulation domain flanks are the segmental DNA of the genomic EcoRII of AcNPV (Summers and Smith, " the method handbook of baculovirus vector and insect cell culture procedure ", Texas's agricultural experiment centre notification number, 1555 (1987)).With SmaI and KpnI excision PCRIIThe clone's of cloning vector HAPCR fragment with agarose gel electrophoresis and Gene Clean test kit purifying, and is connected on the same pMGS12 with SmaI and KpnI digestion.Gained AcNPV transferring plasmid pB879, pB1201 and pB1205 contain the zone of coding HA0, HA1 or HA2 respectively.These zones link to each other in reading frame with the polyhedron promotor with the baculovirus signal peptide of the cleavable that comes from the 61K gene.AcNPV transferring plasmid pB879, pB1201 and pB1205 can be used to HA0, HA1 or HA2 and any interested gene fusion.

Second step that makes up HA-CEA fusion gene transferring plasmid is that the CEA encoding sequence is inserted in the HA coding structure.When plasmid pA9080PCR increases, add the restriction enzyme identification/excision site of SmaI and KpmI at CEA gene two ends.5 ' PCR primer, O-649 originates in from gene 5 ' end 82 base pair places, has deleted original CEA signal peptide sequence.3 ' PCR primer, O-650 have been designed to delete this gene 3 ' end last 72 base pairs of coding C-stub area hydrophobic sequence.30 circulations are carried out in the amplification of CEA gene fragment, comprise 94 ℃ of following sex change 1 minute at every turn, annealed 2 minutes down for 55 ℃, and 72 ℃ prolong 2 minutes down.The CEA gene fragment of the amplification of gained is carried out agarose gel electrophoresis, with GeneClean method purifying, according to the operation instruction of manufacturer, be connected to pCRII ( Invitrogen) on.The gained plasmid, pB806 comprises the CEA gene, and this gene does not contain original signal peptide, the terminal hydrophobic domain of C-or terminator codon, but all contains SmaI and KpnI site at these gene two ends.

A large amount of preparation plasmid pB806 are with SmaI or KpnI dna digestion.With agarose gel electrophoresis and GeneClean test kit purifying CEA encode fragment, then the purifying fragment is connected in each coding HA construct (pB879, pB1201 or pB1205) of cutting with the same restrictions enzyme.For example, the CEA encode fragment that will have SmaI enzyme simple stage property end is connected with HA2-coding construct (pB879, pB1201 and pB1205) with the HA0-that cuts with the SmaI enzyme, HA1-respectively, obtains plasmid pB1250, pB1555 and pB1584 respectively.The CEA encode fragment that will have KpmI enzyme simple stage property end is connected with HA2 coding construct with the HA0-that cuts with the KpnI enzyme, HA1, obtains plasmid pB1264, pB1564 and pB1593.Insert the CEA gene in the SmaI site, make the CEA encoding sequence be positioned at the downstream of HA encoding sequence.To all constructs, design PCR makes during primer CEAGene and HA insert and read in the frame, and the translation of fusion gene will terminate in the general translation termination signal place (TAATTAATTAA) (sequence number No.4) of downstream, KpnI site pMGS12 carrier sequence.

This construct can improve to improve folding and immunogenicity at the amino acid that interleaves between signal peptide as described below and the HAO or between HAO and the fusion gene by deletion.

The preparation of vaccine and packing

Utilize method and material known to the influenza vaccines those skilled in the art, rHA can be prepared and pack separately or with other influenza antigens.In preferred embodiments, will form a kind of polyvalent vaccine from the HA protein binding of two kinds of A strains and a kind of B strain.

In particularly preferred embodiments, with the amount of effective raising, these HA are combined with a kind of adjuvant the proteic immunne response of HA.At present, the unique adjuvant that is widely used in the people is vanadium (aluminum phosphate or an aluminium hydroxide).Be used to study and the saponin(e of animal doctor's application and its purified components OuilA, the complete left agent of Freund ' s and other left agent are because its toxicity has limited their effective application in human vaccine.But, some also should be useful at the chemically new goods that limit, for example Muramyl dipeptide, monophosphoryl lipid A, Goodman-Snitkoff of comprising as this paper reference etc., the described phosphatide conjugate of Journal of Immunology 147:410-415 (1991), Miller of comprising as this paper reference etc., experimental technique magazine 176:1739-1744 (1992) is described to be contained in the intravital protein capsule of proteolipid, be included in lipid microvesicle such as Novasome ^TMLipid microvesicle (MicroVescular Systems, Inc., Nashua, protein capsule NH).

In preferred embodiments, vaccine is used for (being intramuscular, intracutaneous or subcutaneous) medication of non-enteron aisle or nasopharynx (that is, in the nose) medication immunity with unit dose package.As having determined effective dosage described in the embodiment subsequently.Carrier is generally water or buffer saline, contains or do not contain sanitas.Can be with the antigen freeze-drying, resuspended when taking, or make solution.

Carrier also can use macromolecular sustained release system.When the vaccine of antigen release can be effectively controlled in preparation, synthetic macromolecule was particularly useful.An example early is according to Kreuter, the J. report ( Microcapsules and Nanoparticles in Medicine and Pharmacology, M.Donbrow (Ed) .CRC Press.p.125-148) the methyl methylacrylic acid is aggregated into diameter less than 1 micron bead, be called and receive ball.Antibody response and protection to influenza infection are obviously good than taking the antigen that is combined with aluminium hydroxide.Experiment showed, that with what other particulates were done these high molecular booster action effects are relevant with the size and the hydrophobicity of particulate.

Microcapsule have been used to the injection of the medicine that micro-capsule comprises, and controlled drug is discharged.The high molecular selection of many factor affecting microcapsule.The reproducibility of synthetic macromolecule and micro encapsulation process, the expense of microcapsule raw material and production process, toxicity profile, the kinetics requirement of various releases, and polymer and antigenic physical chemistry consistency all are to need the factor considered.Useful high molecular example have polycarbonate, polyester, polyurethane, PolyorthoestersAnd polymeric amide, particularly those are biodegradable.

That medicine often uses, to be used to antigenic carrier recently be poly-(d, 1-Iactide-co-glycolide) (PLGA).This is a kind of biodegradable polyester, medically is used for carrying out degradability stitching, bone carriage and other provisional reparations for a long time, does not show any toxicity.Many medicines comprise that peptide and antigen prepares in the PLGA microcapsule.Relevant PLGA is used for existing accumulation of data that antigenic controllable release changes, as Eldridge, and J.H., the summary CurrentTopics in Microbiology and Immunology that waits, 1989,146:59-66.When oral, it is in 1 to 10 micron the microballoon of PLGA that antigen is wrapped in diameter, has tangible booster action.The PLGA micro encapsulation is through an invert emulsion separation phase.The purpose mixture is made the aqueous solution.PLGA then is dissolved in appropriate organic solvent such as METHYLENE CHLORIDE and ethyl acetate.These two kinds of immiscible solution are emulsification altogether under high speed rotating.Add this high molecular non-solvent then, make to be wrapped in water droplet polymer precipitation on every side, form embryo peptide shape microcapsule.Collect microcapsule, make it stable, remove solvent through vacuum-drying or extracting with one of class reagent (polyvinyl alcohol (PVA), gelatin, alginate, polyvinylpyrrolidone (PVP), methylcellulose gum).

Can further understanding be arranged to the present invention with reference to following non-restrictive example.

Embodiment 1: breeding of influenza virus and purifying

From the FDA of egg allantoic fluid, obtain following influenza vaccines strain:

A/Beijing/353/89-like(H3N2)

A/Beijing/32/92-like(H3N2)

A/Texas/36/91-like(H1N1)

B/Panama/45/90

The influenza virus original seed that obtains from FDA for breeding, at the trypsin Sigma Chemical Co. that TPCK handles, St Louis, MO) and can obtain under the existence of foetal calf serum of optimal concentration of the maximum titre first-generation virus infection mdck cell.Establishing criteria HA analyzes experiment (Rosen, " with the hemoagglutination of animal virus ", in " virusology basic technology ", editor K.Habel and N.P.Salzman, pp.276-28 (academic press, New York 1969)), infect mdck cell with strains of influenza viruses with low infection multiplicity (0.1 to 0.5).Infected cells was cultivated 48 hours down at 33 ℃, with the viral yield in the hemagglutination reaction activation analysis method measurement substratum.Produce the active condition of the highest HA and be used to prepare a large amount of influenza viruses.The suitableeest TPCK trypsinase and foetal calf serum concentration to above influenza virus are listed in table 1.

The suitableeest TPCK trypsinase of table 1. and foetal calf serum concentration

	A/Beijing/353/89	A/Beijing/32/92	A/Texas/36/91	B/Panama/45/90
					The % foetal calf serum	0.25％	0.25％	0.25％	5.0％
The trypsinase amount that TPCK handles	45μ/ml	45μg/ml	45μ/ml	3μ/ml

The purifying of influenza virus: infect after 24-48 hour, the substratum (1,000 * g, 10 minutes) of the mdck cell through clarifying influenza infection is gathered in the crops virus from the 10T175 tissue culture flasks.Centrifugal 1 hour of 100,000 * g, precipitation virus from substratum.Gained virus precipitation is resuspended in the phosphate buffer soln (PBS) of 1ml pH7.4, and is centrifugal in 20ml 20-60% (w/v) PBS saccharose gradient.At 40-45% saccharose gradient district results influenza virus band, with the PBS dilution, 100,000 * g precipitation.The virus precipitation of purifying is resuspended in 0.5ml PBS, preserves down for-70 ℃.

Embodiment 2: the clone of influenza virus A/Texas/36/91HA gene

Fig. 2 has shown the specific embodiment of clone's step of an influenza virus HA gene.Extract the influenza A/Texas/36/91 viral RNA that obtains from CDC as mentioned above.Use and influenza RNA fragment 3 ' end 5 '-AGCAAAAGCAGG-3 ' (SEQ ID NO.1) complementary universal primer, with the synthetic influenza cDNAs of Moloney murine leukemia virus (M-MuLV) reversed transcriptive enzyme.M-MuLV reversed transcriptive enzyme in the first chain cDNA synthetic agent box that provides with Pharmacia Inc. is a template with the viral RNA or the mRNA (5 μ g) of purifying, synthetic cDNA.Be used for primer from the cDNA of influenza virus A strain virus RNA and be synthetic oligonucleotide primer (5 '-AGCAAAAGCAGG-3 ') (SEQ ID NO.1), it is with all virion HA gene fragment 3 ' terminal homologies.

With polymerase chain reaction (PCR) (Gene Amp kits under the standard reaction condition; Cetus/Perkin Elmer, Norwalk, CT), from cDNA amplification HA gene.PCR reaction mixture (100 μ l) contains the 20pmol primer, according to the consensus sequence of finding from GeneBank DNA data information shown in the table 2 determine this primer to influenza virus A (H3) or A (H1) or influenza B strain HA gene 5 ' and 3 ' end have specificity.30 circulations are carried out in amplification, comprise 94 ℃ of following sex change 1 minute at every turn, 55 ℃ of following primer annealings 2 minutes, and 72 ℃ prolong 2 minutes down.The PCR product carries out 0.8% agarose gel electrophoresis before the clone, to determine its correctly size.

In PCR, use HA gene 5 ' end PCR primer: 5 '-GGG GGT ACC CCCGGG AGC AAA AGC AGG GGA AAA TAA AAA-3 ' (SEQ ID NO.2) and HA gene 3 ' end primer: 5 '-GA AAC GTC ACG TCT TAT ACG/T TAG/TACT CCA TGG CCC-3 ' (SEQ ID NO.3), the HA gene of generation total length.

From a kind of new 5 ' PCR primer of gene 5 ' end design: no signal sequence 5 ' end: 5 '-GGGGGT ACC CCC GGG GAC ACA ATA TGT ATA GGC TAC CAT-3 ' (SEQ ID NO.4) and gene 3 ' hold: 5 '-GA AAC GTC ACG TCT TATACG/T TAG/T ACT CCA TGG CCC-3 ' (SEQ ID NO.5).In PCR, use these primers to produce the HA gene of no signal peptide sequence.Be inserted into then in the TA carrier of cutting with the KpnI enzyme.To be used for the 61K signal peptide of baculovirus expression and the TA carrier that the polyhedrin promotor is inserted the HA gene that contains no influenza virus signal peptide sequence then.Gained baculovirus recombinant vectors contains the polyhedrin promotor, the HA gene of 61K baculovirus signal peptide and influenza virus A/Texas/36/91.

From the mdck cell that is infected by influenza virus B strain B/Panama/45/90, extract total messenger RNA(mRNA) (mRNA), therefrom clone HA gene from the influenza virus B strain.The total RNA of preparation from 5 bottles of T175 culturing bottle infected cells.In the presence of guanidine thiocyanate, the cell of cracking results, the total cell RNA of purifying as mentioned above.As mentioned above, from cell RNA, extract total mRNA with oligomerization (dT) Mierocrystalline cellulose column spinner.

Be used for primer from the mRNA of influenza virus B strain and be at random the oligonucleotide dna primer (Pharmacia, Inc.).

There is a routine cNDA synthetic product to use influenza virus A/Texas/36/91 viral RNA to be template.The segmental position of the proteic cDNA of encoding influenza virus can be determined with following steps.With the viral RNA of purifying and general purpose single chain DNA primer 5 '-AGCAAAAGCAGG-3 ' (SEQ IDNO.1) combines in reaction mixture.As mentioned above, the segmental 3 ' end of this primer and influenza virus particles is complementary.The reaction also comprise adding [α- ³²P] dCTP, go up isolating cDNA product to observe at 1.5% basic hydrolysis gel (Maniatis, et al., 1982), and expose to the X-OMAT-A film.

Embodiment 3: clone HA gene is to bacterial plasmid

With the TA cloning system (Invitrogen, Inc.) with the rHA gene clone of pcr amplification to pUC-class plasmid vector.Through with restricted enzyme cutting analysis through standard method (Maniatis, etc., 1982) plasmid DNA of purifying, confirm the existence of HA gene.Then 5 of rHA gene ' end is carried out dna sequence analysis, design the new primer of having removed coding HA protein N terminal hydrophobic signal peptide sequence.Then with specificity listed in the table 25 ' and 3 ' oligonucleotide primer the cDNA product is carried out pcr amplification, and with the standard cloning process be cloned into E.coli TA plasmid vector (Invitrogen, Inc.) on.The gained dna clone comprises the sequence of encoding mature HA.

With standard method (Maniatis etc., 1982) will be from A/Texas/36/91, A/Beijing/353/89, the rHA gene subclone of A/Beijing/32/92 and B/Panama/45/90 is in rhabdovirus expression vector.With suitable Restriction Enzyme the HA gene is excised from the TA cloned plasmids, the HA dna fragmentation of purifying is inserted in the baculovirus recombinant plasmid.The gained bacterial clone screens with amicillin resistance, uses restriction enzyme digestion then, discharges the HA gene that inserts, and exists to determine it.With the csCl-ethidium bromide gradient method (Maniatis, etc., 1982) purifying contains the recombinant plasmid of HA gene.Plasmid 5 ' end is checked order, to determine existing correct baculovirus signal (AcNPV polyhedrin promotor, ATG translation initiation signal and baculovirus signal peptide sequence) and HA encoding sequence to be positioned at correct reading frame.HA gene 5 ' end dna sequence dna and flank AcNPV polyhedrin promotor and baculovirus signal peptide (preceding 18 amino-acid residues of each aminoacid sequence) are listed in the sequence table.

HA gene 5 ' terminal sequence (sequence scope 1-481) of SEQ ID NO.6 coding A/Beijing/32/92.SEQ ID NO.7 is corresponding aminoacid sequence (from the initiator codon " ATG " [Nucleotide 21] of SEQ ID NO.6).The aminoacid sequence of 61K signal peptide is seen 1-18 the amino acid of SEQ ID NO.7.

HA gene 5 ' terminal sequence (sequence scope 1-481) of SEQ ID NO.8 coding A/Texas/36/91.SEQ ID NO.9 is that the aminoacid sequence of corresponding aminoacid sequence (from the initiator codon " ATG " [Nucleotide 21] of SEQ ID NO.8) 61K signal peptide is seen 1-18 the amino acid of SEQ ID NO.9.

HA gene 5 ' terminal sequence (sequence scope 1-434) of SEQ ID NO.10 coding B/Panama/45/90.SEQ ID NO.11 is corresponding aminoacid sequence (from the initiator codon " ATG " [Nucleotide 21] of SEQ ID NO.10).The aminoacid sequence of 61K signal peptide is seen 1-18 the amino acid of SEQID NO.11.

In SEQ ID Nos 6,8 and 10, Nucleotide 1-20 is polyhedrin promotor 3 ' end, Nucleotide 21-74 coding 61K signal peptide, 5 ' end of coding HA gene after the Nucleotide 75.

Embodiment 4: the expression of reorganization HA in insect cell

Purifying contains the chimeric recombinant plasmid of clone HA gene, gets 2 μ g and mixes with 1 μ g AcNPV wild-type DNA.With NDA and calcium co-precipitation, advance the S.frugiperda cell with standard method (Smith, Summers and Fraser, molecule and cytobiology 3:2156-2165 (1983)) transfection.Differentiate recombinant baculovirus by the plaque form, and then be further purified with plaque method of purification several times.The recombinant baculovirus through the plaque purifying of rHA is expressed in screening, selects a rhabdovirus expression vector, further tests.

With the baculovirus vector infection S.frugiperda cell that contains from the HA gene of strains of influenza viruses B/Panama/45/90.Infect after 24,48 and 72 hours, with 25 μ Ci[ ³²S] methionine(Met) pulse 1 * 10 ⁶Cell, mark synthetic albumen.Collecting cell is having protein isolate on the 11% polyacrylamide glue gel of 0.1%SDS.On the X-OMAT-AR film, expose, detect by radiolabeled albumen.Protein standard position and size thereof (kilodalton kd) show that after infecting 48 and 72 hours, 85kd reorganization HA albumen is one of cell synthetic major protein.

Embodiment 5: preparation and the purifying of reorganization HA

Rhabdovirus expression vector A8611 contains the gene of influenza virus A/Beijing/353/89, and its preparation is similar to the preparation of A/Beijing/32/92 hemagglutinin under the control of polyhedrin promotor as mentioned above substantially.Infect the S.frugiperda cell with it.(Gibco BRL, Gaithersburg MD), are 1 * 10 in 27 ℃ of culturing cells to concentration in being added with the TNMFH substratum of 10% foetal calf serum ⁶Cell/mL uses the A8611 recombinate shape virus infection, and infection multiplicity (MOI) is 1.Between period of infection,, produce influenza virus A/Beijing/353/89 hemagglutinin transcribing under the control of baculovirus polyhedrin body protein promotor.Infect after 72 hours, 3, centrifugal 15 minutes of 400xg, harvested cell, resuspended cleaning in the TNMFH of serum-free substratum, then 10, under the 400xg centrifugal 30 minutes.Abandon supernatant, preserve the cell precipitation that infects in-70 ℃.

Formed a kind of under the condition that does not make the antigen sex change, the method for selective extraction reorganization HA from cells infected.Unless indicate, all leaching process all carry out at 4 ℃.The cell precipitation (about 5 * 10 that will from the 0.5L nutrient solution, obtain ⁸Cell) at the ice-cold 30mM Tris-HCl of 40mL, pH8.4,25mM LiCl, 1% (V/V) Tween-20 in the 1mg/mL leupeptin, uses Polytron ^TM(Brinkmann Instruments Inc.Wesbury's homogenizer NY) broke 2 minutes.Homogenate is 9, under the 200xg centrifugal 30 minutes.Abandon supernatant, collecting precipitation.Further from insect cell, removed solubility and film peripheral protein, and kept integral protein as rHA.For extracting rHA, will be deposited in the ice-cold 30mMTris of 40mL, the 10mM thanomin, pH11,25mM LiCl, among the 2%Tween-20 with 4 grades of homogenate 2 minutes.Behind the ice bath 60 minutes, mix well slurries pH to 8.4 with 1N HCl, 9, centrifugal 30 minutes of 200xg removes insoluble substance.Decant contains the supernatant liquor of solubility rHA, surveys pH, if desired, at room temperature transfers pH to 8.4.Resuspended insolubles is analyzed in 40mL water.The HA integral protein at high pH, contain under the condition of Tween-20 washing agent and dissolve, when pH reduces, keep dissolved state.

Albumen is through the sds polyacrylamide gel electrophoresis analysis.In the presence of 2% sodium laurylsulfonate (SDS) and 5% beta-mercaptoethanol,, containing electrophoresis on 11% polyacrylamide gel of 0.1%SDS, use Coomassie blue stain more then with break sample 10 minutes of boiling water bath.

Form a kind of chromatography purification method, be used for purification of Recombinant HA, can obtain high-purity, unmodified, as to be suitable for the influenza vaccines component that uses as people reorganization HA antigen.HA in order to following method purifying A/Beijin/353/89 from the S.frugiperda cell that is infected by recombinant virus A8611.Being used for from the chromatography gel medium of the infected S.frugiperda cell of 0.5L purifying HA is 30mL Pharmacia DEAE Sepharose FastFlow (in Pharmacia C16/20 post) and 4mL Pharmacia LentilLectin sepharose 4B (in Pharmacia C10/10 post).The spout of DEAE post with LentilThe influx of lectin post links to each other, and S/N 2 cell extracts of preparation are as mentioned above crossed post, and flow velocity is 1mL/ minute.Use 30mM Tris-HCl, pH8.4,25mM LiCl, the 0.5%Tween-20 wash-out, extremely LentilThe UV of 280nm place of lectin post effluent liquid absorbs and drops to baseline.With this understanding, most of impurity albumen combines with DEAE, and reorganization HA flows through pillar.Remaining impurities is through the lectin post, and glycosylated rHA is incorporated into LentilOn the lectin affinity medium.The DEAE post of dismantling, with other 40mL 30mM Tris-HCl, pH8.4,25mM LiCl, 0.5%Tween-20 wash the lectin post.Use 40mL 30mM Tris-HCl then, pH8.4,25mM LiCl, 0.4% (V/V) Sodium desoxycholate (DOC) is washed post.This step substitutes the Tween-20 washing agent with another kind of washing agent such as DOC, and DOC can remove from albumen by dialysis.Then with about 20mL is to 40mLContain 0.3M A-D-The 30mM Tris-HCl of methyl mannoside, pH8.4,25mMLiCl, 0.4% (V/V) Sodium desoxycholate wash-out reorganization HA.Elutriant is analyzed with 11%PAGE.

Because the proteic genetic diversity of influenza virus HA, to each special reorganization HA albumen, the details of above purification process can change.For example, rHA may be combined on the DEAE ion exchange column, and does not flow out.If this thing happens, should wash post with the damping fluid of the LiCl that contains greater concn, NaCl or other salt, separate rHA from the DEAE post.

In order to remove DOC washing agent and other damping fluid components, the elutriant of lectin post that contains the rHA of purifying is dialysed in phosphate-buffered saline pH7.5 (PBS).Analyze on sds page, the purity of the reorganization HA of purifying is at least 95%.

Embodiment 6:rHA protease resistant is analyzed

Ripe HA is assembled into the tripolymer structure, and the anti-multiple monomeric proteolytic enzyme of HA of degrading comprises trypsin Murphy and Webster, 1990).Therefore tryptic resistance be can be used to carry out active tripolymer and form analysis.Following method is used to study the resistance of rHA to protease treatment.

With two parts at 30mM Tris-HCl, pH8.4,60 μ g/mL rHA (A/Beijing/353/89) of the purifying among the 150mM NaCl cultivate 30 minutes on ice containing and do not contain to place under the tryptic condition that 50 μ g/mL TPCK handle.Aqueous isopropanol to the final concentration that adds the 57.4mM phenylmethylsulfonyl fluoride is that 1mM comes termination reaction.Under the 3%SDS reductive condition, boiling deactivation each duplicate samples, electrophoresis on 11.5% polyacrylamide gel, and be transferred on the nitrocellulose filter with standard Western blotting.Use anti-HA serum of cavy and goat-anti cavy IgG alkaline phosphatase enzyme conjugates detection HA polypeptide at the rHA preparation of purifying.

Untreated rHA is with the size migration of HA precursor (HA0).Two main bands appear in protease treatment, move with the influenza virus lectin HA1 of prediction and the size of HA2.The result shows that trypsinase cracking rHA albumen once produces two polypeptide that are predicted as HA1 and HA2 size.There is not further protein cleavage processing to take place.These result's proofs are in order to the HA protease inhibitor degraded of last method purifying.This characteristic exists with trimeric form with the rHA of purifying and conforms to.

Embodiment 7: use the standard mouse TireAnalyze the rHA immunogenicity

A kind of method of measuring antigen immune originality is to measure to cause the amount (mouse that the detectable antibody response of mouse is required TireAnalyze).Use the standard mouse TireThe immunogenicity of analysis to measure rHA0 vaccine.With the rHA that contains serial dilution, i.e. 0.500 μ g, 0.1 μ g, the vaccine of the rHA of 0.02 μ g and 0.004 μ g purifying to several groups of mouse immunes once, every group contains 5-10 mouse.Immunity was collected serum after 28 days, used standard in the trace dish of 96 holes Enzyme linked immunological Solid-phase Assay (ELISA)Measure the antigenic antibody of anti-rHA.If the OD450 of 1: 100 diluent of 28 days antiserum(antisera)s greater than mouse immune before three times of standard deviations of serum OD450 mean value, then mouse has seroconversion.Cause 50% mice serum to change required vaccine effective dose (ED ₅₀) be a measurement index of antigen immune originality.

For example, four groups of mouse, every group contains 10 mouse, and with 0.1 μ g, 0.02 μ g, the rHA0 vaccine immunity is once for 0.004 μ g or 0.0008 μ g (5 times of dilutions).Immunity was collected serum after 28 days, to each the rHA0 antigen in the vaccine, measured seroconversion with ELISA.Calculate 50% mice serum and change required dosage (ED ₅₀), set up the antigenic minimum ED of each rHA0 ₅₀

Initial data show that single dose 0.004 μ g rHA0 can make at least 50% mice serum change.

Embodiment 8: with adjuvant bonded rHA take and with the comparisons of existing influenza vaccines

Measure from the mouse of the rHA of influenza virus A/Beijing/353/89 purifying with aluminium or without aluminium (purified) and to tire, and with the commercial stream influenza vaccine that contains influenza virus A/Beijign/353/89 strain, (Connaught Laboratories, Inc.Swiftwater, PA) relatively.Vaccine is with 0.5 μ g, 0.1 μ g, and the dosage of 0.02 μ g and 0.004 μ g is taken.In the time of 28 days, use the rHA dosage booster immunization mouse of purifying as mentioned above.Collect serum in the time of 42 days, measure the titre of the anti-HA antibody of IgG with ELISA.

The results are shown in Fig. 3.During no adjuvant, have only the dosage of 0.5 μ g to produce tangible antibody titers (200,000).When adjuvant was arranged, the low dose of 0.004 μ g rHA0 just can produce tangible antibody.With the animal generation of rHA (purified) immunity and the anti-HA antibody of the roughly the same level of commercial vaccine.Aluminium has increased the immunogenicity of rHA, and the anti-HA antibody titers that is produced is without 10 times of adjuvant or higher.

In a word, with the immunogenicity of the rHA0 of purifying and a kind of influenza complete virion vaccine ( Connaught Laboratories, Inc., Swiftwater PA) compares, and proves that rHA0 made mouse produce similar immunne response during 42 days.Detect with method described in the embodiment 7, the auxiliary rHA0 of aluminium absorbs and can obviously increase the rHA0 of purifying to immunogenicity in mice.Make the IgG hemagglutinin antibody be higher than with combining of aluminium

Influenza vaccines.

Embodiment 9: blood clotting suppresses research

Blood clotting suppresses (HAI) antibodies three in four known epi-positions of hemagglutinin, stop influenza virus aggegation erythrocyte ability (Wilson etc., " The structure of influenza virus hemagglutinin membrane glycoprotein under the resolving power " Nature, 289:366-378 (1981)).These antigenic determinants are gathered in around the sialic acid receptor binding site of hemagglutinin trisome.At the antibody in these sites can neutralize virus infectivity (Weis etc., " with the structure of its acceptor sialic acid compound influenza virus hemagglutinin ", Nature, 333:426-431 (1988)).The titre of HAI antibody and specificity are the important measurement index of influenza virus vaccine at the protectiveness of the susceptible virus strain infection of class Sihe related streams.

The research of in mouse, carrying out compared purifying from the rHA0 of A/Beijing/353/89 with

(Connaught Laboratories, Inc, Swiftwater, the ability that causes the generation of HAI antibody PA).0 and 28 day to several groups of mouse, 5 every group, inject 0.5 μ g, 0.1 μ g, 0.02 μ g or 0.004 μ g rHA0 or triplication

Hemagglutinin is so that use the reorganization or the virus of A/Beijing/353/89 hemagglutinin of par.For example, with 1.5 μ g

Hemagglutinin (from 0.5 μ g hemagglutinin of each strain) and 0.5 μ g rHA0 are to the mouse immune of high dose group. In cause producing cross-reacting antibody from other the antigenic existence of other hemagglutinin of two kinds of strains of influenza viruses.

With the antihemagglutinin antibody (hemagglutinin IgG) of standard dilution ELISA metering needle to the rHA0 of purifying.Following antigen to 4 hemagglutinin units is measured HAI antibody: complete influenza A/Beijing/353/89 virus (A/Bei), the rHA0 A/Beijing/353/89 antigen of purifying and

The HAI titre is meant the inverse that can suppress the sero-fast high dilution of chicken erythrocyte 50% agglutinative.

Mice serum hemagglutinin IgG and HAI titre when table 3 has been summed up 42 days.With reorganization rHA0 production of vaccine high-caliber antihemagglutinin antibody.Contrast

Titre high approximately 10 times.The most meaningfully the rHA0 vaccine can produce the antibody titers that can better suppress the hemaglutination that A/Beijing/353/89 virus and rHA0 antigen causes.Therefore, the HAI antibody that the rHA0 vaccine produces can be discerned immunogen and influenza A/Beijing virus equally preferably.Right

Low HAI titre may be because antiserum(antisera) can not suppress The agglutination of other two strain hemagglutinins in the vaccine.In contrast, when only measuring at himself,

Mice immunized produces high HAI antibody.Influenza A/Beijing/353/89 virus and the antigenic HAI titre of rHA0 are obviously reduced.In the mouse of low dose group, found same result.

Table 3. couple rHA0 and

The HAI titre

These data also show

In influenza A/Beijing/353/89 virus strain exist hereditary difference with egg, passing between the identical strains of influenza viruses of monobasic before the HAI that obtains from FDA analyzes.The antibody that is produced with reorganization HA0 reaction from influenza A/Beijing/353/89 clone suppresses this influenzae strain virus and himself agglutination to erythrocyte, this fact proved in clone's process, and the antigenic sialic acid receptor binding site of rHA0 that hereditary change influences purifying does not take place.

Embodiment 10; Prescription of a kind of 1993/1994 influenza vaccines and clinical effectiveness thereof

Carried out a series of human clinical trials and identified a kind of security and the immunogenicity of experimental influenza vaccines in human body that contains the HA that recombinates, and collect relevant this kind vaccine in influenza season the raw data to the protection effect of natural infection.The result proves; compare according to the Gripovax that method described herein vaccine of producing that contains recombinant influenza hemagglutinin (rHA0) and the standard that obtains from commerce are produced egg; cause local untoward reaction still less astoundingly, and produce suitable or better immunoprotection reaction.

Material and method

Vaccine.The reorganization HA vaccine that uses in this research contains the HA that is not cut (HA0) glycoprotein from the total length of influenza A/Beijing/32/92 (H3N2) virus.Reorganization HA0 (rHA0) produces in Lepidopteran (insect) cell culture, inserts segmental baculovirus vector with the cDNA that contains coding HA gene subsequently and contacts.Through electrophoretic a large amount of antigens on the sodium laurylsulfonate polyacrylamide gel being determined under the non-sex change condition purifying expressing protein extremely＞95% with the quantitative scanning photodensitometry.With amino acid analysis, N-end sequencing with utilize Western engram analysis that anti influenza A/Beijing/32/92 serum carries out to confirm the evaluation of peptide.The rHA0 vaccine comprises the synthetic HA antigen of specified quantitative, is dissolved in the phosphate buffered saline buffer or with the gel suspensions being adsorbed on aluminum phosphate (vanadium) adjuvant.What the trivalent subviral particle vaccine of getting permission that uses in this research contained 15 μ g/ agent (is produced in the FLUZONE of egg from influenza A/Texas/36/91 (H1N1), A/Beijing/32/92 (H3N2) and B/Panama/45/90 virus respectively ^TMGripovax, Connaught Laboratories, Swiftwater, a kind of HA PA).

Clinical study.The Study on Identification method has obtained the approval of Institutional Review Boards of SaintLouis University and University of Rochester.18 to 45 years old normal adults is recruited in two institutes.The testee is accepted one of following five kinds of vaccine preparations by random assignment with double-blind method: (1) 15 μ g rHA0; (2) μ g rHA0 adds aluminium; (3) 90 μ g rHA0; (4) the trivalent inactivated influenza vaccine of getting permission; Or (5) salt solution placebo.Intramuscular injection 0.5ml vaccine.For the security of three kinds of vaccine preparations tentatively determining to contain rHA0, preceding 25 vaccinated experimenters are independent of other experimenter's random packet (being every 5 people of study group), and pay close attention to by telephone contact in back 48 hours in inoculation.Carry out remaining inoculation then.After inoculation preceding 6 days, all experimenters were proposed the report every day card of filling in untoward reaction, comprise part and systemic symptom.Symptom by own by situation be divided into slight, moderate and seriously what.To the experimenter who feels to generate heat, measure and write down its mouth temp.If find that there are local enlargement or erythema in the injection site, be greater than or less than 1/4th I.P.M levels by its diameter respectively.All inoculations are all carried out in November, 1993 in last week and first week of December.After when inoculation, 3 weeks of inoculation and 1994 can three the end of month or April influenza virus no longer in local crowd popular after during at least 2 to 3 weeks, get serum sample.The volunteer of each institute is proposed when influenza pandemic has the influenza similar conditions in season in the winter time and the research centre is got in touch.The influenza similar conditions is meant and continues more than two days or two days that respiratory symptom is arranged, and is attended by heating and/or systemic symptoms such as myalgia or cold.Report that the experimenter that the influenza similar conditions is arranged is got the nasopharynx cotton and wipes away sample, carry out virus culture and evaluation.Clinical sample is numbered, and upsets processing.

Serology.To the serological test of each type, in same independent laboratory, carry out from the sample of two institutes is synthetic a collection of.Remove nonspecific inhibitor with receptor destroying enzyme, and 4 ℃ remove cold agglutinin with hemocyte absorption after by the test of standard droplet degree, measure that the blood clotting at influenza A/Beijing/32/93 (H3N2) virus antigen suppresses (HAI) antibody in the serum.Titre is defined as the required highest serum diluent of hemagglutination that stops 4 virus antigen units to cause fully, and wherein initial dilution is 1: 4.Measure serum HA specific immunoglobulin G (IgG) antibody with enzyme linked immunosorbent assay (ELISA), be used for purifying rHA0 from influenza A/Beijing/32/92 (H3N2) as envelope antigen.Thoughtful interior reagent comprises the rHA0 antigen of (I) purifying successively outside solid phase; (2) serum sample; (3) alkaline phosphatase link coupled goat anti-human igg; (4) right-nitrophenyl Di-Sodium Phosphate substrate.The ELISA titre is being that high dilution when not containing two times of antigenic corresponding reference cell is represented at least when the optical density(OD) that contains the antigen groove.Use former Treanor, J.J. and Betts, the microneutralization detection method that R.F. (transmissible disease magazine 168:455-459 (1993)) describes is measured neutralizing antibody.Briefly, with the serial dilutions of heat-inactivated serum and about 100TCID ₅₀Influenza A/Beijing/32/92 (H3N2) virus is mixed, and cultivates 1 hour at 37 ℃.Under the room temperature, virus-serum mixture is adsorbed in last 1 hour of converging on the 96 hole flat boards of individual layer Madin-Darby dog kidney (MDCK) cell.Washing is dull and stereotyped, to remove residual inoculum, adds again and contains the tryptic serum-free Dulbecco ' of 2 μ g/ml s MEM, at 5%CO ₂In 33 ℃ cultivated 72 hours down.Use the methyl alcohol fixed cell then, be specific to influenza A virus matrix and nucleoprotein (Center for Disease Control, Atlanta, the anti-mouse IgG evaluation of mouse monoclonal antibody GA) and alkaline phosphatase link coupled subsequently virus replication with one group.The whole titre of serum be defined as with non-in compare with reference cell, cause being higher than 50% signal reduces high dilution.

Virusology.Carry out the virus culture that the nasopharynx cotton is wiped away sample in each institute with standard technique.Sample is inoculated in MDCK or the RhMK and cultivated 14 days in 33 ℃.With 0.4% guinea-pig red blood cell monolayer cell is carried out ha test.Adsorb in the positive culture at hemocyte by HAI with H3-specific antisera (viral control center) and to identify influenza virus.

Statistical analysis.Inverse to the titre of HAI, ELISA, IgG and neutralizing antibody is taken the logarithm.Carry out statistical analysis.The significant reaction of inoculating is defined as the preceding antibody titers to inoculation back 3 week serum sample of inoculation has raise 4 times or higher.The experimental evidence of influenza A (H3N2) virus infection is defined as from cavum nasopharyngeum secretory product isolates virus, and/or in the sample of corresponding influenza after season of sample that collect (influenza is before season) December after 3 weeks of inoculation and collection next spring, serum HAI antibody titers has 4 times or higher increase.With the difference between the accurate analysis of experiments vaccine group of Fisher ' s, relatively produce untoward reaction, significant antibody response or the influenza disease of laboratory confirmation or experimenter's ratio of infection, and carry out variance (ANOVA) analysis, inoculating back Iog ₂The mean value of the inverse of antibody titers compares.Be suitable for carrying out multiple possible comparison The improved Bonferroni ' of Shi Jinhang s inequality and Tukey-Kramer test.

The result

Reactionogenicity.The HA0 vaccine that uses in this research is safe and can tolerates.The frequency that produces untoward reaction is not increased to 90 μ g with the rHA0 antigen dose from 15 μ g and changes, but slightly increases with the adding of aluminium.It was reported that in the experimenter who takes the subvirus vaccine of getting permission, the injection site frequency of local erythema, pain and sensitivity occurs obviously than experimenter's height of taking 15 μ g or 90 μ g rHA0 salts solutions.Except a people is occurring medium serious pain, sensitivity and the sclerosis with arm behind the vaccine immunity of getting permission, other symptoms all are gentle, generally continue 1-2 days.Local erythema and/or its scope of hardening of occurring all are no more than 1/4th inches.

Immunogenicity.In 127 experimenters that recruit, 64 (50%) was less than or equal to 1: 8 at the baseline titre of the serum HAI antibody titers of influenza A/Beijing/32/92 (H3N2) virus.In each group of four vaccine group, most subjects has HA specific serum reaction (table 4) through HAI and ELISA measurement.Among all vaccine experimenters after the inoculation of serum HAI antibody titre all more than or equal to 1: 32, except having inoculated two of 15 μ g rHA0 and inoculated the people exception of the vaccine of getting permission.Inoculation is same relevant with the generation of neutralizing antibody in most of volunteer.Compare with antibody titers mean value height behind the vaccine immunity of getting permission with 15 μ g rHA0 immunity, and the seroconversion rate has slight reduction, though these differences are not remarkable statistically.Antibody response to rHA0 does not strengthen with the adding of aluminium.Average HAI that produces after experimenter's immunity with 90 μ g rHA0 immunity and ELISA IgG antibody titers are than high 2 to 5 times in any one of other three vaccine group when serum HAI titre (relatively its difference be significant statistically).

The protection effect.During monitoring, have 26 people and reported 28 similar diseases of influenza altogether.Wherein 4 people (wherein taken placebo for three, another with 15 μ g rHA0 immunity) isolate influenza A (H3N2) virus from cavum nasopharyngeum sample cultivation thing.In the case of four routine cultures proof, have three examples before season and after season the HAI antibody titers in the serum sample at influenza A/Beijing/32/92 be significantly increased, but other reported that the individuality of disease was not like this.The conclusive evidence of appearance experiment subsequently has the experimenter who only accepts rHA0 of influenza illness positive at the culture that immunity obtained after 31 days, and seroconversion is arranged, and becomes 1: 32 that inoculates back (influenza is before season) from inoculating preceding HAI titre less than 1: 4.In addition two volunteers that take the vaccine immunity that placebo and usefulness gets permission have the serological evidence that has infected influenza A (H3N2) virus in influenza pandemic during season, but do not have clinical symptom.Compare as one group with experimenter's (or all accept the experimenter of any rHA0 vaccine) of all immunizations, take influenza A (H3N2) disease that has the experiment conclusive evidence among the experimenter of placebo (p＜.05) or infected (ratio of p＜.005) is obviously bigger.

Above-mentioned discovery shows, and is better by the tolerance of the antigenic influenza vaccines of rHA0 that contain purifying of the described method preparation of the patent application of above qualification, and can cause human experimenter's protective immunological reaction.Even when 90 μ g dosage, the reactionogenicity of the rHA0 that estimates in this research is unlike salt solution placebo height, and by the local untoward reaction that it causes obviously be less than get permission contain half (i.e. 45 μ g) the antigenic trivalent subvirus of HA vaccine always.

With the reaction of the neutralization of 15 μ g rHA0 preparations, HA specific antibody with cause by the subvirus vaccine quite, and when rHA0 dosage is increased to 90 μ g, be significantly improved.

The total infection and the pathogenicity rate that produce owing to contact influenza A (H3N2) viral circulation epidemic strain naturally obviously reduce than the experimenter who takes placebo in the experimenter of inoculation.These data presentation, protective immunity that causes and the vaccine that uses at present be quite or better during particularly with the medication of high dosage for rHA0.

Embodiment 11: the method for the HAO cloning vector that preparation improves

The cloning vector of the ripe HA of expression of an improvement of design, the gene of the HA that wherein encodes is next to the downstream of the sequence of chitinase encoding signal peptide.

Make the linear pMGS27 of tool strand afterbody

In plasmid pMGS27, HA clones Smal or the Kpnl site that into is right after chitinase signal peptide downstream.Nucleic acid and aminoacid sequence are listed in SEQ ID NO.22 and SEQ ID NO.23 respectively:

5 '-chitinase signal peptide Smal Kpnl

TGG TTG GTC GCC GTT TCT AAC GCG ATT CCC GGG GGT ACCTRP LEU VAL ALA VAL SER ASN ALA ILE PRO GLY GLY THR

In this zone the mutagenesis that oligonucleotide instructs taking place, generates pMGS27 (the base underscore of sudden change is represented) (SEQ ID NO.24):

5′TGG TT A GTC GCC GT G TC CTGCAGGCCAGAGAGGCCTT GGTACC Pstl

Cut plasmid pMGS27 linearizing (listing the 6-35 base of SEQ ID NO.24) with the PstI enzyme:

A GTC GCC GTG TCC TGCA 5′GGCCAGAGAGGCC T

T CAG CGG CAC AGG5′ ACGTCCGGTCTCTCCGG A

Handle linear pMGS27 with T4DNA polysaccharase and dATP then, produce strand afterbody as follows (the 23-36 base of SEQ ID NO.24 and the complementary sequence of 6-18 base):

A 5′GGCCAGAGAGGCC T

T CAG CGG CAC AGG 5′ A

With purpose HA gene clone in pMGS27.

Step 1 synthetic pcr primer thing.

Upstream oligonucleotide (SEQ ID NO.25):

5 ' GTC GCC GTG TCC AAC GCG (5 of ripe HA ' 20 bases of end)

Reverse oligonucleotide (complementary sequence of SEQ ID NO.26):

(3 of ripe HA ' 20 bases of end) ATT AAC CGG TCT CTC CGG 5 '

The PCR of HA gene

Purpose HA gene and two oligonucleotides carry out PCR, obtain (SEQ ID NO.25 and SEQ ID NO.26):

5 ' GTC GCC GTG TCC AAC GCG (ripe HA)

CAG CGG CAC AGG TTG CGC (ripe HA)

TAA TTGGCCAGAGAGGCC 3′

ATT AACCGGTCTCTCCGG

With purpose HA gene and pMGS27 annealing, Transformed E .coli

Mixed linear pMGS27 and the HA gene PCR fragment of handling with the T4DNA polysaccharase.The annealing of two molecules forms a circular plasmids, can be used for Transformed E .coli.Comprise SEQ ID NO.25 and SEQ ID NO.26 among the figure, the 23-36 of SEQ ID NO.24 and 6-18 base.

GTCGCCGTGTCCAACGCG (ripe HA) TAATT

TTGCGC (ripe HA) ATTAACCGGTCTCTCCGG

+

A GGCCAGAGAGGCCT

TCAGCGGCACAGG A

Arrive

The chitinase signal peptide stops

GTCGCCGTGTCCAACGCG(ripe HA) TAATTGGCCAGAGAGGCCT

As mentioned above, between signal peptide and ripe HA, there is not unnecessary amino acid.

Embodiment 12: the preparation and the effect thereof of trivalent type A and B 1995-1996 influenza virus vaccine.

Influenza virus vaccine, the reorganization hemagglutinin of purifying, trivalent chromosome, type A and B (A/Texas/36/92 1 (H1N1), A/Johanesburg/33/94 (H3N2), and B/Harbin/7/94) be the non-infectious subunit that the recombinant influenza hemagglutinin antigen (HA) from purifying obtains.The HA gene is cloned from the influenza A and the B virus strain of aforesaid Center for Disease Control/food and the recommendation of medicine office, and determines each cloned genes with the dna sequencing analysis.Use comprises from strains of influenza viruses A/Texas/36/91 (H1N1), and A/Johanesburg/33/94 (H3N2), the rhabdovirus expression vector of B/Harbin/4/94 clone's HA gene produce reorganization HA antigen in the insect cell of cultivating.The HA albumen of reorganization is the hemagglutinin that is not cut (rHA0) of total length, and molecular weight is about 69,000.Produce rHAO in the Spodopterafrugiperda that in serum free medium, keeps (Lepidopteran) clone.Trivalent vaccine contains (purity is greater than 95%, probably greater than the 99%) rHAO from the purifying of two kinds of influenza A strain and a kind of B strain, mixes by same ratio.This vaccine as be dissolved in phosphate buffer soln, the A and the Type B rHAO albumen of the purifying of adding preservative agent are not used for clinical.

Use the experimentation on animals of unit price, divalence and trivalent rHAO vaccine to show that they do not have tangible toxicity.Not having in vaccine can detected poisonous or objectionable impurities.Generally Recognized as safe and immunogenicity to A/Beijing/32/92 and A/Texas/36/91rHAO in mouse and cavy are studied.Do not find untoward reaction.In mouse, inducing high-caliber anti-HA IgG antibody, hemagglutinin to suppress (HAI) antibody and neutralizing antibody in 2 to 3 weeks with the antigenic single immunization of 15 microgram rHAO that does not add adjuvant.

In a research, with immune 10 the every group mouse group of rHAO (rHAO-SF) that originates in the rHAO A/Beijing/32/92 (H3N2) in the cell that adapts to the substratum that contains 10% foetal calf serum or originate in the rHAO of the insect cell that in the substratum that contains 10% foetal calf serum, adapts to or originate in the insect cell that adapts to serum free medium of 15 microgram purifying.After 2 to 3 weeks of injection, mouse is got blood prepare serum sample.Measure the anti-HA IgG and the HAI antibody of each serum sample.RHAO produces identical anti-HA and HAI antibody titers with rHAO-SF antigen.Single immunization is after 2 weeks, and most of mouse produces tangible HAI antibody titers, and the HAI titre of 8/10 mouse is 32 or higher in every group of the 3rd week.These and other biochemistry and immunology studies have shown that the rHAO that originates in the serum-free insect cell culture with in that to contain the rHAO that produces under the fermentation condition of serum as broad as long.

Carried out a research, to 1994-1995 prescription and a kind of purified virus SAV of getting permission of trivalent rHAO influenza vaccines, (a kind of attenuated influenza virus vaccine of producing of cultivating in egg) contrasts.Every kind of per 0.5 microlitre of vaccine contains from A/Texas/36/91 (H1N1), the 15 microgram rHAO or the viral HA of A/Shangdong/9/93 (H3N2) and B/Panama/45/90 strains of influenza viruses.All the reorganization rHAO and Vaccine all is formulated in phosphate buffered saline (PBS).0,2 and 3 weeks after inoculation are that one group array is taken a blood sample and prepared serum with ten mouse.Anti--HA IgG the antibody that just suppresses the Antibody of Influenza of anti-egg-growth, and the just anti-neutralizing antibody test sera sample that comes from the egg-growth influenza virus of each virus strain.

As Fig. 4 a, shown in 4b and the 4c, the influenza vaccines of reorganization rHAO and approval are induced each the serum IgG antibody of hemagglutinin of high-caliber, anti-three kinds of influenza strains that come from vaccine.Compare rHAO is vaccine-induced 4 to 10 times of high IgG antibody titerss with the vaccine of approval.As shown in Figure 5, also prepared the antibody that suppresses the chicken red blood cell hemagglutinin with influenza virus A and B strain virus.Compare with the vaccine of approval, in the mouse of having accepted trivalent rHAO vaccine, at three kinds of influenza virus strains each, the HAI titre is that be equal to or higher.In the test of standard microtitration virus, record this vaccine and also induce the high-caliber anti-special influenza A and the neutralizing antibody of B virus stain.With the mouse and the purified virus SAV of using approval of rHAO immunization,

The mouse of immunization is compared, about 2 times high of its how much NATs.These results show, aspect the serum antibody of trivalent preparation all three kinds of strains in inducing function HAI and the anti-vaccine of neutralization of the rHAO vaccine of 1994-1995 type A and B influenza strain, with the subunit influenza vaccines of approval be that be equal to or more superior.

Table 5: trivalent rHAO vaccine with

Comparison

Trivalent rHAO influenza vaccines

GMT (n=10 mouse) GMT (n=10 mouse)

As antigenic virus strain Anti-HA IgG Anti-HA IgG

0 week of 0 week

A/Texas/36/92(H1N1) ＜1000 103,000 ＜1000 11,200

A/Shangdong/32/92(H ＜1000 162,400 ＜1000 41,000

3N2)

B/Panama/45/90 ＜1000 164,800 ＜1000 26,000

As antigenic virus strain HAI HAI

A/Texas/36/92(H 1N 1) ＜8 1,522 ＜8 1,088

A/Shangdong/32/92(H ＜8 494 ＜8 435

3N2)

B/Panama/45/90 ＜8 174 ＜8 42

As antigenic virus strain In and Ab In and Ab

A/Texas/36/92(H1N1) ＜100 5,800 ＜100 2,720

A/Shangdong/32/92(H ＜100 840 ＜100 360

3N2)

B/Panama/45/90 ＜100 1,300 ＜100 700

Being used to prepare and use the improvement and the change of the method and composition as herein described of recombinant flu vaccines will be conspicuous to those skilled in the art.These improvement and change will be contained in the scope of claim subsequently.

Sequence table

(1) general information

(i) applicant: MicroGeneSys, Inc.

(ii) denomination of invention: a kind of method of producing the influenza hemagglutinin polyvalent vaccine

(iii) sequence number: 32

(iv) contact address:

(A) contact person: Patrea L.Pabst

(B) street: 2800 One Atlantic Center

1201 West Peachetree Street

(C) city: Atlanta

(D) state: GA

(E) country origin: USA

(F) postcode: 30309-3450

(v) computer-reader form:

(A) medium type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOC

(D) software: PatentIn Release#1.0, Version#1.25

(vi) present request for data:

(A) application number: PCT/US95/06750

(B) applying date: May 26 nineteen ninety-five

(C) classification:

(ix) telecommunication information:

(A) phone: (404)-873-8794

(B) fax: (404)-873-8795

(2) SEQ ID NO.1 information:

(i) sequence signature:

(A) length: 12 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(vi) primary source:

(A) organism: influenza virus

(x) published information:

(A) author: Davis etc.

(B) exercise question: contain influenza virus WSN strain (NON1) hemagglutinin gene

The structure of bacterial clone and evaluation

(C) periodical: gene

(D) volume: 10

(F) page or leaf: 205-218

(G) date: 1980

(xi) sequence description: SEQ ID NO.1:AGCAAAAGCA GG 12

(2) SEQ ID NO.2 information:

(i) sequence signature:

(A) length: 39 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(xi) sequence description: SEQ ID NO.2:

GGGGGTACCC CCGGGAGCAA AAGCAGGGGA AAATAAAAA 39

(2) SEQ ID NO.3 information:

(i) sequence signature:

(A) length: 35 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(xi) sequence description: SEQ ID NO.3:

CCCGGTACCT CAKATKCATA TTCTGCACTG CAAAG 35

(2) SEQ ID NO.4 information:

(i) sequence signature:

(A) length: 39 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(xi) sequence description: SEQ ID NO.4:

GGGGGTACCC CC GGGGACAC AATATGTATA GGCTACCAT 39

(2) SEQ ID NO.5 information:

(i) sequence signature:

(A) length: 35 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(xi) sequence description: SEQ ID NO.5:

CCCGGTACCT CAKATKCATA TTCTGCACTG CAAG 35

(2) SEQ ID NO.6 information:

(i) sequence signature:

(A) length: 1793 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(vi) primary source:

(A) organism: influenza virus

(C) the individual separation: A/Beijing/32/92rHA

(ix) feature:

(A) name/symbol: polyhedrin mRNA leading strand (part)

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal sequence encoding district

(B) position: 19 to 72

(ix) feature:

(A) name/symbol: SmaI restriction enzyme site

(B) position: 76 to 81

(ix) feature:

(A) name/symbol: ripe rHA coding region

(B) position: 73 to 1728

(ix) feature:

(A) name/symbol: KpnI restriction enzyme site

(B) position: 1771 to 1777

(ix) feature:

(A) name/symbol: Bg1II restriction enzyme site

(B) position: 1776 to 1782

(ix) feature:

(A) name/symbol: general translation termination signal

(B) position: 1783 to 1793

(xi) sequence description: SEQ ID NO.6:

TAAAAAAACC TATAAATAAT GCCCTTGTAC AAATTGTTAA ACGTTTTGTG GTTGGTCGCC 60

GTTTCTAACG CGATTCCCGG GGACTTTCCA GGAAATGACA ACAGCACAGC AACGCTGTGC 120

CTGGGACATC ATGCAGTGCC AAACGGAACG CTAGTGAAAA CAATCACGAA TGATCAAATT 180

GAAGTGACTA ATGCTACTGA GCTGGTTCAG AGTTCCTCAA CAGGTAGAAT ATGCGACAGT 240

CCTCACCGAA TCCTTGATGG AAAAAACTGC ACACTGATAG ATGCTCTATT GGGAGACCCT 300

CATTGTGATG GCTTCCAAAA TAAGGAATGG GACCTTTTTG TTGAACGCAG CAAAGCTTAC 360

AGCAACTGTT ACCCTTATGA TGTACCGGAT TATGCCTCCC TTAGGTCACT AGTTGCCTCA 420

TCAGGCACCC TGGAGTTTAT CAATGAAGAC TTCAATTGGA CTGGAGTCGC TCAGGATGGG 480

GGAAGCTATG CTTGCAAAAG GGGATCTGTT AACAGTTTCT TTAGTAGATT GAATTGGTTG 540

CACAAATCAG AATACAAATA TCCAGCGCTG AACGTGACTA TGCCAAACAA TGGCAAATTT 600

GACAAATTGT ACATTTGGGG GGTTCACCAC CCGAGCACGG ACAGAGACCA AACCAGCCTA 660

TATGTTCGAG CATCAGGGAG AGTCACAGTC TCTACCAAAA GAAGCCAACA AACTGTAACC 720

CCGAATATCG GGTCTAGACC CTGGGTAAGG GGTCAGTCCA GTAGAATAAG CATCTATTGG 780

ACAATAGTAA AACCGGGAGA CATACTTTTG ATTAATAGCA CAGGGAATCT AATTGCTCCT 840

CGGGGTTACT TCAAAATACG AAATGGGAAA AGCTCAATAA TGAGGTCAGA TGCACCCATT 900

GGCACCTGCA GTTCTGAATG CATCACTCCA AATGGAAGCA TTCCCAATGA CAAACCTTTT 960

CAAAATGTAA ACAGGATCAC ATATGGGGCC TGCCCCAGAT ATGTTAAGCA AAACACTCTG 1020

AAATTGGCAA CAGGGATGCG GAATGTACCA GAGAAACAAA CTAGAGGCAT ATTCGGCGCA 1080

ATCGCAGGTT TCATAGAAAA TGGTTGGGAG GGAATGGTAG ACGGTTGGTA CGGTTTCAGG 1140

CATCAAAATT CTGAGGGCAC AGGACAAGCA GCAGATCTTA AAAGCACTCA AGCAGCAATC 1200

GACCAAATCA ACGGGAAACT GAATAGGTTA ATCGAGAAAA CGAACGAGAA ATTCCATCAA 1260

ATCGAAAAAG AATTCTCAGA AGTAGAAGGG AGAATTCAGG ACCTCGAGAA ATATGTTGAA 1320

GACACTAAAA TAGATCTCTG GTCTTACAAC GCGGAGCTTC TTGTTGCCCT GGAGAACCAA 1380

CATACAATTG ATCTAACTGA CTCAGAAATG AACAAACTGT TTGAAAAAAC AAGGAAGCAA 1440

CTGAGGGAAA ATGCTGAGGA CATGGGCAAT GGTTGCTTCA AAATATACCA CAAATGTGAC 1500

AATGCCTGCA TAGGGTCAAT CAGAAATGGA ACTTATGACC ATGATGTATA CAGAGACGAA 1560

GCATTAAACA ACCGGTTCCA GATCAAAGGT GTTGAGCTGA AGTCAGGATA CAAAGATTGG 1620

ATCCTATGGA TTTCCTTTGC CATATCATGC TTTTTGCTTT GTGTTGTTTT GCTGGGGTTC 1680

ATCATGTGGG CCTGCCAAAA AGGCAACATT AGGTGCAACA TTTGCATTTG AGTGTATTAA 1740

TTAAAAACAC CCTTGTTTCT AGGATGATTC GGTACCAGAT CTTAATTAAT TAA 1793

(2) SEQ ID NO.7 information:

(i) sequence signature:

(A) length: 570 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: polypeptide

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: A/Beijing/32/92rHA

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal sequence

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: ripe rHA

(B) position: 19 to 552

(xi) sequence description: SEQ ID NO.7:

Met Pro Leu Tyr Lys Leu Leu Asn Val Leu Trp Leu Val Ala Val Ser

1 5 10 15

Asn Ala Ile Pro Gly Asp Phe Pro Gly Asn Asp Asn Ser Thr Ala Thr

20 25 30

Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr

35 40 45

Ile Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln

50 55 60

Ser Ser Ser Thr Gly Arg Ile Cys Asp Ser Pro His Arg Ile Leu Asp

65 70 75 80

Gly Lys Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys

85 90 95

Asp Gly Phe Gln Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser Lys

100 105 110

Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu

115 120 125

Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Ile Asn Glu Asp

130 135 140

Phe Asn Trp Thr Gly Val Ala Gln Asp Gly Gly Ser Tyr Ala Cys Lys

145 150 155 160

Arg Gly Ser Val Asn Ser Phe Phe Ser Arg Leu Asn Trp Leu His Lys

165 170 175

Ser Glu Tyr Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Gly

180 185 190

Lys Phe Asp Lys Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asp

195 200 205

Arg Asp Gln Thr Ser Leu Tyr Val Arg Ala Ser Gly Arg Val Thr Val

210 215 220

Ser Thr Lys Arg Ser Gln Gln Thr Val Thr Pro Asn Ile Gly Ser Arg

225 230 235 240

Pro Trp Val Arg Gly Gln Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile

245 250 255

Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile

260 265 270

Ala Pro Arg Gly Tyr Phe Lys Ile Arg Asn Gly Lys Ser Ser Ile Met

275 280 285

Arg Ser Asp Ala Pro Ile Gly Thr Cys Ser Ser Glu Cys Ile Thr Pro

290 295 300

Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile

305 310 315 320

Thr Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu

325 330 335

Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe

340 345 350

Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp

355 360 365

Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala

370 375 380

Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys

385 390 395 400

Leu Asn Arg Leu Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu

405 410 415

Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr

420 425 430

Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu

435 440 445

Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met

450 455 460

Asn Lys Leu Phe Glu Lys Thr Arg Lys Gln Leu Arg Glu Asn Ala Glu

465 470 475 480

Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Ash Ala

485 490 495

Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg

500 505 510

Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys

515 520 525

Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys

530 535 540

Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln

545 550 555 560

Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

565 570

(2) SEQ ID NO.8 information:

(i) sequence signature:

(A) length: 1776 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: A/Texas/36/91 rHA

(ix) feature:

(A) name/symbol: polyhedrin mRNA leading strand (part)

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal peptide-coding region

(B) position: 19 to 72

(ix) feature:

(A) name/symbol: SmaI restriction enzyme site

(B) position: 76 to 81

(ix) feature:

(A) name/symbol: KpnI restriction enzyme site

(B) position: 82 to 87

(ix) feature:

(A) name/symbol: SmaI restriction enzyme site

(B) position: 88 to 93

(ix) feature:

(A) name/symbol: ripe rHA coding region

(B) position: 73 to 1734

(ix) feature:

(A) name/symbol: KpnI restriction enzyme site

(B) position: 1744 to 1749

(ix) feature:

(A) name/symbol: Bg1II restriction enzyme site

(B) position: 1750 to 1755

(ix) feature:

(A) name/symbol: general translation termination signal

(B) position: 1756 to 1766

(xi) sequence description: SEQ ID NO.8:

TAAAAAAACC TATAAATAAT GCCCTTGTAC AAATTGTTAA ACGTTTTGTG GTTGGTCGCC 60

GTTTCTAACG CGATTCCCGG GGGTACCCCC GGGGACACAA TATGTATAGG CTACCATGCG 120

AACAACTCAA CCGACACTGT TGACACAGTA CTTGAGAAGA ACGTGACAGT GACACACTCT 180

GTCAACCTAC TTGAGGACAG TCACAACGGA AAACTATGTC GACTAAAGGG AATAGCCCCA 240

CTACAATTGG GTAATTGCAG CGTTGCCGGA TGGATCTTAG GAAACCCAAA ATGCGAATCA 300

CTGTTTTCTA AGGAATCATG GTCCTACATT GCAGAAACAC CAAACCCTGA GAATGGAACA 360

TGTTACCCAG GGTATTTCGC CGACTATGAG GAACTGAGGG AGCAATTGAG TTCAGTATCA 420

TCATTCGAGA GATTCGAAAT ATTCCCCAAA GAAAGCTCAT GGCCCAACCA CACCGTAACC 480

AAAGGAGTAA CGAGATCATG CTCCCATAAT GGGAAAAGCA GTTTTTACAG AAATTTGCTA 540

TGGCTGACGG AGAAGAATGG CTTGTACCCA AATCTGAGCA AGTCCTATGT AAACAACAAA 600

GAGAAAGAAG TCCTTGTACT ATGGGGTGTT CATCACCCGT CTAACATAAG GGACCAAAGG 660

GCCATCTATC ATACAGAAAA TGCTTATGTC TCTGTAGTGT CTTCACATTA TAGCAGAAGA 720

TTCACCCCAG AAATAGCAAA AAGACCCAAA GTAAGAGATC AAGAAGGAAG AATTAACTAC 780

TACTGGACTC TGCTGGAACC CGGGGACACA ATAATATTTG AGGCAAATGG AAATCTAATA 840

GCGCCATGGT ATGCTTTCGC ACTGAGTAGA GGCTTTGGGT CAGGAATCAT CACCTCAAAC 900

GCATCAATGG ATGAATGTGA CGCGAAGTGT CAAACACCCC AGGGAGCTAT AAACAGTAGT 960

CTTCCTTTCC AGAATGTACA CCCAGTCACA ATAGGAGAGT GTCCAAAGTA TGTCAGGAGT 1020

ACAAAATTAA GGATGGTTAC AGGACTAAGG AACATCCCAT CCATTCAATC CAGAGGTTTG 1080

TTTGGAGCCA TTGCCGGTTT CATTGAAGGG GGGTGGACTG GAATGATAGA TGGATGGTAT 1140

GGTTATCATC ATCAGAATGA ACAAGGATCT GGCTATGCTG CGGACCAAAA AAGCACACAA 1200

AATGCCATTA ACGGGATTAC AAACAAGGTG AATTCTGTAA TCGAGAAAAT GAACACTCAA 1260

TTCACAGCTG TGGGCAAAGA ATTCAACAAA TTAGAAAGAA GGATGGAAAA CTTAAATAAA 1320

AAAGTTGATG ATGGATTTCT GGACATTTGG ACATATAATG CAGAATTGTT GGTTCTACTG 1380

GAAAATGGAA GGACTTTGGA TTTTCATGAC TCAAATGTGA AGAATCTGTA TGAGAAAGTA 1440

AAAAGCCAAT TGAAGAATAA TGCCAAAGAA ATAGGGAACG GGTGTTTTGA ATTCTATCAC 1500

AAGTGTAACA ATGAATGCAT GGAAAGTGTG AAAAATGGAA CTTATGACTA TCCAAAATAT 1560

TCCGAAGAAT CAAAGTTAAA CAGGGGAAAA ATTGATGGAG TGAAATTGGA ATCAATGGGA 1620

GTCTATCAGA TTCTGGCGAT CTACTCAACT GTCGCCAGTT CACTGGTGCT TTTGGTCTCC 1680

CTGGGGGCAA TCAGCTTCTG GATGTGTTCT AATGGGTCTT TGCAGTGCAG AATATGAATC 1740

TGAGGTACCA GATCTTAATT AATTAA 1766

(2) SEQID NO.9 information:

(i) sequence signature:

(A) length: 572 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: peptide

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: A/Texas/36/91rHA

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal sequence

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: ripe rHA

(B) position: 19 to 554

(xi) sequence description: SEQ ID NO.9:

Met Pro Leu Tyr Lys Leu Leu Asn Val Leu Trp Leu Val Ala Val Ser

1 5 10 15

Asn Ala Ile Pro Gly Gly Thr Pro Gly Asp Thr Ile Cys Ile Gly Tyr

20 25 30

His Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn

35 40 45

Val Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly

50 55 60

Lys Leu Cys Arg Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Asn Cys

65 70 75 80

Ser Val Ala Gly Trp Ile Leu Gly Asn Pro Lys Cys Glu Ser Leu Phe

85 90 95

Ser Lys Glu Ser Trp Ser Tyr Ile Ala Glu Thr Pro Asn Pro Glu Asn

100 105 110

Gly Thr Cys Tyr Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu

115 120 125

Gln Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys

130 135 140

Glu Ser Ser Trp Pro Asn His Thr Val Thr Lys Gly Val Thr Arg Ser

145 150 155 160

Cys Ser His Asn Gly Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu

165 170 175

Thr Glu Lys Asn Gly Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn

180 185 190

Asn Lys Glu Lys Glu Val Leu Val Leu Trp Gly Val His His Pro Ser

195 200 205

Asn Ile Arg Asp Gln Arg Ala Ile Tyr His Thr Glu Asn Ala Tyr Val

210 215 220

Ser Val Val Ser Ser His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala

225 230 235 240

Lys Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp

245 250 255

Thr Leu Leu Glu Pro Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn

260 265 270

Leu Ile Ala Pro Trp Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser

275 280 285

Gly Ile Ile Thr Ser Asn Ala Ser Met Asp Glu Cys Asp Ala Lys Cys

290 295 300

Gln Thr Pro Gln Gly Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val

305 310 315 320

His Pro Val Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Thr Lys

325 330 335

Leu Arg Met Val Thr Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Arg

340 345 350

Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly

355 360 365

Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser

370 375 380

Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile

385 390 395 400

Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr

405 410 415

Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu

420 425 430

Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala

435 440 445

Glu Leu Leu Val Leu Leu Glu Asn Gly Arg Thr Leu Asp Phe His Asp

450 455 460

Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn

465 470 475 480

Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys

485 490 495

Asn Asn Glu Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro

500 505 510

Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Gly Lys Ile Asp Gly Val

515 520 525

Lys Leu Glu Ser Met Gly Val Tyr Gln Ile Leu Ala Ile Tyr Ser Thr

530 535 540

Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe

545 550 555 560

Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile

565 570

(2) SEQ ID NO.10 information:

(i) sequence signature:

(A) length: 1799 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: RNA (genome)

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: B/Panama/45/90rHA

(ix) feature:

(A) name/symbol: polyhedrin mRNA leading strand (part)

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: HA signal peptide sequence coding region

(B) position: 19 to 69

(ix) feature:

(A) name/symbol: SmaI restriction enzyme site

(B) position: 22 to 27

(ix) feature:

(A) name/symbol: ripe rHA coding region

(B) position: 70 to 1773

(ix) feature:

(A) name/symbol: KpnI restriction enzyme site

(B) position: 1777 to 1782

(ix) feature:

(A) name/symbol: Bg1II restriction enzyme site

(B) position: 1783 to 1788

(ix) feature:

(A) name/symbol: general translation termination signal

(B) position: 1789 to 1799

(xi) sequence description: SEQ ID NO.10:

TAAAAAAACC TATAAATAAT GCCCGGGAAG GCAATAATTG TACTACTCAT GGTAGTAACA 60

TCCAACGCAG ATCGAATCTG CACTGGGATA ACATCTTCAA ACTCACCTCA TGTGGTCAAA 120

ACAGCTACTC AAGGGGAAGT CAATGTGACT GGTGTGATAC CACTGACAAC AACACCAACA 180

AAATCTCATT TTGCAAATCT AAAAGGAACA AAGACCAGAG GGAAACTATG CCCAAACTGT 240

CTCAACTGCA CAGATCTGGA TGTGGCCTTG GGCAGACCAA TGTGTGTGGG GACCACACCT 300

TCGGCAAAAG CTTCAATACT CCACGAAGTC AGACCTGTTA CATCCGGGTG CTTTCCTATA 360

ATGCACGACA GAACAAAAAT CAGACAGCTA CCCAATCTTC TCAGAGGATA TGAAAATATC 420

AGATTATCAA CCCAAAACGT TATCAACGCA GAAAGAGCAC CAGGAGGACC CTACAGACTT 480

GGAACCTCAG GATCTTGCCC TAACGTTACC AGTAGAGACG GATTCTTCGC AACAATGGCT 540

TGGGCTGTCC CAAGGGACAA CAAAACAGCA ACGAATCCAC TAACAGTAGA AGTACCATAC 600

ATTTGTACCA AAGGAGAAGA CCAAATTACT GTTTGGGGGT TCCATTCTGA TAACAAAATC 660

CAAATGAAAA ACCTCTATGG AGACTCAAAT CCTCAAAAGT TCACCTCATC TGCCAATGGA 720

GTAACCACAC ATTATGTTTC TCAGATTGGT GGCTTCCCAA ATCAAACAGA AGACGGAGGG 780

CTACCACAAA GCGGCAGAAT TGTTGTTGAT TACATGGTGC AAAAACCTGG GAAAACAGGA 840

ACAATTGTCT ATCAAAGAGG TGTTTTGTTG CCTCAAAAGG TGTGGTGCGC AAGTGGCAGG 900

AGCAAGGTAA TAAAAGGGTC CTTGCCTTTA ATTGGTGAAG CAGATTGCCT TCACGAAAAA 960

TACGGTGGAT TAAACAAAAG CAAGCCTTAC TACACAGGAG AACATGCAAA AGCCATAGGA 1020

AATTGCCCAA TATGGGTGAA AACACCTTTG AAGCTTGCCA ATGGAACCAA ATATAGACCT 1080

CCTGCAAAAC TATTAAAGGA AAGGGGTTTC TTCGGAGCTA TTGCTGGTTT CTTAGAAGGA 1140

GGATGGGAAG GAATGATTGC AGGTTGGCAC GGATACACAT CTCATGGAGC ACATGGAGTG 1200

GCAGTGGCAG CAGACCTTAA GAGTACGCAA GAAGCCATAA ACAAGATAAC AAAAAATCTC 1260

AATTCTTTGA GTGAGCTAGA AGTAAAGAAT CTTCAAAGAC TAAGTGGTGC CATGGATGAA 1320

CTCCACAACG AAATACTCGA GCTGGATGAG AAAGTGGATG ATCTCAGAGC TGACACAATA 1380

AGCTCGCAAA TAGAGCTTGC AGTCTTGCTT TCCAACGAAG GAATAATAAA CAGTGAAGAT 1440

GAGCATCTAT TGGCACTTGA GAGAAAACTA AAGAAAATGC TGGGTCCCTC TGCTGTAGAC 1500

ATAGGGAATG GATGCTTCGA AACCAAACAC AAGTGCAACC AGACCTGCTT AGACAGGATA 1560

GCTGCTGGCA CCTTTAATGC AGGAGAATTT TCTCTTCCCA CTTTTGATTC ACTGAATATT 1620

ACTGCTGCAT CTTTAAATGA TGATGGATTG GATAATCATA CTATACTGCT CTACTACTCA 1680

ACTGCTGCTT CTAGTTTGGC TGTAACATTG ATGATAGCTA TTTTTATTGT TTATATGGTC 1740

TCCAGAGACA ATGTTTCTTG TTCCATCTGT CTGTGAGGTA CCAGATCTTA ATTAATTAA 1799

(2) SEQ ID NO.11 information:

(i) sequence signature:

(A) length: 585 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: polypeptide

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: B/Panama/45/90rHA

(ix) feature:

(A) name/symbol: HA signal peptide

(B) position: 1 to 17

(ix) feature:

(A) name/symbol: ripe rHA

(B) position: 18 to 568

(xi) sequence description: SEQ ID NO.11:

Met Pro Gly Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn

1 5 10 15

Ala Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val

20 25 30

Val Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro

35 40 45

Leu Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr

50 55 60

Lys Thr Arg Gly Lys Leu Cys Pro Asn Cys Leu Asn Cys Thr Asp Leu

65 70 75 80

Asp Val Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala

85 90 95

Lys Ala Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe

100 105 110

Pro Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu

115 120 125

Arg Gly Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asn Ala

130 135 140

Glu Arg Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys

145 150 155 160

Pro Asn Val Thr Ser Arg Asp Gly Phe Phe Ala Thr Met Ala Trp Ala

165 170 175

Val Pro Arg Asp Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu Val

180 185 190

Pro Tyr Ile Cys Thr Lys Gly Glu Asp Gln Ile Thr Val Trp Gly Phe

195 200 205

His Ser Asp Asn Lys Ile Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn

210 215 220

Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val

225 230 235 240

Ser Gln Ile Gly Gly Phe Pro Asn Gln Thr Glu Asp Gly Gly Leu Pro

245 250 255

Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Pro Gly Lys

260 265 270

Thr Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val

275 280 285

Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu

290 295 300

Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys

305 310 315 320

Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys

325 330 335

Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr

340 345 350

Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile

355 360 365

Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His

370 375 380

Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu

385 390 395 400

Lys Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser

405 410 415

Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met

420 425 430

Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp

435 440 445

Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu

450 455 460

Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu

465 470 475 480

Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile Gly

485 490 495

Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp

500 505 510

Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr

515 520 525

Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu

530 535 540

Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu

545 550 555 560

Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg

565 570 575

Asp Asn Val Ser Cys Ser Ile Cys Leu

580 585

(2) SEQ ID NO.12 information:

(i) sequence signature:

(A) length: 1811 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: B/Netherlands/13/94rHA

(ix) feature:

(A) name/symbol: polyhedrin mRNA leading strand (part)

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal sequence encoding district

(B) position: 19 to 72

(ix) feature:

(A) name/symbol: SmaI restriction enzyme site

(B) position: 76 to 81

(ix) feature:

(A) name/symbol: ripe rHA coding region

(B) position: 73 to 1785

(ix) feature:

(A) name/symbol: KpnI restriction enzyme site

(B) position: 1789 to 1794

(ix) feature:

(A) name/symbol: Bg1II restriction enzyme site

(B) position: 1795 to 1800

(ix) feature:

(A) name/symbol: general translation termination signal

(B) position: 1801 to 1811

(xi) sequence description: SEQ ID NO.12:

TAAAAAAACC TATAAATAAT GCCCTTGTAC AAATTGTTAA ACGTTTTGTG GTTGGTCGCC 60

GTTTCTAACG CGATTCCCGG GGATCGAATC TGCACTGGGA TAACATCTTC AAAATCACCT 120

CATGTAGTCA AAACAGCTAC TCAAGGGGAG GTCAATGTGA CTGGTGTGAT ACCACTGACG 180

ACAACACCAA CAAAATCTCA TTTTGCAAAT CTCAAAGGAA CAAAGACCAG AGGGAAACTA 240

TGCCCAAACT GTCTCAACTG CACAGATCTG GATGTGGCCT TGGGCAGACC AATGTGTGTG 300

GGGATCACAC CTTCGGCAAA AGCTTCAATA CTCCACGAAG TCAGACCTGT TACATCCGGG 360

TGCTTTCCTA TAATGCATGA CAGAACAAAA ATCAGACAGC TACCCAATCT TCTCAGAGGA 420

TATGAAAACA TCAGACTATC AACCCAAAAC GTTATCAACG CAGAAAAGGC ACCAGGAGGA 480

CCCTACAGAC TTGGAACCTC AGGATCTTGC CCTAACGTTA CCAGTAGAAC CGGATTCTTC 540

GCAACAATGG CTTGGGCTGT CCCAAGGGAC AACAAAACAG CAACGAATCC ACTAACAGTA 600

GAAGTACCAT ACATTTGTAC GAAAGGAGAA GACCAAATTA CTGTTTGGGG GTTCCATTCT 660

GATAACAAAA CCCAAATGAA AAACCTCTAT GGAGACTCAA ATCCTCAAAA GTTCACCTCA 720

TCTGCCAATG GAGTAACCAC ACATTATGTT TCTCAGATTG GTGGCTTCCC AGATCAAACA 780

GAAGACGGAG GACTACCACA AAGCGGCAGA ATTGTTGTTG ATTACATGGT GCAAAAACCT 840

GGGAAAACAG GAACAATTGT CTATCAAAGA GGTATTTTGT TGCCTCAAAA GGTGTGGTGC 900

GCAAGTGGCA GGAGCAAGGT AATAAAAGGG TCCTTGCCTT TAATTGGTGA AGCAGATTGC 960

CTTCACGAAA AATACGGTGG ATTAAACAAA AGCAAGCCTT ACTACACAGG AGAACATGCA 1020

AAAGCCATAG GAAATTGCCC AATATGGGTG AAAACACCTT TGAAGCTTGC CAATGGAACC 1080

AGATATAGAC CTCCTGCAAA ACTATTAAAG GAAAGGGGTT TCTTCGGAGC TATTGCTGGT 1140

TTCTTAGAAG GAGGATGGGA AGGAATGATT GCAGGTTGGC ACGGATACAC ATCTCACGGG 1200

GCACATGGAG TGGCAGTGGC AGCAGACCTT AAGAGTACGC AAGAAGCCAT AAACAAGATA 1260

ACAAAAAATC TCAATTCTTT GAGTGAGCTA GAAGTAAAGA ACCTTCAAAG ACTAAGTGGT 1320

GCCATGGATG AACTCCACAA CGAAATACTC GAGCTGGATG AGAAAGTGGA TGATCTCAGA 1380

GCTGACACAA TAAGCTCGCA AATAGAGCTT GCAGTCTTAC TTTCCAACGA AGGAATAATA 1440

AACAGTGAAG ATGAGCATCT ATTGGCACTT GAGAGAAAAC TAAAGAAAAT GCTGGGTCCC 1500

TCTGCTGTAG ACATAGGGAA TGGATGCTTC GAAACAAAAC ACAAGTGCAA CCAGACCTGC 1560

TTAGACAGGA TAGCTGCTGG CACCTTTAAT GCAGGAGAAT TTTCTCTTCC CACTTTTGAT 1620

TCACTGAATA TTACTGCTGC ATCTTTAAAT GATGATGGAT TGGATAATCA TACTATACTG 1680

CTCTACTACT CAACTGCTGC TTCTAGTTTG GCTGTAACAT TGATGATAGC TATTTTTATT 1740

GTTTATATGG TCTCCAGAGA CAATGTTTCT TGTTCCATCT GTCTGTGAGG TACCAGATCT 1800

TAATTAATTA A 1811

(2) SEQ ID NO.13 information:

(i) sequence signature:

(A) length: 589 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: peptide

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: B/Netherlands/13/94rHA

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal sequence

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: ripe rHA

(B) position: 19 to 571

(xi) sequence description: SEQ ID NO.13:

Met Pro Leu Tyr Lys Leu Leu Asn Val Leu Trp Leu Val Ala Val Ser

1 5 10 15

Asn Ala Ile Pro Gly Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Lys

20 25 30

Ser Pro His Val Val Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr

35 40 45

Gly Val Ile Pro Leu Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn

50 55 60

Leu Lys Gly Thr Lys Thr Arg Gly Lys Leu Cys Pro Asn Cys Leu Asn

65 70 75 80

Cys Thr Asp Leu Asp Val Ala Leu Gly Arg Pro Met Cys Val Gly Ile

85 90 95

Thr Pro Ser Ala Lys Ala Ser Ile Leu His Glu Val Arg Pro Val Thr

100 105 110

Ser Gly Cys Phe Pro Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu

115 120 125

Pro Asn Leu Leu Arg Gly Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn

130 135 140

Val Ile Asn Ala Glu Lys Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr

145 150 155 160

Ser Gly Ser Cys Pro Asn Val Thr Ser Arg Thr Gly Phe Phe Ala Thr

165 170 175

Met Ala Trp Ala Val Pro Arg Asp Asn Lys Thr Ala Thr Asn Pro Leu

180 185 190

Thr Val Glu Val Pro Tyr Ile Cys Thr Lys Gly Glu Asp Gln Ile Thr

195 200 205

Val Trp Gly Phe His Ser Asp Asn Lys Thr Gln Met Lys Asn Leu Tyr

210 215 220

Gly Asp Ser Asn Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr

225 230 235 240

Thr His Tyr Val Ser Gln Ile Gly Gly Phe Pro Asp Gln Thr Glu Asp

245 250 255

Gly Gly Leu Pro Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln

260 265 270

Lys Pro Gly Lys Thr Gly Thr Ile Val Tyr Gln Arg Gly Ile Leu Leu

275 280 285

Pro Gln Lys Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly

290 295 300

Ser Leu Pro Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly

305 310 315 320

Gly Leu Asn Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala

325 330 335

Ile Gly Asn Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn

340 345 350

Gly Thr Arg Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe

355 360 365

Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile

370 375 380

Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val

385 390 395 400

Ala Ala Asp Leu Lys Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys

405 410 415

Asn Leu Asn Ser Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu

420 425 430

Ser Gly Ala Met Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu

435 440 445

Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu

450 455 460

Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His

465 470 475 480

Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala

485 490 495

Val Asp Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln

500 505 510

Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe

515 520 525

Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn

530 535 540

Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala

545 550 555 560

Ala Ser Ser Leu Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr

565 570 575

Met Val Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu

580 585

(2) SEQ ID NO.14 information:

(i) sequence signature:

(A) length: 1757 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: A/Shandong/9/93rHA

(ix) feature:

(A) name/symbol: polyhedrin mRNA leading strand (part)

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal sequence encoding district

(B) position: 19 to 72

(ix) feature:

(A) name/symbol: SmaI restriction enzyme site

(B) position: 76 to 81

(ix) feature:

(A) name/symbol: ripe rHA coding region

(B) position: 73 to 1728

(ix) feature:

(A) name/symbol: KpnI restriction enzyme site

(B) position: 1735 to 1740

(ix) feature:

(A) name/symbol: Bg1II restriction enzyme site

(B) position: 1741 to 1746

(ix) feature:

(A) name/symbol: general translation termination signal

(B) position: 1747 to 1757

(xi) sequence description: SEQ ID NO.14:

TAAAAAAACC TATAAATAAT GCCCTTGTAC AAATTGTTAA ACGTTTTGTG GTTGGTCGCC 60

GTTTCTAACG CGATTCCCGG GCAAGACCTT CCAGGAAATG ACAACAGCAC AGCAACGCTG 120

TGCCTGGGAC ATCATGCAGT GCCAAACGGA ACGCTAGTGA AAACAATCAC GAATGATCAA 180

ATTGAAGTGA CTAATGCTAC TGAGTTGGTT CAGAGTTCCT CAACAGGTAG AATATGCGGC 240

AGTCCTCACC GAATCCTTGA TGGAAAAAAC TGCACACTGA TAGATGCTCT ATTGGGAGAC 300

CCTCATTGTG ATGGCTTCCA AAATAAGGAA TGGGACCTTT TTGTTGAACG CAGCAAAGCT 360

TACAGCAACT GTTACCCTTA TGATGTGCCG GATTATGCCT CCCTTAGGTC ACTAGTTGCC 420

TCATCAGGCA CCCTGGAGTT TATCAATGAA GACTTCAATT GGACTGGAGT CGCTCAGGAT 480

GGGGGAAGCT ATGCTTGCAA AAGAGGATCT GTTAACAGTT TCTTTAGTAG ATTGAATTGG 540

TTGCACAAAT TAGAATACAA ATATCCAGCG CTGAACGTGA CTATGCCAAA CAATGGCAAA 600

TTTGACAAAT TGTACATTTG GGGGGTTCAC CACCCGAGCA CGGACAGTGA CCAAACCAGC 660

CTATATGTTC GAGCATCAGG GAGAGTCACA GTCTCTACCA AAAGAAGCCA ACAAACTGTA 720

ACCCCGAATA TCGGGTCTAG ACCCTGGGTA AGGGGTCAGT CCAGTAGAAT AAGCATCTAT 780

TGGACAATAG TAAAACCGGG AGACATACTT TTGATTGATA GCACAGGGAA TCTAATTGCT 840

CCTCGGGGTT ACTTCAAAAT ACGAAATGGG AAAAGCTCAA TAATGAGGTC AGATGCACCC 900

ATTGGCAACT GCAGTTCTGA ATGCATCACT CCAAATGGAA GCATTCCCAA TGACAAACCT 960

TTTCAAAATG TAAACAGAAT CACATATGGG GCCTGCCCCA GATATGTTAA GCAAAACACT 1020

CTGAAATTGG CAACAGGGAT GCGGAATGTA CCAGAGAAAC AAACTAGAGG CATATTCGGC 1080

GCAATCGCAG GTTTCATAGA AAATGGTTGG GAGGGAATGG TAGACGGTTG GTACGGTTTC 1140

AGGCATCAAA ATTCTGAGGG CACAGGACAA GCAGCAGATC TTAAAAGCAC TCAAGCAGCA 1200

ATCGACCAAA TCAACGGGAA ACTGAATAGG TTAATCGAGA AAACGAACGA GAAATTCCAT 1260

CAAATCGAAA AAGAATTCTC AGAAGTAGAA GGGAGAATTC AGGACCTCGA GAAATATGTT 1320

GAAGACACTA AAATAGATCT CTGGTCTTAC AACGCGGAGC TTCTTGTTGC CCTGGAGAAC 1380

CAACATACAA TTGATCTAAC TGACTCAGAA ATGAACAAAC TGTTTGAAAA AACAAGGAAG 1440

CAACTGAGGG AAAATGCTGA GGACATGGGC AATGGTTGCT TCAAAATATA CCACAAATGT 1500

GACAATGCCT GCATAGGGTC AATCAGAAAT GGAACTTATG ACCATGATGT ATACAGAGAC 1560

GAAGCATTAA ACAACCGGTT CCAGATCAAA GGTGTTGAGC TGAAGTCAGG ATACAAAGAT 1620

TGGATCCTAT GGATTTCCTT TGCCATATCA TGCTTTTTGC TTTGTGTTGT TTTGCTGGGG 1680

TTCATCATGT GGGCCTGCCA AAAAGGCAAC ATTAGGTGCA ACATTTGCAT TTGAGGTACC 1740

AGATCTTAAT TAATTAA 1757

(2) SEQ ID NO.15 information:

(i) sequence signature:

(A) length: 571 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: polypeptide

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: A/Shandong/9/93rHA

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal sequence

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: ripe rHA

(B) position: 19 to 553

(xi) sequence description: SEQ ID NO.15:

Met Pro Leu Tyr Lys Leu Leu Asn Val Leu Trp Leu Val Ala Val Ser

1 5 10 15

Asn Ala Ile Pro Gly Gln Asp Leu Pro Gly Asn Asp Asn Ser Thr Ala

20 25 30

Thr Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr Leu Val Lys

35 40 45

Thr Ile Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val

50 55 60

Gln Ser Ser Ser Thr Gly Arg Ile Cys Gly Ser Pro His Arg Ile Leu

65 70 75 80

Asp Gly Lys Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His

85 90 95

Cys Asp Gly Phe Gln Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser

100 105 110

Lys Ala Tyr Sar Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser

115 120 125

Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Ile Asn Glu

130 135 140

Asp Phe Asn Trp Thr Gly Val Ala Gln Asp Gly Gly Ser Tyr Ala Cys

145 150 155 160

Lys Arg Gly Ser Val Asn Ser Phe Phe Ser Arg Leu Asn Trp Leu His

165 170 175

Lys Leu Glu Tyr Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn

180 185 190

Gly Lys Phe Asp Lys Leu Tyr Ile Trp Gly Val His His Pro Ser Thr

195 200 205

Asp Ser Asp Gln Thr Ser Leu Tyr Val Arg Ala Ser Gly Arg Val Thr

210 215 220

Val Ser Thr Lys Arg Ser Gln Gln Thr Val Thr Pro Asn Ile Gly Ser

225 230 235 240

Arg Pro Trp Val Arg Gly Gln Ser Ser Arg Ile Ser Ile Tyr Trp Thr

245 250 255

Ile Val Lys Pro Gly Asp Ile Leu Leu Ile Asp Ser Thr Gly Asn Leu

260 265 270

Ile Ala Pro Arg Gly Tyr Phe Lys Ile Arg Asn Gly Lys Ser Ser Ile

275 280 285

Met Arg Ser Asp Ala Pro Ile Gly Asn Cys Ser Ser Glu Cys Ile Thr

290 295 300

Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg

305 310 315 320

Ile Thr Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys

325 330 335

Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile

340 345 350

Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val

355 360 365

Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln

370 375 380

Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly

385 390 395 400

Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile

405 410 415

Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys

420 425 430

Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu

435 440 445

Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu

450 455 460

Met Asn Lys Leu Phe Glu Lys Thr Arg Lys Gln Leu Arg Glu Asn Ala

465 470 475 480

Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn

485 490 495

Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr

500 505 510

Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu

515 520 525

Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser

530 535 540

Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys

545 550 555 560

Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

565 570

(2) SEQ ID NO.16 information:

(i) sequence signature:

(A) length: 1814 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: B/Shanhai/4/94rHA

(ix) feature:

(A) name/symbol: polyhedrin mRNA leading strand (part)

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal sequence encoding district

(B) position: 19 to 72

(ix) feature:

(A) name/symbol: SmaI restriction enzyme site

(B) position: 76 to 81

(ix) feature:

(A) name/symbol: KpnI restriction enzyme site

(B) position: 82 to 87

(ix) feature:

(A) name/symbol: ripe rHA coding region

(B) position: 73 to 1794

(ix) feature:

(A) name/symbol: general translation termination signal

(B) position: 1804 to 1814

(xi) sequence description: SEQ ID NO.16:

TAAAAAAACC TATAAATAAT GCCCTTGTAC AAATTGTTAA ACGTTTTGTG GTTGGTCGCC 60

GTTTCTAACG CGATTCCCGG GGGTACCGAT CGAATCTGCA CTGGGATAAC ATCTTCAAAC 120

TCACCTCATG TGGTCAAAAC AGCTACTCAA GGGGAGGTCA ATGTGACTGG TGTGATACCA 180

CTGACAACAA CACCAACAAA ATCTCATTTT GCAAATCTCA AAGGAACAAA GACCAGAGGG 240

AAACTATGCC CAAACTGTCT CAACTGCACA GATCTGGATG TGGCCTTGGG CAGACCAATG 300

TGTGTGGGGA CCACACCTTC GGCAAAAGCT TCAATACTCC ACGAAGTCAG ACCTGTTACA 360

TCCGGGTGCT TTCCTATAAT GCACGACAGA ACAAAAATCA GACAGCTACC CAATCTTCTC 420

AGAGGATATG AAAATATCAG ATTATCAACC CAAAACGTTA TCAACGCAGA AAAGGCACCA 480

GGAGGACCCT ACAGACTTGG AACCTCAGGA TCTTGCCCTA ACGCTACCAG TAGAAGCGGA 540

TTTTTCGCAA CAATGGCTTG GGCTGTCCCA AGGGACAACA ACAAAACAGC AACGAATCCA 600

CTAACAGTAG AAGTACCATA CATTTGCACA AAAGGAGAAG ACCAAATTAC TGTTTGGGGG 660

TTCCATTCTG ATAACAAACC CCAAATGAAA AACCTCTATG GAGACTCAAA TCCTCAAAAG 720

TTCACCTCAT CTGCTAATGG AGTAACCACA CATTATGTTT CTCAGATTGG CGGCTTCCCA 780

GATCAAACAG AAGACGGAGG GCTACCACAA AGCGGCAGAA TTGTTGTTGA TTACATGGTG 840

CAAAAACCTG GGAAGACAGG AACAATTGTC TATCAGAGAG GTGTTTTGTT GCCTCAAAAG 900

GTGTGGTGCG CTAGTGGCAG GAGCAAAGTA ATAAAAGGGT CCTTGCCTTT AATTGGTGAA 960

GCAGATTGCC TTCACGAAAA ATACGGTGGA TTAAACAAAA GCAAGCCTTA CTACACAGGA 1020

GAACATGCAA AAGCCATAGG AAATTGCCCA ATATGGGTGA AAACACCTTT GAAGCTTGCC 1080

AATGGAACCA AATATAGACC TCCTGCAAAA CTATTAAAGG AAAGGGGTTT CTTCGGAGCT 1140

ATTGCTGGTT TCTTAGAAGG AGGATGGGAA GGAATGATTG CAGGTTGGCA CGGATACACA 1200

TCTCACGGAG CACATGGAGT GGCAGTGGCA GCAGACCTTA AGAGTACGCA AGAAGCCATA 1260

AACAAGATAA CAAAAAATCT CAATTCTTTG AGTGAGCTAG AAGTAAAGAA TCTTCAAAGG 1320

CTAAGTGGTG CCATGGATGA ACTCCACAAC GAAATACTCG AGCTGGATGA GAAAGTGGAT 1380

GATCTCAGAG CTGACACAAT AAGCTCGCAA ATAGAACTTG CAGTCTTGCT TTCCAACGAA 1440

GGAATAATAA ACAGTGAAGA TGAGCATCTA TTGGCACTTG AGAGAAAACT AAAGAAAATG 1500

CTGGGTCCCT CTGCTGTAGA CATAGGAAAT GGATGCTTCG AAACCAAACA CAAGTGCAAC 1560

CAGACCTGCT TAGACAGGAT AGCTGCTGGC ACCTTTAATG CGGGAGAATT TTCTCTTCCC 1620

ACTTTTGATT CACTGAATAT TACTGCTGCA TCTTTAAATG ATGATGGATT GGATAACCAT 1680

ACTATACTGC TCTACTACTC AACTGCTGCT TCTAGTTTGG CGGTAACATT GATGATAGCT 1740

ATTTTTATTG TTTATATGGT CTCCAGAGAC AATGTTTCTT GCTCCATCTG TCTGTGAGGA 1800

TCTTAATTAA TTAA 1814

(2) SEQ ID NO.17 information:

(i) sequence signature:

(A) length: 592 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: polypeptide

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: B/Shanhai/4/94rHA

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal peptide

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: ripe rHA

(B) position: 19 to 574

(xi) sequence description: SEQ ID NO.17:

Met Pro Leu Tyr Lys Leu Leu Asn Val Leu Trp Leu Val Ala Val Ser

1 5 10 15

Asn Ala Ile Pro Gly Gly Thr Asp Arg Ile Cys Thr Gly Ile Thr Ser

20 25 30

Ser Asn Ser Pro His Val Val Lys Thr Ala Thr Gln Gly Glu Val Asn

35 40 45

Val Thr Gly Val Ile Pro Leu Thr Thr Thr Pro Thr Lys Ser His Phe

50 55 60

Ala Asn Leu Lys Gly Thr Lys Thr Arg Gly Lys Leu Cys Pro Asn Cys

65 70 75 80

Leu Asn Cys Thr Asp Leu Asp Val Ala Leu Gly Arg Pro Met Cys Val

85 90 95

Gly Thr Thr Pro Ser Ala Lys Ala Ser Ile Leu His Glu Val Arg Pro

100 105 110

Val Thr Ser Gly Cys Phe Pro Ile Met His Asp Arg Thr Lys Ile Arg

115 120 125

Gln Leu Pro Asn Leu Leu Arg Gly Tyr Glu Asn Ile Arg Leu Ser Thr

130 135 140

Gln Asn Val Ile Asn Ala Glu Lys Ala Pro Gly Gly Pro Tyr Arg Leu

145 150 155 160

Gly Thr Ser Gly Ser Cys Pro Asn Ala Thr Ser Arg Ser Gly Phe Phe

165 170 175

Ala Thr Met Ala Trp Ala Val Pro Arg Asp Asn Asn Lys Thr Ala Thr

180 185 190

Asn Pro Leu Thr Val Glu Val Pro Tyr Ile Cys Thr Lys Gly Glu Asp

195 200 205

Gln Ile Thr Val Trp Gly Phe His Ser Asp Asn Lys Pro Gln Met Lys

210 215 220

Asn Leu Tyr Gly Asp Ser Asn Pro Gln Lys Phe Thr Ser Ser Ala Asn

225 230 235 240

Gly Val Thr Thr His Tyr Val Ser Gln Ile Gly Gly Phe Pro Asp Gln

245 250 255

Thr Glu Asp Gly Gly Leu Pro Gln Ser Gly Arg Ile Val Val Asp Tyr

260 265 270

Met Val Gln Lys Pro Gly Lys Thr Gly Thr Ile Val Tyr Gln Arg Gly

275 280 285

Val Leu Leu Pro Gln Lys Val Trp Cys Ala Ser Gly Arg Ser Lys Val

290 295 300

Ile Lys Gly Ser Leu Pro Leu Ile Gly Glu Ala Asp Cys Leu His Glu

305 310 315 320

Lys Tyr Gly Gly Leu Asn Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His

325 330 335

Ala Lys Ala Ile Gly Asn Cys Pro Ile Trp Val Lys Thr Pro Leu Lys

340 345 350

Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu

355 360 365

Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu

370 375 380

Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly

385 390 395 400

Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gln Glu Ala Ile Asn Lys

405 410 415

Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu Leu Glu Val Lys Asn Leu

420 425 430

Gln Arg Leu Ser Gly Ala Met Asp Glu Leu His Asn Glu Ile Leu Glu

435 440 445

Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gln

450 455 460

Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu

465 470 475 480

Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly

485 490 495

Pro Ser Ala Val Asp Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys

500 505 510

Cys Asn Gln Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala

515 520 525

Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala

530 535 540

Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr

545 550 555 560

Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Ile Ala Ile Phe

565 570 575

Ile Val Tyr Met Val Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu

580 585 590

(2) SEQ ID NO.18 information:

(i) sequence signature:

(A) length: 1802 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: B/Harbin/7/94rHA

(ix) feature:

(A) name/symbol: polyhedrin mRNA leading strand (part)

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: HA signal peptide sequence coding region

(B) position: 19 to 69

(ix) feature:

(A) name/symbol: SmaI restriction enzyme site

(B) position: 22 to 27

(ix) feature:

(A) name/symbol: ripe rHA coding region

(B) position: 70 to 1776

(ix) feature:

(A) name/symbol: KpnI restriction enzyme site

(B) position: 1780 to 1785

(ix) feature:

(A) name/symbol: Bg1II restriction enzyme site

(B) position: 1786 to 1791

(ix) feature:

(A) name/symbol: general translation termination signal

(B) position: 1792 to 1802

(xi) sequence description: SEQ ID NO.18:

TAAAAAAACC TATAAATAAT GCCCGGGAAG GCAATAATTG TACTACTCAT GGTAGTAACA 60

TCCAACGCAG ATCGAATCTG CACTGGGATA ACATCTTCAA ACTCACCTCA TGTGGTCAAA 120

ACAGCTACTC AAGGGGAAGT CAATGTGACT GGTGTGATAC CACTGACAAC AACACCAACA 180

AAATCTCATT TTGCAAATCT AAAAGGAACA AAGACCAGAG GGAAACTATG CCCAAACTGT 240

CTCAACTGCA CAGATCTGGA TGTGGCCTTG GGCAGACCAA TGTGTGTGGG GACCACACCT 300

TCGGCAAAAG CTTCAATACT CCACGAAGTC AGACCTGTTA CATCCGGGTG CTTTCCTATA 360

ATGCACGACA GAACAAAAAT CAGACAGCTA CCCAATCTTC TCAGAGGATA TGAAAATATC 420

AGATTATCAA CCCAAAACGT TATCAATGCA GAAAAAGCAC CAGGAGGACC CTACAGACTT 480

GGAACCTCAG GATCTTGCCC TAACGCTACC AGTAGAAGCG GATTTTTTGC AACAATGGCT 540

TGGGCTGTCC CAAGGGACGA CAACAAAACA GCAACGAATC CACTAACAGT AGAAGTACCA 600

TACGTTTGTA CAGAAGGAGA AGACCAAATT ACTGTTTGGG GGTTCCATTC TGATAACAAA 660

GCCCAAATGA AAAACCTCTA TGGAGACTCA AATCCTCAAA AGTTCACCTC ATCTGCTAAT 720

GGAGTAACCA CACATTATGT TTCTCAGATT GGCGGCTTCC CAGATCAAAC AGAAGACGGA 780

GGGCTACCAC AAAGCGGCAG AATTGTTGTT GATTACATGG TGCAAAAACC TGGGAAAACA 840

GGAACAATTG TCTATCAAAG AGGTGTTTTG TTGCCTCAAA AGGTGTGGTG CGCGAGTGGC 900

AGGAGCAAAG TAATAAAAGG GTCCTTGCCT TTAATTGGTG AAGCAGATTG CCTTCACGAA 960

AAATACGGTG GATTAAACAA AAGCAAGCCT TACTACACAG GAGAACATGC AAAAGCCATA 1020

GGAAATTGCC CAATATGGGT GAAAACACCT TTGAAGCTTG CCAATGGAAC CAAATATAGA 1080

CCTCCTGCAA AACTATTAAA GGAAAGGGGT TTCTTCGGAG CTATTGCTGG TTTCTTAGAA 1140

GGAGGATGGG AAGGAATGAT TGCAGGTTGG CACGGATACA CATCTCACGG AGCACATGGA 1200

GTGGCAGTGG CAGCAGACCT TAAGAGTACG CAAGAAGCCA TAAACAAGAT AACAAAAAAT 1260

CTCAATTCTT TGAGTGAGCT AGAAGTAAAG AATCTTCAAA GACTAAGTGG TGCCATGGAT 1320

GAACTCCATA ACGAAATACT CGAGCTGGAT GAGAAAGTGG ATGATCTCAG AGCTGACACT 1380

ATAAGCTCGC AAATAGAACT TGCAGTCTTG CTTTCCAACG AAGGAATAAT AAACAGTGAA 1440

GATGAGCATC TATTGGCACT TGAGAGAAAA CTAAAGAAAA TGCTGGGTCC CTCTGCTGTA 1500

GACATAGGGA ATGGATGCTT CGAAACCAAA CACAAGTGCA ACCAGACCTG CTTAGACAGG 1560

ATAGCTGCTG GCACCTTTAA TGCAGGAGAA TTTTCTCTCC CCACTTTTGA TTCACTGAAT 1620

ATTACTGCTG CATCTTTAAA TGATGATGGA TTGGATAATC ATACTATACT GCTCTACTAC 1680

TCAACTGCTG CTTCTAGTTT GGCTGTAACA TTGATGATAG CTATTTTTAT TGTTTATATG 1740

GTCTCCAGAG ACAATGTTTC ATGCTCCATC TGTCTGTGAG GTACCAGATC TTAATTAATT 1800

AA 1802

(2) SEQ ID NO.19 information:

(i) sequence signature:

(A) length: 586 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: polypeptide

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: B/Harbin/7/94rHA

(ix) feature:

(A) name/symbol: HA signal peptide

(B) position: 1 to 17

(ix) feature:

(A) name/symbol: ripe rHA

(B) position: 18 to 569

(xi) sequence description: SEQ ID NO.19:

Met Pro Gly Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn

1 5 10 15

Ala Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val

20 25 30

Val Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro

35 40 45

Leu Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr

50 55 60

Lys Thr Arg Gly Lys Leu Cys Pro Asn Cys Leu Asn Cys Thr Asp Leu

65 70 75 80

Asp Val Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala

85 90 95

Lys Ala Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe

100 105 110

Pro Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu

115 120 125

Arg Gly Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asn Ala

130 135 140

Glu Lys Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys

145 150 155 160

Pro Asn Ala Thr Ser Arg Ser Gly Phe Phe Ala Thr Met Ala Trp Ala

165 170 175

Val Pro Arg Asp Asp Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu

180 185 190

Val Pro Tyr Val Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly

195 200 205

Phe His Ser Asp Asn Lys Ala Gln Met Lys Asn Leu Tyr Gly Asp Ser

210 215 220

Asn Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr

225 230 235 240

Val Ser Gln Ile Gly Gly Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu

245 250 255

Pro Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Pro Gly

260 265 270

Lys Thr Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys

275 280 285

Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro

290 295 300

Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn

305 310 315 320

Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn

325 330 335

Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys

340 345 350

Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala

355 360 365

Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp

370 375 380

His Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp

385 390 395 400

Leu Lys Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn

405 410 415

Ser Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala

420 425 430

Met Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp

435 440 445

Asp Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu

450 455 460

Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala

465 470 475 480

Leu Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile

485 490 495

Gly Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu

500 505 510

Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro

515 520 525

Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly

530 535 540

Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser

545 550 555 560

Leu Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser

565 570 575

Arg Asp Asn Val Ser Cys Ser Ile Cys Leu

580 585

(2) SEQ ID NO.20 information:

(i) sequence signature:

(A) length: 1757 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: A/Johannesburg/33/94rHA

(ix) feature:

(A) name/symbol: polyhedrin mRNA leading strand (part)

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal peptide-coding region

(B) position: 19 to 72

(ix) feature:

(A) name/symbol: SmaI restriction enzyme site

(B) position: 76 to 81

(ix) feature:

(A) name/symbol: ripe rHA coding region

(B) position: 73 to 1731

(ix) feature:

(A) name/symbol: KpnI restriction enzyme site

(B) position: 1735 to 1740

(ix) feature:

(A) name/symbol: Bg1II restriction enzyme site

(B) position: 1741 to 1747

(ix) feature:

(A) name/symbol: general translation termination signal

(B) position: 1747 to 1757

(xi) sequence description: SEQ ID NO.20:

TAAAAAAACC TATAAATAAT GCCCTTGTAC AAATTGTTAA ACGTTTTGTG GTTGGTCGCC 60

GTTTCTAACG CGATTCCCGG GCAGGACCTT CCAGGAAATG ACAACAGCAC AGCAACGCTG 120

TGCCTGGGAC ACCATGCAGT GCCAAACGGA ACGCTAGTGA AAACAATCAC GAATGATCAA 180

ATTGAAGTGA CTAATGCTAC TGAGCTGGTT CAGAGTTCCC CAACAGGTAG AATATGCGAC 240

AGTCCTCACC GAATCCTTGA TGGAAAGAAC TGCACACTGA TAGATGCTCT ATTGGGAGAC 300

CCTCATTGTG ATGGCTTCCA AAATAAGGAA TGGGACCTTT TTGTTGAACG CAGCAAAGCT 360

TACAGCAACT GTTACCCTTA TGATGTGCCG GATTATGCCT CCCTTAGGTC ACTAGTTGCC 420

TCATCAGGCA CCCTGGAGTT TATCAACGAA AACTTCAATT GGACTGGAGT CGCTCAGGAT 480

GGGAAAAGCT ATGCTTGCAA AAGGGGATCT GTTAACAGTT TCTTTAGTAG ATTGAATTGG 540

TTGCACAAAT TAGAATACAA ATATCCAGCG CTGAACGTGA CTATGCCAAA CAATGGCAAA 600

TTTGACAAAT TGTACATTTG GGGGGTTCAC CACCCGAGCA CGGACAGTGA CCAAACCAGC 660

CTATATGTCC GAGCATCAGG GAGAGTCACA GTCTCTACCA AAAGAAGCCA ACAAACTGTA 720

ATCCCGGATA TCGGGTATAG ACCATGGGTA AGGGGTCAGT CCAGTAGAAT AGGCATCTAT 780

TGGACAATAG TAAAACCGGG AGACATACTT TTGATTAATA GCACAGGGAA TCTAATTGCT 840

CCTCGGGGTT ACTTCAAAAT ACGAAATGGG AAAAGCTCAA TAATGAGGTC AGATGCACCC 900

ATTGGCAACT GCAGTTCTGA ATGCATCACT CCAAATGGAA GCATTCCCAA TGACAAACCT 960

TTTCAAAATG TAAACAGGAT CACATATGGG GCCTGCCCCA GATATGTTAA GCAAAACACT 1020

CTGAAATTGG CAACAGGGAT GCGGAATGTA CCAGAGAAAC AAACTAGAGG CATATTCGGC 1080

GCAATCGCAG GTTTCATAGA AAATGGTTGG GAGGGAATGG TAGACGGTTG GTACGGTTTC 1140

AGGCATCAAA ATTCTGAGGG CACAGGACAA GCTGCAGATC TTAAAAGCAC TCAAGCAGCA 1200

ATCGACCAAA TCAACGGGAA ACTGAATAGG TTAGTCGAGA AAACGAACGA GAAATTCCAT 1260

CAAATCGAAA AAGAATTCTC AGAAGTAGAA GGGAGAATTC AGGACCTCGA GAAATATGTT 1320

GAAGACACTA AAATAGATCT CTGGTCTTAC AATGCGGAGC TTCTTGTTGC TCTGGAGAAC 1380

CAACATACAA TTGATCTAAC TGACTCAGAA ATGAACAAAC TGTTTGAAAG AACAAGGAAG 1440

CAACTGAGGG AAAATGCTGA GGACATGGGC AATGGTTGTT TCAAAATATA CCACAAATGT 1500

GACAATGCCT GCATAGGGTC AATCAGAAAT GGAACTTATG ACCATGATGT ATACAGAGAC 1560

GAAGCATTAA ACAACCGGTT CCAGATCAAA GGTGTTGAGC TGAAGTCAGG ATACAAAGAT 1620

TGGATTCTAT GGATTTCCTT TGCCATATCA TGCTTTTTGC TTTGTGTTGT TTTGCTTGGG 1680

TTCATCATGT GGGCCTGCCA AAAAGGCAAC ATTAGGTGCA ACATTTGCAT TTGAGGTACC 1740

AGATCTTAAT TAATTAA 1757

(2) SEQ ID NO.21 information:

(i) sequence signature:

(A) length: 571 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: polypeptide

(iii) hypothetical structure: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) organism: influenza virus

(C) individual strain isolated: A/Johannesburg/33/94rHA

(ix) feature:

(A) name/symbol: AcNPV 61K protein signal sequence

(B) position: 1 to 18

(ix) feature:

(A) name/symbol: ripe rHA

(B) position: 19 to 569

(xi) sequence description: SEQ ID NO.21:

Met Pro Leu Tyr Lys Leu Leu Asn Val Leu Trp Leu Val Ala Val Ser

1 5 10 15

Asn Ala Ile Pro Gly Gln Asp Leu Pro Gly Asn Asp Asn Ser Thr Ala

20 25 30

Thr Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr Leu Val Lys

35 40 45

Thr Ile Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val

50 55 60

Gln Ser Ser Pro Thr Gly Arg Ile Cys Asp Ser Pro His Arg Ile Leu

65 70 75 80

Asp Gly Lys Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His

85 90 95

Cys Asp Gly Phe Gln Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser

100 105 110

Lys Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser

115 120 125

Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Ile Asn Glu

130 135 140

Asn Phe Asn Trp Thr Gly Val Ala Gln Asp Gly Lys Ser Tyr Ala Cys

145 150 155 160

Lys Arg Gly Ser Val Asn Ser Phe Phe Ser Arg Leu Asn Trp Leu His

165 170 175

Lys Leu Glu Tyr Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn

180 185 190

Gly Lys Phe Asp Lys Leu Tyr Ile Trp Gly Val His His Pro Ser Thr

195 200 205

Asp Ser Asp Gln Thr Ser Leu Tyr Val Arg Ala Ser Gly Arg Val Thr

210 215 220

Val Ser Thr Lys Arg Ser Gln Gln Thr Val Ile Pro Asp Ile Gly Tyr

225 230 235 240

Arg Pro Trp Val Arg Gly Gln Ser Ser Arg Ile Gly Ile Tyr Trp Thr

245 250 255

Ile Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu

260 265 270

Ile Ala Pro Arg Gly Tyr Phe Lys Ile Arg Asn Gly Lys Ser Ser Ile

275 280 285

Met Arg Ser Asp Ala Pro Ile Gly Asn Cys Ser Ser Glu Cys Ile Thr

290 295 300

Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg

305 310 315 320

Ile Thr Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys

325 330 335

Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile

340 345 350

Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val

355 360 365

Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln

370 375 380

Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly

385 390 395 400

Lys Leu Asn Arg Leu Val Glu Lys Thr Asn Glu Lys Phe His Gln Ile

405 410 415

Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys

420 425 430

Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu

435 440 445

Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu

450 455 460

Met Asn Lys Leu Phe Glu Arg Thr Arg Lys Gln Leu Arg Glu Asn Ala

465 470 475 480

Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn

485 490 495

Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr

500 505 510

Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu

515 520 525

Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser

530 535 540

Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys

545 550 555 560

Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

565 570

(2) SEQ ID NO.22 information:

(i) sequence signature:

(A) length: 39 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO.22:

TGGTTGGTCG CCGTTTCTAA CGCGATTCCC GGGGGTACC 39

(2) SEQ ID NO.23 information:

(i) sequence signature:

(A) length: 13 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: polypeptide

(xi) sequence description: SEQ ID NO.23:

Trp Leu Val Ala Val Ser Asn Ala Ile Pro Gly Gly Thr

1 5 10

(2) SEQ ID NO.24 information:

(i) sequence signature:

(A) length: 43 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO.24:

TGGTTAGTCG CCTGTGCCTG CAGGCCAGAG AGGCCTTGGT ACC 43

(2) SEQ ID NO.25 information:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO.25:

GTCGCCGTGT CCAACGCG 18

(2) SEQ ID NO.26 information:

(i) sequence signature:

(A) length: 18 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO.26:

TAATTGGCCA GAGAGGCC 18

(2) SEQ ID NO.27 information:

(i) sequence signature:

(A) length: 39 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO.27:

GGGGGATCCG GTACCAGCAA AAGCAGGGGA TAATTCTAT 39

(2) SEQ ID NO.28 information:

(i) sequence signature:

(A) length: 38 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO.28:

GGGGGTACCC CCGGGGACTT TCCAGGAAAT GACAACAG 38

(2) SEQ ID NO.29 information:

(i) sequence signature:

(A) length: 44 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO.29:

CCCGGTACCG AATCATCCTA GAAACAAGGG TGTTTTTAAT TAAT 44

(2) SEQ ID NO.30 information:

(i) sequence signature:

(A) length: 47 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO.30:

GGGGAATTCG GTACCCCCGG GAAGGCAATA ATTGTACTAC

TCATGGT 47

(2) SEQ ID NO.31 information:

(i) sequence signature:

(A) length: 36 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO.31:

GGTACCCCCG GGGATCGAAT CTGCACTGGG ATAACA 36

(2) SEQ ID NO.32 information:

(i) sequence signature:

(A) length: 50 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(xi) sequence description: SEQ ID NO.32:

GGGGAATTCG GATCCGGTAC CTCACAGACA GATGGARCAA

GAAACATTGT 50

Claims (26)

1. the recombinant influenza HAO hemagglutinin of expressing in the baculovirus expression system in the insect cell of cultivating.

2. the albumen of claim 1 further comprises directly being coupled on the HAO albumen, does not contain to interleave amino acid whose baculovirus signal peptide.

3. the albumen of claim 1 comprises that further the medicinal acceptable carrier of going up is with as the vaccine medication.

4. the albumen of claim 1 further comprises adjuvant and the medicinal acceptable carrier of going up, as the vaccine medication.

5. the albumen of claim 3, wherein medicinal to go up acceptable carrier be the polymer transfer system.

6. the albumen of claim 1, wherein influenza virus is selected from influenza virus A strain and influenza virus B strain.

7. the albumen of claim 6, wherein the influenza infection mankind.

8. the albumen of claim 1 further comprises the another kind of albumen that merges with hemagglutinin.

9. the albumen of claim 8 is selected from hepatitis B virus albumen, HIV albumen, carcinomebryonic antigen and neuraminidase.

10. carrier that is used to make recombinant influenza HAO hemagglutinin, comprise following 5 '-＞3 ' sequence: from the polyhedrin promotor of baculovirus, the ATG translation initiation codon, signal peptide, coding is from the sequence of the ripe hemagglutinin of an influenzae strain virus, translation stop codon, and polyhedrin RNA polyadenylation signal.

11. the carrier of claim 10, wherein signal peptide is the baculovirus protein that molecular weight is about 61K, and aminoacid sequence is listed in preceding 18 amino acid of sequence table ID NO.7.

12. the carrier of claim 10, wherein signal peptide is the influenza hemagglutinin protein promotor.

13. the carrier of claim 10, the wherein sequence of coded signal peptide and hemagglutinin any amino acid that interleaves of not encoding.

14. the carrier of claim 10 further comprises second kind of proteic sequence of coding, this albumen and hemagglutinin are with the formal representation of fusion rotein.

15. the carrier of claim 10 is in the insect cell that transfection advances to cultivate.

16. method of making the recombinant influenza hemagglutinin, comprise with carrier and infect the insect cell of cultivating, that this carrier comprises is following 5 '-＞3 ' sequence: from the polyhedrin promotor of baculovirus, the ATG translation initiation codon, signal peptide, coding is from the sequence of the hemagglutinin of the influenza virus that is selected from influenza virus A strain and influenza virus B strain, translation stop codon and polyhedrin RNA polyadenylic acid signal, and in nutritional medium culturing cell.

17. the method for claim 16 further comprises and separate the HAO influenza hemagglutinin protein from cell, makes its purity be at least 95%.

18. the method for claim 17, wherein pass through under alkaline pH, from non-membranin, separate hemagglutinin, wash embrane-associated protein with the wash-out hemagglutinin, be incorporated into other albumen of anionite-exchange resin from other and separate hemagglutinin, and be incorporated into the albumen of Zeo-karb by changing salt concn from other and separate blood clotting and usually separate this albumen by changing change pH values.

19. method of making carrier, that this carrier comprises is following 5 '-＞3 ' sequence: from the polyhedrin promotor of baculovirus, the ATG translation initiation codon, signal peptide, coding is selected good strains in the field for seed from the sequence of the hemagglutinin of the influenza virus of influenza virus A strain and influenza virus B strain from one, translation stop codon and polyhedrin RNA polyadenylic acid signal, this method comprises

Results virus from cell culture medium, with regard to influenza virus A strain isolated viral RNA, or with regard to influenza virus B strain separating mRNA;

Synthetic cDNA, to viral RNA from the influenza virus A strain, the usefulness universal primer (5 '-AGCAAAAGCAGG-3 ' (SEQ ID NO.1)), to mRNA from the influenza virus B strain, use random primer, wherein 5 ' and 3 ' primer end contain the restriction enzyme site that does not have in the hemagglutinin gene;

Amplification is mixed with the segmental influenza virus A of hemagglutinin gene or B primer and influenza virus cDNA, produces the double chain DNA fragment that contains complete ripe hemagglutinin encoding sequence;

Identify the signal peptide of hemagglutinin gene, the hemagglutinin gene of the no signal peptide that increases then; With

The hemagglutinin gene of no signal peptide is cloned in the carrier that contains AcNPV polyhedrin promotor.

20. the method for claim 19 wherein is cloned into hemagglutinin gene on the carrier with PCR, thereby the signal peptide of this vector encoded is directly connected on the hemagglutinin, does not contain any amino acid that interleaves.

21. the method for claim 19 further comprises insect cell is advanced in the carrier transfection, selects to express the cell of hemagglutinin.

22. the method at influenza virus inoculation animal comprises to animal and uses the recombinant influenza hemagglutinin HAO that expresses in the baculovirus expression system in the insect cell of cultivating of significant quantity.

23. the method for claim 22 further comprises and uses the albumen that is present in the polymer transfer system.

24. the method for claim 22, wherein influenza virus is selected from influenza virus A strain and influenza virus B strain.

25. the method for claim 22, wherein animal is selected from Mammals and birds.

26. the method for claim 22, wherein animal is the people.

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