CN1791427A - Method and composition of a novel vaccine design for the prevention and treatment of SARS - Google Patents

Abstract

The invention disclosed a pharmaceutical composition that can induce immune response against SARS virus, and a method for using such pharmaceutical composition. Vaccines were designed to express multiple SARS gene products, including S1 domain, extracellular domain of orf8, N protein and others. It was demonstrated that the vaccines could effectively induce immune reaction and protect primates from SARS virus infection.

Description

The method for designing and the composition thereof of the new generation vaccine of prevention and treatment SARS

The application requires U.S. Provisional Patent Application No.60/474,507 priority, and the applying date is on May 31st, 2003.

Invention field

The present invention relates to viral vaccine, relate more specifically to the disease that causes is infected in SARS virus vaccine and prevention or treatment by SARS virus method.

Background of invention

SARS is a kind of respiratory system disease, and in November, 2002, reported first was found in the Guangdong Province of SOUTHERN CHINA.From then on this disease begins to extend, and the morbidity scope has surpassed 12 countries, and the whole world has 8,000 people of surpassing infected.Estimate that SARS patient's general mortality rate is 14-15%, and in the patient of over-65s, general mortality rate then surpasses 50%.

The general symptom of SARS comprises: heating surpasses 100.4F ⁰, dry cough, physical distress, dyspnea.Generally believe that SARS passes through interpersonal contact transmission closely, but also may be because people's contact stain article, touch nose and mouth again and catch.

The Center for Disease Control and some other breadboard scientist detect the coronavirus of not seeing before a kind of in SARS patient's body, and this new virus may be the main cause that causes SARS.

Summary of the invention

The invention provides the SARS virus vaccine of the nucleotide sequence that comprises one section encoding SARS coronavirus gene outcome.In concrete enforcement, the formation recombinant vector can link to each other the nucleotide sequence of the one or more SARS virus gene outcomes of coding with regulating and controlling sequence on the pharmacy compatibility carrier.

When concrete the application, the sars coronavirus gene is selected from S albumen, Orf8 albumen, M albumen, N albumen, E albumen, Orf1a albumen, Orf1b albumen and its association.Ingredient comprises a regulating and controlling sequence, is often referred to a promoter.For example, with the CMV promoter as promoter.

Pharmaceutical compatible carrier can be viral vector, plasmid vector or RNA carrier.For example, viral vector is selected replication-defective adenoviral for use.In concrete the application, employed adenovirus is 5 type replication-defective adenovirals.

In one embodiment, the SARS virus vaccine is made up of one section nucleic acid molecules that is cloned in two the sars coronavirus gene outcomes of coding on the pharmacy compatibility carrier, wherein first gene outcome comprises a sars coronavirus gene cell external structure territory, or derivatives thereof at least.Another gene outcome comprises an intracellular protein, or derivatives thereof.When concrete the application, first gene is selected from a SARS-CoV gene group, comprises S, M, Orf8 and E, and second gene is selected from the gene group of the SARS-CoV that comprises N, Orf1a and Orf1b.

In another embodiment, first gene outcome can be the proteic part of sars coronavirus S, the perhaps antigenic determinant of its domain, and perhaps its analog, wherein S albumen has the C-terminal of a truncate, has removed and has striden film grappling and cell intracellular domain.

In a relevant embodiment, first gene outcome comprises two sars coronavirus gene outcome fragments, first fragment comprises the antigenic determinant of the proteic part of sars coronavirus S or its domain, perhaps its analog, wherein S albumen has the C-terminal of a truncate, to remove striping grappling and cell intracellular domain; Another fragment comprises the proteic part of sars coronavirus Orf8, the perhaps antigenic determinant of its domain, and perhaps its homologue, wherein Orf8 albumen has the C-terminal of individual truncate, strides film grappling and cell intracellular domain to remove.

In another embodiment, the nucleic acid molecules of SARS vaccine is made up of the codon of optimizing, and these optimizations are to carry out according to the frequency of utilization of mammal cance high-expression gene codon.In a specific embodiment, through containing 50% GC base at least in the gene after optimizing.In another embodiment, the codon after at least 50% optimization is G or C base on the 3rd.

In another embodiment, the sars coronavirus gene outcome also comprises an excretory signal peptide fragment.When concrete the application, the signal peptide fragment can be the Igk signal sequence.

The SARS vaccine provides with the form of polypeptide.These polypeptide can be a kind of of above-mentioned any gene outcome, and these peptides have passed through abundant purification external.

The invention still further relates to a kind of in certain individuality, inducing, become to assign to realize by offering this individual said medicine at certain antigenic immunoreactive method.

In one embodiment, about 1 week of ingredient administration, 2 weeks, 3 weeks, 4 week or 6 weeks, perhaps, after the first administration, at different time administering drug combinations again.

In another embodiment, ingredient and an adjuvant administering drug combinations.

When concrete the application, the dosage of the ingredient of plasmid vector is per injection 1mg to 5mg.When Another Application, the dosage of the ingredient of viral vector is per injection 1 * 10 ⁶-1 * 10 ¹³Individual virion.

In one embodiment, ingredient is carried out the intramuscular injection administration.Perhaps, ingredient is carried out Intradermal, subcutaneous, intranasal or oral administration.

In another embodiment, induce the administration that comprises ingredient at a certain antigenic immunoreactive method, use the ingredient of different carriers to carry out the administration first time, to excite to above-mentioned antigenic immunoreation.

Description of drawings

Fig. 1 is the structural representation of synthetic genetic fragment.

Fig. 2 is that sars coronavirus, HPV and people's codon bias compares, and the G/C content of optimized gene is the meansigma methods of the G/C percentage ratio of two codon optimized back synthetic genes.

Fig. 3 is sketch map and the full length DNA sequence with fusogenic peptide of secretion signal.Fusion rotein comprises S1 domain and the proteic extracellular domain of Orf8.Two domains link by a flexible connexon.The sequence of encoding fusion protein is optimized at the optimal expression of primate.

Fig. 4 is S1-orf8 fusion rotein sketch map and the aminoacid sequence with secretion signal.

Fig. 5 is N proteose intention and full length DNA sequence.The sequence of this fusion rotein of encoding is optimized at the optimal expression of primate.

Fig. 6 is the proteic aminoacid sequence of N.

Fig. 7 is the mice T cells with antigenic specificity reaction after plasmid SV1000 immunity.Female Balb/c mice was gathered in the crops whole splenocytes 0 day, 14 days and immunity in 28 days at the 42nd day.With the length that covers whole gene 1 and gene 2 is that the peptide library of 20 aminoacid (it is overlapping that wherein each polypeptide and previous polypeptide have 10 aminoacid) stimulates splenocyte 20hr.Each peptide storehouse has 12 kinds of peptides approximately, and the final concentration of every kind of peptide is 1uM in the hole.

Fig. 8 is the antibody response of mice after the immunity of single injection SV8000 plasmid.

SARS coronary virus resistant IgG antibody titer is measured the sars coronavirus particle bag of use deactivation by elisa plate, carries out direct ELISA reaction.

Fig. 9 is to use recombinant adenovirus SV8000 to carry out immunity back mice IFNg t cell responses.The SV8000 of the cumulative dosage of intramuscular injection comes immune mouse, gathers in the crops whole lymphocytes after 3 weeks, and stimulates splenocyte with peptide library.The mice of using placebo group is with comparing (data not shown).

Figure 10 shows that the method for immune induction associating booster immunization has strengthened antibody response greatly.Use separately DNA, adenovirus, or DNA and adenovirus are united the use immune mouse.Before and after exciting, collects adenovirus blood serum sample.

Figure 11 is the situation that the single injection recombinant adenovirus vaccine is effectively induced the neutralizing antibody reaction in Rhesus Macacus.Three monkeys are injected placebo and 1E12 recombinant adenovirus granule (SV8000) respectively.Collect blood serum sample weekly, carry out the standard neutralization test, measure in it and titre with the serial dilution blood serum sample and the SARS virus incubation postoperative infection Vero E6 cell of living.

Figure 12 is the time situation of change of Rhesus Macacus antibody response.Before the ELISA test, the blood serum sample of different time points is done 40 times of dilutions.The enzyme of mensuration HRP is applied flexibly and is represented relative antibody titer.

Figure 13 is the response situation of total IgG antibody before and after the virus attack.Collect the animal serum sample of virus attack before 2 days and after 7 days respectively, carry out TPPA with 96 orifice plates of deactivation sars coronavirus granule bag quilt.

Figure 14 is the situation that detects sars coronavirus in the monkey that infects the PUMC-1 Strain.The 2nd day, the 5th day and the 7th day collection throat swab sample after virus attack.At first with every duplicate samples and DMEM incubation, and the filter with 0.22um filters before inoculation Vero cell.Negative value represents do not have visible cytopathic effect (CPE) after going down to posterity for continuous 3 times.Amount with the viral RNA that exists in the definite throat swab sample of quantitative RT-PCR reaction.According to workbook drawing standard curve to measure genomic copy number.

Figure 15 is the become celestial Histochemical studies result of lung tissue section of the Rhesus Macacus of sars coronavirus that infect to live.After virus attack, animal implemented euthanasia in the 7th day, the sample of taking from multiple organ is handled according to the described method of method part.

Lung tissue section lists at this as representative data, a. matched group; B/c. high dose group; D. low dose group; E/f. placebo group.The bubble position of arrow indication has been full of protein sample liquid, the transparent plasma membrane of arrow points placebo group.

Figure 16 is the immunohistochemical analysis result that SARS virus alive is attacked the lung tissue section that becomes celestial of monkey.Determine the antigen of lung tissue section, a. negative control with the monoclonal antibody of SARS coronary virus resistant; B. high dose group; C. low dose group; D. placebo group.Arrow points be SARS antigen presentation positive cells.

Detailed Description Of The Invention

Narrating these albumen and nucleotide sequence before the method for application in vaccination and the treatment and application, we should understand method, scheme, cell strain, carrier and reagent that the present invention is not limited in concrete narration here, because these can difference.Below describe in detail and be interpreted as term as used herein just in order to describe specific embodiment yet, its purpose is not in order to limit the scope of the invention, and claimed scope only is described in additional claim.

Must be noted that, unless tangible explanation is arranged, herein with accessory claim in employed singulative comprise plural form.Therefore, for example, a described host cell comprises the plural form of this host cell, and described antibody refers to one or more antibody, and its implication is that industry technical staff is known.

Unless otherwise indicated, all technology used in the present invention are identical with the general implication of understanding of industry those of ordinary skill with scientific terminology.Although any similar or be equal to method described herein and material and can be used for implementing, perhaps detect the present invention, now description preferred certain methods, equipment and material.Mentioned herein and publication all be in order to describe and illustrate cell strain, carrier and method, these all have report in publication, and related to the present invention.

Any point described here all can not be as the uncommitted reason that is disclosed in the content of first to file in advance of the present invention.

Definition

" nucleotide sequence " used herein is meant a kind of oligonucleotide, nucleotide or polynucleotide, and wherein fragment or part, derives from DNA or rna gene group or synthetic strand or two strands, both can be that positive-sense strand also can be an antisense strand.

Similarly, " aminoacid sequence " used herein is meant a kind of oligopeptide, peptide, polypeptide or protein sequence and a fragment or a part, and what can be that nature exists also can be synthetic molecule.

Here " aminoacid sequence " mentioned is meant a kind of aminoacid sequence of the protein molecule that exists naturally, " aminoacid sequence " and similar term, as " polypeptide " or " protein ", be not limited only to complete, the natural aminoacid sequence relevant with the protein molecular of being narrated.

" SARS gene " used herein is meant gene or the genetic fragment that sars coronavirus is coded.Some genes are by Marra, and MA et al (2003) described among the 1-16 at Sciencexpress.These genes include but not limited to following listed gene: Spike albumen (S), memebrane protein (M), nucleocapsid protein (N), replicative enzyme 1A, replicative enzyme 1B, little envelope E protein, orf3, orf4, orf6, orf8, orf9, orf10, orf11, orf13 and orf14.

" SARS albumen " used herein is meant by SARS gene or genetic fragment encoded polypeptides, these SARS albumen can fully purification be next from various species, mammal particularly, comprise cattle, sheep, pig, Mus, horse, especially human, the source can be natural, synthetic, semisynthetic or reorganization.

SARS used herein proteic " mutant " is meant the aminoacid sequence that wherein one or more aminoacid change.This " mutant " can contain the change of " guarding ", and promptly alternate aminoacid has on the similar structure and characteristic chemically, such as replacing leucine with isoleucine.Under the more rare situation, this mutant also can contain the change of " non-conservation ", such as replacing glycine with tryptophan.Similarly small sudden change also can be amino acid whose deletion or insertion, or deletes simultaneously and insert.Determine which amino acid residue should be replaced, insert or deletion and don't the method that influences biology or immunologic competence can obtain in existing computer software, for example, DNASTAR software.

" deletion " used herein is meant the variation of aminoacid or nucleotide sequence, and promptly one or more aminoacid or nucleic acid residue lack respectively.

" insertion " used herein or " interpolation " be meant with naturally occurring molecule and compare, the variation of aminoacid or nucleotide sequence and caused the increase of one or more aminoacid or nucleic acid residue respectively.

" substituting " used herein is meant that one or more aminoacid or nucleic acid are substituted by different aminoacid or nucleic acid respectively.

Term " biologic activity " is that finger protein has the function of naturally occurring molecule at aspects such as structure, adjusting or biochemistrys.Similarly, " immunologic competence " be meant natural, reorganization or synthetic SARS albumen or any relevant polypeptide cause corresponding animal or cell-specific immunoreation and with the ability of specific antibodies.

Term used herein " agonist " is meant a kind of molecule, thereby regulates these proteic activity when this molecule can cause these proteic changes with by the protein binding of SARS gene code the time.Agonist can be albumen, nucleic acid, carbohydrate or any other can with SARS albumen or the bonded molecule of protein fragments.

Term used herein " antagonist " or " inhibitor " are meant a kind of molecule, can suppress or regulate proteic biology of SARS or immunologic competence when this molecule combines with SARS albumen or protein fragments.Antagonist and inhibitor can be albumen, nucleic acid, carbohydrate or any other can with the protein bound molecule of SARS.

Term used herein " adjusting " is meant the change or the variation of the proteic biologic activity of SARS.Adjusting can be the enhancing or the reduction of protein active, or the change of binding characteristic, also can be SARS albumen in any other change of biology, function or immunological properties.

Term used herein " analogies " is meant a kind of molecule, the structure of this molecule be by SARS albumen or wherein the knowledge development of structure of a part come, and become class SARS protein molecular with part or all of SARS protein active.

Term used herein " derivant " is meant the chemical modification on the SARS gene that occurs in proteic nucleic acid of encoding SARS or coding.Illustrate, this chemical modification can be that hydrogen is replaced by hydroxyl, acyl group or amino group.This nucleic acid derivative peptide species of can encoding, and this polypeptide has kept the biological characteristics of natural molecule necessity.

Term used herein " purification fully " is meant that the nucleic acid that is separated to or aminoacid sequence are to separate or separate with the composition of relevant naturally other from natural environment, its purity is 60% at least, better reaches 75%, preferably can reach 90%.

" amplification " used herein is meant the production of the more multicopy of nucleotide sequence, (the Dieffenbach that realizes by well-known polymerase chain reaction (PCR) technology normally, C.W.and G.S.Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

" hybridization " used herein is meant that any nucleic acid chains and a complementary strand are by the bonded process of base pairing.

Term used herein " hybridization complex " is meant that two nucleotide sequences are by forming the complex that hydrogen bond is combined between complementary bases G and the C and between complementary base A and the T.These hydrogen bonds also can become more stable by the base stacking effect.Article two, the hydrogen bond of complementary nucleotide sequence has a kind of antiparallel conformation.This hybridization complex can form (as C.sub.Ot or R.sub.Ot analysis) in solution, also can be that a nucleotide sequence is in solution and another nucleotide sequence is fixed on (as film, filter, chip, needle point or microscope slide etc., cell is fixed and carries out in situ hybridization) on a kind of solid support.

Term used herein " complementary " or " complementarity " are meant that many nucleotidess are natural combination by base pairing under the condition of suitable salinity and temperature.For example, sequence " A-G-T " combine with complementary series " T-C-A ".Article two, the complementarity between the single chain molecule can be " part ", promptly has only some nucleic acid combinations, and this complementarity also can be complete, and is promptly fully complementary between two single chain molecules.The interchain complementary degree of nucleic acid greatly affects the efficient and the intensity of their hybridization.This is even more important in amplified reaction, because amplified reaction relies on the combination of two complementary strands.

Term used herein " homology " is meant complementary degree.Can be homeologous, also can be complete homology (as, concordance).Article one, the complementary sequence of part can partly suppress the hybridization of a same sequence and target nucleic acid at least; This sequence also can be represented with functional term " fully homology ".The hybridization of fully-complementary sequence and target sequence is suppressed situation can detect (Southern or northern blot, solution hybridization and other similar experiments) by the hybrid experiment of low preciseness.Under low strict degree condition, abundant homologous sequence or probe can and suppress combine (hybridization just) of complete homologous sequence with complete homologous sequence and probe competition.This is not to allow nonspecific bonded generation under the condition of low strict degree; It is specific (just, optionally) reaction that low strict degree condition needs the combination between two sequences.Whether do not have nonspecific reaction needed to detect with the second aim sequence, this target sequence should be (being lower than 30% as similarity) of having only the complementarity of fraction; Owing to there is not non-specific binding, probe just can not hybridized with the non-complementary target sequence of second so.

As everyone knows, many suitable condition elements can be used for constituting the condition of low strict degree or high strict degree.The length of many factors such as sequence and character (DNA, RNA, base complex), as the existence of Methanamide, dextran sulfate and/or polyethylene glycol whether the character of target sequence (DNA, RNA, base complex, exist in solution still fixing etc.), salt and other component concentrations () etc. all should be taken into account, though the difference of hybridization solution can produce the different suitable low strict degree with above-mentioned condition or the condition of high strict degree.

Term used herein " preciseness " is meant " from being lower than Tm-5 ℃ (being lower than 5 ℃ of probe melting temperatures) to being lower than Tm20-25 ℃ " strict degree ".The personnel that possess Professional knowledge can both understand, and the strict degree of hybridization can change, and are used for differentiating or detect the sweet acid sequence of multinuclear same or that be correlated with.

Term used herein " antisense " is meant and a specific DNA or the complementary nucleotide sequence of RNA sequence.Term used herein " antisense strand " is meant and the complementary nucleic acid chains of " justice " chain.Antisense molecule can generate by any method, comprises by supporting the synthetic viral promotors of complementary strand oppositely to be connected to come synthetic with one interested gene.In case enter cell, this transcribes chain will be combined into dimer with the native sequences that generates in the cell.These dimers will suppress further to transcribe or translate then.Just can produce the phenotype of sudden change in this way." feminine gender " is used to refer to antisense strand sometimes, and " positive " is used to refer to positive-sense strand sometimes.

Term used herein " part " is meant this proteic fragment at a kind of albumen (as " part of certain specific protein ").This segmental size can be from 4 amino acid residues to only lacking an aminoacid than whole aminoacid sequence.Therefore be somebody's turn to do the albumen of " containing the proteic a part of aminoacid sequence of SARS at least " and comprise total length SARS albumen and fragment thereof.

Here Ding Yi " conversion " is meant that foreign DNA enters and change the process of recipient cell.This process can realize by well-known variety of way under natural or artificial condition.Conversion can depend on and anyly existing exogenous nucleic acid sequences is inserted into method in protokaryon or the eukaryotic host cell.This method is based on that situation that host cell transformed selects, and can include but are not limited to following these methods: virus infection method, electroporation, lipofection and particle bombardment method.The cell of this " conversion " comprises stable transformant, and promptly the DNA of Cha Ruing can duplicate as plasmid that can self-replicating or as a chromosomal part.Also comprise simultaneously the only DNA of transient expression insertion or the transformant of RNA in the limited time.

Term used herein " antigenic determinant " is meant the part (i.e. epitope) of the molecule that connects with specific antibody.When coming immune host animal with an a kind of albumen or a proteic part, production of antibodies can be caused in these proteic many zones, and these antibody can combine with this proteic specific region or three dimensions conformation; These zones or conformation are exactly said antigenic determinant.Antigenic determinant can with the complete antigen immunogen of immune response stimulating (promptly be used for) competitively with antibodies.

Term used herein " special combination " or " specificity in conjunction with " are that to refer in particular to this interaction when the interaction of mentioning between antibody and albumen or the polypeptide be to depend on a kind of specific structure/conformation of the existence on the protein molecular (antigenic determinant or epitope); In other words be exactly that antibody is in identification and is not only albumen itself in conjunction with a kind of particular proteins structure/conformation.For example, if a kind of antibody is special to epi-position A, in the reaction system that contains " A " that be labeled and antibody, adds a kind of albumen (unlabelled A) that has epi-position A so and will reduce the A of labelling and the level of antibodies.

Term used herein " sample " is a generalized concept, be meant any biological specimen that may contain encoding SARS albumen and segmental nucleic acid thereof, comprise cell, from cell the extract of isolating chromosome (as the chromosome of metaphase in cell division), genomic DNA (be in the solution or and be incorporated on the solid support), RNA (be in the solution or picture analyze be incorporated on the solid support in testing), cDNA (be in the solution or be incorporated on the solid support), cell or tissue at Northern as analyzing in the experiment at Southern, or the like.

Term used herein " is expressed relevant with polynucleotide " to be meant to analyze to detect in the experiment at Northern and is indicated in the sample with the ribonucleic acid similar by the SARS virus polynucleotide encoding and to exist the proteic mRNA of encoding SARS, thereby relevant with the expression of the transcript of these proteic polynucleotide of encoding.

" change " of the polynucleotide by the SARS gene code used herein comprised any change of the sweet acid sequence of multinuclear of SARS gene code, the point mutation that comprises deletion, inserts and can detect with hybrid experiment.Also include the detection, SARS virus polynucleotide encoding fragment of the change of the proteic genomic dna sequence of encoding SARS and the disappearance (as using homotopic special oligonucleotide) and the incorrect or unexpected hybridization of the ability of genomic DNA sample hybridization in this definition, in addition site of the normal chromosomal locus that occurs in the proteic polynucleotide sequence of encoding SARS as hybridization (as to metaphase of cell division chromosome carry out fluorescence in situ hybridization).

Term used herein " antibody " is meant complete antibody molecule and fragment thereof, as can with the bonded Fab fragment of antigenic determinant, F (ab ') ₂Fragment and Fv fragment.Originally prepared as immunity with enough complete polypeptide of the protein bound antibody capable of SARS or little peptide section.It is can be with corresponding RNA translation synthetic or directly synthesize with the method for chemistry to be used for the polypeptide of immune animal or peptide, if necessary, also can combine with carrier protein.Normally used carrier connects by chemical bond with polypeptide, comprises bovine serum albumin and Elityran.The polypeptide of Lian Jieing just can be used for immune animal (as mice, rat or rabbit) then.

Term used herein " humanized antibody " is meant the replaced antibody molecule of aminoacid of non-antigen binding domain, makes its more approaching mankind's antibody with this, but still keeps primary binding characteristic.

Term used herein " carrier " be meant any can be under adapt circumstance the nucleotide or the nucleotide sequence of (as cell and external not celliferous transcribing/translation system) express polypeptide.Carrier includes but are not limited to DNA plasmid, virus expression carrier and rna replicon.

Preferred embodiment is described in detail

The invention provides the design of the new generation vaccine of a kind of prevention and treatment SARS, comprise single or polygenes and genetic fragment, gene outcome, albumen and the protein fragments of sars coronavirus.

The genome of SARS virus is the sub-thread positive chain RNA, because the Lo-Fi of the RNA polymerase that RNA relies on, the SARS genome may experience quick sudden change.In fact, several in the world research institutions check order to the genome of sars coronavirus, and the result is committed to the data base, as GenBank.The sequence alignment of standard shows, exists the minority sequence to change between the different virus separated strain.

For the high virus of mutation rate, there is huge challenge in the exploitation of its effective vaccine, because the immunoreation of a certain strain virus is improved, may other variant not had effect.SARS virus also same problem can occur.For the influence that virus variation is brought drops to minimumly, can be simultaneously elect the genomic polygenes of SARS virus or albumen the target of vaccine as.

In one embodiment of the invention, can express one or more SARS gene encoding productions, or the carrier of one section fragment or derivatives thereof wherein, can be used as vaccine with prevention or treatment SARS.These genes comprise spike glycoprotein (S), membrane glycoprotein (M), nucleocapsid protein (N), replicative enzyme 1A, replicative enzyme 1B, little envelope E protein, orf3, orf4, orf6, orf8, orf9, orf10, orf11, orf13 and orf14, but are not limited thereto.These genes are called SARS gene (seeing definition).Wherein portion gene is existing describes (Marra, MA etc., 2003, Sciencexpress.1-5).The manufacture method of vaccine also have description (Raz, E etc. 1994, Proc.Natl.Acad.Sci.USA, 91,9519-9523; Donnellly, J.J etc., 1996, J.Infect.Dis., 173,314-320).In a specific embodiment, can express S and N for one, or its fragment, the carrier of or derivatives thereof can be as the vaccine of prevention or treatment SARS.

In another embodiment, two or more SARS albumen (seeing definition) or fragment or derivatives thereof can be expressed in a suitable hosts, can be used as the vaccine of prevention and treatment SARS.

In another embodiment, the carrier that can express the fusion rotein of two or more SARS genetic fragment or derivatives thereofs can be as the vaccine of prevention and treatment SARS.These genes can directly connect, or connect by one or more amino acid whose connexons.

In another embodiment, the fusion rotein carrier that can express virus surface proteins extracellular domain or derivatives thereof, the vaccine that can be used as prevention and treat SARS.These virus surface proteins comprise spike glycoprotein (S), membrane glycoprotein (M), little envelope protein (E), orf3 and orf8, but do not limit therewith.In a specific embodiment, can express S and orf8 extracellular domain for one, or genetic fragment, the fusion rotein carrier of or derivatives thereof can be as the vaccine of prevention or treatment SARS.

In another embodiment, the extracellular domain of two or more genes of S, M, E, orf8 and orf3, or its fragment, or derivatives thereof can merge, in suitable body,, can be used as the vaccine of prevention or treatment SARS with the formal representation of fusion rotein.In specific embodiment, the extracellular domain of S and orf8, perhaps a fragment or derivatives thereof can merge, and can give expression to fusion rotein, is used for prevention or treatment SARS.

In another embodiment, the combination that can express the SARS fusion rotein, or fragment, the carrier of or derivatives thereof and single SARS albumen or protein fragments can be used for prevention and treatment SARS.

The nucleotide sequence of any encoding SARS gene, or its fragment can be expressed SARS gene or its segmental recombinant molecule as producing.In specific embodiment, the present invention includes the application of the polynucleotide of coding S, orf8 and N.

Industry technical staff appreciates the merger of genetic code very much, because can produce the nucleotide sequence of the very little encoding SARS gene of numerous and any dna homologs known and that exist naturally like this.Therefore, the present invention fully takes into account each possible variant nucleic acid sequence, selects according to possible codon, reaches goal of the invention by selecting various combination nucleic acid codon.The state that establishing criteria triplet genetic code is applied to the SARS gene of nature existence comes the pin design sub-portfolio, below specific descriptions will be arranged to these variations of considering.

Under appropriate strict degree condition, the nucleotide sequence of encoding SARS gene and its variant can be well and spontaneous SARS genetic fragment hybridization, but some the time select use to be different from spontaneous codon may be more favourable.In indivedual protokaryons and eukaryotic cell host, can select unique codon according to the frequency of utilization of host's codon, thereby increase its expression rate.The other reasons that changes the nucleotide sequence of SARS gene and its derivant and do not change amino acid sequence coded comprises the rna transcription product that obtained by it than having better characteristic by the product that exists sequence to transcribe naturally to obtain, such as the half-life longer.

The DNA sequence of encoding SARS gene or its partial sequence or derivatives thereof can obtain by chemical synthesis process, and synthetic sequence can be inserted any available expression vector and cell system.In addition, with the method for chemosynthesis the sequence of encoding SARS gene is introduced sudden change, comprise disappearance in the present invention, insert or replace different nucleoside, the result can produce coding albumen identical or that function is identical.Encoded protein also may comprise the replacement of disappearance, insertion or amino acid residue, and this will produce nonsense mutation, and the result produces the SARS gene of functional equivalent.Polarity, electric charge, dissolubility, hydrophobicity, hydrophilic and/or amphiphilic characteristics according to amino acid residue, can carry out purposive aminoacid replaces, as long as the corresponding biologic activity of these albumen can keep,, but be not restricted to this as immunogenicity.For example, the aminoacid of positively charged can be aspartic acid and glutamic acid, and electronegative aminoacid can be lysine; The polar aminoacid group of neutral has similar hydrophilic, comprises leucine, isoleucine and valine, glycine and alanine, glutamate, Glu, serine and threonine, phenylalanine and tyrosine.

Scope of the present invention also comprises the allelic use of encoding SARS gene.Erect image is used herein to be arrived, and one " allele " or " allelic sequence " is the form that gene can be selected, and this may be because at least one sudden change of nucleotide sequence causes.Allele may cause the change of mRNA or polypeptide, and its structure or function may change also and may not change.Any known gene may not exist, and may have one or more allelic forms yet.Can cause allelic routine suddenly change nucleoside normally disappearance naturally, add or replace.These mutant forms can take place separately, also can take place with other, can come across in the known array by one or many.

The method of dna sequencing is the method that the industry is known and used always, and can be applied to the bright any embodiment of we.The enzyme that this method is used has Klenow fragment dna polymerase i, SEQUENCE archaeal dna polymerase (U.S. BiochemicalCorp, Cleveland, Ohio), Taq polymerase (Perkin Elmer), heat-resisting T7 polymerase (Amersham, Chicago Ill) or use in conjunction reorganization polymerase and have the exonuclease of calibration function, as ELONGASE amplification system (GIBCO/BRL, Gaithersburg, Md).Preferably, the order-checking process can be carried out on machine automatically, as HamiltonMICROLAB 2200 (Hamilton, Reno, Nev.), Peltier thermal cycler (PTC200, MJ Research, Watertown is Mass.) with ABI 377DNA sequenator (Perkin Elmer).

Polymerized nucleoside acid sequence or its fragment of encoding SARS gene, gene outcome, fusion rotein or its functional similarity thing can be used for recombinant DNA molecules, in appropriate host cell, can instruct protein expression.Owing to genetic code annexs property, may produce the DNA sequence of the aminoacid sequence of other encode quite similar or functional equivalents, these sequences can be used for the clone and express these albumen.

The positive copy industry technology personnel understood, and the codon that uses non-natural to exist has the nucleotide sequences that helps produce the encoding SARS gene.For example special protokaryon and eucaryon host may more preferred codons, with the efficient that increases protein expression or produce optimize the recombinant RNA transcription, for example its half-life is longer than the transcription that naturally occurring sequence produces.

For the sequence of different purpose change SARS gene codes, can use the known technological transformation nucleotide sequences of industry technical staff, comprise expression, but be not restricted to this clone's modification, processing and/or gene outcome.Random fragment DNA resets, and the PCR of genetic fragment recombinates and synthesizing of oligonucleotide can be used to transform nucleotide sequence.For example rite-directed mutagenesis can be used for inserting new restriction endonuclease sites, changes glycosylation pattern, changes codon bias, produces to shear variant or introduce sudden change etc.

In another embodiment of invention, encoding SARS gene natural, nucleotide sequence after the modification or reorganization can be connected in the xenogenesis sequence, with encoding fusion protein, is used for the diagnosis of SARS, prevention and treatment.For example, screen supressor protein from peptide library, the SARS dna chimeric gene may be useful, because commercial antibody can cognitive this fusion rotein.During construction of fusion protein, can add shearing site between SARS gene coded sequence and heterologous sequence, S or ORF8 can shear from allos part and purification comes out like this.

Can adopt chemical method well known in the art completely or partially the sequence of composite coding SARS gene (referring to Caruthers, M H. etc., 1980, Nucl.Acids Res.Symp.7:2315-223; Horn, T. etc., 1980, Nucl.AcidsRes.Symp, 7:225-232).Also can select the aminoacid sequence of the synthetic SARS gene of chemical method or its part.For example use different solid phase techniques (Roberge, JY., 1995, Science.269:202-204) and automatically synthetic technology is carried out the synthetic of peptide, for example uses the peptide synthesizer (Perkin Elmer) of ABI company.

New synthetic peptide can be highly purified by high-efficient liquid phase chromatogram technology (as, Creighton, T.1983, Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.).The composition of synthetic peptide can confirm by amino acid analysis or order-checking (as the Edman edman degradation Edman, Creghton, supra).In addition, when directly synthetic, and/or be connected with other albumen complete sequences or partial sequence with chemical method, when producing a variant peptides, SARS aminopeptidase gene acid sequence or its any part all may change.

In order to express a SARS gene with biologic activity, the nucleotide sequences of encoding SARS gene or its functional similarity thing can be inserted suitable expression, carrier comprises that insertion sequence is necessary and transcribes and translate element.

Use the known method of industry technical staff, can make up the expression vector that comprises the encoding SARS gene order and suitably transcribe and translate control element.These methods comprise the extracorporeal recombinant DNA technology, genetic recombination techniques in synthetic technology and the body.Said method can be with reference to Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F.M.et al. (1989) Current Protocols inMolecular Biology, John Wiley﹠amp; Sons, New York, N.Y..

The expression vector and the host system that can hold and express the sequence of encoding SARS gene or gene outcome have many kinds; comprise microorganism; as transformed recombinant phage; the antibacterial of plasmid or cosmid DNA expression vector; the yeast that Yeast expression carrier transforms; the insect cell system of infective virus expression vector (as adenovirus or baculovirus); the transforming virus expression vector is (as Brassica oleracea L. var. botrytis L. mosaics virus; tobacco mosaic virus (TMV)) plant cell system; or the plant cell system of transform bacteria expression vector (as Ti or Pbr322 plasmid); or the zooblast system, but be not limited thereto.

" control sequence " or " regulating and controlling sequence " is carrier enhancer, promoter, 5 ' and 3 ' untranslated region, and these zones interact with host cell proteins, to transcribe and to translate.The intensity of these elements may be different with specificity.Different according to employed carrier system with the host, can use the suitable element of transcribing and translate, these elements comprise the basis and inducible promoter.For example, when being cloned into bacterial system, (Calif) or PSPORT1 plasmid (Gibco BRL), or its similar products can use for Stratagene, LaJolla as the heterozygosis lacZ inducible promoter of BLUESCRIPT phagemid.In insect cell, can use bacilliform virus promoter.Derive from the genomic promoter of plant cell, enhancer (as heat shock, RUBISCO and storage protein gene) or plant virus (as viral promotors or leader) and can be cloned into carrier.In the mammal cell line system, preferably derive from the promoter of mammalian genes or mammalian virus.If must obtain to comprise a plurality of copies of encoding SARS gene order cell strain, can use the carrier that has suitable selected marker based on SV40 or EBV.

In bacterial system,, can select multiple expression vector for use according to the purpose that the SARS gene uses.For example, when a large amount of gene induced antibody of SARS of needs, can use easy purification, directly influence the carrier of fusion rotein high level expression.Similar substrates has multi-functional E.coli clonal expression carrier, as BLUESCRIPT phagemid (Stragene), can be with the sequence insertion vector of encoding SARS gene, the N-terminal methionine of SARS gene is in the reading frame of carrier, and the C-end follows the beta galactosidase of 7 residues closely.Also can select pIN carrier (Van Heeke for use, G.and S.M.Schuster (1989) J.Biol.Chem.264:5503-5509) and similar PGEX carrier (Promega, Madison, Wis.) have the hybrid protein of glutathione transferase (GST) with the formal representation of fusion rotein, but be not limited thereto.Generally speaking, these albumen are solubilities, can obtain from cell lysis easily, can make it be adsorbed in the glutathione agarose gel beads with it by pillar, get final product eluting when no glutathion exists.The albumen that is designed for this system's generation has heparin, thrombin, XA factor protein enzyme shearing site, and like this, destination protein can discharge from heterozygote easily.

At yeast, in the beer yeast, there are many carriers that carry composing type or inducible promoter can be used the such promoter such as a factor, Alcohol oxidase and PGH etc.Referring to Ausubel etc., (supra) and Grant etc., (1987), Methods Enzymol.153:516-544.

In the example that uses plant expression vector, can use most promoteres to start the expression of the gene order of encoding SARS.Viral promotors for example, the 35S of CaMV and 19S promoter can be used separately, also can unite use (Takamatsu, N. (1987) EMBO are J.6:307-311) with the ω leader of TMV.Also can use plant promoter in addition, as the little subunit of RUBISCO, or the heat shock promoter.(Coruzzi, G etc., 1984, EMBO is J.3:1671-1680; Broglie, R. etc., 1984, Science 224:838-843; Winter, J. etc., 1991, Results Probl.Cell Differ.17:85-105).These carriers that build can transform or the transfection of pathogen mediation and introduced plant cell by direct DNA.These technology in some summaries, have description (referring to as Hobbs, S or Murry, L.E.inMcGraw Hill Yearbook do Science and Technology (1992) McGraw Hill, New York, N.Y.; Pp.191-196).

The insecticide system also can be used for expressing the SARS gene.For example, in such system, autographa california nuclear polyhedrosis virus (AcNPV) is as carrier, can be in fall army worm cell or powder mosquito moth larvae expression alien gene.The sequence of encoding SARS gene can be cloned into the nonessential position of virus, and for example romote n gene puts it under the control of romoten gene.The successful insertion of SARS gene will make and produce the recombinant virus that lacks envelope protein by romote n inactivation of gene.With recombinant virus infected spodoptera frugiperda cell or powder mosquito moth larvae, to express S or ORF8 (Engelhard, E.K.et al. (1994) Proc.Nat.Acad.Sci.91:3224-3227).

In mammalian host cell, can adopt multiple expression system based on virus.In using the example of adenovirus as carrier, the sequence of coding S or ORF8 can be transcribed/translate complex with the adenovirus of being made up of late promoter and three leaders and is connected.Can obtain a great-hearted virus at nonessential E1 of viral genome and E3 position insertion fragment, this virus can be expressed SARS gene (Logan, J.and Shenk, T in the host cell that infects, 1984, Proc.Natl.Acad.Sci.81:3655-3659).In addition, transcriptional enhancer, for example Rous sarcoma virus (RSV) enhancer can be used for increasing the expression of mammalian host cell.

Special initial signal can improve the efficient of encoding SARS gene order translation, and these signals comprise ATG start codon and contiguous sequence.In the example of encoding SARS gene order, its start codon and upstream sequence are inserted suitable expression, do not need other to transcribe or translate control signal.Yet, when having only coded sequence or part to insert, need provide the external source translation that comprises start codon ATG control signal.In addition, start codon must be positioned at correct reading frame, to guarantee the correct translation of whole insertion sequence.External source translation element may be different with the start codon source, but all are natural or synthetic.Described in the erect image document, in specific expression system, adding enhancer, the efficient of its expression can increase (Scharf, D. etc., 1994, Results Probl.Cell Differ.20:125-162).

In addition, can process proteic ability, select host cell strain according to the expression of regulation and control insertion sequence or according to desired mode.The modification of these polypeptide comprises, acetylation, and carboxylation, glycosylation, phosphorylation, fatization, and acidylate, but be not limited thereto.The translation post-treatment can be the shear protein precursor, also will help proofreading and correct inserting, folding and/or function.Different host cells as CHO, HeLa, MDCK, HEK293 and WI38, at the post translational modification activity, all has its unique organelle and mechanism features, can select as required, with modification and the processing of guaranteeing that foreign protein is correct.

On long terms, recombiant protein output height, and can stably express be first-selected.For example, the cell strain of stably express SARS gene can comprise the reproduction element and/or the internal representations element of viral source by conversion, and a selected marker's expression vector obtains, and these elements and marker gene are present in same carrier or independently in the carrier.After introducing these carriers, cell was cultivated 1-2 days in abundant culture medium, was transferred in the selective medium afterwards.The purpose of selected marker is that resistance is selected, and there be the growth and the acquisition of cell of calling sequence that allowed successful expression in it.As long as use the tissue culture technique that is fit to cell type, the resistance clone of stable transformed cells can amplification culture.

In order to obtain the transformant strain, most selective systems can be used, comprise herpes simplex virus thymidine kinase (Wigler, M. etc., 1977, Cell.11:223-32) and adenine phosphoribosyl transferase (Lowy, I. etc., 1980, Cell.22:817-23), be respectively applied for tk or aprt cell, antimetabolite, antibiotic, or herbicide also can be used as the resistance selected marker, for example dhfr is to resistance (Wigler, M. etc., 1980 of methotrexate, Proc.Natl.Acad.Sci.77:3567-70), npt is to resistance (Colbere-Garapin, F. etc., 1981 of aminoglycoside neomycin and G-418, J.Mol.Biol.150:1-14), (Murry supra), but is not restricted to this to the resistance of grand to chlorine the sulphur respectively and PPT Acetylase of als or pat.Other gene that can select also has description, as the trpB gene, can make cell substitute tryptolphan with indole; The hisD gene can make cell use histinol alternate sets propylhomoserin (Hartman, S.C.and R.C.Mulligan (1988) Proc.Nat.Acad.Sci.85:8047-51).Recently, everybody likes using visual labelling, as the pattern glucosides, β-Pu Taotanggansuanmei and its substrate GUS, luciferase and its substrate fluorescein, it uses extensively, not still in order to identify transformant, and be in order to determine a certain specific support system instantaneous or the proteic quantity of stably express (Rhodes, C.A. etc., 1995, Methods Mol.Biol.55:121-131).

Can show whether genes of interest exists although whether marker gene is expressed, its existence and expression need to confirm equally.For example, if the sequence of encoding SARS gene is inserted in the marker gene, the disappearance of marker gene function then can be used for identifying that can reconstitution cell express the SARS gene.Perhaps, in a promoter downstream, then under drug-induced and selectivity culture condition, the expression of marker gene has also indicated the SARS expression of gene usually with the S of SARS or ORF8 gene and marker gene arranged in series.

In addition, the authentication method that comprises the host cell expression SARS gene of encoding SARS gene nucleotide series is that industry technical staff is known, comprise DNA-DNA or DNA-RNA hybridization, measure or immunoassay as the protein biology of film and solution etc., and nucleic acid or Protein Detection and/or quantitative chip technology, but be not restricted to this.

The polynucleotide of encoding SARS gene order can be hybridized by DNA-DNA, DNA-RNA hybridization, or detects with the method that polymerized nucleoside acid probe, its partial sequence or the fragment of encoding SARS gene increases.Use the oligonucleotide or the oligomer of coding S or ORF8 sequence to carry out the nucleic acid amplification test, comprise coding S or the DNA of ORF8 or the transformant of RNA with detection.Used herein, " oligonucleotide " or " oligomer " refers to comprise at least the sequence of 10 nucleotide, is about the sequence of 60 nucleotide at most, preferably about 15-30 nucleoside, be most preferably 20-25 nucleoside, these " oligonucleotide " or " oligomer " can be used as probe or amplimer.

The scheme that detects and measure S or ORF8 expression has a variety of, typically use the known scheme of the industry, for example at certain proteic polyclone or monoclonal antibody. elisa (ELISA), radioimmunoassay (RIA), flow cytometer (FACS).The first-selected immune analysis method of using respectively cognitive two the non-interfering SARS gene epi-positions of two monoclonal antibodies, the also method that can test with competitive birding.Above-mentioned analytical method is seen Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D.E.et al. (1983; J.Exp.Med.158:1211-1216).

The known diversified labelling of the technical staff of the industry and be coupled technology can be used for different nucleic acid and test amino acid.The probe mark method comprises the labelling oligomer, nick translation, and end labelling or carry out pcr amplification with a kind of nucleotide of labelling, these probes are used to detect the sequence relevant with the polynucleotide of encoding SARS gene for hybridization or PCR probe.In addition, SARS gene or other any protein gene can be cloned into carrier, obtain the mRNA probe.So carrier commercialization gives suitable R NA polymerase such as T7, behind the nucleotide of T3 or SP6 and labelling, and can be at the vitro synthesized RNA probe.The visible commercial reagents box explanation of preparation method (Pharmacia﹠amp; Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); And U.S.Biochemical Corp., Cleveland, Ohio).Suitable label and reporter molecules comprise radionuclide, enzyme, fluorescein, chemiluminescent substance, chromophore and substrate, cofactor, mortifier, magnetic bead etc.

Change the condition of culture of the host cell of encoding SARS gene nucleotide series over to, should be suitable for proteic expression, and be suitable for from cell culture fluid, gathering in the crops albumen.According to the difference of employed sequence and/or carrier, the albumen that reconstitution cell produces can excretory form or the form of inclusion body exist.Personnel are known for the positive copy industry technology, and the polynucleotide expression vector of encoding SARS gene contains signal sequence more, and it can instruct protein excretion to arrive outside protokaryon or the eukaryotic cell membrane.Other recombinants can link to each other the sequence of encoding SARS gene with the sequence of one section coded polypeptide domain, this polypeptide structure territory helps the purification of soluble protein, these domains comprise metal chelating peptide, as histidine-tryptophan molecule can be on solidified metal purification, protein A can be on solidified immunoglobulin purification, or utilize FLAG extension/affine system purification (Immunex Corp., Seattle, but be not restricted to this Wash.).The connexon sequence that can shear between the domain of purification and the SARS gene helps purification, and the such sequence such as the special XA factor and enterokinase (Invitrogen, San Diego, Calif.).Similarly expression vector can provide the Expression of Fusion Protein that comprises SARS gene and 6 histidine residues of a coding, and these 6 histidine residues can be positioned at before thioredoxin or the enterokinase shearing site.Histidine residues help IMIAC (the fixing metal ions affinity chromatography is seen Porath, J. etc., 1992, purification Prot.Exp.Purif.3:263-281), yet, the enterokinase shearing site provide a kind of from fusion rotein the method for purification SARS gene.Kroll is seen in the discussion of fusion rotein carrier, (1993, DNA Cell Biol.12:441-453) such as D J..

Except the generation of recombiant protein, the SARS genetic fragment can use solid phase synthesis technique directly carry out peptide synthetic (Merrifield J., 1963, J.Am.Chem.Soc.85:2149-2154).The synthetic method that can use operating guidance to provide of albumen, or automatization's synthetic technology.Automated system has many kinds, and as using ABI 43 IA peptide synthesizers (PerkinElmer), different SARS genetic fragments can be carried out chemosynthesis respectively, and uses chemical method to connect, to produce full-length molecule.

The specific antibody of SARS gene can be used as target or means of transportation, and the guiding pharmaceutical agents is expressed S or ORF8 to cell or tissue.

In another embodiment, any human cytokines, antagonist, antibody, agonist, antisense sequences or aforesaid carrier can be united use with other suitable treatment reagent.According to conventional pharmacy principle, in therapeutic alliance, suitably the selection of reagent is one of general technology in the industry.Treatment reagent is united the different disorderly phenomenon that use can act synergistically on treatment or prevention stage mutually.Make in this way, every kind of reagent adopts lower dosage therapeutic effect will occur, has therefore reduced potential side reaction.

Can high level expression S or segmental carrier transformant of ORF8 or tissue, the SARS expression of gene may be closed.Such carrier may have been introduced the justice or the antisense sequences of untranslated, and even without being integrated into DNA sequence, carrier also can continue to transcribe out the RNA molecule, up to them by endogenous nuclease inactivation.When having nonreplication vector, transient expression may continue one month or the longer time, if when suitable reproduction element is arranged in the carrier system, the persistent period may be longer.

The method of carrier being introduced cell or tissue has many kinds, and these methods to be equally applicable to body interior, external or in vitro.For in vitro treatment, carrier can be introduced the patient stem cell, again through autotransplantation, same patient is gone in reverse pumping behind the clonal expansion.Use the known method of the industry, can inject by transfection and liposome and implement transmission.

Above-mentioned any Therapeutic Method need can be applied to any object of treatment, comprises, and mammal such as Canis familiaris L., cat, cattle, horse, rabbit, monkey, first-selected human.

At above-mentioned any therapeutic effect, another embodiment of invention is relevant with the carrier administering drug combinations of ingredient and medicine.These ingredients comprise antibody, analog, agonist, antagonist or the S of S or ORF8, S or ORF8 or the inhibitor of ORF8.These compositions can be individually dosed, or with at least a other reagent administering drug combinations, as stabilizing agent.These stable elementss can administration in the carrier of any aseptic, biocompatible, and these carriers comprise salt, buffer salt, glucose and water, but are not restricted to this.These can be individually dosed, perhaps with other reagent, medicine or hormons administration.

The route of administration of ingredient used in the present invention has multiple, comprise in oral, intravenous, intramuscular, intra-arterial, the medullary substance, in the sheath, in the ventricle, the method for Intradermal, subcutaneous, intraperitoneal, intranasal, intestinal, part, Sublingual or rectally, but be not restricted to this.

Except active component, these ingredients may comprise suitable pharmaceutical compatible carrier, comprise excipient, adminicle, and these compositions help ingredient is processed into the preparation that pharmaceutically uses.About prescription and the detail of medicine-feeding technology can (Maack Publishing Co., Easton find in Pa.) at the Remington pharmaceutical science.

The recipe design of the ingredient of oral administration can be used the known pharmaceutical compatible carrier of the industry, according to the suitable dosed administration of oral administration.These ingredients can be designed to tablet, pill, dragee, capsule, liquid, gelinite, syrup, slurry, float, and perhaps homologue makes things convenient for the patient to absorb.

The oral drugs preparation can obtain by the mode of active component and solid excipient coupling, chooses wantonly, wears into the form of mixture, and the process mixture granule adds suitable adminicle, if desired, can be processed into tablet or dragee core.Suitable adminicle is carbohydrate or protein fill, as sugar, comprise lactose, sucrose, mannose or sorbitol, corn, Semen Tritici aestivi, rice, Rhizoma Solani tuber osi or other plant amylum, cellulose such as methylcellulose, hydroxypropyl emthylcellulose or sodium carboxymethyl cellulose, rubber comprises romot and tragacanth, and albumen comprises gel and collagen.If desired, can add and decompose or solubilising reagent, as cross linked polyvinyl pyrrolidone, agar, alginic acid, or its salt are as Sargassum sodium.

The dragee core can link to each other with suitable peplos when using, and as spissated sugar juice, may also be full of glue romot, Talcum, polyvinylpyrrolidone, CBP glue, polyethylene glycol and/or titanium dioxide, lacquer solution and suitable organic solvent or solvent mixture.Can add dyestuff or pigment at tablet or dragee peplos, for the medicine identification or for the quantity of effective ingredient is described, as dosage.

The medicament preparation that can orally use comprises the capsule of being made by gel of taking easily, and the capsule and the peplos that also have gel soft, sealing to make are as glycerol or sorbitol.The capsule of taking easily may comprise active component and filler or binding agent, for example lactose or starch, lubricant such as Talcum or magnesium stearate and optional stabilizing agent.Whether in soft capsule, active component can dissolve or be suspended in the suitable liquid, as oils and fats, liquid or polyethylene glycol liquid, exist stabilizing agent all can.

The pharmaceutical formulation design of parenterai administration can be with the form of aqueous solution, especially compatible solution with physiology, and as Hawks solution, Ringer solution or physiological buffer salt solution.Also can add the material that increases viscosity in the aqueous solution, as carboxycellulose sodium, sorbitol or dextran.In addition, the active component float can be prepared as suitable oily injection float, or synthetic fatty acid ester, as ethane oleate, triglyceride or liposome.Optionally, float also can comprise suitable stabilizing agent maybe can increase solable matter, is convenient to prepare the higher solution of concentration.

For part or nasal-cavity administration, in recipe design, allow to use suitable penetrating agent at concrete barrier.These penetrating agent are general uses in the industry.

Ingredient among the present invention can be according to known a kind of method preparation in the industry, as mixing, dissolving, granulation, dragee making, grinding, the emulsifying of routine, incapsulate, embedding or lyophilizing handle.

Ingredient can provide with the form of salt, can form with many acid, comprises hydrochloric acid, sulphuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid etc., but is not restricted to this.Compare with corresponding radical form, in aqueous solution or in other proton solutions, the easier dissolving of salt.In other example, preferred prepared product is a lyophilized powder, comprises any/or all following materials: the 1-50mM histidine, and the mannitol of 0.1%-2% sucrose and 2-7%, the pH scope is united use at 4.5-5.5 with aforementioned buffer.

After ingredient prepares, can put into proper container, and stamp label, point out indication.For the administration of S or ORF8, these labels should comprise the method for quantity, frequency and administration.

The ingredient of using among the present invention has comprised the effective dose of active component.The mensuration of effective dose is that the technical staff is known in the industry.

To any composition, in cell culture test (as oncocyte) or animal model (normally mice, rabbit, Canis familiaris L. or pig), can estimate at first the treatment effective dose.Also can use animal model to measure proper concentration or route of administration.These data also can be used as the reference of measuring human effective dose and route of administration.

The effective dose of treatment refers to the quantity of active component, comprises SARS gene or its fragment, the antibody of SARS gene, agonist, antagonist, and the inhibitor of S or ORF8, these effective ingredient can improve symptom.Therapeutic effect and toxicity can be carried out cell culture and experimental animal test by the drug test method of standard, as ED50 (median effective dose) and LD50 (median lethal dose(LD 50)).The dosage ratio of treatment and toxic effect is a therapeutic index, can be expressed as LD50/ED50.Usually can select the ingredient of high therapeutic index for use.The data that obtain from cell culture test and animal experiment can be used for the designer and use dosage range.The effective dose of the medicine in circulation should reach ED50 amount and have seldom or do not have toxicity.According to employed dosage form, patient's sensitivity, and route of administration, effective dose can change usually to some extent.

The concrete condition of treatment target is determined definite dosage by the doctor as required.Dosage and administration will be through suitably adjusting, so that obtain enough active parts or desirable therapeutic effects.Should consider following factors: severity of disease, the overall health of object, age, body weight and sex, diet, time of administration and frequency, medication combined thing, reaction sensibility and tolerance/reaction to treating.Different with clearance rate according to the survival of the half of concrete prescription, depot drug product can be every administration in 3-4 days, every other week or every two all administrations.

Because the difference of route of administration, normal dosage is from 0.1-100, and 000ug does not wait, sometimes can be up to 1g.About the concrete dosage and the method for administration, the doctor of the industry can obtain from general document.The technical staff of the industry will use different nucleotide medicine prescriptions, rather than albumen or inhibitor.Equally, the administering mode of polymerized nucleoside or polypeptide should wait to determine according to concrete cell, state and position.

For advantage of the present invention more fully is described, provide the following example.Should be understood that the following example only is used to illustrate the present invention, rather than the restriction scope of invention.

According to following two principles design synthetic gene: 1) comprise may be relevant with virus absorption and infection surface protein; 2) comprise a conservative relatively albumen at least.

According to the SARS-CoV genome sequence of prediction, two surface proteins of S1 and orf8 have been selected.The S1 territory regulation and control virus of spike protein adheres to cell surface, the unknown of orf8 protein function, but have a profound extracellular domain, also may be relevant with viral infection.N albumen is relatively very conservative albumen, has shown that it can activate the t helper cell function strongly, and therefore, this gene is contained in the gene packing.

In order to express 3 albumen simultaneously, connexon is warm flexibly for the domain of S1 and orf8 and one, and this connexon helps albumen independently folding.

By a ribosome entry site(RES) (IRES), N albumen when transcribing and the warm albumen of S1-orf8 warm.As shown in Figure 1, by a CMV promoter, two albumen can be expressed simultaneously.

As mentioned above, virus has a great difference with people's codon bias.Many viruses, as HIV and HPV, in its coded sequence, G-C content is lower.On the contrary, human gene's G-C content is higher, and the 3rd bit base of codon is especially true.When being partial to have the codon of higher G-C content, may be that albumen can more effectively be translated like this because the concentration of the interior tRNA of the cell of codon is higher separately.In addition, high G-C content is high relevant with mRNA stability, and this can increase the expression of destination protein.

Use preferred people's codon that the target antigen coded sequence is optimized, effectively transcribe guaranteeing, as shown in Figure 2, SARS-CoV S and N gene G-C content are lower, are less than 40% at the 3rd base position G-C content, and typical viral skewed popularity is arranged; On the contrary, G-C percentage ratio is 77% in the human protein that efficiently expresses.

The 3rd base position, the average G-C content of preferred gene is 66%, makes it to meet human average percent.In order further to optimize synthetic gene, use intron-exon bonding pad rule that potential selectivity mRNA shearing site is identified.By silent mutation these sites are removed, to guarantee only to express correct target protein.

In a preferred embodiment, S1 domain and orf8 extracellular domain are warm, as shown in Figure 3.Add an IgK secretion signal at warm proteic N-terminal, fusion rotein can be secreted out like this, produces how potential antibody response.As shown in Figure 3, the sequence of these fusion rotein is through optimizing, to the primates optimization expression.The aminoacid sequence of these fusion rotein is seen Fig. 4.

In another preferred embodiment, the N protein sequence is seen Fig. 5 through optimizing.Amino acid sequence corresponding as shown in Figure 6.

These synthetic genes are cloned into different carriers, comprise virus and plasmid vector.

The effectiveness that in a test, has shown DNA plasmid vector (SV1000) vaccine.Use 100ug not have endotoxic DNA plasmid SV1000, in the 0th week, in 2 weeks, the female Balb/c mice in 5 all immune 6-8 weeks is collected serum sample in the 3rd week and the 6th week.Collect total lymphocyte in the 6th week that experiment stops.The serum conversion phenomena appears in most of mices before 3 weeks, although antibody titer is relatively low.In the 6th when week, antibody titer rises to 1: 640, this with document in other dna vaccinations of reporting induce the scope of antibody response consistent.

Detected in a blood serum sample simultaneously and the ability of SARS virus infection Vero E6 cell alive.Before infecting Vero E6 cell, serum is made 2 times of serial dilutions, hatch with virus.Through calculating, final in and titre be about 1: 300.

Using the special ELISPOT test of IFNg that T cells with antigenic specificity is reacted carries out quantitatively.A synthetic polypeptide that covers 2 full-length genes and 10 amino acid whose eclipsed 20-mer are arranged.Use whole lymphocyte reactions of 12 peptide storehouse immune stimulatory mices respectively.As shown in Figure 7, at a plurality of zones of vaccine, SV1000 can induce potential Th1 t cell responses.Importantly, the zone of S1-orf8 and N is identified, and shows that these two genes all express in vivo, and participates in producing special immunoreation.

Reorganization 5 type adenovirus, i.e. SV8000 that have synthetic gene have also been made up simultaneously.As shown in Figure 8, single injection SV8000 has caused the very strong immunoreation of mice, and antibody titer was above 1: 1000.This reaction also is dose-dependent, has induced very strong antibody response in the 1E9 virion, surpasses the 1E8 virion.

The IFNg t cell responses is also analyzed by ELISOPT.As shown in Figure 9, single injection SV8000, potential t cell responses has been induced in numerous zones of synthetic gene.The degree of reaction is obviously induced the result greater than the SV1000 dna vaccination.What is interesting is there is not the linear correlation relation between vaccine dose and the t cell responses.The inductive t cell responses of IE8 particle is the strongest, yet, no matter be 1E9 or 1E7 particle, behind the immune mouse, immunoreation is all more weak.

Nearest studies show that, different primers-enhancing mode can strengthen the immunoreation that any single vaccine composition produces greatly.When dna vaccination was used as initiation antigen, this saying was especially correct.It is said that because dna vaccination is lower than antigen concentration in the recombinant virus, and lacks " noise " antigen that has usually in the recombinant virus, so DNA causes and can activate and enlarge the T cells with antigenic specificity reaction, at induction period, has very high affinity.Virus strengthens and has further enlarged thesaurus, compares with single vaccine, has produced very strong immunoreation.

As shown in figure 10, compare with single DNA or recombinant adenovirus, initiation-enhancing (prime boost) method has been induced and has been higher than its antibody response of 10 times at least.

According to the literature, single injection recombinant adenovirus vaccine can protect non-human primates to avoid the attack of Ebola virus fully.When the permanent immunity plan was infeasible, this was for handling Public Health Emergencies, as SARS outburst, advantageous particularly.Single injection, the vaccine of snap action can provide very fast protection, can reduce the potential danger that trigger reaction working group faces.

Use the 1E12 of SV8000 vaccine that two Rhesus Macacus have been carried out immunity.The 3rd monkey only carries out immunity with culture medium.Before immunity, collect blood serum sample, in immunity 10 weeks of back, collect blood serum sample weekly.Separate PBMCs, and be stored in the liquid nitrogen, to carry out follow-up batch analysis.Blood serum sample is used for the virus neutralization experiment of standard.Can observe immunoreactive visible trend from data.As shown in figure 11, neutralizing antibody the peak occurs in the 4th week, and later 3 weeks are steady relatively.Average NAT that it should be noted that the reconvalescent only is about 1: 64.Although this may show, inject SV8000 separately protective effect may be provided, and do not know whether only have neutralizing antibody just can play a protective role.The T cell, especially CTLs as IBV and MHV, plays an important role in other coronavirus infection animal models.And, because the history of SARS-CoV is shorter, do not know whether this virus can invade once more with different S albumen, in this particle, the T cell activation is very crucial to the protection of intersection strain.

Effect to vaccine in monkey detects.In the 0th week and the 4th week, intramuscular injection SV8000 immunity Rhesus Macacus.In 4 weeks of pro-, antibody response is relatively low.Behind booster immunization, the very fast increase of antibody response (Figure 12).In the 8th week, the monkey that all immunity are crossed has produced the IgG antibody of high titre.

In order to detect the protection efficient of vaccine, use the monkey of high dose, low dosage and placebo group to use 10 respectively ⁵TCID ₅₀The live virus intranasal is attacked, and two monkeys of matched group are raised in same animal raising equipment, but does not infect live virus, as the negative control of experiment.Behind the virus inoculation three days, it is drowsiness that 4 monkeys of placebo group become, and feed significantly reduces, and two monkeys demonstrate the respiratory distress symptom.On the contrary, immune animal or the control animals of crossing all do not demonstrate visible clinical symptoms.

With attack before compare, the concentration of the special IgG of immune group sars coronavirus significantly increases (Figure 13) after attack.After the virus attack 7 days, the scope of increase changes between 3 times to nearly 30 times, show vaccine injection after, effectively caused humoral immune reaction.

Behind challenge virus the 2nd day, collected throat swab and serum sample in the 5th day and the 7th day.Detect the existence of sars coronavirus by the method for RT-PCR or Virus culture.In 4 animals of high dose group, 2 can detect viral RNA (Figure 14) in 2 days after virus attack, and viral RNA disappears in the time of 5 days.Other two all do not detect viral RNA at any time.4 animals of low dose group can not detect viral RNA at the 2nd day or the 5th day, had only an animal can detect the RNA of reduced levels at the 7th day.On the contrary, 4 animals of placebo group are after virus attack, and viral RNA detects all positive.

In order after attacking, to separate live virus again, with throat swab sample inoculation Vero cell.As shown in figure 14, in 4 animals of placebo group, 3 only to isolate live virus.High dose group and low dose group can watch for animals and avoid viral infection.

The animal of placebo group, pulmonary's well damage, alveolar wall damage widely, the albumen sample liquid buildup (Figure 15 e) of alveolar space.Because mononuclear cell seriously permeates, alveolar wall thickens, and transparent plasma membrane is linear arrangement in alveolar wall.(Figure 15 f).In the most serious example, can observe epithelium layer extensively damage and local bleeding.Although in the only a few case, found less edema and mononuclear cell infiltration (Figure 15 c) in alveolar wall, (Figure 15 a) has a tangible difference on the histology for high dose group (Figure 15 b) and matched group.The Phenotype of low dose group is (Figure 15 d) between high dose group and placebo group.

In order to find the relation between disease pathology and virus replication, the lung tissue section that adopted the SABC methods analyst simultaneously, as shown in figure 16.The positive staining of minute quantity is arranged in the high dose group section, very similar to negative control group.On the contrary, the lung tissue section of placebo group demonstrates many positive cells, and tangible kytoplasm dyeing is arranged, and shows successive antigen presentation in these cells.Low dose group also shows middle type, antigen-positive cell occurred in alveolar wall and epithelial cell.

Claims (25)

1. ingredient comprises the nucleic acid molecules of the one or more sars coronavirus gene outcomes of coding, and this nucleic acid molecules and a regulating and controlling sequence link, and are present in the pharmaceutical compatible carrier.

2. ingredient according to claim 1, wherein, the sars coronavirus gene is selected from S albumen, Orf8 albumen, M albumen, N albumen, E albumen, Orf1a albumen, Orf1b albumen and its association.

3. ingredient according to claim 1, wherein, regulating and controlling sequence is a promoter.

4. ingredient according to claim 3, wherein, promoter is the CMV promoter.

5. ingredient according to claim 1, wherein, pharmaceutical compatible carrier is viral vector, plasmid vector or RNA carrier.

6. according to the ingredient of claim 5, wherein, viral vector is a kind of adenovirus of replication defect type.

7. ingredient according to claim 6, wherein, viral vector is a kind of 5 type replication-defective adenovirals.

8. ingredient according to claim 1, wherein, two sars coronavirus gene outcomes of nucleic acid molecule encoding, in a pharmaceutical compatible carrier, first gene outcome comprises at least one SARS-CoV gene cell external structure territory, or derivatives thereof, second gene outcome comprises an intracellular protein, or derivatives thereof.

9. ingredient according to claim 8, wherein, first gene is selected from a SARS-CoV gene group, comprises S, M, Orf8 and E, and second gene is selected from the gene group of the SARS-CoV that comprises N, Orf1a and Orf1b.

10. ingredient according to claim 8, wherein, first gene outcome comprises the proteic part of SARS-CoV S, or its antigenic determinant binding structural domain, or its homologue.Wherein, S albumen has the C-terminal of a truncate, removes and strides film grappling and cell intracellular domain.

11. ingredient according to claim 8, wherein, first gene outcome comprises first genetic fragment, the proteic part of sars coronavirus S or its antigenic determinant binding structural domain, or its homologue, wherein S albumen has the C-terminal of a truncate, strides film grappling and cell born of the same parents intracellular domain with removal; Second genetic fragment comprises the proteic part of SARS-CoV Orf8 or its antigenic determinant binding structural domain or its homologue, and wherein Orf8 albumen has the C-terminal of a truncate, removed and striden film grappling and cell intracellular domain.

12. ingredient according to claim 1, wherein, nucleic acid molecules is made up of the mammal codon utilization rate preferred amino acids codon based on high expressed.

13. ingredient according to claim 12, wherein, the GC content of preferred codon is at least 50%.

14. ingredient according to claim 12, wherein, the G-C content of the 3rd base position of preferred codon is at least 50%.

15. ingredient according to claim 1, wherein, the SARS-CoV gene outcome also comprises a secretion signal fragments of peptides.

16. ingredient according to claim 8, wherein, first gene outcome also comprises the secretion signal fragments of peptides.

17. ingredient according to claim 16, wherein, the secretion signal fragments of peptides is an Igk signal sequence.

18. peptide or peptide association are selected from the arbitrary described gene outcome as claim 1-17, wherein these peptides are at external purification.

19. in body, induce at certain antigenic immunoreactive method for one kind, comprise offering this individual arbitrary described ingredient as claim 1-17.

20. as claimed in claim 19 inducing at certain antigenic immunoreactive method, wherein, after the ingredient first administration, respectively in about 1 week, 2 weeks, 3 weeks, 4 week or 6 all administrations, or administering drug combinations.

21. as claimed in claim 19 inducing at certain antigenic immunoreactive method, wherein, the administration of ingredient associating adjuvant.

22. as claimed in claim 19 inducing at certain antigenic immunoreactive method, wherein, the dosage of plasmid vector ingredient is per injection 1-5mg..

23. as claimed in claim 19 inducing at certain antigenic immunoreactive method, wherein, the dosage of viral vector ingredient is per injection 1 * 10 ⁶-1 * 10 ¹³Individual virion.

24. as claimed in claim 19 inducing at certain antigenic immunoreactive method, wherein, ingredient is by intramuscular, Intradermal, subcutaneous, intranasal or oral administration.

25. as claimed in claim 19 inducing at certain antigenic immunoreactive method also is included in the first time to behind the medicine composition, gives the ingredient of different carriers again, to cause above-mentioned antigenic immunoreation.

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