CN101297029A - Methods and compositions for expressing a heterologous protease - Google Patents

Abstract

The present invention provides methods and compositions for expressing a protease or pro-protease in cells that do not naturally express the protease or pro-protease. The invention further provides methods of producing viruses, e.g., influenza viruses, in such cells. The invention also provides methods for increasing the titer of influenza viruses grown in cells that express such a heterologous protease or pro-protease. Additionally, the invention provides a protease from Streptomyces griseus useful in the methods and compositions.

Description

The method and composition of expressing heterologous proteolytic enzyme

1. invention field

In one aspect, the invention provides at natural not expressing protein enzyme or the cell inner expression proteolytic enzyme of pepsinogen (pro-protease) or the method and composition of pepsinogen.In other respects, the invention provides the viral method of production in this cell as influenza virus.In other respects, the invention provides the method for tiring that improves the intracellular influenza virus that is grown in this heterologous protease of expression or pepsinogen.Again on the other hand, the invention provides the heterologous protease that is used for described method and composition from streptomyces griseus (Streptomyces griseus).

2. background of invention

The definition of flu outbreak is rapidly to increase because of global M ﹠ M that influenza disease causes.Severity and scope that several factor affecting disease popularities are arranged are comprising the low and viral infection efficient in the crowd of crowd's immunity programm.As a rule, the latter not only is subjected to the influence of virus itself, and is subjected to the influence of crowd's density with the difficulty or ease of turnover this area.Causing pandemic virus all is the antigenic shift strain that occurs recently usually, and the great majority among the crowd are not contacted this mutant strain in the past, thereby does not produce or seldom produce the immunity to this mutant strain.In addition, interpersonal effect spread is arranged is quick popular prerequisite, and under the situation that the crowd propagates, virus must adapt to duplicates in human body, and effect spread can be arranged in animality virus.

The influenza velocity of propagation is very fast, influence that can damaging property.Twentieth century is the most serious is very popular is be very popular in 1918, in the U.S. 500,000 citizen's death are arranged, and the death toll in the global range is between 2 to 4,000 ten thousand.Pandemic influenza starts relatively apace and propagates is that the global outbreak of tackling this degree has proposed several problems, has applied great burden for acute respondent and working healthily person.Rapid identification and being very popular of occurring reacted clearly is the prerequisite of dealing with problems; Set up a few cover programs at present in the world and be used to monitor emerging influenza virus, comprising seldom causing the ill avian influenza virus of people.These monitoring data can be determined the possibility that threatens and provide guidance for successfully managing in conjunction with the predefined early warning level that is very popular.

Immunization is the most important public health measure of popular caused disease of flu-prevention year.Potential pandemic determine to begin obviously to raise with ill level between of short duration interval be a big challenge protecting most of crowd for preparing effective vaccine.Deposit vaccine production technology and production basis facility are very crucial for the appearance that reduces a large amount of diseases and death before the appearance of next time being very popular.The needed short reaction times of preparation " vaccine is very popular " will not allow long research or handle the development time so that effective reaction to be provided.

Up to the present, all containing in the embryo egg at all commercial influenza vaccines of the non-strain of being very popular in the U.S. and breeding.Though influenza virus can well grow in egg, the production of vaccine will rely on the operability of egg.The egg supply must be organized, and the virus strain that is used for production of vaccine will screen at the earlier month that arrives season of influenza next time, and this can limit the handiness of this method, therefore usually can cause the delay and the shortage of production of vaccine and distribution.Unfortunately, some influenza vaccines virus strain as 2003-04 season popular A/Fujian/411/02 prototype-strain, can not be duplicated in containing the embryo egg well, therefore have to utilize cell cultures to separate this virus strain, this method not only costliness but also production process is consuming time very long.

Developed recent years in cell culture the system that produces influenza virus (referring to, as, Furminger, Vaccine Production, volumes such as Nicholson, Textbook of InfluenzaThe 324-332 page or leaf; Merten etc. (1996), Production of influenza virus in cell cultures for vaccine preparation, Cohen ﹠amp; Shafferman (volume), Novel Strategies in Design and Production of VaccinesThe 141-151 page or leaf).In general, these methods all comprise with the suitable immortal host cell of virus strain infection that filters out.Though can overcome many difficulties with respect to production vaccine in egg, not all influenza virus pathogenic strain can be according to good growth of existing tissue culture method and preparation.In addition, many have a desired characteristics, for example, attenuation characteristic, temperature sensitivity and acclimatization to cold, the virus strain that is suitable for preparing attenuated live vaccine utilize existent method in tissue culture, not grow.

So need: 1) produce required producing apparatus and the method and 2 of influenza vaccines from cell culture) develop from cell culture and make the effective technology of vaccine to prevent by the popular disease that causes of seasonal influenza.These methods and technology can be applied to make and distribute the prevailing disease vaccine rapidly when flu outbreak on the horizon.

For one of many obstacles of influenza vaccines based on cell culture being got the Green Light must overcome are to need proteolysis excision hemagglutinin (HA) albumen so that the new virus that forms effectively infects new cell.During zoogenetic infection, by the endogenous trypsin-like serine protease excision HA albumen of animal.When cultivating, the common deficiency of endopeptidase activity so that influenza virus duplicate effectively.Therefore, after with interested influenza infection culturing cell, in substratum, add proteolytic enzyme such as trypsinase usually to improve viral yield.Referring to, for example, U.S. Patent number 5,698,433.Yet, in cell culture medium, add exogenous protease and can in substratum, introduce extra composition, increase complicacy and need to accept extra management.In addition, adding external source trypsin trypin) increased cost with cell cultures manufactured vaccine.Therefore, need or not to add the novel method and the composition of growth influenza virus in culture of exogenous protease.In addition, this method and composition must overcome the intrinsic toxicity at the cell inner expression active protease.The present invention can satisfy these and other unsatisfied demand.

The reference that this paper quoted or discussed also is not equal to and admits that these are prior aries of the present invention.In addition, patent quotes also to be not equal to and admits its validity.

3. summary of the invention

The invention provides the cell that is referred to herein as " cell of the present invention ", it comprises the nucleic acid of proteins encoded enzyme or pepsinogen, thereby the level of this proteolytic enzyme of this cell expressing or pepsinogen will be higher than the common expression level of the cell that lacks this nucleic acid.In some embodiments, do not express this proteolytic enzyme or pepsinogen under the described cell normal circumstances.In some embodiments, described cell is with lower level, and for example to be lower than best or this proteolytic enzyme of the active horizontal expression of required particular organisms or pepsinogen, described particular organisms activity is for example cultivated the virus such as influenza virus.In some aspects, the invention provides a kind of cell that comprises the nucleic acid of proteins encoded enzyme or pepsinogen, the nucleic acid of wherein said proteins encoded enzyme or pepsinogen is stabilized in the genome that is integrated into this cell.In other respects, the invention provides a kind of cell that comprises the nucleic acid of proteins encoded enzyme or pepsinogen, the nucleic acid of wherein said proteins encoded enzyme or pepsinogen is present in outside the karyomit(e).In other respects, the invention provides a kind of cell that comprises the nucleic acid of proteins encoded enzyme or pepsinogen, the nucleic acid of wherein said proteins encoded enzyme or pepsinogen transient expression in this cell.In some embodiments, this proteolytic enzyme of described cell expressing or pepsinogen.In some embodiments, described this proteolytic enzyme of cell constitutive expression or pepsinogen.In some embodiments, described cell induction type is expressed this proteolytic enzyme or pepsinogen.In some embodiments, this proteolytic enzyme of described emiocytosis or pepsinogen.In some embodiments, described cell is expressed this proteolytic enzyme or pepsinogen in the cytosol of cell.

In some embodiments, described proteolytic enzyme is serine protease.In some embodiments, described serine protease is a S1 family protein enzyme.In some embodiments, described proteolytic enzyme is trypsinase.In some embodiments, described serine protease is the bacterium subtilisin.In some embodiments, described proteolytic enzyme is the SPRT of streptomyces griseus.In some embodiments, described proteolytic enzyme is the listed proteolytic enzyme of table 1.In some embodiments, described proteolytic enzyme is pepsinogen.In some embodiments, described pepsinogen is a trypsinogen.In some embodiments, described pepsinogen is processed to active protease.

In some embodiments, described proteolytic enzyme or pepsinogen are expressed under inducible promoter control.In some embodiments, described inducible promoter is induced by Interferon, rabbit or interferon-induced downstream signaling molecule.In some embodiments, described inducible promoter is induced by the expression system of tsiklomitsin-regulation and control.In some embodiments, described proteolytic enzyme or pepsinogen are expressed under the control of constitutive activity promotor.In some embodiments, the nucleic acid of described proteins encoded enzyme or pepsinogen comprises the encoding sequence that instructs this proteolytic enzyme or pepsinogen excretory secretion signal.

In some embodiments, the about 0.1ng of described cell expressing is to about 50 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, the about 1ng of described cell expressing is to about 50 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, the about 10ng of described cell expressing is to about 50 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, the about 100ng of described cell expressing is to about 50 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, the about 1 μ g of described cell expressing is to about 50 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, the about 0.1ng of described cell expressing is to about 5 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, the about 0.1ng of described cell expressing is to about 100ng proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, the about 0.1ng of described cell expressing is to about 10ng proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, the about 0.1ng of described cell expressing is to about 1ng proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, the amount of the proteolytic enzyme of described cell expressing or pepsinogen is enough to improve tiring of the virus of growing in the cell culture of expressing this proteolytic enzyme or pepsinogen.In some embodiments, the molecular weight of described proteolytic enzyme or pepsinogen calculates based on the pepsinogen form of this enzyme.In some embodiments, the molecular weight of described proteolytic enzyme or pepsinogen calculates based on maturation, the activity form of this proteolytic enzyme.

In some embodiments, described cell expressing is at least about 0.1ng proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, described cell expressing is at least about 1ng proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, described cell expressing is at least about 10ng proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, described cell expressing is at least about 100ng proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, described cell expressing is at least about 1 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, described cell expressing is at least about 10 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, described cell expressing is at least about 20 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, described cell expressing is at least about 30 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, described cell expressing is at least about 40 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, described cell expressing is at least about 50 μ g proteolytic enzyme or pepsinogen/ml cell culture.

In some embodiments, described cell is a bacterial cell.In some embodiments, described bacterial cell is intestinal bacteria (E.coli) cells.In some embodiments, described cell is a mammalian cell.In some embodiments, described mammalian cell is a canine cells.In some embodiments, described canine cells is a mdck cell.In some embodiments, described mdck cell is non-tumorigenesis.In some embodiments, described mammalian cell is a primate cell.In some embodiments, described primate cell is African grivet or human cell.In some embodiments, described cell is the birds cells.In some embodiments, described birds cell is a chicken cell.

On the other hand, the invention provides a kind of method of producing virus, this method comprises with a kind of virus infection cell of the present invention, cultivates this cell under the condition that allows this virus replication, and collects virus from cell culture.In an embodiment, described virus is influenza virus.

On the other hand, the invention provides a kind of method of producing virus, described method comprises with comprising virus genomic nucleic acid transfection cell of the present invention, cultivates this cell under the condition that allows this virus replication, and collects virus from cell culture.In an embodiment, described viral genome is the influenza genome, and described virus is influenza virus.In some embodiments, described influenza virus is corresponding to Influenza B virus.In some embodiments, described influenza virus is corresponding to influenza A virus.In some embodiments, described virus comprises attenuated influenza virus, acclimatization to cold influenza virus, temperature sensitive influenza virus or has the virus of the arbitrary combination of these desired characteristic.In one embodiment, described influenza virus is a B/Ann Arbor/1/66 strains of influenza viruses, for example acclimatization to cold of B/AnnArbor/1/66, temperature sensitive, attenuation strain.In another embodiment, described influenza virus is an A/Ann Arbor/6/60 strains of influenza viruses, for example, and the acclimatization to cold of A/Ann Arbor/6/60, temperature sensitive, attenuation strain.

In some embodiments, described method comprises that reclaiming influenza virus also prepares such as immunogenic compositions such as vaccines with this virus.In one embodiment, described virus can cause immunne response give object in for example being applied to nose after.In some embodiments, the virus that is used to prepare vaccine through deactivation, in other embodiments, prepares vaccine with the attenuated virus of living before using.In some embodiments, make the recombinant chou and the reprovision body (reassortant) of first type and Influenza B virus according to method of the present invention.In one embodiment, the vaccine of preparation comprises, virus deactivation or that kill that live from the viral deutero-by the inventive method manufacturing.In one embodiment, the virus by the inventive method manufacturing is used to duplicate other virus in cell culture or egg.In one embodiment, the vaccine that provides comprises the viral deutero-immunogenic polypeptide of making from by the inventive method.

In some embodiments, described cell expressing pepsinogen, and described method also is included in and adds exogenous protease in the substratum.In some embodiments, the maximum of described exogenous protease is added the about 0.1 μ g/ml of concentration.In some embodiments, described exogenous protease is a trypsinase.

In some embodiments, described virus is dna virus.In some embodiments, described virus is RNA viruses.In some embodiments, described virus is single-stranded DNA viruses.In some embodiments, described virus is double-stranded DNA virus.In some embodiments, described virus is sense single stranded rna virus.In some embodiments, described virus is negative adopted single strand RNA virus.In some embodiments, described virus is diplornavirus.In some embodiments, described virus is retrovirus.

On the other hand, the invention provides a kind of method of replicating influenza virus, this method comprises cultivates this cell with influenza infection cell of the present invention under the condition that allows this influenza virus to duplicate, and collects influenza virus from cell culture.In some embodiments, this condition does not comprise proteolytic enzyme or the pepsinogen that external source is added, for example, and trypsinase or trypsinogen.

Again in yet another aspect, the invention provides a kind of method of replicating influenza virus, this method comprises with the genomic nucleic acid transfection cell of encoding stream sense, cultivates this cell under the condition that allows this influenza virus to duplicate, and collects influenza virus from cell culture.In some embodiments, this condition does not comprise proteolytic enzyme or the pepsinogen that external source is added, for example, and trypsinase or trypsinogen.

In some embodiments, not expressed proteins proenzyme or zymoprotein enzyme of this cell under the described cell expressing normal circumstances.In some embodiments, described cell is a mammalian cell.In some embodiments, described cell is the birds cells.In some embodiments, described cell is primate cell, canine cells, hamster cell, mouse cell or rat cell.In some embodiments, described cell is a mdck cell.In some embodiments, described cell is the Vero cell.In some embodiments, described cell is a chicken cell.

Again in yet another aspect, the invention provides a kind of raising and be grown in the method that the influenza virus in the cell culture is tired, this method is included in and cultivates described influenza virus in the cell culture, cell expressing proteolytic enzyme or pepsinogen in the wherein said cell culture, this proteolytic enzyme or pepsinogen i) to described cell allos, ii) excised the hemagglutinin of described influenza virus, thereby, with respect to by for cultivating tiring that influenza virus obtains in the cell of not expressing heterologous proteolytic enzyme or pepsinogen, improved the influenza virus that is grown in the described cell culture and tired.

In some embodiments, described cytotostatic is expressed this heterologous protease.In some embodiments, described this heterologous protease of cell constitutive expression.In some embodiments, described cell induction type is expressed this heterologous protease.In some embodiments, described heterologous protease is a trypsinase.In some embodiments, this heterologous protein proenzyme of described cell constitutive expression.In some embodiments, described cell induction type is expressed this heterologous protein proenzyme.In some embodiments, described pepsinogen is a trypsinogen.In some embodiments, described proteolytic enzyme is the SPRT proteolytic enzyme of streptomyces griseus.In some embodiments, described pepsinogen is preceding-SPRT pepsinogen (prepro-SPRT protease) of streptomyces griseus.

Cell can support that an evidence of virus replication is to obtain virus from the cell culture that infects.Can measure viral yield by many methods known to those skilled in the art.For example, can be according to the half tissue culture infective dose (TCID that measures the infectious virus body ₅₀) thereby the concentration of the virus that exists in the test determination sample quantizes viral yield.TCID ₅₀Value is typically expressed as log ₁₀TCID ₅₀/ mL.In one embodiment, the cell of described expressing heterologous proteolytic enzyme supports influenza virus (for example, ca/ts strain) to copy to following log ₁₀TCID ₅₀/ mL: at least 6.0 or at least 6.2 or at least 6.4 or at least 6.6 or at least 6.8 or at least 7.0 or at least 7.2 or at least 7.4 or at least 7.6 or at least 7.8 or at least 8.0 or at least 8.2 or at least 8.4 or at least 8.6 or at least 8.8 or at least 9.0 or at least 9.2 or at least 9.4 or at least 9.6 or at least 9.8.In an embodiment, the cell of described expressing heterologous proteolytic enzyme is supported influenza virus (for example, ca/ts strain) to copy to commercialization and is reasonably tired (＞10 ⁷Log TCID ₅₀/ mL).

In some embodiments, the influenza virus that in the cell culture of the cell of expressing heterologous proteolytic enzyme, produces tire with the corresponding cell of expressing heterologous proteolytic enzyme not and do not add exogenous protease such as tryptic culture in the tiring of influenza virus that produce compare its log ₁₀TCID ₅₀/ mL improves at least about 0.1, or at least about 0.2, or at least about 0.3, or at least about 0.4, or at least about 0.5, or at least about 0.6, or at least about 0.7, or at least about 0.8, or at least about 0.9, or at least about 1.0, or at least about 1.2, or at least about 1.4, or at least about 1.6, or at least about 1.8, or at least about 2.0, or at least about 2.2, or at least about 2.4, or at least about 2.6, or at least about 2.6, or at least about 2.8, or at least about 3.0, or at least about 3.2, or at least about 3.4, or at least about 3.6, or at least about 3.8, or at least about 4.0, or at least about 4.2, or at least about 4.4, or at least about 4.6, or at least about 4.8, or at least about 5.0.

In some embodiments, the influenza virus that in the cell culture of the cell of expressing heterologous proteolytic enzyme, produces tire with the corresponding cell of expressing heterologous proteolytic enzyme not and do not add exogenous protease such as tryptic culture in the tiring of influenza virus that produce compare raising at least about 10%.In some embodiments, tiring of the influenza virus that produces in the cell culture of the cell of expressing heterologous proteolytic enzyme improved at least about 25%.In some embodiments, tiring of the influenza virus that produces in the cell culture of the cell of expressing heterologous proteolytic enzyme improved at least about 50%.In some embodiments, tiring of the influenza virus that produces in the cell culture of the cell of expressing heterologous proteolytic enzyme improved at least about 100%.In some embodiments, tiring of the influenza virus that produces in the cell culture of the cell of expressing heterologous proteolytic enzyme improved at least about 500%.In some embodiments, tiring of the influenza virus that produces in the cell culture of the cell of expressing heterologous proteolytic enzyme improved at least about 1000%.In some embodiments, tiring of the influenza virus that produces in the cell culture of the cell of expressing heterologous proteolytic enzyme improved at least about 5000%.In some embodiments, tiring of the influenza virus that produces in the cell culture of the cell of expressing heterologous proteolytic enzyme improved at least about 10,000%.In some embodiments, tiring of the influenza virus that produces in the cell culture of the cell of expressing heterologous proteolytic enzyme improved at least about 30,000%.In some embodiments, tiring of the influenza virus that produces in the cell culture of the cell of expressing heterologous proteolytic enzyme improved at least about 50,000%.In some embodiments, tiring of the influenza virus that produces in the cell culture of the cell of expressing heterologous proteolytic enzyme improved at least about 70,000%.In some embodiments, tiring of the influenza virus that produces in the cell culture of the cell of expressing heterologous proteolytic enzyme improved at least about 100,000%.

In some embodiments, the cell culture medium that can cultivate cell of the present invention is a serum-free.In some embodiments, the cell culture medium that can cultivate cell of the present invention contains serum (for example, foetal calf serum).In some embodiments, the cell culture medium that can cultivate cell of the present invention does not contain the animal protein that external source is added, and this substratum is commonly called " animal protein-free " or " APF " substratum.In some embodiments, the cell culture medium that can cultivate cell of the present invention contains exogenous protease (for example, pig trypsinase).

Again in yet another aspect, the invention provides a kind of method of in cell, making heterologous protease or pepsinogen, wherein said cell can support influenza virus to duplicate, described method is included in cultivates the cell that comprises under the normal circumstances not at the coding nucleic acid of the proteolytic enzyme of cell inner expression or pepsinogen under the condition that allows described proteolytic enzyme or pepsinogen to express, thereby makes this proteolytic enzyme or pepsinogen in described cell.

In some embodiments, described cell expressing proteolytic enzyme.In some embodiments, described cytotostatic expressing protein enzyme.In some embodiments, described cell expressing pepsinogen.In some embodiments, described cytotostatic expressing protein proenzyme.In some embodiments, described cell is secreted into cell culture medium with proteolytic enzyme or pepsinogen.In some embodiments, the about 0.1ng of described cell expressing is to about 50 μ g proteolytic enzyme or pepsinogen/ml cell culture.In some embodiments, the amount of the proteolytic enzyme of described cell expressing or pepsinogen is enough to improve tiring of the virus that is grown in the cell culture of expressing this proteolytic enzyme or pepsinogen.In some embodiments, the expression of described proteolytic enzyme or pepsinogen is an induction type.In some embodiments, the expression of described proteolytic enzyme or pepsinogen is a composing type.

Again in yet another aspect, the invention provides a kind of method of making the cell of expressing under this cell normal circumstances expressed proteins enzyme not or pepsinogen, this method comprises and operability is connected in this proteolytic enzyme of coding of influencing the controlling element that proteolytic enzyme or pepsinogen express or the nucleic acid of pepsinogen is introduced cell (this proteolytic enzyme or pepsinogen can not be secreted or discharge to this cell can maybe), thereby makes under the cell expressing normal circumstances this cell not expressed proteins enzyme or pepsinogen.

Can adopt any appropriate technology well known by persons skilled in the art and/or carrier that nucleic acid is introduced cell, be not limited in this respect.In some embodiments, described nucleic acid is introduced cell with plasmid, clay, virus vector, phage, phagemid, transposon or artificial chromosome form.In some embodiments, described nucleic acid is introduced cell with the retroviral vector form.In some embodiments, described nucleic acid stability maintains in the cell.In other embodiments, described nucleic acid is temporarily kept.

In some embodiments, the nucleic acid of described proteins encoded enzyme or pepsinogen also comprises selectable marker.It is well known by persons skilled in the art utilizing selectable marker to select the method for cell of the nucleic acid of those stably express proteins encoded enzymes or pepsinogen.Well known by persons skilled in the artly can affected any selectable marker in the cell of having introduced this nucleic acid all can be used for this embodiment.Therefore, in some embodiments, described selectable marker is an antibiotics resistance gene.In some embodiments, described selectable marker is the gene that remedies in the metabolic pathway of synthesizing of this cell defect.For example, the nucleic acid of described proteins encoded enzyme or pepsinogen can be introduced for example synthetic defective cell of amino acid.This nucleic acid can comprise the gene that remedies this cell defect.Just can select the nucleic acid that comprises this synthetic gene by in not containing this amino acid whose substratum, cultivating this cell.Any this genoid well known by persons skilled in the art all can be used for the present invention, is not limited in this respect.

Again in yet another aspect, the invention provides with SEQ ID NO.:1 at least about 90% identical isolating nucleic acid.In some embodiments, described isolating nucleic acid comprises SEQ ID NO.:1 or is made of SEQ ID NO.:1.In some embodiments, described isolating nucleic acid is hybridized with the nucleic acid (or its complementary strand) of coding SEQ ID NO.:1 under hybridization conditions.In some embodiments, described hybridization conditions is a stringent hybridization condition.In some embodiments, described hybridization conditions is highly strict hybridization conditions.

Again in yet another aspect, the invention provides a kind of isolated polypeptide that comprises aminoacid sequence SEQ ID NO.:2 or constitute by aminoacid sequence SEQ ID NO.:2.

Again in yet another aspect, the invention provides that a kind of coding comprises aminoacid sequence SEQ ID NO.:2 or the isolating nucleic acid of the polypeptide that is made of aminoacid sequence SEQ ID NO.:2.

Again in yet another aspect, the invention provides a kind of expression of nucleic acids carrier of the present invention that comprises.

Again in yet another aspect, the invention provides a kind of usefulness expression vector cells transfected of the present invention.

4. accompanying drawing summary

Fig. 1 has represented ca A/Vietnam/1203/2004 (H5N1) duplicating in mdck cell.

Fig. 2 is the form of the amount of observed luciferase activity in different retroviral vector cells transfected.

Fig. 3 is shown in the comparison of observed luciferase activity in the mdck cell and the retroviral particle concentration that is used for infecting mdck cell.

Fig. 4 is for showing the form of 12 kinds of luciferase expression in the different MDCK clone with two kinds of the single MDCK clone of the difference mixture.

Form among Fig. 5 has been summed up from making two kinds of Different Package clones and cloning with the MDCK that the virion of three kinds of different carriers transfections obtains.

Fig. 6 A-6C is the diagram of tiring by the influenza virus that obtains with MDV-A or the different MDCK clones of ca A/NC influenza virus strain infection (figure A is the contrast clone, and figure B is Ampho trypsinogen clone, and figure C is GP2 trypsinogen clone).The external source trypsinase that adds: 0.0 μ g/ml, hollow triangle; The 1st day 0.1 μ g/ml, solid circles; 1-5 days 1.0 μ g/ml, square hollow.

The western trace that Fig. 7 A-7B expresses for the trypsinogen that shows 12 clones' 6xHis-mark among 16 kinds of MDCK clones.

Fig. 8 is for showing the form of derivable luciferase expression among 15 kinds of different MDCK clones.

Fig. 9 is the nucleotide sequence (SEQ ID NO:1) of the sprT gene of coding streptomyces griseus serine protease.

Figure 10 is the aminoacid sequence (SEQ ID NO:2) of the streptomyces griseus serine protease of sprT genes encoding.

Figure 11 is the nucleotide sequence (SEQ ID NO:3) of coding trypsinogen.

Figure 12 is forward primer and the reverse primer (being respectively SEQ ID NO:5 and 5) that is used to clone the sprT gene.

Figure 13 is transfected into R3/7 clone's the pT-Rex-DEST30/ luciferase and the synoptic diagram of pT-Rex-DEST30/ trypsinase plasmid.

5. detailed Description Of The Invention

5.1 definition

Unless otherwise defined, the implication of all Science and Technology terms is all identical with its normally used implication of association area institute. For the purposes of the present invention, following term is defined as follows.

Term " nucleic acid ", " polynucleotides ", " polynucleotide sequence " and " nucleotide sequence " refer to strand or double-stranded DNA nucleotides or ribonucleotide acid polymer or its block polymer or analog. In this article, term can also comprise the polymer of the natural nucleus glycoside acid-like substance with natural nucleotide fundamental characteristics, the mode that wherein they can be similar to natural nucleotide and single-chain nucleic acid hybridization (for example, peptide nucleic acid). Unless otherwise indicated, specific nucleic acid sequence of the present invention also comprises complementary series except comprising the sequence of clearly describing.

Being of wide application of term " gene " refers to any nucleic acid relevant with biological function. Therefore, gene comprises that coded sequence and/or its express needed adjusting sequence. Term " gene " is applicable to concrete genome sequence and coded cDNA and the mRNA of this genome sequence.

Gene also comprises the nucleic acid fragment of non-expression, for example, forms the nucleic acid fragment of the recognition sequence of other albumen. The adjusting sequence of non-expression comprises " promoter " and " enhancer ", regulates albumen such as transcription factor with it in conjunction with causing near that close on or sequence to be transcribed. " tissue-specific " promoter or enhancer refer to regulate promoter or the enhancer of transcribing in one or more particular tissue type or cell type.

Term " carrier " refers to plasmid, viral vectors, recombinant nucleic acid or cDNA. Carrier can also be DNA of the DNA of the DNA of naked RNA polynucleotides, naked DNA polynucleotides that can not self-replicating, the polynucleotides that are comprised of the DNA in the same chain and RNA, poly-D-lysine combination or RNA, peptide combination or RNA, liposome combination etc. Modal is that carrier of the present invention is plasmid.

" expression vector " is the carrier that can drive its expression of nucleic acid that comprises and copy, such as plasmid. In general, the nucleic acid that express " operability is connected in " promoter and/or enhancer, and controlled by the transcriptional regulatory of promoter and/or enhancer.

The feature of " two-way expression vector " normally contains two promoters, the two is opposite direction for nucleic acid between two promoters, can both start expression like this on both direction, causes, for example, transcribing of (+) or positive-sense strand and negative (-) or antisense strand RNA just. In addition, two-way expression vector can be the ambisense carrier, and wherein viral mRNA and virus genome RNA (cRNA) are expressed from the same chain.

In the context of the present invention, term " separation " refer to substantially not be included in its natural surroundings with its in conjunction with or the biomaterial of interactional component, such as nucleic acid or protein. Parting material can also be included in its natural surroundings, for example, cell, in undiscovered material. For example, if material is in its natural surroundings, for example in the cell, material is non-natural the material of residing position (for example, genome or genetic elements) in this environment in cell so. For example; natural acid (for example; coded sequence, promoter, enhancer etc.) if by the non-natural mode be directed to concerning this nucleic acid be non-natural genomic locus (for example; carrier such as plasmid or viral vectors; or amplicon) in, so this natural acid just becomes separation. This nucleic acid is also referred to as " allos " nucleic acid.

Term " recombinant " refers to by artificial interference manual change or the synthetic material (for example, nucleic acid or protein) that changes (non-natural) occur. Change can be finished by the material in its natural surroundings or state, also can finish at the material of separating from its natural surroundings or state. Especially, if refer to virus, for example, and influenza virus, when virus is by expressing recombinant nucleic acid when preparing, virus is recombinant.

Term " reprovision body " refers to that virus comprises gene and/or the polypeptide fractions that derives from a plurality of parental virus strains or resource when being used for virus. For example, 7: 1 reprovision bodies comprise 7 the viral genome fragments (or genetic fragment) that derive from the first parental virus and derive from a complementary viral genome fragment of the second parental virus, for example, and coding hemagglutinin or neuraminidase. The reprovision body comprised 6 genomic fragments that derive from the first parental virus in 6: 2, modal 6 internal gene, and two complementary fragments that derive from different parental virus, for example, hemagglutinin and neuraminidase.

Term " introducing " refers to when being used in reference to heterologous nucleic acids or isolating nucleic acid that nucleic acid is inserted in eucaryon or the prokaryotic, its amplifying nucleic acid (for example can be inserted in the genome of cell, chromosome, plasmid, plastid or mitochondrial DNA), change into self-replicating or transient expression (for example, the mRNA of transfection). This term comprises that these methods are such as " infection ", " transfection ", " conversion " and " transduction ". In the context of the present invention, the whole bag of tricks all can be used for nucleic acid is incorporated in the prokaryotic, comprising the transfection (lipofection) of electroporation, calcium phosphate precipitation, lipid mediation etc.

Term " host cell " refers to contain the heterologous nucleic acids such as carrier, support this nucleic acid replication and/or expression, and the optional cell that comprises polypeptide and/or viral one or more coded products of making. Host cell can be prokaryotic such as Escherichia coli, or eukaryotic such as yeast, insect, amphibian, birds or mammalian cell, comprising people's cell. In the context of the present invention, typical host cell (for example comprises Vero (African green monkey kidney) cell, Per.C6 cell (people's Embryonic Retina cell), BHK (young hamster kidney) cell, former generation chicken kidney (PCK) cell, Ma-Da Shi dog kidney (MDCK) cell, Ma-Da Shi ox kidney (MDBK) cell, 293 cells, the 293T cell) and COS cell (for example, COS1, COS7 cell). The term host cell comprises combination or the mixture of cell, comprising, for example, the mixed culture of different cell types or clone (for example, Vero and CEK cell). The common cultivation of the sf Vero cell of electroporation exists, and for example, among the PCT/US04/42669 that on December 22nd, 2004 submitted to description is arranged, and this paper is complete including in as a reference.

Term " engineered " is for referring to virus, viral nucleic acid or encoding viral product herein, and for example, polypeptide, vaccine comprise the sudden change that at least one is introduced by recombination method, for example, and directed mutagenesis, PCR mutagenesis etc. Term " engineered " refers to when being used in reference to the virus (or virus component or product) that comprises one or more coding mutations and/or amino acid replacement that viral genome or the genomic fragment of coding virus (or virus component or product) are not to derive from natural resources, for example by the natural viral strain of non-recombination method (for example going down to posterity 25 ℃ of progressions) preparation or the in the past just zoo virus strain of existence, for example, the A/Ann Arbor/6/60 of wild type or Cold tolerance or B/Ann Arbor/1/66 strain.

Term " % sequence homogeny " can use with term " % homogeny " is mutual in this article, when referring to utilize the sequence alignment program to compare, the level of the amino acid sequence homogeny between two or more peptide sequences, the perhaps level of the nucleotide sequence homogeny between two or more nucleotide sequences. For example, in this article, 80% homogeny refers to refer to that by the thing that the algorithm of determining is determined and 80% sequence homogeny is same a given sequence has at least 80% to be consistent with another sequence of another length. Typical sequence homogeny level includes, without being limited to have 60,70,80,85,90,95 with given sequence, 98% or higher sequence homogeny.

Term " % sequence homology " can use with term " % homology " is mutual in this article, when referring to utilize the sequence alignment program to compare, the level of the amino acid sequence homology between two or more peptide sequences, the perhaps level of the nucleotide sequence homology between two or more nucleotide sequences. For example, in this article, 80% homology refers to by the thing that the algorithm of determining is determined and 80% sequence homology is same, and the homologue of corresponding given sequence has in the length of given sequence and surpasses 80% sequence homology. Typical sequence homology level includes, without being limited to have 60,70,80,85,90,95 with given sequence, 98% or higher sequence homology.

Can be used for determining that the normatron degree of homogeny between two sequences includes, without being limited to a cover blast program, for example, BLASTN, BLASTX and TBLASTX, BLASTP and TBLASTN, open on the NCBI website, can find on the internet. Also can be referring to Altschul etc., 1990, J.Mol.Biol. 215:403-10 (can be with particular reference to disclosed default setting, that is, and parameter w=4, t=17) and Altschul etc., 1997, Nucleic Acids Res., 25:3389-3402. If when estimating a given amino acid sequence with respect to the homogeny of the amino acid sequence in GenBank protein sequence database and other public databases, general using BLASTP program is carried out sequence retrieval. The BLASTX program is preferred for retrieving the nucleotide sequence of all reading frames being translated into the amino acid sequence in GenBank protein sequence database and other public databases. BLASTP and BLASTX can adopt the default parameters operation, and namely open breach point penalty is 11.0, and extending the breach point penalty is 1.0, and utilizes the BLOSUM-62 matrix. The same.

The preferred comparison of the selected sequence of carrying out for " the % homogeny " determined between two or more sequences can utilize the CLUSTAL-W program of MacVector 6.5 versions to finish, open breach point penalty comprising 10.0,0.1 extension breach point penalty, and BLOSUM 30 similarity matrixs.

" specifically hybridization " or " specific hybrid " or " optionally hybridization " refer to that complex mixture (for example, total cell) when having specific nucleotide sequence among DNA or the RNA, nucleic acid molecules is preferential under stringent condition be combined, forms two strands with this nucleotide sequence or hybridize.

Term " stringent condition " refers to probe and its target sequence is preferentially hybridized, the condition low or that do not hybridize with other sequence hybridization degree. " strict hybridization " and " strictly hybridizing wash conditions " are sequence dependent when being used for the nucleic acid hybridization test such as southern blotting technique hybridization and RNA blot hybridization, are distinguishing under different ambient parameters. Instructing more widely of nucleic acid hybridization seen Tijssen, 1993, " biochemistry and Molecular Biology Lab's technology-use nucleic acid probe hybridization " Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part i, the 2nd chapter, " hybridization principle and the general introduction of nucleic acid probe analysis strategy " (Overview of principles of hybridization and the strategy of nucleic acid probe assays), New York Ai Siweier (Elsevier, NY); Sambrook etc., 2001, " molecular cloning: laboratory manual " (Molecular Cloning:A Laboratory Manual), cold spring harbor laboratory (Cold Spring Harbor Laboratory), the third edition, New York; And " contemporary molecular biology operation manual " (Current Protocols in Molecular Biology) of the latest edition compiled such as Ausubel, New York Ge Lien cooperation in publishing and Willie international scientific publishing house (Greene Publishing Associates and Wiley Interscience, NY).

In general, high strict hybridization and the selected temperature of wash conditions are than low about 5 ℃ of the pyrolysis chain temperature (Tm) of specificity sequence under specific ion intensity and pH. Temperature (under specific ionic strength and pH) when Tm is 50% target sequence with the Probe Hybridization of complete coupling. Very strict condition refers to equal the Tm of particular probe.

For the hybridization of complementary nucleic acid on filter membrane that contains the complementary residue more than 100 of having an appointment in southern blotting technique or the RNA trace, an example of stringent hybridization condition be 42 ℃, contain 50% formaldehyde of 1mg heparin, hybridization is spent the night. An example of high strict wash conditions is about 15 minutes of hybridization in 72 ℃, 0.15M NaCl. An example of strict wash conditions is washing 15 minutes in 65 ℃, 0.2 * SSC. Referring to the description to the SSC buffer solution such as Sambrook. Before the strict washing of height, can hang down first strict washing to remove the background probe signals. For, the double-strandednucleic acid of about 100 above nucleotides for example, a kind of typical medium strict wash conditions is washing 15 minutes in 45 ℃, 1 * SSC. For, the double-strandednucleic acid of about 100 above nucleotides for example, a kind of typical low strict wash conditions is washing 15 minutes in 40 ℃, 4-6 * SSC. In general, 2 times (or higher) that SNR is higher than the SNR that uncorrelated probe has in specific cross test illustrate that what detect is specific hybrid.

In this article, unless specialize, term " about " refer to that a numerical value is not more than or the numerical value modified less than this term 10%. For example, term " about 5 μ g/kg " refers to the scope from 4.5 μ g/kg to 5.5 μ g/kg. Another example, " about 1 hour " refers to from 48 minutes to 72 minutes scope.

In the literary composition, when mentioning nucleic acid, term " stable integration " refers to that therefore nucleic acid also become the part of cellular genome with the genomic nucleic acids restructuring of host cell. The nucleic acid of stable integration can comprise selectable marker continues to become cellular genome with the nucleic acid of guaranteeing stable integration a part. The nucleic acid of stable integration needn't continue to be integrated into genome in single position; Nucleic acid can be integrated in more than one position and can be from a position movement to another position in genome.

5.2 the cell of expressing heterologous protease or pepsinogen

The influenza virus that precursor hemagglutinin molecule (HA0) is contained on the surface can not and be caused infection with Fusion of Cells. Must carry out proteolysis cutting and separating HA1 and HA2 subunit to HA0, to obtain its activity form. The virion that the surface has ripe HA can actively merge and cause infection with host cell. Some cell types in the body comprise the cell in the air flue, contain the protease that can activate HA; Yet the cell type of many external uses comprises MDCK, does not contain the active protease that can effectively cut HA. For these cells, the tryptic concentration of external source that adds when cultivating can not cause negative effect to cell but can be with HA cutting and activation. Referring to, for example, U.S. Patent number 5,698,433 and 5,756,341.

Pig trypsase has shown and can effectively activate HA and be that some influenza researchers are normally used by reaching this purpose. In the following embodiments, estimated the recombinant cell of expressing pig trypsase for example or trypsinogen and the ability that they support influenza virus to copy screened in mdck cell for example.

Therefore, in some embodiments, estimate that active protease or the pepsinogen of recombination system or cell oneself expression is relevant with the present invention. Can reduce or remove the animal derived product from protease or the pepsinogen of recombinant cell lines expression cloning, and original position provides protease or pepsinogen can cut most effectively the HA molecule. Perhaps, can express protease or the pepsinogen by the cytogene group coding that cell is not expressed under normal circumstances by changing to the regulation and control of protease or pepsinogen expression. For example, can introduce the promoter that instructs protease or pepsinogen translation and transcribe by the homologous recombination of for example regulating the cellular genome zone that protease or pepsinogen express, such as inducible promoter. By be chosen in activated promoter in the selected cell type (and/or other regulate sequences) can be under normal circumstances not this protease of the cells of expressing protein enzyme or pepsinogen or pepsinogen.

Any known cell that is used for cultivating influenza virus well known by persons skilled in the art can be used for producing cell of the present invention, is not limited in this respect. For example, the suitable host cell for replicating influenza virus comprises that for example, Vero cell, Per.C6 cell, bhk cell, mdck cell, 293 cells and COS cell are comprising 293T cell, COS7 cell. In addition, the coculture that contains two or more above-mentioned clones can improve duplicating efficiency, and ratio that for example can 1: 1 is used mdck cell and 293T or COS cell. In this embodiment, one of cell of cultivating altogether or both can express this heterologous protease.

5.2.1 the protease of cellular expression

Cell of the present invention can be expressed any proteolytic enzyme that is used to cut the HA0 influenza virus protein well known by persons skilled in the art.

Recombination system be can estimate and the ability of some proteolytic enzyme and the ability that some proteolytic enzyme engineerings is built into cell such as mdck cell itself made.Suitable proteolytic enzyme and pepsinogen include, but not limited to trypsinase, trypsinogen and SPRT.Operable other exemplary proteolytic enzyme are listed in following table 1.In addition, described protease activities fragment also can be used for cell of the present invention and method.

Those skilled in the art can determine whether certain specific proteases is applicable to cell of the present invention and method according to routine.Usually, this test comprises the cutting of estimating viral protein, and this cutting is an important step in the viral life cycle.Can directly or indirectly estimate this cutting, directly evaluation Example is as passing through monitoring than larger protein to two a less proteinic generation, and Indirect evaluation is for example by monitoring the virus titer that produces when this proteolytic enzyme exists.For example, can identify and be applicable to the cell of cultivating influenza virus and/or the proteolytic enzyme of method by for example estimating the proteic cutting of HA0 or comprising virus titer in the cell culture of this proteolytic enzyme by monitoring.

Table 1: proteolytic enzyme

S1 family-SA branch	Enzyme classification	Length (AA)	The SwissProt accession number	The GenBank accession number
					Achelase I proteolytic enzyme: Io moth, saturnid moth	3.4.21.-	213	(P23604) ACH1_LONAC	N/A
Achelase II proteolytic enzyme: Io moth, saturnid moth	3.4.21.-	214	(P23605) ACH2_LONAC	N/A
					Acrosin: goat	3.4.21.10	60	(P10626) ACRO_CAPHI	N/A
Acrosin: people	3.4.21.10	421	(P10323) ACRO_HUMAN	Y00970
					Acrosin: mouse	3.4.21.10	436	(P23578) ACRO_MOUSE	S66245
Acrosin: pig	3.4.21.10	415	(P08001)ACRO_PIG	J04950
					Acrosin: rabbit	3.4.21.10	431	(P48038) ACRO_RABIT	U05204

Acrosin: rat	3.4.21.10	437	(P29293) ACRO_RAT	X59254
					Viprinex: Malaya abdomen snake	3.4.21.74	234	(P26324) ANC1_AGKRH	N/A
Viprinex: Malaya abdomen snake	3.4.21.74	258	(P47797) ANC2_AGKRH	L07308
					Viprinex: Cantil, tropical mocassin	3.4.21.74	20	(P33588) ANCR_AGKBI	N/A
Viprinex: southern copperhead (southern copperhead)	3.4.21.74	231	(P09872) ANCR_AGKCO	N/A
					Lipophorin (A): people	3.4.21.-	4548	(P08519) APOA_HUMAN	X06290
Lipophorin (A): macaque	3.4.21.-	1420	(P14417) APOA_MACMU	J04635
					Thrombin-like enzyme: barba amaril, fer-de-lance	3.4.21.74	255	(P04971) BATX_BOTAT	J02684
Complement C1R component: people	3.4.21.41	705	(P00736) C1R_HUMAN	X04701
					Complement C1S component: people	3.4.21.42	688	(P09871) C1S_HUMAN	X06596
Pepsinogen E (proproteinase) (proearboxypeptidase A complex body: ox	N/A	253	(P05805) CAC3_BOVIN	N/A
					Reddish black element (azurocidin) (cationic antimicrobial albumen) that kills: people	N/A	251	(P20160) CAP7_HUMAN	M96326
The reddish black element (cationic antimicrobial PROTEIN C AP37) that kills: pig	N/A	219	(P80015)CAP7_PIG	N/A
					Ca-dependent serine protease: golden hamster	3.4.21.-	695	(P15156) CASP_MESAU	X16160
Cathepsin G: people	3.4.21.20	255	(P08311) CATG_HUMAN	M16117
					Cathepsin G: mouse	3.4.21.20	261	(P28293) CATG_MOUSE	M96801
Cathepsin G: rat	3.4.21.20	26	(P17977) CATG_RAT	N/A
					Cerastotin: desert, tool angle adder (horned desert viper)	3.4.21.-	98	(P81038) CERA_CERCE	N/A
Cerastobin: the husky viper in the Sahara	3.4.21.-	35	(P18692) CERA_CERVI	N/A
					Cercaria proteolytic enzyme: schistosomicide	3.4.21.-	264	(P12546) CERC_SCHMA	J03946
Complement factor B: ox	3.4.21.47	16	(P81187) CFAB_BOVIN	N/A
					Complement factor B: people	3.4.21.47	764	(P00751) CFAB_HUMAN	X72875
Complement factor B: mouse	3.4.21.47	761	(P04186) CFAB_MOUSE	M60646
					Complement factor B: pig	3.4.21.47	151	(Q03710)CFAB_PIG	M59240
Complement Factor D: people	3.4.21.46	253	(P00746) CFAD_HUMAN	M84526
					Complement Factor D: mouse	3.4.21.46	259	(P03953) CFAD_MOUSE	M11768

Complement Factor D: pig	3.4.21.46	259	(P51779)CFAD_PIG	U29948
					Complement Factor D: rat	3.4.21.46	263	(P32038) CFAD_RAT	S73894
Complement factor I: people	3.4.21.45	583	(P05156) CFAI_HUMAN	Y00318
					Complement factor B sample proteolytic enzyme: chicken	3.4.21.-	250	(P81475) CFBL_CHICK	N/A
The calcium enzyme falls: rat	3.4.21.-	268	(P55091) CLCR_RAT	S80379
					Complement C2: people	3.4.21.43	752	(P06681) CO2_HUMAN	M15082
Complement C2: mouse	3.4.21.43	760	(P21180) CO2_MOUSE	M60579
					Cocoon enzyme: Atlantic Ocean king crab	3.4.21.-	14	(P35586) COCO_LIMPO	N/A
Colloidal sol pepsinogen 25KD II/III: crab-beetle	3.4.21.32	20	(P34153) COG1_CHIOP	N/A
					Colloidal sol pepsinogen 28KD: red King Crab	3.4.21.32	20	(P20731) COG1_PARCM	N/A
Colloidal sol pepsinogen 35KD II: crab-beetle	3.4.21.32	20	(P34154) COG2_CHIOP	N/A
					Colloidal sol pepsinogen 36KD: crab-beetle	3.4.21.32	20	(P34155) COG3_CHIOP	N/A
Colloidal sol pepsinogen 36KDA: red King Crab	3.4.21.32	20	(P20732) COGA_PARCM	N/A
					Colloidal sol pepsinogen 36KDB: red King Crab	3.4.21.32	20	(P20733) COGB_PARCM	N/A
Colloidal sol pepsinogen 36KDC: red King Crab	3.4.21.32	20	(P20734) COGC_PARCM	N/A
					Collagenase: bomb fly	3.4.21.-	260	(P08897) COGS_HYPLI	X74306
Brachyurin: Atlantic Ocean fiddler crab (atlantic sand fiddler crab)	3.4.21.32	226	(P00771) COGS_UCAPU	N/A
					The complement activation component of the reactive factor of RA-: people	N/A	699	(P48740) CRAR_HUMAN	D17525
The complement activation component of the reactive factor of RA-: mouse	N/A	704	(P98064) CRAR_MOUSE	D16492
					Quimotrase 1: African malarial mosquito	3.4.21.1	259	(Q27289) CTR1_ANOGA	Z18887
Quimotrase BI: prawn, the white shrimp in Europe	3.4.21.1	271	(Q00871) CTR1_PENVA	X66415
					Quimotrase 2: African malarial mosquito	3.4.21.1	258	(Q17025) CTR2_ANOGA	Z18888
Quimotrase 2: dog	3.4.21.1	263	(P04813) CTR2_CANFA	K01173
					Quimotrase BII: prawn, the white shrimp in Europe	3.4.21.1	271	(P36178) CTR2_PENVA	N/A
Quimotrase II: European hornet	3.4.21.1	218	(P00769) CTR2_VESCR	N/A
					Quimotrase II: east hornet	3.4.21.1	216	(P00768) CTR2_VESOR	N/A

Quimotrase A: ox	3.4.21.1	245	(P00766) CTRA_BOVIN	N/A
					Quimotrase A: Atlantic Ocean cod	3.4.21.1	263	(P47796) CTRA_GADMO	X78490
Quimotrase B: ox	3.4.21.1	245	(P00767) CTRB_BOVIN	N/A
					Quimotrase B: Atlantic Ocean cod	3.4.21.1	245	(P80646) CTRB_GADMO	N/A
Quimotrase B: people	3.4.21.1	263	(P17538) CTRB_HUMAN	M24400
					Quimotrase B: rat	3.4.21.1	263	(P07338) CTRB_RAT	K02298
Chymotrypsin-like serine protease: the red abalone in California	3.4.21.-	254	(P35003) CTRL_HALRU	X71438
					Chymotrypsin-like proteolytic enzyme CTRL-1: people	3.4.21.-	264	(P40313) CTRL_HUMAN	X71874
Quimotrase: prawn	3.4.21.1	31	(P35002) CTRP_PENMO	N/A
					Duodenum proteolytic enzyme (duodenase) I: ox	3.4.21.-	226	(P80219) DDN1_BOVIN	N/A
Mite allergen der.F 3: dermatophagoides pteronyssinus	3.4.21.-	259	(P49275) DEF3_DERFA	D63858
					Mite allergen der.F 6: dermatophagoides pteronyssinus	3.4.21.-	20	(P49276) DEF6_DERFA	N/A
Mite allergen der.P 3: dermatophagoides pteronyssinus	3.4.21.-	261	(P39675) DER3_DERPT	U11719
					Mite allergen der.P 6: dermatophagoides pteronyssinus	3.4.21.-	20	(P49277) DER6_DERPT	N/A
Serine protease easter: fruit bat	3.4.21.-	392	(P13582) EAST_DROME	J03154
					Elastoser 1: ox	3.4.21.36	266	(Q28153) EL1_BOVIN	M80838
Elastoser 1: people	3.4.21.36	68	(P11423) EL1_HUMAN	N/A
					Elastoser 1: pig	3.4.21.36	266	(P00772)EL1_PIG	X04036
Elastoser 1: rat	3.4.21.36	266	(P00773)EL1_RAT	V01234
					Bite NE 2A: horse	3.4.21.-	85	(P37357) EL2A_HORSE	N/A
Elastoser 2A: people	3.4.21.71	269	(P08217) EL2A_HUMAN	M16631
					Bite NE 2B: horse	3.4.21.-	73	(P37358) EL2B_HORSE	N/A
Elastoser 2B: people	3.4.21.71	269	(P08218) EL2B_HUMAN	M16653
					Elastoser 2: ox	3.4.21.71	269	(Q29461) EL2_BOVIN	X97635
Elastoser 2: mouse	3.4.21.71	271	(P05208) EL2_MOUSE	X04573
					Elastoser 2: pig	3.4.21.71	269	(P08419)EL2_PIG	M16651
Elastoser 2: rat	3.4.21.71	271	(P00774)EL2_RAT	V01233

Elastoser IIIA: people	3.4.21.70	270	(P09093)EL3A_HUMAN	M18700
					Elastoser IIIB: people	3.4.21.70	270	(P08861)EL3B_HUMAN	M16630
Elastoser: Atlantic Ocean cod	3.4.21.-	20	(P32197)ELAS_GADMO	N/A
					Leukocyte elastase: people	3.4.21.37	267	(P08246)ELNE_HUMAN	J03545
Erepsin: ox	3.4.21.9	1035	(P98072)ENTK_BOVIN	U09859
					Erepsin: people	3.4.21.9	1019	(P98073)ENTK_HUMAN	U09860
Erepsin: mouse	3.4.21.9	1069	(P97435)ENTK_MOUSE	U73378
					Erepsin: pig	3.4.21.9	1034	(P98074)ENTK_PIG	D30799
Arginine esterase: dog	3.4.21.35	260	(P09582)ESTA_CANFA	Y00751
					Factor X: ox	3.4.21.6	492	(P00743)FA10_BOVIN	X00673
Factor X: chicken	3.4.21.6	475	(P25155)FA10_CHICK	D00844
					Factor X: people	3.4.21.6	488	(P00742)FA10_HUMAN	K03194
Factor X: rabbit	3.4.21.6	490	(O19045)FA10_RABIT	AF003200
					Plasma thromboplastin antecedent: people	3.4.21.27	625	(P03951)FA11_HUMAN	M13142
Hageman factor: ox	3.4.21.38	593	(P98140)FA12_BOVIN	S70164
					Hageman factor: cavy	3.4.21.38	603	(Q04962)FA12_CAVPO	X68615
Hageman factor: people	3.4.21.38	615	(P00748)FA12_HUMAN	M31315
					Proconvertin: ox	3.4.21.21	407	(P22457)FA7_BOVIN	N/A
Proconvertin: people	3.4.21.21	466	(P08709)FA7_HUMAN	M13232
					Proconvertin: mouse	3.4.21.21	446	(P70375)FA7_MOUSE	U66079
Proconvertin: rabbit	3.4.21.21	444	(P98139)FA7_RABIT	U77477
					Plasma thromboplastin component: ox	3.4.21.22	416	(P00741)FA9_BOVIN	J00007
Plasma thromboplastin component: dog	3.4.21.22	452	(P19540)FA9_CANFA	M21757
					Plasma thromboplastin component: cavy	3.4.21.22	285	(P16295)FA9_CAVPO	M26237
Plasma thromboplastin component: people	3.4.21.22	461	(P00740)FA9_HUMAN	K02402
					Plasma thromboplastin component: mouse	3.4.21.22	459	(P16294)FA9_MOUSE	M23109

Plasma thromboplastin component: pig	3.4.21.22	271	(P16293)FA9_PIG	M26235
					Plasma thromboplastin component: rabbit	3.4.21.22	275	(P16292) FA9_RABIT	M26234
Plasma thromboplastin component: rat	3.4.21.22	282	(P16296)FA9_RAT	M26247
					Plasma thromboplastin component: sheep	3.4.21.22	274	(P16291) FA9_SHEEP	M26233
Flavoxobin: habu	3.4.21.-	260	(P05620) FLVB_TRIFL	D67078
					Gilatoxin: pearl lizard (beaded lizard)	3.4.21.-	245	(P43685) GILX_HELHO	N/A
Granzyme A: people	3.4.21.78	262	(P12544) GRAA_HUMAN	M18737
					Granzyme A: mouse	3.4.21.78	260	(P11032) GRAA_MOUSE	X14799
Granzyme B: people	3.4.21.79	247	(P10144) GRAB_HUMAN	M17016
					Granzyme B (G, H): mouse	3.4.21.79	247	(P04187) GRAB_MOUSE	X04072
Granzyme C: mouse	3.4.21.-	248	(P08882) GRAC_MOUSE	M22527
					Granzyme D: mouse	3.4.21.-	248	(P11033) GRAD_MOUSE	J03255
Granzyme E: mouse	3.4.21.-	248	(P08884) GRAE_MOUSE	M36901
					Granzyme F: mouse	3.4.21.-	248	(P08883) GRAF_MOUSE	M36902
Granzyme G: mouse	3.4.21.-	248	(P13366) GRAG_MOUSE	M36900
					Granzyme H: people	3.4.21.-	246	(P20718) GRAH_HUMAN	J02907
Granzyme K: people	3.4.21.-	264	(P49863) GRAK_HUMAN	U35237
					Granzyme K: mouse	3.4.21.-	263	(O35205) GRAK_MOUSE	AF011446
Granzyme K: rat	3.4.21.-	258	(P49864) GRAK_RAT	L19694
					Granzyme M: people	3.4.21.-	257	(P51124) GRAM_HUMAN	L36936
Granzyme M: rat	3.4.21.-	258	(Q03238) GRAM_RAT	L05175
					Granzyme sample protein I: rat	3.4.21.-	248	(Q06605) GRL1_RAT	X66693
Granzyme sample protein I I: rat	3.4.21.-	248	(Q06606) GRL2_RAT	X68657
					Halistase: Japanese Pallas pit viper	3.4.21.-	238	(P81176) HAYS_AGKHA	N/A
Serine protease hepsin: people	3.4.21.-	417	(P05981) HEPS_HUMAN	M18930
					Serine protease hepsin: mouse	3.4.21.-	416	(O35453) HEPS_MOUSE	AF030065
Serine protease hepsin: rat	3.4.21.-	416	(Q05511)	X70900

			HEPS_RAT
					PHGF activator: people	3.4.21.-	655	(Q04756) HGFA_HUMAN	D14012
Hepatocyte growth factor-like protein: people	N/A	711	(P26927) HGFL_HUMAN	M74178
					Hepatocyte growth factor-like protein: mouse	N/A	716	(P26928) HGFL_MOUSE	M74180
PHGF (dispersion factor): people	N/A	728	(P14210) HGF_HUMAN	D90334
					PHGF (dispersion factor): mouse	N/A	728	(Q08048) HGF_MOUSE	D10212
PHGF (dispersion factor): rat	N/A	728	(P17945)HGF_RAT	D90102
					Hypodermin A: bomb fly	3.4.21.-	256	(P35587) HYPA_HYPLI	X74303
Hypodermin B: bomb fly	3.4.21.-	256	(P35588) HYPB_HYPLI	L24915
					Plasma kallikrein: people	3.4.21.34	638	(P03952) KAL_HUMAN	M13143
Plasma kallikrein: mouse	3.4.21.34	638	(P26262) KAL_MOUSE	M58588
					Plasma kallikrein: rat	3.4.21.34	638	(P14272)KAL_RAT	M62357
Glandular kallikrein, inframandibular: cavy	3.4.21.35	31	(P12322) KLK1_CAVPO	N/A
					Glandular kallikrein 1: people	3.4.21.35	262	(P06870) KLK1_HUMAN	M25629
Glandular kallikrein 1: cynomolgus monkey	3.4.21.35	257	(Q07276) KLK1_MACFA	L10039
					Glandular kallikrein K1: mouse	3.4.21.35	261	(P15947) KLK1_MOUSE	M13500
Glandular kallikrein 1: sacredbaboon	3.4.21.35	258	(Q28773) KLK1_PAPHA	L43121
					Glandular kallikrein, 1 of pancreas: rat	3.4.21.35	261	(P00758)KLK1_RAT	J00758
Glandular kallikrein, prostatic: cavy	3.4.21.35	239	(P12323) KLK2_CAVPO	N/A
					Glandular kallikrein 2: people	3.4.21.35	261	(P20151) KLK2_HUMAN	M18156
Tonin (tonin): rat	3.4.21.35	259	(P00759)KLK2_RAT	M11565
					Glandular kallikrein K3: mouse	3.4.21.35	261	(P00756) KLK3_MOUSE	X01389
Glandular kallikrein 3, inframandibular: rat	3.4.21.35	188	(P15950)KLK3_RAT	M26534
					7S nerve growth factor α chain: mouse	N/A	256	(P00757) KLK4_MOUSE	X01800
Glandular kallikrein K5: mouse	3.4.21.35	261	(P15945) KLK5_MOUSE	Y00500
					Glandular kallikrein K6: mouse	3.4.21.35	261	(P00755) KLK6_MOUSE	V00829
Glandular kallikrein 7, inframandibular/kidney: rat	3.4.21.35	261	(P36373)KLK7_RAT	M19647
					Glandular kallikrein K8: mouse	3.4.21.35	261	(P07628) KLK8_MOUSE	X03994

Glandular kallikrein 8, prostatic: rat	3.4.21.35	261	(P36374)KLK8_RAT	M27217
					Glandular kallikrein K9: mouse	3.4.21.35	261	(P15949) KLK9_MOUSE	M17962
Glandular kallikrein 9, inframandibular: rat	3.4.21.35	259	(P07647)KLK9_RAT	M11566
					Glandular kallikrein K11: mouse	3.4.21.35	261	(P15946) KLKA_MOUSE	X13215
Glandular kallikrein 10: rat	3.4.21.35	244	(P36375) KLKA_RAT	S48142
					Glandular kallikrein 12, inframandibular/kidney: rat	3.4.21.35	259	(P36376) KLKB_RAT	M19648
Glandular kallikrein K13: mouse	3.4.21.35	261	(P36368) KLKC_MOUSE	M17982
					γ-feritin, glandula submandibularis: mouse	3.4.21.54	261	(P04071) KLKF_MOUSE	J03877
Glandular kallikrein K22: mouse	3.4.21.35	259	(P15948) KLKL_MOUSE	M17979
					Glandular kallikrein, kidney: African fur rat	3.4.21.35	263	(P32824) KLKR_PRANA	X17352
Glandular kallikrein K26: mouse	3.4.21.35	261	(P36369) KLKZ_MOUSE	K01831
					Glandular kallikrein: pig	3.4.21.35	232	(P00752)KLK_PIG	N/A
Chymotrypsin-like serine protease: people	3.4.21.-	26	(P34168) LCLP_HUMAN	N/A
					King crab coagulation factors C: Japanese king crab	3.4.21.84	1019	(P28175) LFC_TACTR	D90271
Chymotrypsin: dog	3.4.21.39	249	(P21842) MCT1_CANFA	J02904
					Chymotrypsin: people	3.4.21.39	247	(P23946) MCT1_HUMAN	M69137
Chymotrypsin: cynomolgus monkey	3.4.21.39	247	(P56435) MCT1_MACFA	AB000823
					Mast cell protease 1: mongolian gerbil	3.4.21.-	246	(P50340) MCT1_MERUN	D45173
Mast cell protease 1: mouse	3.4.21.-	246	(P11034) MCT1_MOUSE	S44609
					Chymotrypsin: sacredbaboon	3.4.21.39	247	(P52195) MCT1_PAPHA	U38521
Mast cell protease 1 I: rat	3.4.21.39	260	(P09650) MCT1_RAT	U67915
					Mast cell protease 1 A: sheep	3.4.21.-	245	(P80931) MCT1_SHEEP	Y14654
Mast cell protease 12: mongolian gerbil	3.4.21.-	247	(P50341) MCT2_MERUN	D45174
					Mast cell protease 12: mouse	3.4.21.-	244	(P15119) MCT2_MOUSE	J05177
Mast cell protease 1 II: rat	3.4.21.-	247	(P00770) MCT2_RAT	J02712
					Mast cell protease 13: mouse	3.4.21.-	21	(P21843) MCT3_MOUSE	N/A
Mast cell protease 1 III: rat	3.4.21.-	247	(P50339) MCT3_RAT	D38495

Mast cell protease 14: mouse	3.4.21.-	246	(P21812) MCT4_MOUSE	M55617
					Mast cell protease 15: mouse	3.4.21.-	247	(P21844) MCT5_MOUSE	X68805
Mast cell protease 16: mouse	3.4.21.-	276	(P21845) MCT6_MOUSE	M57626
					Mast cell protease 17: mouse	3.4.21.-	273	(Q02844) MCT7_MOUSE	L00654
Mast cell protease 17: rat	3.4.21.-	273	(P27435) MCT7_RAT	U67910
					Mast cell protease 18: mouse	3.4.21.-	247	(P43430) MCT8_MOUSE	X78545
Mast cell protease 19: mouse	3.4.21.-	246	(O35164) MCT9_MOUSE	AF007119
					Mast cell protease 1 sample albumen: mouse	3.4.21.-	246	(Q00356) MCTX_MOUSE	M57401
Serine protease nudel: fruit bat	3.4.21.-	2616	(P98159) NDL_DROME	U29153
					Neutral killer cell proteolytic enzyme 1: rat	3.4.21.-	248	(P18291)NKP1_RAT	M34097
Bite neutrophilic leukocyte proteolytic enzyme 4: the people	3.4.21.-	25	(P18078) NP4_HUMAN	N/A
					Okimaxobin I: hime habu	3.4.21.-	20	(P20005) OKI1_TRIOK	N/A
Proclotting enzyme: Japanese king crab	3.4.21.86	375	(P21902) PCE_TACTR	M58366
					Plasmin: ox	3.4.21.7	812	(P06868) PLMN_BOVIN	X79402
Plasmin: dog	3.4.21.7	333	(P80009) PLMN_CANFA	N/A
					Plasmin: West Europe hedgehog	3.4.21.7	810	(Q29485) PLMN_ERIEU	U33171
Plasmin: horse	3.4.21.7	338	(P80010) PLMN_HORSE	N/A
					Plasmin: people	3.4.21.7	810	(P00747) PLMN_HUMAN	X05199
Plasmin: macaque	3.4.21.7	810	(P12545) PLMN_MACMU	J04697
					Plasmin: mouse	3.4.21.7	812	(P20918) PLMN_MOUSE	J04766
Plasmin: conger pile	3.4.21.7	325	(P33574) PLMN_PETMA	N/A
					Plasmin: pig	3.4.21.7	790	(P06867)PLMN_PIG	N/A
Plasmin: rat	3.4.21.7	169	(Q01177) PLMN_RAT	M62832
					Plasmin: sheep	3.4.21.7	343	(P81286) PLMN_SHEEP	N/A
Myeloblastin (myeloblastin): people	3.4.21.76	256	(P24158) PRN3_HUMAN	X56132
					Prostate specific antigen: people	3.4.21.77	261	(P07288) PROS_HUMAN	X14810
Prostate specific antigen: macaque	3.4.21.35	261	(P33619)	X73560

			PROS_MACMU
					VITAMIN-K dependence protein C: ox	3.4.21.69	456	(P00745) PRTC_BOVIN	K02435
VITAMIN-K dependence protein C: dog	3.4.21.69	157	(Q28278) PRTC_CANFA	D43751
					VITAMIN-K dependence protein C: goat	3.4.21.69	157	(Q28315) PRTC_CAPHI	D43752
VITAMIN-K dependence protein C: cat	3.4.21.69	157	(Q28412) PRTC_FELCA	D43750
					VITAMIN-K dependence protein C: horse	3.4.21.69	157	(Q28380) PRTC_HORSE	D43753
VITAMIN-K dependence protein C: people	3.4.21.69	461	(P04070) PRTC_HUMAN	M11228
					VITAMIN-K dependence protein C: macaque	3.4.21.69	161	(Q28506) PRTC_MACMU	D43754
VITAMIN-K dependence protein C: mouse	3.4.21.69	461	(P33587) PRTC_MOUSE	D10445
					VITAMIN-K dependence protein C: rabbit	3.4.21.69	458	(Q28661) PRTC_RABIT	U49933
VITAMIN-K dependence protein C: rat	3.4.21.69	461	(P31394)PRTC_RAT	X64336
					Serine sample proteolytic enzyme 1: people	3.4.21.-	276	(O43240) PSL1_HUMAN	AF024605
Prostasin: people	3.4.21.-	343	(Q16651) PSS8_HUMAN	LA1351
					Proteolytic enzyme M: people	3.4.21.-	244	(Q92876) PSS9_HUMAN	U62801
Proteolytic enzyme RVV-V α: adder	3.4.21.95	236	(P18964) RVVA_DABRU	N/A
					Proteolytic enzyme RVV-V γ: adder	3.4.21.95	236	(P18965) RVVG_DABRU	N/A
Serine protease SP24D: African malarial mosquito	3.4.21.-	271	(Q17004) S24D_ANOGA	U21917
					Stratum corneum Quimotrase: people	3.4.21.-	253	(P49862) SCCE_ HUMAN	L33404
Serine protease S1 and 2: fruit bat	3.4.21.-	265	(P17205) SER1_DROME	M24379
					Serine protease 3: fruit bat	3.4.21.-	61	(P17207) SER3_DROME	M24380
Serine protease snake: fruit bat	3.4.21.-	430	(P05049) SNAK_DROME	X04513
					Serine protease stubble: fruit bat	3.4.21.-	786	(Q05319) STUB_DROME	L11451
Subtilisin: subtilis	3.4.21.-	378	N/A	M28537
					Zymoplasm: ox	3.4.21.5	625	(P00735) THRB_BOVIN	V00135
Zymoplasm: people	3.4.21.5	622	(P00734) THRB_HUMAN	M17262
					Zymoplasm: mouse	3.4.21.5	618	(P19221) THRB_MOUSE	X52308
Zymoplasm: rat	3.4.21.5	617	(P18292) THRB_RAT	X52835

Gyroxin analogue: pallas pit viper	3.4.21.74	228	(P33589) THRL_LACMU	N/A
					The transmembrane protein enzyme, Serine 2: people	3.4.21.-	492	(O15393) TMS2_HUMAN	U75329
Trypsinase 1: African malarial mosquito	3.4.21.4	274	(P35035) TRY1_ANOGA	Z18889
					Trypsinase, cationic: ox	3.4.21.4	243	(P00760) TRY1_BOVIN	D38507
Trypsinase, cationic: dog	3.4.21.4	246	(P06871) TRY1_CANFA	M11590
					Trypsinase I-P1: chicken	N/A	248	(Q90627) TRY1_CHICK	U15155
Trypsinase I: Atlantic Ocean cod	3.4.21.4	241	(P16049) TRY1_GADMO	X76886
					Trypsinase I: people	3.4.21.4	247	(P07477) TRY1_HUMAN	M22612
Trypsinase I, anionic: rat	3.4.21.4	246	(P00762)TRY1_RAT	V01273
					Trypsinase I: Atlantic salmon	3.4.21.4	242	(P35031) TRY1_SALSA	X70075
Trypsinase: yellow hot mosquito	3.4.21.4	243	(P19799) TRY1_XENLA	X53458
					Trypsinase 2: African malarial mosquito	3.4.21.4	277	(P35036) TRY2_ANOGA	Z18890
Trypsinase, anionic: ox	3.4.21.4	247	(Q29463) TRY2_BOVIN	X54703
					Trypsinase, anionic: dog	3.4.21.4	247	(P06872) TRY2_CANFA	M11589
Trypsinase I-P38: chicken	N/A	248	(Q90628) TRY2_CHICK	U15156
					Trypsinase II: people	3.4.21.4	247	(P07478) TRY2_HUMAN	M27602
Trypsinase II, anionic: rat	3.4.21.4	246	(P00763)TRY2_RAT	V01274
					Trypsinase II: Atlantic salmon	3.4.21.4	231	(P35032) TRY2_SALSA	X70073
Trypsinase: yellow hot mosquito	N/A	244	(P70059) TRY2_XENLA	U72330
					Trypsinase 3A1: yellow hot mosquito	3.4.21.4	254	(P29786) TRY3_AEDAE	X64362
Trypsinase 3: African malarial mosquito	3.4.21.4	275	(P35037) TRY3_ANOGA	Z22930
					Trypsinase II-P29: chicken	N/A	248	(Q90629) TRY3_CHICK	U15157
Trypsinase III: people	3.4.21.4	247	(P15951) TRY3_HUMAN	X15505
					Trypsinase α-3: calliphorid, Australia lucilia cuprina	3.4.21.4	165	(P35043) TRY3_LUCCU	L15632
Trypsinase III, cationic: rat	3.4.21.4	247	(P08426)TRY3_RAT	M16624
					Trypsinase III: Atlantic salmon	3.4.21.4	238	(P35033) TRY3_SALSA	X70074
Trypsinase 4: African malarial mosquito	3.4.21.4	275	(P35038) TRY4_ANOGA	Z22930

Trypsinase 4A: people	3.4.21.4	304	(P35030) TRY4_HUMAN	X72781
					Trypsinase α-4: calliphorid, Australia lucilia cuprina	3.4.21.4	255	(P35044) TRY4_LUCCU	L15632
Trypsinase IV: rat	3.4.21.4	247	(P12788)TRY4_RAT	X15679
					Trypsinase 5G1: yellow hot mosquito	3.4.21.4	238	(P29787) TRY5_AEDAE	X64363
Trypsinase 5: African malarial mosquito	3.4.21.4	274	(P35039) TRY5_ANOGA	Z22930
					Trypsinase 6: African malarial mosquito	3.4.21.4	273	(P35040) TRY6_ANOGA	Z22930
Trypsinase 7: African malarial mosquito	3.4.21.4	267	(P35041) TRY7_ANOGA	Z22930
					Trypsinase α: fruit bat	3.4.21.4	256	(P54624) TRYA_DROER	U40653
Trypsinase α: fruit bat	3.4.21.4	256	(P04814) TRYA_DROME	X02989
					α-tryptase: people	3.4.21.59	275	(P15157) TRYA_HUMAN	M30038
Alkalescence trypsinase A: maduca sexta	N/A	256	(P35045) TRYA_MANSE	L16805
					Trypsinase V-A: rat	3.4.21.4	246	(P32821)TRYA_RAT	X59012
Trypsinase β: fruit bat	3.4.21.4	253	(P54625) TRYB_DROER	U40653
					Trypsinase β: fruit bat	3.4.21.4	253	(P35004) TRYB_DROME	M96372
β-tryptase: people	3.4.21.59	275	(P20231) TRYB_HUMAN	M37488
					Alkalescence trypsinase B: maduca sexta	N/A	256	(P35046) TRYB_MANSE	L16806
Trypsinase V-B: rat	3.4.21.4	246	(P32822) TRYB_RAT	X59013
					Alkalescence trypsinase C: maduca sexta	N/A	256	(P35047) TRYC_MANSE	L16807
Trypsinase δ/γ: fruit bat	3.4.21.4	253	(P54626) TRYD_DROER	U40653
					Trypsinase δ: fruit bat	3.4.21.4	253	(P42276) TRYD_DROME	U04853
Trypsinase ε: fruit bat	3.4.21.4	256	(P54627) TRYE_DROER	U40653
					Trypsinase ε: fruit bat	3.4.21.4	256	(P35005) TRYE_DROME	M96372
Trypsinase γ: fruit bat	3.4.21.4	253	(P42277) TRYG_DROME	U04853
					Trypsinase ι: fruit bat	3.4.21.4	252	(P52905) TRYI_DROME	U41476
Trypsinase precursor: streptomyces griseus	3.4.21.-	259	(P00775) TRYP_STRGR	N/A
					Tryptase sample proteolytic enzyme: rat	3.4.21.59	23	(P27436)TRYL_RAT	N/A
Mastocytoma proteolytic enzyme: dog	3.4.21.-	269	(P19236) TRYM_CANFA	M24665

Mast cell tryptase: rat	3.4.21.59	274	(P50343) TRYM_RAT	D38455
					Trypsinase I: wide finger lobster	3.4.21.4	237	(P00765) TRYP_ASTFL	N/A
Trypsinase CFT-1: dragon spruce budworm	3.4.21.4	256	(P35042) TRYP_CHOFU	L04749
					Trypsinase: cat	3.4.21.4	16	(P81071) TRYP_FELCA	N/A
Trypsinase: sharp sickle spore	3.4.21.4	248	(P35049) TRYP_FUSOX	S63827
					Trypsinase: mouse	3.4.21.4	246	(P07146) TRYP_MOUSE	X04574
Trypsinase: prawn	3.4.21.4	32	(P35050) TRYP_PENMO	N/A
					Trypsinase: pig	3.4.21.4	231	(P00761)TRYP_PIG	N/A
Trypsinase: plaise	3.4.21.4	250	(P35034) TRYP_PLEPL	X56744
					Trypsinase: marble grain lung fish (marbledlungfish)	3.4.21.4	21	(P35051) TRYP_PROAT	N/A
Trypsinase: streptomyces erythareus (Streptomyceserythraeus)	3.4.21.4	227	(P24664) TRYP_SACER	N/A
					Trypsinase: grey sarcophagid	3.4.21.4	254	(P51588) TRYP_SARBU	X94691
Trypsinase: black fly	3.4.21.4	247	(P35048) TRYP_SIMVI	L08428
					Trypsinase: sping dogfish	3.4.21.4	229	(P00764) TRYP_SQUAC	N/A
Trypsin-like proteolytic enzyme: light greenish blue streptomycete	3.4.21.-	268	(Q54179) TRYP_STRGA	U13770
					Trypsinase: streptomyces griseus	3.4.21.4	259	(P00775) TRYP_STRGR	M64471
Tryptase: dog	3.4.21.59	275	(P15944) TRYT_CANFA	M24664
					Trypsinase θ: fruit bat	3.4.21.4	262	(P54628) TRYT_DROER	U40653
Trypsinase θ: fruit bat	3.4.21.4	262	(P42278) TRYT_DROME	U04853
					Mast cell tryptase: mongolian gerbil	3.4.21.59	270	(P50342) TRYT_MERUN	D31789
Trypsinase η: fruit bat	3.4.21.4	258	(P54629) TRYU_DROER	U40653
					Trypsinase η: fruit bat	3.4.21.4	262	(P42279) TRYU_DROME	U04853
Trypsinase X: Atlantic Ocean cod	3.4.21.4	241	(Q91041) TRYX_GADMO	X76887
					Trypsinase ζ: fruit bat	3.4.21.4	281	(P54630) TRYZ_DROER	U40653
Trypsinase ζ: fruit bat	3.4.21.4	280	(P42280) TRYZ_DROME	U04853
					Urokinase-type fibrinolysin activator: ox	3.4.21.73	433	(Q05589) UROK_BOVIN	L03546

Urokinase-type fibrinolysin activator: chicken	3.4.21.73	434	(P15120) UROK_CHICK	J05187
					Urokinase-type fibrinolysin activator: people	3.4.21.73	431	(P00749) UROK_HUMAN	X02419
Urokinase-type fibrinolysin activator: mouse	3.4.21.73	433	(P06869) UROK_MOUSE	X02389
					Urokinase-type fibrinolysin activator: gold hair baboon	3.4.21.73	433	(P16227) UROK_PAPCY	X51935
Urokinase-type fibrinolysin activator: pig	3.4.21.73	442	(P04185)UROK_PIG	X01648
					Urokinase-type fibrinolysin activator: rat	3.4.21.73	432	(P29598) UROK_RAT	X63434
Organize fibrinolysin activator: ox	3.4.21.68	566	(Q28198) UROT_BOVIN	X85800
					Organize fibrinolysin activator: the people	3.4.21.68	562	(P00750) UROT_HUMAN	X07393
Organize fibrinolysin activator: mouse	3.4.21.68	559	(P11214) UROT_MOUSE	J03520
					Organize fibrinolysin activator: rat	3.4.21.68	559	(P19637) UROT_RAT	M23697
Saliva fibrinolysin activator α 1: vampire	3.4.21.68	477	(P98119) URT1_DESRO	M63987
					Saliva fibrinolysin activator α 2: vampire	3.4.21.68	477	(P15638) URT2_DESRO	M63988
Saliva fibrinolysin activator β: vampire	3.4.21.68	431	(P98121) URTB_DESRO	M63989
					Saliva fibrinolysin activator γ: vampire	3.4.21.68	394	(P49150) URTG_DESRO	M63990
Vitellin-degrading proteinase: silkworm moth	3.4.21.-	264	(Q07943) VDP_BOMMO	D16232

5.2.2 the carrier of proteins encoded enzyme

Except, well known by persons skilled in the art being used for all can be used to expressing heterologous proteolytic enzyme or pepsinogen in any appropriate method of cell inner expression heterologous gene.Usually, the recombinant nucleic acid construction that contains the nucleic acid of proteins encoded enzyme or pepsinogen is to adopt conventional Protocols in Molecular Biology to make up, and then this construction is introduced interested host cell and is.Evaluation comprises the cell of required construction, screens then to identify with the proteic cell of desired concn expressing heterologous.The method of carrying out each step in these operations is very many.

The many carriers that are applicable to this purpose are that the public can obtain, comprising but be not limited to: plasmid, phage, virus vector, retroviral vector, artificial chromosome and additive type (episomal) carrier.It is well known to those skilled in the art selecting construction and using the method for this carrier.This carrier can be used for simple clone and mutagenesis; Yet, when the nucleic acid of proteins encoded enzyme or pepsinogen is introduced suitable cell, should adopt expression vector.Can select to hold length carrier usually from 0.25 kilobase (kb) to 40kb or the proteolytic enzyme of bigger any required size or pepsinogen encoding sequence.

Carrier contains various function ingredients usually, comprises clone's (or " polylinker ") site, replication orgin and at least one selectable marker gene.In addition, for expressing this proteolytic enzyme or pepsinogen, carrier has one or more following elements usually: enhancer element, promotor, Transcription Termination and signal sequence, and they are positioned near the cloning site separately, thus operability is connected in the nucleic acid of proteins encoded enzyme or pepsinogen.

Any promotor known in the art or enhancer element be may command proteolytic enzyme or pepsinogen protein expression all.Adoptable suitable promotor includes but not limited to: SV40 early promoter zone (Bernoist and Chambon, 1981, Nature 290:304-310), contained promotor (Yamamoto etc. in the Rous sarcoma virus 3 ' long terminal repeat, 1980, Cell 22:787-797), herpes thymidine kinase promoter (Wagner etc., 1981, Proc.Natl.Acad.Sci.U.S.A.78:1441-1445), adjusting sequence (the Brinster etc. of metallothionein gene, 1982, Nature 296:39-42), tsiklomitsin (Tet) promotor (Gossen etc., 1995, Proc.Nat.Acad.Sci.USA 89:5547-5551) or CMV promotor (as the early stage immediately CMV promotor of people).In addition, temporarily control the expression of proteolytic enzyme or pepsinogen if desired, can adopt any inducible promoter well known by persons skilled in the art according to the present invention, be not limited in this respect.For example, described controlling element can be Interferon, rabbit-reactive inducible promoter or controlling element.Referring to, for example, Levy etc., 1988, Genes ﹠amp; Devel.2:383-393, Reich etc., 1987, P.N.A.S.USA 84:6394-6398 and Pellegrini etc., 1989, Mol Cell Biol.9:4605-4612.Carrier also can be owing to containing estrogen responsive element such as glucocorticoid responsive element (GRE) and estrogen response element (ERE) has inducibility, and described estrogen responsive element can provide the hormone inducibility when containing the cell inner expression of corresponding hormone receptor when carrier is used to.The background level of expressing for reducing can use element that moulting hormone (a kind of insect hormone) is responded as an alternative, with the ecdysone receptor coexpression.

Carrier contains the nucleotide sequence that carrier is duplicated usually in one or more selected host cells.Yet do not need can self-replicating for carrier when carrier attempts to be integrated into the genome of host cell, and does not wish that in fact also their can self-replicating.Usually, this sequence chromosomal DNA of making carrier can be independent of the host duplicates and comprises replication orgin or autonomously replicating sequence.This sequence of various bacteriums, yeast, virus and mammalian cell is known.

The replication orgin of plasmid pBR322 is fit to most of Gram-negative bacterias, and this 2 microns plasmid starting point is fit to yeast, and various viral starting points (for example SV40, adenovirus) can be used for the cloning vector in the Mammals.Usually, mammalian expression vector does not need replication orgin, unless they are used for duplicating the mammalian cell of high-level DNA, as the COS cell.

Advantageously, carrier can contain the selection gene that is called as selectable marker.This genes encoding is grown in the transformed host cells survival of selecting in the substratum or the necessary protein of growing.Therefore not contained the carrier transformed host cells of selecting gene can not survive in this substratum.The protein of typical selection genes encoding can be given the resistance to microbiotic or other toxin such as penbritin, Xin Meisu, methotrexate or tsiklomitsin, remedies the auxotroph defective, or the critical nutrients that can not obtain in the growth medium is provided.Although modal is that the carrier of proteins encoded enzyme or pepsinogen is introduced mammalian cell, also can advantageously adopt Mammals selectable marker, for example G418 resistant gene.

Many screening systems can use, comprising and be not limited to herpes simplex virus thymidine kinase (Wigler etc., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyl transferase (Szybalska ﹠amp; Szybalski, 1962, Proc.Natl.Acad.Sci.USA 48:2026) and VITAMIN B4 nucleic acid ribose transferring enzyme (Lowy etc., 1980, Cell22:817) gene, these genes can be respectively applied in tk-, hgprt-or the aprt-cell.In addition, the metabolic antagonist tolerance also can be used as the basis of screening following gene: the dhfr gene, give the tolerance of cell to ammonia first dish purine (Wigler etc., 1980, Natl.Acad.Sci.USA 77:3567; O ' Hare etc., 1981, Proc.Natl.Acad.Sci.USA78:1527); The gpt gene is given the tolerance of cell to mycophenolic acid (Mulligan ﹠amp; Berg, 1981, Proc.Natl.Acad.Sci.USA 78:2072); The neo gene, give the tolerance of cell to Glucosaminitol G-418 (Colberre-Garapin etc., 1981, J.Mol.Biol.150:1); And the hygro gene, give the tolerance of cell to Totomycin (Santerre etc., 1984, Gene 30:147).Other antibiotic-resistant genes, as the gene of giving penbritin, cefotaxime, gentamicin, G418, Totomycin, Rifampin, kantlex, Xin Meisu, spectinomycin or tetracyclin resistance also can be used as selectable marker.

Expression vector contains the promotor that is connected in encoding sequence interested by host living beings identification and operability usually.This promotor can be induction type or composing type.Term " operability connects " refers to that the arrangement mode of described component allows them to play a role in the mode of their expections.Control sequence " operability is connected in " thus encoding sequence is to interconnect by this way to express this encoding sequence under the condition that is complementary with this control sequence.

Adopt conventional interconnection technique to make up the carrier of proteins encoded enzyme or pepsinogen.With the mode that will produce required carrier cut, cutting and reconnect isolated vectors or dna fragmentation.It is correct sequence that the words that need can be analyzed in a known way with what exist in the carrier that confirms to make up.Construction of expression vector, prepare the in-vitro transcription thing, DNA introduced host cell and the appropriate method analyzed with evaluation expression and function is well known by persons skilled in the art.By whether having gene order in the following routine techniques test sample or its amplification and/or expression being carried out quantitatively: Southern or Northern analyze, the Western trace, DNA, RNA or plaque of protein dot blotting, in situ hybridization, the sequential analysis of immunocytochemistry or nucleic acid or protein molecule.Those skilled in the art will understand easily if desired and how these methods be improved.

When adenovirus is used as expression vector, can makes protein of interest enzyme or pepsinogen encoding sequence connect adenovirus and transcribe/translate control mixture such as late promoter and tripartite leader[.Can this mosaic gene be inserted the adenoviral gene group by reorganization in external or the body then.Insert that virus genomic nonessential zone (for example, E1 or E3 district) will cause surviving and can be in infected host the recombinant virus of expressing protein enzyme or pepsinogen (for example, referring to Logan; Shenk, 1984, Proc.Natl.Acad.Sci.USA 8 1:355-359).Perhaps can adopt retrovirus expression vector, it comprises Harvey murine sarcoma virus (Ha-MSV) long terminal repeat (LTR), the nucleic acid of the abc transport polypeptide that this long terminal repeat side joint promotor and coding are modified.Retroviral vector can duplicate competence or replication defect type.Under the latter event, virus goes down to posterity and will occur over just in the complementary host cell usually.

Artificial chromosome also can be used for transporting the long dna fragmentation of dna fragmentation that can comprise and express than plasmid or retroviral vector.Can make up about 6kb to the artificial chromosome of 10Mb and transport method (liposome, polycation amino polymer or vesica) by routine and transport to be used for the treatment of purpose.(referring to, for example, Harrington, J.J. etc. (1997) Nat.Genet.15:345-355.)

Also may need specific start signal for effectively translating the proteolytic enzyme or the pepsinogen encoding sequence that insert.These signals comprise ATG initiator codon and flanking sequence.In addition, initiator codon should be positioned at the reading frame of required encoding sequence to guarantee to translate complete insertion fragment.These external source translation control signals and initiator codon can be from various natural and synthetic sources.Comprise suitable transcriptional enhancer element, transcription terminator etc. can improve expression efficiency (referring to, for example, Bittner etc., 1987, Methods in Enzymol.153:516-544).

The cell of stably express proteolytic enzyme or pepsinogen is provided in some embodiments.For making this clone but do not adopt the expression vector that contains the virus replication starting point, the available DNA transformed host cell that is subjected to suitable expression controlling elements (for example, promotor, enhanser, sequence, transcription terminator, polyamides thuja acid site etc.) and selectable marker control.Introduce after the foreign DNA, the cell of through engineering approaches was grown in enriched medium 1-2 days, transfer to the selection substratum then.Selectable marker in the recombinant plasmid is given and is selected resistance and allow cell the plasmid stable integration to be gone in their genome and growth formation colony, can clone colony then and it is extended to clone.

5.2.3 make the method for the cell of expressing heterologous proteolytic enzyme

The selected method that is used for the carrier of proteins encoded enzyme or pepsinogen is introduced required cell will depend on carrier and cell usually.

The additive method that transforms and nucleic acid is introduced host cell (for example, in conjunction with, protoplast transformation or fusion, transfection, electroporation, liposome transport, film integration technology, high speed DNA bag merged by particle, virus infection and protoplastis) can realize by the whole bag of tricks well known in the art (referring to, for example, Ausubel, the same, with Sambrook etc., the same).Expression vectors such as available plasmid, clay transform or transfection bacterium, yeast, plant or mammalian cell, and as mentioned above, wherein said expression vector comprises interested nucleic acid.Perhaps, the available virus expression carrier cells infected that comprises nucleic acid interested.According to the method for transformation of host cell, carrier and employing, the transient expression of polypeptide or stably express will be composing type or induction type.As mentioned above, one of ordinary skill in the art can determine transient expression or stably express polypeptide, and be constitutive expression or inducible expression protein.

Mammalian cell can be packaged the virus vector direct infection or by chemistry or electrical method transfection.For carrying out chemical transfection, available CaPO ₄Co-precipitation DNA or use based on the reagent of liposome lipid or non-liposome lipid and introduce DNA.Can obtain to carry out CaPO ₄Commercial reagent box (the CalPhos of transfection ^TMThe Mammals transfection reagent, clone's science and technology laboratory company of California, USA Paro atropic (Clontech Laboratories, Palo Alto, Calif., USA)), and the transfection of lipid mediation, described reagent such as LIPOFECTAMIN are carried out in available commercial reagent 2000, LIPOFECTAMINE ^TMReagent, CELLFECTIN Reagent, LIPOFECTIN

Reagent (California, USA Ka Ersibaide because of the dimension Qu Gen (Invitrogen of company, Carlsbad, Calif., USA)), DOTAP lipofectamine, FuGENE 6, X-tremeGENE Q2, DOSPER ((the Roche Molecular Biochemicals of Luo Shi molecular biochemistry company of Indianapolis, IN, USA, Indianapolis, Ind.USA)), Effectene ^TM, PolyFect

And Superfect (the proper root company of California, USA Valencia (Qiagen, Inc., Valencia, Calif., USA)).The method of electroporation of mammalian cells can for example find in the following document: Norton etc. (volume), Gene Transfer Methods:Introducing DNA into Living Cells and Organisms, BioTechniques Books, Eton publishing company (Eaton Publishing Co.) (2000).Other rotaring dyeing technologies comprise partickle bombardment transfection and microinjection.Referring to, for example, Cheng etc., Proc.Natl.Acad.Sci.USA90 (10): 4455-9 (1993); Yang etc., Proc.Natl.Acad.Sci.USA 87 (24): 9568-72 (1990).

5.2.4 the cell of culture expression heterologous protease

The cell of expressing heterologous proteolytic enzyme can be cultivated in any suitable medium well known by persons skilled in the art, is not limited in this respect.In general, cell is cultivated in the commercial substratum of standard, (for example for example add serum, 10% foetal calf serum) the improved Eagle substratum of Dulbecco, perhaps in serum free medium, cultivate, culture condition is for being suitable for keeping the controlled humidity and the CO of neutral buffered pH (for example, pH is between 7.0 to 7.2) ₂Concentration.Optionally, substratum can comprise microbiotic to prevent bacterial growth, for example, penicillin, Streptomycin sulphate etc., and/or other nutrition, for example L-glutaminate, Sodium.alpha.-ketopropionate, non-essential amino acid, impel other additives that produce good growth characteristics, for example, trypsinase, beta-mercaptoethanol etc.

The existing a lot of reports of method for culturing mammalian cells also are well-known to those skilled in the art in culture.Following books provide universal method, for example, and Freshney (1983) Culture of Animal Cells:Manualof Basic Technique, New York Alan A Lisi (Alan R.Liss, New York); Paul (1975) Cell andTissue Culture, the 5th edition, the Edinburg Reeves pause (Livingston, Edinburgh); Adams (1980) Laboratory Techniques in Biochemistry and Molecular Biology-Cell Culture forBiochemists, Work and Burdon (volume), Amsterdam Ai Siweier (Elsevier, Amsterdam).In the external preparation process of influenza virus, with comprise for other relevant details of the tissue culture method of specific purpose, for example, Merten etc., (1996), Production of influenza virus in cell cultures for vaccinepreparation is selected from Cohen and Shafferman (volume) Novel Strategies in Design and Production ofVaccines, and this paper is complete to be included in as a reference.In addition, can determine to be fit to the variation of this method of the present invention at an easy rate by routine test.

In one embodiment, cell of the present invention is used as attached cell and cultivates on their accompanying surfaces.The adherent surface that the tissue culture cell can be grown in the above is well known in the art.Adherent surface includes but not limited to: the polystyrene plastic of finishing, protein coating surface are (for example, the glass/plastic of fibronectin and/or collagen coating) and a large amount of commercially available microcarrier (for example, the DEAE-Dextran microcarrier bead, as Dormacell, Pfeifer ﹠amp; Langen; Superbead, Flow Laboratories; Styrol copolymer-three-methylamine pearl, as Hillex, SoloHill, AnnArbor).

In one embodiment, cell of the present invention is used as suspension cell culture, does not need carrier.Make the cell adapted method that does not need the suspension growth of carrier be known in the art (referring to, for example, U.S. Patent number 6,825036 and 6,455,298).Optional or randomly, the proteolytic enzyme of adjustable abridged edition invention cell expressing or the level of pepsinogen just can make the cell suspension growth so that need not any adaptation.In some embodiments, thus but the proteolytic enzyme of cell expressing adequate level of the present invention or pepsinogen need not to adapt to just suspension growth.In other embodiments, the cell of the present invention that need not to adapt to as suspension cell growth is used to replication-competent virus.In an embodiment, the cell of the present invention that need not to adapt to as suspension cell growth is used to replicating influenza virus.

In one embodiment, because energy expressing protein enzyme or pepsinogen, therefore cell of the present invention need not to adapt to and just can be used as suspension cell growth.In some embodiments, described proteolytic enzyme or pepsinogen are serine proteases.In other embodiments, described proteolytic enzyme or pepsinogen are trypsinase or trypsinogen.Again in other embodiments, described proteolytic enzyme or pepsinogen are Mammals trypsinase or trypsinogen.Again in another embodiment, described proteolytic enzyme or pepsinogen are bacteriotrypsin or trypsinogen.

In some embodiments, be that about 0.1ng is to about 50 μ g/ml cell cultures by the proteolytic enzyme of the cell expressing of the present invention of suspension growth or the level of pepsinogen.In other embodiments, be 0.1ng or 0.5ng or 1.0ng or 5.0ng or 10ng or 20ng or 30ng or 40ng or 50ng or 60ng or 70ng or 80ng or 90ng or at least 1 μ g or at least 2 μ g or at least 5 μ g or at least 10 μ g or at least 20 μ g or at least 30 μ g or at least 40 μ g or at least 50 μ g/ml cell cultures at least at least at least at least at least at least at least at least at least at least at least at least at least by the level of the proteolytic enzyme of the cell expressing of the present invention of suspension growth or pepsinogen.

The cell that is used for making influenza virus can be cultivated and contain serum or serum free medium.Under the certain situation, for example, host cell is grown under the serum-free condition for making the virus of purifying.Suitable serum free medium is described in the U.S. Provisional Application submitted on December 23rd, 2004 number 60/638,166, the U.S. Provisional Application of submitting on January 5th, 2005 number 60/641, the Application No. 11/304 that on December 16th, 139 and 2005 submitted to, 589, this paper is included in each application by reference in full in.

Those skilled in the art will understand, when tissue culture, adopt serum or animal extracts may have shortcoming (Lambert, K.J. etc., be selected from: Animal Cell Biotechnology, the 1st volume, Spier, R.E. etc., compile the academic press, New York (Academic Pres New York), 85-122 page or leaf (1985)).For example, the chemical constitution of these additives may there are differences between each batch, even if also be like this from same manufacturers.In addition, the additive in animal or human source also may be polluted (for example, mycoplasma, virus and Protein virus) by foreign matter.When in cell culture medium is cultivated, containing these contaminated additives, but the health of these reagent havoc culturing cells.In addition, when the material that produces in by the culture of external reagent contamination was used to cell therapy and other clinical applications, these reagent may bring health risk.What worry most is that existence can cause spongiform encephalopathy of animal and the sick Protein virus of Ke-Ya of people (Creutzfeld-Jakob).Therefore, in some embodiments, described substratum does not contain serum fully.Advantageously, described substratum can not contain animal product fully.Therefore, in some embodiments, described substratum is animal protein-free substratum (APF).In some embodiments, in substratum, do not add the proteolytic enzyme of external source animal derived.In some embodiments, in substratum, do not add the animal derived product.Concrete culture medium prescription is described in, for example, and the Application No. 11/304,589 that on December 16th, 2005 submitted to.

Cell can be in small-scale, for example, be less than in substratum, culture tube or the culturing bottle of 25ml and cultivate, perhaps the vibration big culturing bottle, revolving bottle, perhaps the microcarrier bead in culturing bottle, culture tank or reactor (for example, diethylin ethyl dextran microcarrier pearl, as Dormacell, Pfeifer ﹠amp; Langen; Superbead, FlowLaboratories; Styrol copolymer-three-methylamine pearl, as Hillex, SoloHill, Ann Arbor) go up and cultivate.Microcarrier bead is bead (diameter is between the 100-200 micron), can provide huge surface-area in the cell culture of unit volume so that the adherent cell growth.For example, 1 liter of substratum can comprise and surpass 2,000 ten thousand microcarrier beads, provides to surpass 8000 square centimeters growth surface.For the commercial production of virus, for example, be used for production of vaccine, culturing cell is normally more satisfactory in bio-reactor or fermentor tank.The volume of prior biological reactor from below 1 liter to not waiting more than 100 liters, for example, the Cyto3 bio-reactor (this nimonic company of Austria of Minnesota State Ming Nitangka (and Osmonics, Minnetonka, MN); The NBS bio-reactor (the new cloth Lanace Wei Ke scientific ﹠ technical corporation of New York Madison (New Brunswick Scientific, Edison, N.J.)); The laboratory scale of Braun biotechnology international corporation and commercial-scale bio-reactor (the Braun biotechnology international corporation of German Mai Xungen (B.Braun Biotech, Melsungen, Germany)).

No matter volume of culture is much, in some embodiments, culture is maintained under the temperature of being less than or equal to 35 ℃ with the reorganization of guaranteeing acclimatization to cold to a certain extent and/or the efficient recovery of reprovision influenza virus.For example, in some embodiments, described cell is cultivated between about 32 ℃ to 35 ℃, and temperature is generally between about 32 ℃ to about 34 ℃, usually at about 33 ℃.

In general, can use can perception and keep the setter of cell culture system temperature, for example, thermostatted, or other devices guarantee that temperature can be above 35 ℃ in virus replication.

5.3 the SPRT proteolytic enzyme of streptomyces griseus

Except above-mentioned proteolytic enzyme, the invention provides the new bacteria proteolytic enzyme of a kind of SPRT of being called.As described in the following examples, cloned the gene of this proteolytic enzyme of encoding from the grey Streptomycin sulphate.As mentioned above, SPRT proteolytic enzyme is adapted at being used for cultivating the cell inner expression of virus.

The nucleotides sequence of sprT gene is shown in Fig. 9 (SEQ ID NO:1), and the aminoacid sequence of SPRT proteolytic enzyme is shown in Figure 10 (SEQ ID NO:2).Except the natural acid of coding sprT gene, also predicted nucleotide sequence homologous nucleic acid coding sequence with the sprT gene according to the present invention.Therefore, in some embodiments, the invention provides the have an appointment nucleic acid of 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55% or about 50% identical nucleotide sequence of coding and the nucleotide sequence of Fig. 9.On the other hand, the invention provides the coding nucleic acid of certain polypeptide, it is 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55% or about 50% identical that the sequence that this polypeptide had and the peptide sequence of nucleic acid sequence encoding shown in Figure 9 are had an appointment.

In another embodiment, the invention provides the nucleic acid of hybridizing with the nucleic acid (or nucleic acid of polypeptide shown in the coding schedule 1) that comprises nucleotide sequence shown in Figure 9.In some embodiments, described nucleic acid under the hybridization conditions of regulation with comprise the nucleic acid hybridization of nucleotide sequence shown in Figure 9.In some embodiments, described hybridization conditions is a stringent hybridization condition.In some embodiments, described hybridization conditions is highly strict hybridization conditions.

In addition, nucleic acid of the present invention also comprises the derivative form of the nucleic acid of coding SPRT proteolytic enzyme.This derivative can make by any method known to those skilled in the art, is not limited in this respect.For example, can prepare derivative, substituting, inserting or disappearance comprising 1,2,3,5,10 or more a plurality of Nucleotide of nucleic acid by site-directed mutagenesis.Perhaps, derivative can prepare by random mutagenesis.A kind of method of random mutagenesis nucleic acid is included in and has 0.1mM MnCl ₂With under the condition of unbalanced nucleotide concentration in the PCR reaction amplification of nucleic acid.These conditions have increased the probability that the polysaccharase mistake is mixed in the PCR reaction, cause the nucleic acid generation random mutagenesis that is amplified.

In some embodiments, compare with the wild-type enzyme described in the literary composition, the derivative nucleic acids of described coding SPRT proteolytic enzyme derivative has improved characteristic.For example, in some embodiments, the SPRT proteolytic enzyme of deriving of described derivative nucleic acids coding has stronger activity, for example, and than living greater than wild-type enzyme.In some embodiments, the described SPRT of deriving proteolytic enzyme has with respect to wild-type protease can improve the SPRT proteolytic enzyme excretory secretion signal of deriving.In some embodiments, the described SPRT of deriving proteolytic enzyme can have with respect to wild-type protease and can improve from the former propetide of deriving of SPRT proteolytic cleavage propetide former (prepropeptide) sequence.In some embodiments, the pH when the described SPRT of deriving proteolytic enzyme demonstrates maximum activity is different from wild-type protease.In some embodiments, described pH cultivates the preferred pH of virus as influenza virus.

In other respects, the invention provides a kind of SPRT polypeptide.In some embodiments, described SPRT amino acid sequence of polypeptide is identical at least about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55% or about 50% with the aminoacid sequence of Figure 10.In other respects, the invention provides a kind of protein, its amino acid sequence of polypeptide has at least about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55% or about 50% identical with the aminoacid sequence of table 1.In addition, the invention provides SPRT polypeptide of the present invention or protease activities fragment.In some embodiments, described active fragments comprise aminoacid sequence shown in Figure 10 (or table 1) at least about 10,20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310 or 320 contiguous amino acids, kept protease activity simultaneously.Those skilled in the art can identify this active fragments by ordinary method, for example adopt routine techniques to express this fragment and detect protease activity.

Therefore, on the other hand, the present invention relates in mature polypeptide shown in the SEQ ID NO:2 (or table 1) or its homologous sequence, comprise the artificial variant that one or more amino acid whose conservative propertys replace, lack and/or insert.In an embodiment, minimum on described amino acid whose the transnaturing, promptly be that not remarkably influenced protein folding and/or active conservative amino acid replace or insertion; Little deletion is generally about 30 amino acid of 1-; Little amino-or carboxyl-end extension, as amino-terminal methionine residues; To little joint peptide up to about 20-25 residue; Or help to carry out the little extension of purifying by changing net charge or other functions, described other functions such as polyhistidyl passage (poly-histidine tract), epitope or in conjunction with the territory.

The example that conservative property replaces has the interior replacement of group amino acid down: basic aminoacids (arginine, Methionin and Histidine), acidic amino acid (L-glutamic acid and aspartic acid), polare Aminosaeren (glutamine and l-asparagine), hydrophobic amino acid (leucine, Isoleucine and Xie Ansuan), aromatic amino acid (phenylalanine, tryptophane and tyrosine) and p1 amino acid (glycine, L-Ala, Serine, Threonine and methionine(Met)).Usually it is well known in the art not changing than the aminoacid replacement of living, and is described in, for example, H.Neurath and R.L.Hill, 1979, " protein " (TheProteins), New York press (Academic Press, New York).The change of common generation is Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.

Except 20 kinds of standard amino acids, also available non-standard amino acid (as 4-Hydroxyproline, 6-M-methyllysine, 2-aminoisobutyric acid, isovaline and Alpha-Methyl Serine) replaces the amino-acid residue of wild type peptide.Available a limited number of non--conservative amino acid, can't help genetic code amino acids coding and alpha-non-natural amino acid substituted amino acid residue." alpha-non-natural amino acid " modified behind protein synthesis, and/or has the chemical structure that is different from standard amino acid at its side chain.Alpha-non-natural amino acid can be chemosynthesis and can obtain by commercial, comprising pipecolinic acid, Thiazolidinecarboxylacid acid, dehydroproline, 3-and 4-methylproline and 3,3-dimethyl proline(Pro).

Perhaps, the character of the described amino acid change physical-chemical characteristic of polypeptide for a change.For example, amino acid change can improve the thermostability of polypeptide, changes substrate specificity, changes best pH etc.

Can identify indispensable amino acid in parent's polypeptide according to methods known in the art such as site-directed mutagenesis or L-Ala-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085).In a kind of technology in back, in each residue of molecule, all introduced alanine mutation, and detected the amino-acid residue of the biologic activity (that is lipase activity) of gained mutating molecule with this molecular activity of evaluation decision.Also can referring to, Hilton etc., 1996, J.Biol.Chem.271:4699-4708.Also can determine the interactional avtive spot of enzyme or other biological by the amino acid mutation that structure is carried out physical analysis and supposition contact site, described structure is determined by the technology such as nucleus magnetic resonance, crystallography, electron diffraction or photoaffinity labeling.Referring to, for example, de Vos etc., 1992, Science 255:306-312; Smith etc., 1992, J.Mol.Biol.224:899-904; Wlodaver etc., 1992, FEBS Lett.309:59-64.Also can identify indispensable amino acid by analyzing with the homogeny of polypeptide related polypeptide of the present invention.

Can adopt known mutagenesis, reorganization and/or reorganization method, the screening method of being correlated with then is to make and to test single or multiple aminoacid replacement, described screening method such as Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc.Natl.Acad.Sci.USA 86:2152-2156; WO 95/17413; Or the method for describing among the WO 95/22625.Adoptable additive method comprises fallibility PCR, phage display (for example, Lowman etc., 1991, Biochem.30:10832-10837; U.S. Patent number 5,223,409; WO 92/06204) and fixed regional mutagenesis (Derbyshire etc., 1986, Gene 46:145; Ner etc., 1988, DNA 7:127).

Mutagenesis/reorganization method can make up to detect the activity clone, the mutagenesis polypeptide (Ness etc., 1999, Nature Biotechnology 17:893-896) of host cell expression with high-throughput automatic screening method.Can from host cell reclaim the coding active polypeptide mutagenesis dna molecular and check order fast with the standard method of this area.These methods can the importance of each amino-acid residue of rapid test in polypeptide of interest, and can be used in the polypeptide of structure the unknown.

5.4 expression vector

Again in yet another aspect, the invention provides the expression vector of expressing SPRT proteolytic enzyme or other proteolytic enzyme of the present invention (referring to, for example, table 1).Usually, expression vector is to comprise the recombination of polynucleotide molecule of expression control sequenc that operability is connected in the nucleotide sequence of coded polypeptide.By comprising suitable promotor, replication sequence, selectable marker etc., expression vector can be brought into play function easily in prokaryotic organism or eukaryote, thereby causes the stable mRNA that transcribes and translate.Construction of expression vector and be well known in the art in the technology of the cell inner expression gene that comprises this expression vector.Referring to, for example, Sambrook etc., 2001, " molecular cloning: laboratory manual ", the third edition, cold spring harbor laboratory, " the contemporary molecular biology operational manual " of the latest edition that cold spring port, New York and Ausubel etc. compile, New York Ge Lien publishes cooperation and Willie international scientific press.

The useful promotor that is used for expression vector includes but not limited to: metallothionein promoter, composing type adenovirus major late promoter, the derivable MMTV promotor of dexamethasone, SV40 promotor, MRP pol III promotor, composing type MPSV promotor, the derivable CMV promotor of tsiklomitsin (as the early stage immediately CMV promotor of people), composing type CMV promotor and ifn response type promotor.

Expression vector should comprise expression and the reproducing signals with the cytocompatibility of expressing SPRT proteolytic enzyme.Suitable expression vector includes but not limited to: virus vector such as retrovirus, adenovirus and adeno associated virus, plasmid vector, clay etc.Preferred virus and plasmid vector when expression vector is transfected into mammalian cell.For example, (San Diego, California can provide good transfection and express the speed (rate) of this cell because of dimension Qu Gen company (Invitrogen, San Diego, CA)) to comprise the expression vector pcDNAl of CMV promotor in the expression control sequenc.Other examples of spendable expression vector provide in the following embodiments.

Can by any method known to those skilled in the art expression vector be introduced cell, be not limited in this respect.This method includes but not limited to: for example, directly absorb this molecule by cell from solution; For example adopt liposome or immunoliposome by the fat transfection to help picked-up; The transfection of particle mediation; Or the like.Referring to, for example, U.S. Patent number 5,272,065; Volumes such as Goeddel, 1990, Methods in Enzymology, the 185th volume, academic press, California company limited (Academic Press, Inc., CA); Krieger, 1990, Gene Transfer andExpression--A Laboratory Manual, New York Si Tuketong press (Stockton Press, NY); Sambrook etc., 1989, " molecular cloning: laboratory manual ", New York cold spring harbor laboratory; " the contemporary molecular biology operational manual " of the latest edition of compiling with Ausubel etc., New York Ge Lien publishes cooperation and Willie international scientific press.

Expression vector also can contain can simplify the isolating purification part of sending construction.For example, the poly-histamine part that comprises such as 6 histidine residues can be impregnated in proteinic N-terminal.Should poly-histamine partly allow by nickel chelating chromatography isolated protein easily in a step.In some embodiments, can be after purifying from sending the described purification part of remainder excision of construction.In other embodiments, described part can not hinder the function of sending the construction functional domain therefore to need not excision.

5.5 operation nucleic acid and proteinic additive method

In literary composition of the present invention, can comprise the nucleic acid of the nucleic acid etc. of viral nucleic acid, proteins encoded enzyme or pepsinogen according to the operation of the Protocols in Molecular Biology known.The detailed process that comprises numerous this methods of amplification, clone, mutagenesis, conversion etc. is described in, for example, and Ausubel etc., " contemporary molecular biology operational manual " (augmenting), (the John Wiley ﹠amp of John Wiley Publishing Company in 2006; Sons), New York (" Ausubel "); Sambrook etc., " molecule Clone: laboratory manual "(third edition), the 1-3 volume, cold spring harbor laboratory, the cold spring port, New York, 2001 (" Sambrook "), and Berger and Kimmel " molecule clone technology guide, Enzymology method " (Guide to Molecular Cloning Techniques, Methods in Enzymology), the 152nd volume, academic press, San Diego, California company limited (Academic Press, Inc., San Diego, CA) (" Berger ").

Except above-mentioned reference, (for example can in following document, find such as the technology of polymerase chain reaction (PCR), ligase chain reaction (LCR) (LCR), Q β-replicative enzyme amplification and the mediation of other RNA polymerase, NASBA) etc. the amplification in vitro technological method of cDNA probe of the present invention is used for for example increasing: Mullis etc. (1987) U.S. Patent number 4,683,202; PCR Protocols A Guide to Methods and Applications(volume such as Innis) academic press, San Diego, California company limited (Academic Press Inc.San Diego, CA) (1990) (" Innis "); Arnheim and Levinson (1990) C﹠amp; EN36; The Journal Of NIH Research(1991) 3:81; Kwoh etc. (1989) Proc Natl Acad Sci USA 86, 1173; Guatelli etc. (1990) Proc Natl Acad Sci USA87:1874; Lomell etc. (1989) J Clin Chem35:1826; Landegren etc. (1988) Science241:1077; Van Brunt (1990) Biotechnology8:291; Wu and Wallace (1989) Gene4:560; Barringer etc. (1990) Gene89:117, and Sooknanan and Malek (1995) Biotechnology13:563.In literary composition of the present invention, the additive method that is used for cloning nucleic acid comprises the U.S. Patent number 5,426,039 of Wallace etc.Be summarized in (1994) such as Cheng by improving one's methods of the huge nucleic acid of pcr amplification Nature369:684 and bibliography wherein.

Some polynucleotide of the present invention such as oligonucleotide can utilize the various solid phase strategies that comprise based on the phosphoramidite coupling chemistry of mononucleotide and/or trinucleotide synthetic.For example, can come synthetic nucleic acid sequence by in the polynucleotide chain that is extending, adding activatory monomer activation trisome successively.Referring to for example, Caruthers, M.H. etc. (1992) Meth Enzymol211:3.

Except synthetic required sequence, any in fact nucleic acid all can customize in any one source from many commercial source, these commercial source such as qualified reagent company (The Midland Certified Reagent Company), Pan American genome company (The Great American Gene Company), (ExpressGen of genetic expression company limited interiorly, Inc.), (the Operon Technologies of operon technology company limited, Inc.), or the like.

In addition, can replace selected amino-acid residue in viral polypeptide, proteolytic enzyme or the pepsinogen by for example site-directed mutagenesis.For example, for manufacturing has with desired phenotype feature such as attenuation phenotype, acclimatization to cold, temperature sensitive, by the viral polypeptide of the relevant aminoacid replacement of functions such as specific proteases shearing, specific sudden change can be introduced the viral nucleic acid sections of this polypeptide of coding.Site-directed mutagenesis method is well known in the art and is described in, for example, Ausubel, Sambrook and Berger, the same.The various test kits that carry out site-directed mutagenesis can obtain by commercial, for example, Chameleon site-directed mutagenesis the test kit ((Stratagene of Lars Jia Te genome company of Le Zhula, and can use in the genome segment of first type or second type influenza virus polypeptide of encoding respectively or in the nucleic acid of proteins encoded enzyme or pepsinogen, to introduce for example one or more aminoacid replacement La Jolla)), according to the explanation of manufacturers.

Cultivate influenza virus 5.6 adopt the cell of expressing heterologous proteolytic enzyme

The method of replicating influenza virus is (referring to above being entitled as " cell of culture expression heterologous protease " joint) well known to those skilled in the art in cell culture.Usually, these methods comprise with the virus strain infection proper host cell of selecting.Perhaps, can adopt recombination method that viral genome is introduced host's (for example, hereinafter being entitled as the plasmid rescue of describing in detail in the joint of " the influenza genome carrier in the host cell of expressing protein enzyme or pepsinogen ").Expounding adequately as mentioned, usually in substratum, add exogenous protease with in the cell culture of the cell of not expressing the proteic proteolytic enzyme of effective excision HA0, effectively make influenza virus (referring to, for example, Appleyard etc., 1974, J.Gen Virol.25:351-357; United States Patent (USP) 5,824,536; 4,500,513; With patent disclosure WO96/15232).The cell that an object of the present invention is to provide expressing heterologous proteolytic enzyme is with replicating influenza virus, and in an embodiment, purpose is to improve the efficiency of infection of influenza virus to improve the virus titer in the cell culture.

Therefore, in some embodiments, in the cell culture medium of wanting replication-competent virus such as influenza virus, do not add such as exogenous protease such as pig trypsinase.In some embodiments, the tiring of cell cultures deposits yields of the not expressing heterologous proteolytic enzyme that equals or equal substantially to have added exogenous protease of tiring of influenza virus of cell cultures deposits yields that does not contain the expressing heterologous proteolytic enzyme of exogenous protease.In other embodiments, the tiring of tiring of influenza virus of cell cultures deposits yields that does not contain the expressing heterologous proteolytic enzyme of exogenous protease greater than the cell cultures deposits yields of the not expressing heterologous proteolytic enzyme that has added exogenous protease.Again in other embodiments, the cell culture of expressing heterologous proteolytic enzyme can be bred influenza virus and reached commercial and reasonably tire (＞10 ⁷Log TCID ₅₀/ mL).

Again in another embodiment, can in the cell culture medium of wanting replication-competent virus such as influenza virus, add such as exogenous protease such as pig trypsinase or SPRT proteolytic enzyme.Such embodiment can be used for, for example, and with low-level as be not enough to cultivate virus in the cell of the present invention that propagative viruses reaches commercial horizontal expression proteolytic enzyme of rationally tiring.Such embodiment also can be used for cell expressing wherein of the present invention and cuts and the method for activatory pepsinogen by proteolysis.

In one embodiment, provide the method that in culturing cell, produces the infectious viral particle of minus-stranded rna virus, wherein in cell culture medium, do not added such as exogenous protease such as trypsinase.In an embodiment, (the log that tires that provides the reproducible minus-stranded rna virus that it is reached in cell mass ₁₀TCID ₅₀/ mL) be at least about 6.0 or at least 6.2 or at least 6.4 or at least 6.6 or at least 6.8 or at least 7.0 or at least 7.2 or at least 7.4 or at least 7.6 or at least 7.8 or at least 8.0 or at least 8.2 or at least 8.4 or at least 8.6 or at least 8.8 or at least 9.0 or at least 9.2 or at least 9.4 or at least 9.6 or at least 9.8 method, wherein in this cell culture medium, do not add exogenous protease.

In some embodiments, do not add (the log that tires of influenza virus of cell cultures deposits yields of cell of the expressing heterologous proteolytic enzyme of exogenous protease ₁₀TCID ₅₀/ mL) be at least 6.0 or at least 6.2 or at least 6.4 or at least 6.6 or at least 6.8 or at least 7.0 or at least 7.2 or at least 7.4 or at least 7.6 or at least 7.8 or at least 8.0 or at least 8.2 or at least 8.4 or at least 8.6 or at least 8.8 or at least 9.0 or at least 9.2 or at least 9.4 or at least 9.6 or at least 9.8.

In an embodiment, do not add the log that tires of influenza virus of cell cultures deposits yields of cell of the expressing heterologous proteolytic enzyme of exogenous protease ₁₀TCID ₅₀/ mL tires greatly at least about 0.1 than the influenza virus that produces in the culture that does not add such as the corresponding cell of the not expressing heterologous proteolytic enzyme of exogenous protease such as trypsinase, or at least about 0.2, or at least about 0.3, or at least about 0.4, or at least about 0.5, or at least about 0.6, or at least about 0.7, or at least about 0.8, or at least about 0.9, or at least about 1.0, or at least about 1.2, or at least about 1.4, or at least about 1.6, or at least about 1.8, or at least about 2.0, or at least about 2.2, or at least about 2.4, or at least about 2.6, or at least about 2.6, or at least about 2.8, or at least about 3.0, or at least about 3.2, or at least about 3.4, or at least about 3.6, or at least about 3.8, or at least about 4.0, or at least about 4.2, or at least about 4.4, or at least about 4.6, or at least about 4.8, or at least about 5.0.

In other embodiments, the proteolytic enzyme that can add in cell culture medium is less than otherwise the proteolytic enzyme that should add in cell culture medium.Therefore, in some embodiments, provide in culturing cell the method for the infectious viral particle that produces minus-stranded rna virus, wherein in the cell culture medium that is provided, added minimum such as exogenous protease such as trypsinase.In an embodiment, (the log that tires that provides the reproducible minus-stranded rna virus that it is reached in cell mass ₁₀TCID ₅₀/ mL) be at least about 6.0 or at least 6.2 or at least 6.4 or at least 6.6 or at least 6.8 or at least 7.0 or at least 7.2 or at least 7.4 or at least 7.6 or at least 7.8 or at least 8.0 or at least 8.2 or at least 8.4 or at least 8.6 or at least 8.8 or at least 9.0 or at least 9.2 or at least 9.4 or at least 9.6 or at least 9.8 method, wherein in this cell culture medium, added about 0.1ng/ml to about 100 μ g/ml exogenous protease.

In another specific implementations, (the log that tires that provides the reproducible minus-stranded rna virus that it is reached in cell mass ₁₀TCID ₅₀/ mL) be at least about 6.0 or at least 6.2 or at least 6.4 or at least 6.6 or at least 6.8 or at least 7.0 or at least 7.2 or at least 7.4 or at least 7.6 or at least 7.8 or at least 8.0 or at least 8.2 or at least 8.4 or at least 8.6 or at least 8.8 or at least 9.0 or at least 9.2 or at least 9.4 or at least 9.6 or at least 9.8 method, wherein in this cell culture medium, added about 1mU/ml to about 5000mU/ml exogenous protease.

Again in another embodiment, added the tire (log of about 0.1ng/ml to the influenza virus of the cell cultures deposits yields of the cell of the expressing heterologous proteolytic enzyme of about 100 μ g/ml exogenous protease ₁₀TCID ₅₀/ mL) be at least 6.0 or at least 6.2 or at least 6.4 or at least 6.6 or at least 6.8 or at least 7.0 or at least 7.2 or at least 7.4 or at least 7.6 or at least 7.8 or at least 8.0 or at least 8.2 or at least 8.4 or at least 8.6 or at least 8.8 or at least 9.0 or at least 9.2 or at least 9.4 or at least 9.6 or at least 9.8.In an embodiment, added the tire (log of about 0.1ng/ml to the influenza virus of the cell cultures deposits yields of the cell of the expressing heterologous proteolytic enzyme of about 10 μ g/ml exogenous protease ₁₀TCID ₅₀/ mL) be at least 7.0 or at least 7.2 or at least 7.4 or at least 7.6 or at least 7.8 or at least 8.0 or at least 8.2 or at least 8.4 or at least 8.6 or at least 8.8 or at least 9.0 or at least 9.2 or at least 9.4 or at least 9.6 or at least 9.8.In another embodiment, added the tire (log of about 0.1ng/ml to the influenza virus of the cell cultures deposits yields of the cell of the expressing heterologous proteolytic enzyme of about 1.0 μ g/ml exogenous protease ₁₀TCID ₅₀/ mL) be at least 7.0 or at least 7.2 or at least 7.4 or at least 7.6 or at least 7.8 or at least 8.0 or at least 8.2 or at least 8.4 or at least 8.6 or at least 8.8 or at least 9.0 or at least 9.2 or at least 9.4 or at least 9.6 or at least 9.8.

In some embodiments, added the tire (log of about 1mU/ml to the influenza virus of the cell cultures deposits yields of the cell of the expressing heterologous proteolytic enzyme of about 5000mU/ml exogenous protease ₁₀TCID ₅₀/ mL) be at least 6.0 or at least 6.2 or at least 6.4 or at least 6.6 or at least 6.8 or at least 7.0 or at least 7.2 or at least 7.4 or at least 7.6 or at least 7.8 or at least 8.0 or at least 8.2 or at least 8.4 or at least 8.6 or at least 8.8 or at least 9.0 or at least 9.2 or at least 9.4 or at least 9.6 or at least 9.8.In an embodiment, added the tire (log of about 1mU/ml to the influenza virus of the cell cultures deposits yields of the cell of the expressing heterologous proteolytic enzyme of about 1000mU/ml exogenous protease ₁₀TCID ₅₀/ mL) be at least 7.0 or at least 7.2 or at least 7.4 or at least 7.6 or at least 7.8 or at least 8.0 or at least 8.2 or at least 8.4 or at least 8.6 or at least 8.8 or at least 9.0 or at least 9.2 or at least 9.4 or at least 9.6 or at least 9.8.In another embodiment, added the tire (log of about 1mU/ml to the influenza virus of the cell cultures deposits yields of the cell of the expressing heterologous proteolytic enzyme of about 500mU/ml exogenous protease ₁₀TCID ₅₀/ mL) be at least 7.0 or at least 7.2 or at least 7.4 or at least 7.6 or at least 7.8 or at least 8.0 or at least 8.2 or at least 8.4 or at least 8.6 or at least 8.8 or at least 9.0 or at least 9.2 or at least 9.4 or at least 9.6 or at least 9.8.

In some embodiments, described virus titer obtains incubated cell about 2 days to about 10 days or after optional about 3 days to about 7 days.In an embodiment, described virus titer obtains after 2 days or 3 days or 4 days or 5 days or 6 days or 7 days or 8 days or 9 days or 10 days or 12 days or 14 days at incubated cell.

In some embodiments, add the final concentration of the exogenous protease in the cell culture medium to less than about 0.1ng/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 0.1ng/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 0.5ng/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 1ng/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 5ng/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 10ng/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 50ng/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 100ng/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 250ng/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 500ng/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 750ng/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 1 μ g/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 5 μ g/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 10 μ g/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 25 μ g/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 50 μ g/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 100 μ g/ml.

In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 0.01ng/ml about 100 μ g/ml extremely.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 1ng/ml about 100 μ g/ml extremely.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 10ng/ml about 100 μ g/ml extremely.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 100ng/ml about 100 μ g/ml extremely.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is that about 1 μ g/ml is to about 100 μ g/ml.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is that about 10 μ g/ml are to about 100 μ g/ml.

In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 0.01ng/ml about 10 μ g/ml extremely.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 0.01ng/ml about 1 μ g/ml extremely.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 0.01ng/ml about 100ng/ml extremely.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 0.01ng/ml about 10ng/ml extremely.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 0.01ng/ml about 1ng/ml extremely.In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 0.01ng/ml about 0.1ng/ml extremely.

In some embodiments, the final concentration that adds the exogenous protease in the cell culture medium to is about 1-5000mU/ml, or 5-1000mU/ml, or 100-500mU/ml.In one embodiment, the final concentration that adds the exogenous protease in the cell culture medium to is less than about 1mU/ml.In one embodiment, the final concentration that adds the exogenous protease in the cell culture medium to is less than about 5mU/ml.In one embodiment, the final concentration that adds the exogenous protease in the cell culture medium to is less than about 10mU/ml.In one embodiment, the final concentration that adds the exogenous protease in the cell culture medium to is less than about 25mU/ml.In one embodiment, the final concentration that adds the exogenous protease in the cell culture medium to is less than about 50mU/ml.In one embodiment, the final concentration that adds the exogenous protease in the cell culture medium to is less than about 100mU/ml.In one embodiment, the final concentration that adds the exogenous protease in the cell culture medium to is less than about 250mU/ml.In one embodiment, the final concentration that adds the exogenous protease in the cell culture medium to is less than about 500mU/ml.In one embodiment, the final concentration that adds the exogenous protease in the cell culture medium to is less than about 1000mU/ml.

When cultured cells expressing protein proenzyme, these embodiments that add the proteolytic enzyme that lacks than the common required proteolytic enzyme of effective generation influenza virus in substratum are useful especially.In substratum, add exogenous protease and can shear protected propetide, pepsinogen is changed into its activity form, thus " sensitization (prime) " pepsinogen.Afterwards, activatory proteolytic enzyme can be sheared subsequently the pepsinogen by cell expressing, thereby the pepsinogen of these new generations is changed into active protease.Therefore such embodiment can reduce proteolytic enzyme such as the tryptic consumption of pig that external source is added in a large number.

Therefore, in some embodiments, in cell culture, only add exogenous protease one time.In some embodiments, described exogenous protease simultaneously or almost simultaneously adds cell culture with the virus that is used for cells infected.In some embodiments, described exogenous protease is adding cell culture on the same day with the virus that is used for cells infected.In other embodiments, described exogenous protease is used in adding adding cell culture before the virus of cells infected.In some embodiments, described exogenous protease simultaneously or almost simultaneously adds the cell culture of making virus with introducing carrier in cell.In some embodiments, described exogenous protease is being introduced carrier the cell culture that adds manufacturing virus on the same day of cell.In other embodiments, before adding the carrier of introducing cell, described exogenous protease is added the cell culture of making virus.

Perhaps, in some embodiments, described cell not only expressing protein enzyme but also expressing protein proenzyme.In some such embodiment, can under inducible promoter control, express described proteolytic enzyme to allow described proteolytic enzyme transient expression.Such embodiment allows the proteolytic enzyme transient expression, thus the activator proenzyme.Afterwards, the activatory pepsinogen can continue to activate expressed proteins proenzyme subsequently.Perhaps, in some embodiments, described cell can be expressed two or more proteolytic enzyme or pepsinogen.In some embodiment, described two or more pepsinogens can be expressed under identical or different regulator control system control, as composing type or inducible expression.For the embodiment of inducible expression, but but the inducible system of selecting for the proteolytic enzyme of every kind of abduction delivering or pepsinogen can be identical or different.

In some embodiments, control in time that the expression of heterologous protease is useful in the cell culture.For example, at the appointed time, for example be proved to be useful with inducing about 1,2,3,4 or 5 day the time described proteolytic enzyme to express behind the influenza infection cell culture.In other embodiments, before with the influenza infection cell culture, induce described proteolytic enzyme to express.Again in other embodiments, express with induce described proteolytic enzyme simultaneously with the influenza infection cell culture.Again in another embodiment, before the carrier that will introduce cell add to be made the cell culture of virus, simultaneously or induce described proteolytic enzyme to express afterwards.Therefore, in some embodiments, described heterologous protease can be subjected to inducible promoter control or controlled by the genetic regulation element, as mentioned above.

5.7 the influenza genome carrier in the host cell of expressing protein enzyme or pepsinogen

Except depending on the method based on cell culture with live virus cells infected culture, available recombinant DNA technology such as plasmid rescue are made full infectious influenza virus in cell culture.Referring to, for example, Neumann etc. (1999) Generation of influenza A virus entirely from cloned cDNAs. Proc Natl Acad Sci USA96:9345-9350; Fodor etc. (1999) Rescue of influenza A virus from recombinant DNA. J.Virol73:9679-9682; Hoffmann etc. (2000) A DNA transfection system for generation ofinfluenza A virus from eight plasmids Proc Natl Acad Sci USA97:6108-6113; WO01/83794; Hoffmann and Webster (2000), Unidirectional RNA polymerase I-polymerase IItranscription system for the generation of influenza A virus from eight plasmids, 81:2843-2847; Hoffmann etc. (2002), Rescue of influenza B viruses from 8 plasmids, 99 (17): 11411-11416; U.S. Patent number 6,649,372 and 6,951,754; US publication 20050003349 and 20050037487, these documents are included this paper by reference in.

In some embodiments, the inventive method comprises the cell that a plurality of carriers are introduced expressing heterologous proteolytic enzyme or pepsinogen with the acquisition influenza virus, wherein each vector encoded part influenza virus gene group.Then at the cell that allows culture expression heterologous protease under the condition of viral growth or pepsinogen and reclaim influenza virus.In some embodiments, described influenza virus is attenuated virus, acclimatization to cold venereal disease poison and/or temperature sensitive virus.For example, in some embodiments, described carrier deutero-recombinant influenza can be attenuation, acclimatization to cold, temperature sensitive virus, for example is suitable as attenuated live vaccine, as using with nose intradermal vaccine preparation.In an illustrative embodiments, make virus by introducing a plurality of carriers, described carrier comprises all or part of influenza B/Ann Arbor/1/66 viral genome, for example ca B/Ann Arbor/1/66 viral genome.

In some embodiments, comprise the cDNA of at least 6 internal gene groups of a kind of influenza strain of coding sections and a plurality of carriers of the cDNA of the coding one or more genome segments of different influenza strains (for example, HA and NA vRNA sections) and can be introduced into the cell of expressing heterologous proteolytic enzyme or pepsinogen to obtain influenza virus.For example, described from first type attenuation, acclimatization to cold and/or temperature sensitive or second type influenza virus strain, for example at least 6 internal gene group sections (" main chain ") of the ca of B/Ann Arbor/1/66, att, ts strain can be introduced into the cell of expressing heterologous proteolytic enzyme or pepsinogen with coding derived from the antigenic one or more sections of the immunogenicity of other virus strain.Described immunogenicity surface antigen generally includes one of hemagglutinin (HA) and/or neuraminidase (NA) antigen or both.In the embodiment of the single sections of introducing coding immunogenicity surface antigen, 7 complementary sections of selected virus also are introduced into this host cell.

In some embodiments, comprise the cDNA of 1-7 internal gene group of a kind of influenza strain of coding sections and a plurality of carriers of the cDNA of coding 1-7 genome segments of different influenza strains (for example, HA and NA vRNA sections) and can be introduced into the cell of expressing heterologous proteolytic enzyme or pepsinogen to obtain influenza virus.

In some embodiments, the expression vector of coding influenza vRNA can be gone into the cell of expressing heterologous proteolytic enzyme or pepsinogen by the electroporation cotransfection.In some embodiments, by when having the liposome transfection agent, being transfected into cell or passing through the cell that the calcium phosphate precipitation method is introduced described expression vector expressing heterologous proteolytic enzyme or pepsinogen.In some embodiments, described expression vector is a plasmid.In some embodiments, described expression vector comprises that isolating expression vector is to express each the geneome RNA sections or the respective coding RNA of described virus.

In some embodiments, encode a plurality of plasmid vectors of each influenza virus vRNA are introduced into a group host cell.For example, in some embodiments, can utilize 8 plasmids that complete influenza genome is introduced host cell, the vRNA sections that each own coding of described plasmid is different.In one embodiment, also the encode mRNA of at least a influenza polypeptide of plasmid vector.Perhaps can adopt the more a plurality of plasmids that comprise less genome subsequence.

According to the present invention, can be inserted into recombinant expression vector with operation and production influenza virus with each corresponding virus genome RNA in 8 genome segments of influenza virus.In literary composition of the present invention, can adopt the variety carrier that comprises virus vector, plasmid, clay, phage and artificial chromosome.Usually, for the purpose of easy and simple to handle, the viral genome sections is inserted into plasmid vector, so that one or more replication orgin and optional marks of being convenient to screen or select to be mixed with the cell of plasmid sequence that have function in bacterium and eukaryotic cell to be provided.These carriers can be introduced the cell of expressing heterologous proteolytic enzyme or pepsinogen then to obtain influenza virus.

In another embodiment, the invention provides a kind of method of making recombinant influenza, this method is included in and introduces a plurality of expression vectors in the cell of the present invention, described expression vector comprises the RNA pol I promotor that operability is connected in the cDNA of each influenza geneome RNA of one or more codings, and the expression vector of one or more expression virus mRNA, described virus mRNA coding following one or more influenza polypeptide: PB2, PB1, PA, HA, NP, NA, M1, M2 and NS2; And, promptly gather in the crops described recombinant influenza from described cellular segregation.

In one embodiment, the invention provides the method that produces infectious recombinant influenza in host cell of the present invention, this method adopts expression vector to express vRNA sections or corresponding cRNA and influenza virus protein, especially PB1, PB2, PA and NA.According to this embodiment, can comprise or not comprise helper virus to produce infectious recombinant influenza.

The invention provides a kind of method that in culturing cell of the present invention, produces the infectious viral particle of minus-stranded rna virus, described method comprises: (a) introduce one group of expression vector in the described cell of a group, this expression vector can be at described cell inner expression genome vRNA providing the complete genome group vRNA of described virus, and can express the mRNA of described one or more polypeptide of virus of coding simultaneously; (b) cultivate described cell, thereby make described virion.In some embodiments, described cell is a canine cells.In some embodiments, described cell is a mdck cell.In some embodiments, described virus is Influenza B virus.In some embodiments, this group expression vector is comprised in 1-17 the plasmid.In some embodiments, this group expression vector is contained in 1-8 the plasmid.In some embodiments, this group expression vector is contained in 1-3 the plasmid.In some embodiments, this group expression vector is introduced by electroporation.In some embodiments, each vRNA sections of this group expression vector codes influenza virus.In some embodiments, the mRNA of these one or more influenza polypeptide of group expression vector codes.In some embodiments, each the vRNA sections of this group expression vector codes influenza virus and the mRNA of one or more influenza polypeptide.In some embodiments, the vRNA or the mRNA of second kind of virus of this group expression vector codes.For example, this group carrier comprises the encode HA of second kind of influenza virus and/or one or more carriers of NA mRNA and/or vRNA.In one embodiment, adopted helper virus in the described method.In one embodiment, the described culturing cell that is used for this method is a canine cells.

The present invention also provides a kind of method that produces the infectious recombinant virus particle of minus-stranded rna virus in culturing cell of the present invention, described method comprises: (a) introduce first group of expression vector in the described cell of a group, this expression vector can be at described cell inner expression genome vRNA to provide the complete genome group vRNA of described virus; (b) introduce second group of expression vector in described cell, this expression vector can be expressed the mRNA of described one or more polypeptide of virus of coding; (c) cultivate described cell, thereby make described virion.In some embodiments, described cell is a canine cells.In some embodiments, described cell is a mdck cell.In some embodiments, described virus is Influenza B virus.In some embodiments, described first group of expression vector is contained in 1-8 the plasmid.In some embodiments, described first group of expression vector is contained in 1 plasmid.In some embodiments, described second group of expression vector is contained in 1-8 plasmid.In some embodiments, described second group of expression vector is contained in 1 plasmid.In some embodiments, described first group, second group or these two groups of expression vectors are introduced by electroporation.In some embodiments, each vRNA sections of described first group of expression vector codes influenza virus.In some embodiments, the mRNA of described second group of one or more influenza polypeptide of expression vector codes.In some embodiments, described first group or second group of expression vector (or these two groups) comprise nucleic acid of the present invention.In one embodiment, adopted helper virus in the described method.

The present invention also provides a kind of method that has the infectious recombinant virus particle of the segmented minus-stranded rna virus that surpasses 3 genome vRNA sections such as influenza virus, as influenza A virus etc. that produces in culturing cell of the present invention, described method comprises: (a) introduce first group of expression vector in the described cell of a group, this expression vector can be at described cell inner expression genome vRNA sections to provide the complete genome group vRNA sections of described virus; (b) introduce second group of expression vector in described cell, this expression vector can be expressed the mRNA of described one or more polypeptide of virus of coding; (c) cultivate described cell, thereby make described virion.In some embodiments, described cell is a canine cells.In some embodiments, described cell is a mdck cell.In some embodiments, described recombinant virus is first type or Influenza B virus.In some embodiments, described first group of expression vector is contained in 1-8 the plasmid.In some embodiments, described first group of expression vector is contained in 1 plasmid.In some embodiments, described second group of expression vector is contained in 1-8 the plasmid.In some embodiments, described second group of expression vector is contained in 1 plasmid.In some embodiments, described first group, second group or these two groups of expression vectors are introduced by electroporation.In some embodiments, each vRNA sections of described first group of expression vector codes influenza virus.In some embodiments, the mRNA of one or more or all influenza polypeptide of described second group of expression vector codes.In some embodiments, the vRNA or the mRNA of second kind of virus of described first group or second group of expression vector (or these two groups) coding.For example, one group of carrier comprises HA of second kind of influenza virus of one or more codings and/or the carrier of NA mRNA and/or vRNA.In one embodiment, adopted helper virus in the described method.

The present invention also provides a kind of method that has the infectious recombinant virus particle of the segmented minus-stranded rna virus that surpasses 3 genome vRNA sections such as influenza virus, as influenza A virus etc. that produces in culturing cell of the present invention, described method comprises: (a) introduce one group of expression vector in the described cell of a group, this expression vector can be at described cell inner expression genome vRNA sections providing the complete genome group vRNA sections of described virus, and can express the mRNA of described one or more polypeptide of virus of coding simultaneously; (b) cultivate described cell, thereby make described virion.In some embodiments, described cell is a canine cells.In some embodiments, described cell is a mdck cell.In some embodiments, described virus is first type or Influenza B virus.In some embodiments, this group expression vector is contained in 1-17 the plasmid.In some embodiments, this group expression vector is contained in 1-8 the plasmid.In some embodiments, this group expression vector is contained in 1-3 the plasmid.In some embodiments, this group expression vector is contained in 1 plasmid.In some embodiments, this group expression vector is introduced by electroporation.In some embodiments, each vRNA sections of this group expression vector codes influenza virus.In some embodiments, the mRNA of these one or more influenza polypeptide of group expression vector codes.In some embodiments, each the vRNA sections of this group expression vector codes influenza virus and the mRNA of one or more influenza polypeptide.In some embodiments, this group expression vector comprises nucleic acid of the present invention.In some embodiments, the vRNA or the mRNA of second kind of virus of this group expression vector codes.For example, this group carrier comprises the encode HA of second kind of influenza virus and/or one or more carriers of NA mRNA and/or vRNA.In some embodiments, the vRNA or the mRNA of second kind of virus of described first group or second group of expression vector (or these two groups) coding.For example, one group of carrier comprises HA of second kind of influenza virus of one or more codings and/or the carrier of NA mRNA and/or vRNA.In one embodiment, adopted helper virus in the described method.

Described plasmid expression vector can be the two-way expression vector that can make viral genome sections transcriptional start (that is to say generation (+) chain and (-) chain viral RNA molecule) on any one direction of insertion.In order to carry out bidirectional transcription effectively, each viral genome sections all is inserted in the carrier that contains two independent startup at least, the copy of virus genome RNA can be transcribed out from a chain by first rna polymerase promoter (for example RNA pol I promotor) like this, and virus mRNA is synthetic from second rna polymerase promoter (for example, can start RNA Pol II promotor or other promotors of transcribing that RNA pol II is mediated in cell).Therefore, these two promotors can be arranged relatively, and (that is) both sides, restriction endonuclease recognition sequence preferably are suitable for inserting unique cloning site of virus genome RNA sections to be positioned at least one cloning site.Perhaps, also can use " ambisense (ambisense) " carrier, wherein (+) chain mRNA and (-) chain viral RNA (cRNA) are transcribed from same carrier chain.

VRNA or cRNA in order to ensure each expression have 3 ' correct end, and each vRNA or cRNA expression vector can insert ribozyme sequence or suitable terminator sequence in RNA encoding sequence downstream.This sequence can be, for example, and hepatitis δ viral genome ribozyme sequence or its functional deriv, perhaps mouse rDNA terminator sequence (Genbank accession number M12074).In addition, for example, can use Pol I terminator (Neumann etc., 1994, Virology 202:477-479).The rna expression carrier can utilize the mode identical with the described vRNA expression vector of following document to make up: Pleschka etc., 1996, J.Virol.70:4188-4192; Hoffmann and Webster, 2000, J.Gen Virol.81:2843-2847; Hoffmann etc., 2002, Vaccine 20:3165-3170; Fodor etc., 1999, J.Virol.73:9679-9682; Neumann etc., 1999, P.N.A.S.USA 96:9345-9350; And Hoffmann etc., 2000, Virology 267:310-317; U.S. Patent number 6,649,372 and 6,951,754; US publication 20050003349 and 20050037487, this paper is complete to be included in as a reference.

In other system, pol I can transcribe from different expression vectors with the virus sequence that pol II promotor is transcribed.In these embodiments, can use coding to be in the carrier of each the viral genome sections under the control of pol I promotor and the carrier that coding is in PA at least, PB1, PB2 and NP under the control of pol II promotor.

Under each situation relevant with pol II promotor, the influenza virus gene group sections operability that express is connected in suitable transcriptional control sequence (promotor) to instruct synthesizing of mRNA.Many promotors all are applicable in the expression vector to regulate transcribing of influenza virus gene group sections.In some embodiments, utilization is rna plymerase ii (Pol II) promotor that cytomegalovirus (CMV) DNA relies on.If desired, for example, express for adjusting condition, the promotor that can replace other is to induce under special conditions, perhaps at particular tissues or intracellular, rna transcription.Can obtain a lot of viruses and mammalian promoter now, for example, people's promotor perhaps can be separated these promotors according to the special purpose of being paid close attention to.For example, other promotors that derive from animal and human's virus comprise such promotor, as adenovirus (as adenovirus 2), papillomavirus, hepatitis B virus and polyomavirus, and the miscellaneous retroviruses promotor.Mammalian promoter includes, without being limited to actin promoter, immunoglobulin promoter, heat-shocked promotor etc.In an embodiment, regulate sequence and comprise adenovirus 2 major late promoters that link to each other with the tripartite leader[of the montage of adenovirus hominis 2, as Berg etc., Bio Techniques 14:972-978 is described.In addition, phage promoter also can be united use with the homologous RNA polymerase, for example, and the T7 promotor.

Be used to express viral protein, particularly form the expression vector preferred expression and the purpose virus homologous viral protein of the viral protein of RNP mixture.The expressed viral protein of these expression vectors can be regulated by any adjusting sequence well known by persons skilled in the art.Regulating sequence can be constitutive promoter, inducible promoter or tissue-specific promoter.

Other examples that can be used for controlling the promotor of viral protein expression in the protein expression vector include, without being limited to: SV40 early promoter district (Bernoist and Chambon, 1981, Nature 290:304-310), promotor (the Yamamoto etc. that the sarcoma viral 3 ' long terminal repeat of Rous is comprised, 1980, Cell 22:787-797), herpes thymidine kinase promoter (Wagner etc., 1981, Proc.Natl.Acad.Sci.USA 78:1441-1445), adjusting sequence (the Brinster etc. of metallothionein gene, 1982, Nature 296:39-42); Prokaryotic expression carrier such as β-Nei Xiananmei promotor (Villa-Kamaroff etc., 1978, Proc.Natl.Acad.Sci.USA 75:3727-3731), or tac promotor (DeBoer etc., 1983, Proc.Natl.Acad.Sci.USA 80:21-25); Also can be referring to " useful proteins in recombinant bacteria source " (" Useful proteins fromrecombinant bacteria " the in Scientific American) among " Scientific Beauty compatriots " (Scientific American), 1980,242:74-94; Comprise courage fat alkali synthase promoter district (Herrera-Estrella etc., Nature 303:209-213) or cauliflower mosaic virus 35S RNA promotor (Gardner etc., 1981, Nucl.Acids Res.9:2871) plant expression vector, and the promotor (Herrera-Estrella etc. of photosynthetic enzyme ribulose hydrophosphate carboxylase, 1984, Nature 310:115-120); The promoter element such as Gal 4 promotors, ADC (ethanol dehydrogenase) promotor, PGK (phosphoglycerokinase) promotor, alkaline phosphatase promoter and the following animal transcripting controling area that come from yeast or other fungies, these transcripting controling areas all have tissue specificity, be used in the transgenic animal: activated elastase I gene-controlled area (Swift etc. in pancreatic acinar cell, 1984, Cell 38:639-646; Omitz etc., 1986, Cold Spring Harbor Symp.Quant.Biol.50:399-409; MacDonald, 1987, Hepatology 7:425-515); Activated insulin gene control region in pancreatic beta cell (Hanahan, 1985, Nature 315:115-122), activated immunoglobulin gene control region (Grosschedl etc., 1984, Cell 38:647-658 in lymphoidocyte; Adames etc., 1985, Nature 318:533-538; Alexander etc., 1987, Mol.Cell.Biol.7:1436-1444), activated mouse papilloma virus control region (Leder etc. in testicular cell, mammary gland cell, lymphoidocyte and mastocyte, 1986, Cell 45:485-495), activated albumin gene control region (Pinkert etc. in liver, 1987, Genes and Devel.1:268-276), activated a-fetoprotein gene control region (Krumlauf etc., 1985, Mol.Cell.Biol.5:1639-1648 in liver; Hammer etc., 1987, Science 235:53-58), activated alpha1-antitrypsin gene-controlled area (Kelsey etc. in liver, 1987, Genes and Devel.1:161-171), activated beta globin gene control region (Mogram etc. in medullary cell, 1985, Nature 315:338-340; KoIlias etc., 1986, Cell 46:89-94); Activated myelin basic protein gene-controlled area (Readhead etc. in the oligodendrocyte of brain, 1987, Cell 48:703-712), activated myosin light chain 2 gene-controlled area (Sani in skeletal muscle, 1985, Nature 314:283-286), activated gonadotropin releasing hormone gene-controlled area (Mason etc. and in hypothalamus, 1986, Science 234:1372-1378).

In an embodiment, described protein expression vector comprise with nucleotide sequence can control the promotor that is connected, one or more replication orgin and, randomly, one or more selectable markers (for example, antibiotics resistance gene).In another embodiment, protein expression vector can produce bicistronic mRNA, and this carrier can prepare by inserting the bicistronic mRNA sequence.Can utilize some internal ribosome entry site (IRES) sequence).Preferred IRES element includes, without being limited to Mammals BiP IRES and hepatitis C virus IRES.

Can pass through, for example, three kinds of universal methods identify that comprising gene inserts segmental carrier: (a) nucleic acid hybridization; (b) there is and do not exist " mark " gene function; And (c) expression of insertion sequence.In first method, can detect the virogene that whether has insertion in the carrier by nucleic acid hybridization, used probe comprises and the sequence of inserting dna homolog.In the second approach, can be according to whether existing, gene identifies and screens recombinant vectors/host system because being inserted into some " mark " gene function of causing in the carrier (for example, to antibiotics resistance and transform phenotype).In the third method, can identify expression vector by analyzing the gene product of expressing.This analysis can based on, for example, physical property or the functional performance of viral protein in the analyzed in vitro system, for example, viral protein combines with antibody.

In an embodiment, one or more protein expression vector codings and expression RNP mixture form necessary viral protein.In another embodiment, one or more protein expression vector codings and expression form the necessary viral protein of virion.In another embodiment, all viral proteins of one or more protein expression vector codings and expression certain negative strand rna virus.

Optionally, can strengthen transcribing of expression vector by inserting enhancer sequence.In general, enhanser is a kind of short, and for example, 10-500bp can strengthen the cis-acting DNA element of transcribing with the promotor synergy.From mammalian genes (oxyphorase, Proteinase, bone marrow serine, albumin, alpha-fetoprotein and Regular Insulin) and eukaryotic cell virus, isolate many enhancer sequence.Enhanser can be by 5 ' or 3 ' position of montage allogeneic coding sequence in the carrier, but is inserted into 5 ' position of promotor usually.In general, selected promotor, and if desired, also have other to transcribe enhancement sequences and can optimize the interior expression of host cell, wherein allogeneic dna sequence DNA is introduced in (Scharf etc. (1994), " heat stress promotor and transcription factor (Heat stress promoters andtranscription factors) in this cell Results Probl Cell Differ20:125-62; Kriegler etc. (1990), assembling enhanser, promotor and splicing signal are transferred expression of gene (Assembly of enhancers with control, promoters, and splice signals to control expression of transferred genes) Methods in Enzymol185:512-27).Optionally, amplicon can also comprise ribosome bind site or the internal ribosome entry site (IRES) that is used for translation initiation.

Described carrier can also comprise sequence that is used for Transcription Termination and the sequence that is used for stable mRNA, as polyamides thuja acid site or terminator sequence.This sequence be present in usually eukaryotic cell or viral DNA or cDNA 5 ' and, be 3 ' once in a while, non-translational region.In some embodiments, SV40 polyamides thuja acid sequence provides polyamides thuja acid signal.

In addition, as mentioned above, described expression vector is optional to comprise one or more selectable marker genes to be provided for screening the phenotypic characteristic of transformed host cells, except top listed gene, mark such as Tetrahydrofolate dehydrogenase or neomycin resistance also are applicable to the screening in the eukaryotic cell culture.

The expression vector that comprises above-mentioned suitable dna sequence dna and suitable promotor or control sequence can be used for transforming the host cell that allows protein expression.Though expression vector of the present invention can duplicate in bacterial cell, for the purpose of expressing, modal is to be introduced in the mammalian cell, for example, and Vero cell, bhk cell, mdck cell, 293 cells, COS cell.In an embodiment, use mdck cell for the purpose of expressing.

Described expression vector can be used for instructing genome vRNA or the expression of cRNA accordingly that comprises one or more sudden changes.These sudden changes can cause the attenuation of virus.For example, the vRNA sections can be the vRNA sections that contains attenuation base pair alternate influenza A virus on the panhandle double stranded promoter subarea, particularly, for example, on the double stranded region of NA specificity vRNA the C of 11-12 ' position to A and G to the alternative (Fodor etc. of the known attenuation base pair of U, 1998, J.Virol.6923-6290).By utilizing the method for preparing recombinant negative strand rna virus of the present invention, can identify the attenuation sudden change that makes new advances.

In addition, U.S. Patent number 6,951,754,6,887,699,6,649,372,6,544,785,6,001,634,5,854,037,5,824,536,5,840,520,5,820,871,5,786,199 and 5,166,057 and U.S. Patent Application Publication No. 20060019350,20050158342,20050037487,20050266026,20050186563,20050221489,20050032043,20040142003,20030035814 and 20020164770 described any expression vectors all can be used for the present invention.

In addition, this expression vector also can be used to make the embedded virus of expressing with the allogenic sequence of viral genome.Expression vector instructs the expression of vRNA or corresponding cRNA, and is introduced into host cell with the expression vector that instructs viral protein expression, to produce new infectious recombinant negative strand rna virus or embedded virus.Can introduce these viral heterologous sequences by engineering method and comprise antisense nucleic acid and nucleic acid, as ribozyme.Perhaps, can in these viruses, introduce the heterologous sequence of expression of peptides or polypeptide by engineering method.Can in these viruses, introduce the heterologous sequence of following peptide of coding or polypeptide by engineering method: 1) the distinctive antigen of pathogenic agent; 2) the distinctive antigen of autoimmune disorder; 3) the distinctive antigen of allergen; And 4) the distinctive antigen of tumour.For example, can include but not limited to by the heterologous gene sequence that engineering method is introduced embedded virus of the present invention: human immunodeficiency virus's (HIV) epi-position, as gp160; Hepatitis B virus surface antigen (HBsAg); The glycoprotein of simplexvirus (for example gD, gE); The VP1 of poliovirus; And non-viral pathogenic agent such as bacterium and parasitic antigenic determinant.

In addition, also can modify comprising some aspects of those skilled in the art's currently known methods, thereby improve the rescue effect of infectious viral particle method of the present invention.For example, the reverse genetic technology comprises that preparation contains the synthetic recombinant virus RNA of negative strand viruses RNA non-coding region, and the essential packaging signal of mature virion is discerned and produced in this zone by varial polymerases necessary.Recombinant RNA is from recombinant DNA template synthetic, and rebuilds the reorganization ribonucleoprotein (RNP) that can be used to cut off cell with formation at external varial polymerases complex body with purifying.No matter be external or in vivo, if having varial polymerases albumen during the synthetic rna transcription then can carry out more effectively transfection.Synthetic reorganization RNP can be gone into infectious viral particle by rescue.The U.S. Patent number 5,166,057 that above-mentioned technical description was authorized: on November 24th, 1992; The U.S. Patent number 5,854,037 that on December 29th, 1998 authorized; The U.S. Patent number 5,789,229 that on August 4th, 1998 authorized; The open EP 0702085A1 of the European patent of announcing on February 20th, 1996; U.S. Patent Application Serial Number 09/152,845; The open PCR WO97/12032 of the international monopoly of announcing on April 3rd, 1997; The WO96/34625 that on November 7th, 1996 announced; The open EP-A780475 of European patent; The WO99/02657 that on January 21st, 1999 announced; The WO98/53078 that on November 26th, 1998 announced; The WO98/02530 that on January 22nd, 1998 announced; The WO99/15672 that on April 1st, 1999 announced; The WO98/13501 that on April 2nd, 1998 announced; The WO97/06720 that on February 20th, 1997 announced; And EPO 780 47SA1 of announcement on June 25th, 1997, its content is separately included this paper by reference in full in.

Heterologous nucleic acids is introduced eukaryotic method the carrier that comprises the influenza genome segment (for example can be introduced according to well known in the art, be transfected into) host cell of the present invention, these methods comprise, for example, the transfection of coprecipitation of calcium phosphate, electroporation, microinjection, fat transfection and employing polyamine transfection agents.For example, adopt polyamine transfection agents TransIT-LT1 (Mirus) carriers such as plasmid can be transfected into host cells such as combination such as COS cell, 293T cell, mdck cell or COS or 293T cell and mdck cell according to the explanation of manufacturers.Each carrier that about 1 μ g will be introduced the host cell group mixes with about 2 μ l TransIT-LT1, and with the dilution of 160 μ l substratum (as serum free medium), making cumulative volume is 200 μ l.With DNA: transfection reagent mixture adds 800 μ l substratum then incubated at room 45 minutes.Transfection mixture added host cell and by above-mentioned culturing cell.Therefore, be in cell culture, to make reorganization or reprovision virus, will have mixed in 8 kinds of genome segments (PB2, PB1, PA, NP, M, NS, HA and NA) carrier of each and mix with about 20 μ l TransIT-LT1 and be transfected into host cell.Randomly, available serum free medium such as Opti-MEM I replace containing the substratum of serum before transfection, and cultivate 4-6 hour.

In addition, the electroporation carrier that can be used for comprising influenza virus gene group sections is incorporated in the host cell of the present invention.For example, the plasmid vector that comprises first type or Influenza B virus can be introduced the Vero cell according to following processes by electroporation.In brief, 5 * 10 in the improved Eagle substratum (MEM) that adds 10% foetal calf serum (FBS) will for example be grown in ⁶Individual Vero cell suspension is added in the electroporation groove then in 0.4ml OptiMEM.The 20 μ g DNA that are dissolved in maximum 25 μ l volumes are added in the interior cell of electroporation groove, by knocking gentle the mixing.Specification sheets (for example, being connected with the BioRad Gene Pulser II of Capacitance Extender Plus) according to producer carries out electroporation, and parameter is that 300 volts, 950 micro farads, time constant are between the 28-33 millisecond.Beat cell mixing by gentleness, the 0.7ml MEM that will contain 10%FBS behind the electroporation in about 1-2 minute directly is added in the electroporation groove.Then with cell transfer in two holes of standard 6 hole tissue culture plates, every hole contains the OPTI-MEM of 2ml MEM, 10%FBS or serum-free.Washing electroporation groove is to reclaim all remaining cells, and the washing suspension is assigned in two holes.Final volume is about 3.5ml.Cell is hatched under the condition that allows viral growth then, for example, is about 33 ℃ for acclimatization to cold venereal disease strain.

5.8 reclaim virus from cell culture

Virus usually has from growing the substratum of (transfection) cell of infection and reclaims.Usually before concentrating influenza virus, to clarify thick substratum.Method commonly used comprises filtration, ultrafiltration, absorption and wash-out and centrifugal on barium sulfate.For example, come from the thick substratum that infects culture and at first pass through centrifugal clarification, for example, the centrifugal time enough of 1000-2000 * g, for example centrifugal 10 to 30 minutes, to remove cell debris and other large particulate matters.Perhaps, substratum is filtered the complete cell of removal and other large particulate matters by 0.8 μ m acetate cellulose filters.Randomly, the substratum supernatant of centrifugal clarification is so that influenza virus is agglomerating subsequently, for example, and centrifugal about 3 to 5 hours of 15,000 * g.With suitable damping fluid such as STE (0.01M Tris-HCl; 0.15M NaCl; 0.0001M EDTA) or the phosphate-buffered saline of pH7.4 (PBS) with virus roll into a ball resuspended after, by sucrose (60%-12%) or soluble tartrate (50%-10%) density gradient centrifugation virus is concentrated.Continuously or the substep gradient all be suitable, the substep gradient is as 12% to 60% saccharose gradient with 4 12% steppings.Gradient is centrifugal so that virally be condensed into the visible band so that reclaim with enough speed and time.In addition, for large-scale commercial applications, need utilize the band centrifugation rotor with continuous-mode operation so that from density gradient, eluriate virus.Other details that following document provides are enough to the work that the guidance technology personnel finish from tissue culture the preparation influenza virus, and for example, the Vaccine Production (vaccine production) of Furminger sees Nicholson's etc. (volume) Textbook of Influenza (influenza virus textbook), the 324-332 page or leaf; Merten etc. (1996), Production of influenza virus in cell cultures for vaccinepreparation (preparation is used for the influenza virus of production of vaccine in cell culture) sees Cohen and Shafferman (volume) Novel Strategies in Design and Production of Vaccines (Vaccine design and production New Policy), 141-151 page or leaf, and U.S. Patent number 5,690,937.If desired, the virus of recovery can be stored in-80 ℃, adds sucrose-phosphoric acid-L-glutamic acid (SPG) as stablizer.

In one embodiment, composition provided by the invention is included in the virus (or its part) of duplicating in the cell of the present invention, and described cell has used benzene Zuo Neisi chemicals treatment such as (benzonase) to eliminate the potential oncogene.Therefore, embodiments of the present invention are particularly including the vaccine composition that does not contain oncogene.

5.9 other viruses

Except above-mentioned influenza virus, cell of the present invention and method also can be used to cultivate other virus.In some embodiments, one or more polypeptide of described virus can be cut by the protease protein hydrolysis of host cell on some point of viral life cycle.In concrete embodiment, adopt cell of the present invention or method will improve other required biological natures of viral yield, viral infection or the known virus of raising one of ordinary skill in the art.

Therefore, in some embodiments, the virus of described cultivation is dna virus.In some embodiments, described virus is RNA viruses.In some embodiments, described virus is single-stranded DNA viruses.In some embodiments, described virus is double-stranded DNA virus.In some embodiments, described virus is sense single stranded rna virus.In some embodiments, described virus is negative adopted single strand RNA virus.In some embodiments, described virus is diplornavirus.In some embodiments, described virus is retrovirus.

In some embodiments, described virus is the member who is selected from down in the virales of organizing: Caudoviradles (Caudovirales), Mononegavirales (Mononegavirales) and nest virales (Nidovirales).

In some embodiments, described virus is to be selected from down the Viraceae of group or a member in the subfamily: Myoviridae (Myoviridae), Siphoviridae, Podoviridae (Podoviridae), Rudiviridae (Rudiviridae), Tectiviridae (Tectiviridae), Corticoviridae (Corticoviridae), Lipothrixviridae, Plasmaviridae (Plasmaviridae), Fuselloviridae, Phycodnaviridae, Guttaviridae, Poxviridae (Poxviridae), Chordopoxvirinae (Chordopoxvirinae), Entomopoxvirinae (Entomopoxvirinae), Iridoviridae (Iridoviridae), Polydnaviridae, herpetoviridae (Herpesviridae), Alphaherpesvirinae (Alphaherpesvirinae), Betaherperesvirinae (Betaherpesvirinae), Gammaherpesvirinae (Gammaherpesvirinae), Polyomaviridae, Papillomaviridae (Papillomaviridae), Adenoviridae (Adenoviridae), Ascoviridae (Ascoviridae), Rhabdoviridae (Baculoviridae), Nimaviridae and Asfarviridae.

In some embodiments, described virus is a member that is selected from down in the Tobamovirus of group: T4 sample Tobamovirus, P1 sample Tobamovirus, P2 sample Tobamovirus, Mu sample Tobamovirus, SP01 sample Tobamovirus, Φ H sample Tobamovirus, λ sample Tobamovirus, T1 sample Tobamovirus, T5 sample Tobamovirus, c2 sample Tobamovirus, L5 sample Tobamovirus, ψ M1 sample Tobamovirus, T7 sample Tobamovirus,

29 sample Tobamovirus, P22 sample Tobamovirus, Rudivirus (Rudivirus), Tectivirus (Tectivirus), Corticovirus (Corticovirus), the α lipothrixvirus belongs to (Alphalipothrixvirus), the β lipothrixvirus belongs to (Betalipothrixvirus), the γ lipothrixvirus belongs to (Gammalipothrixvirus), plasmavirus belongs to (Plasmavirus), fusellovirus (Fusellovirus), Chlorovirus (Chlorovirus), Prasinovirus, Prymnesiovirus (Prymnesiovirus), Phaeovirus, Raphidovirus, Coccolithovirus, Guttavirus, orthopoxvirus (Orthopoxvirus), parapoxvirus belongs to (Parapoxvirus), Avipoxvirus (Avipoxvirus), Capripoxvirus (Capripoxvirus), rabbitpox virus belongs to (Leporipoxvirus), Suipoxvirus (Suipoxvirus), Molluscipoxvirus (Molluscipoxvirus), Yatapoxvirus (Yatapoxvirus), α Entomopoxvirus (Alphaentomopoxvirus, β Entomopoxvirus (Betaentomopoxvirus), γ Entomopoxvirus (Gammaentomopoxvirus), iridescent virus (Iridovirus), Chloriridovirus belongs to (Chloriridovirus), Ranavirus (Ranavirus), Lymphocystivirus (Lymphocystivirus), Ichnovirus (Ichnovirus), Bracovirus (Bracovirus), (Ictalurivirus), Simplexvirus (Simplexvirus), Varicellavirus (Varicellovirus), (Mardivirus), (Iltovirus), cytomegalovirus belongs to (Cytomegalovirus), Muromegalovirus (Muromegalovirus), Roseolovirus (Roseolovirus), Lymphocryptovirus belongs to (Lymphocryptovirus), (Rhadinovirus), Polyomavirus (Polyomavirus), Papillomavirus (Papillomavirus), mastadenovirus genus (Mastadenovirus), Aviadenovirus (Aviadenovirus), (Atadenovirus), (Siadenovirus), Ascovirus (Ascovirus), (Mimivirus), Nucleopolyhedrovirus (Nucleopolyhedrovirus), Granulovirus (Granulovirus), (Whispovirus), (Asfivirus) and Rhizidiovirus (Rhizidiovirus).

In some embodiments, described virus is selected from down group: enterobacteria phage T4, enterobacteria phage P1, enterobacteria phage P2, enterobacteria phage Mu, bacillus phage SP01, halophilic bacterium phage Φ H, enterobacteria phage, enterobacteria phage T1, enterobacteria phage T5, lactococcus phage c2, mycobacteriophage L5, methagen ψ M1, enterobacteria phage t7, bacillus phage

29, enterobacteria phage P22, sulfolobus virus SIRV1, enterobacteria phage PRD1, replace Zymomonas mobilis phage PM2, thermoproteus virus 1, Sulfolobus islandicus filamentous virus), (Acidianus filamentous virus 1), acholeplasma phage L2, sulfolobus virus SSV1, paramecium bursaria Chlorella virus 1, micromonas pusilla virus SP1, chrysophyceae virus PW1, (Ectocarpus siliculosis virus 1), (Heterosigma akashiwo virus 01), (Emilianiahuxleyi virus 86), (Sulfolobus neozealandicus droplet-shaped virus), vaccinia virus, blue tongue virus, fowlpox virus, capripox virus, myxoma virus, pig pox virus, mollascus contagiosum virus, yaba monkey tumour virus, melolontha melolontha entomopoxvirus, amsacta moorei entomopoxvirus, chironomus entomopoxviruses, invertebrate iridescent virus 6, invertebrate iridescent virus 3, frog virus 3, lymphocystis disease virus 1, (Campoletissonorensis ichnovirus), (Cotesia melanoscela bracovirus), (Ictalurid simplexvirus 1), the human herpesvirus 1, the herpes virus hominis 3, avian herpetoviruses 2, avian herpetoviruses 1, the human herpesvirus 5, murid herpesvirus 1, the herpes virus hominis 6, the human herpesvirus 4, Saimiriine simplexvirus 2), simian virus 40, cottontail rabbit papillomavirus, adenovirus hominis C, fowl adenovirus A, ovine adenovirus D, turkey adenovirus B, spodoptera frugiperda ascovirus, (Acanthamoeba polyphaga mimivirus), (Autographa californicanucleopolyhedrovirus), cydia pomonella granulovirus, (White spot syndrome virus 1), African swine fever virus and rhizidiomyces virus.

In some embodiments, described virus is to be selected from down the Viraceae of group or a member in the subfamily: Inoviridae (Inoviridae), Microviridae (Microviridae), Geminiviridae (Geminiviridae), Circoviridae (Circoviridae), (Nanoviridae), Parvoviridae (Parvoviridae), (Parvovirinae) and Densovirinae.

In some embodiments, described virus is a member that is selected from down in the Tobamovirus of organizing: inovirus belongs to (Inovirus), Plectrovirus (Plectrovirus), microvirus belongs to (Microvirus), Spiromicrovirus (Spiromicrovirus), Bdellomicrovirus (Bdellomicrovirus), Chlamydiamicrovirus (Chlamydiamicrovirus), Mastrevirus (Mastrevirus), (Curtovirus), Begomovirus (Begomovirus), Circovirus (Circovirus), (Gyrovirus), (Nanovirus), (Babuvirus), parvovirus belongs to (Parvovirus), Erythrovirus (Erythrovirus), dependovirus (Dependovirus), Densovirus (Densovirus), (Iteravirus) and Brevidensovirus (Brevidensovirus).

In some embodiments, described virus is selected from down group: enterobacteria phage M13, acholeplasma phage MV-L51, enterobacteria phage

X174, spiroplasma phage 4, bdellovibrio phage MAC1, chlamydia phage 1, corn virus 2 (Maize streak virus), Beet curly top virus, bean golden mosaic virus-Puerto Rico, porcine circovirus virus, chicken anaemia virus, subterranean clover stunt virus, abaca bunchy top virus (Babana bunchy topvirus), minute virus of mice, B19 virus, adeno associated virus 2, junonia coenia densovirus, bombyx mori densovirus and aedes aegypti densovirus.

In some embodiments, described virus is to be selected from down the Viraceae of group or a member in the subfamily: Hepadnaviridae (Hepadnaviridae) and Caulimoviridae (Caulimoviridae).

In some embodiments, described virus is a member that is selected from down in the Tobamovirus of organizing: positive Hepadnavirus (Orthohepadnavirus), Avihepadnavirus (Avihepadnavirus), Badnavirus (Badnavirus), Caulimovirus (Caulimovirus), (Tungrovirus), (Soymovirus), (Cavemovirus) and Petuvirus.

In some embodiments, described virus is selected from down group: hepatitis B virus, duck hepatitis B virus, commelina yellow mottle virus, cauliflower mosaic virus, rice tungro bacilliform virus, soybean chlorotic mottle virus, cassava vein mosaic virus and petunia vein clearing virus.

In some embodiments, described virus is to be selected from down the Viraceae of group or a member in the subfamily: Pseudoviridae (Pseudoviridae), Metaviridae (Metaviridae) and Retroviridae (Retroviridae).

In some embodiments, described virus is a member that is selected from down in the Tobamovirus of organizing: pseudovirus belongs to (Pseudovirus), Hemivirus (Hemivirus), Metavirus (Metavirus), Errantivirus (Errantivirus), Alpharetrovirus (Alpharetrovirus), Betaretrovirus (Betaretrovirus), Gammaretrovirus (Gammaretrovirus), Deltaretrovirus (Deltaretrovirus), ε Epsilonretrovirus (Epsilonretrovirus), lentivirus (Lentivirus) and Spumavirus (Spumavirus).

In some embodiments, described virus is selected from down group: Saccharomyces cerevisiae Ty1 virus, black-tailed fruit flies copia virus, Saccharomyces cerevisiae Ty3 virus, Drosophila melanogaster gypsy virus, avian leukosis viruses, mouse mammary tumour virus, murine leukemia virus, bovine leukemia virus, walleye dermal sarcoma virus, human immunodeficiency virus 1 and chimpanzee foamy virus.

In some embodiments, described virus is to be selected from down the Viraceae of group or a member in the subfamily: Cystoviridae (Cystoviridae), Reoviridae (Reoviridae), (Birnaviridae), (Totiviridae), (Chrysoviridae), (Partitiviridae and Hypoviridae (Hypoviridae).

In some embodiments, described virus is a member that is selected from down in the Tobamovirus of organizing: Cystovirus belongs to (Cystovirus), Orthoreovirus (Orthoreovirus), Orbivirus (Orbivirus), rotavirus (Rotavirus), (Coltivirus), Aquareovirus (Aquareovirus), cypovirus (Cypovirus), Fijivirus belongs to (Fijivirus), Phytoreovirus (Phytoreovirus), Oryzavirus (Oryzavirus), Aquabirnavirus (Aquabirnavirus), Avibirnavirus (Avibirnavirus), (Entomobirnavirus), Totivirus (Totivirus), Giardiavirus (Giardiavirus), Leishmaniavirus (Leishmaniavirus), chrysovirus (Chrysovirus), Partitivirus (Partitivirus), Alphacryptovirus (Alphacryptovirus), Betacryptovirus (Betacryptovirus), Hypovirus (Hypovirus) and Varicosavirus (Varicosavirus).

In some embodiments, described virus is selected from down group: pseudomonas phage, mammalian orthoreovirus, blue tongue rims, rotavirus A, colorado tick fever virus, Aquareovirus A, cypovirus 1, Fiji's virus, fractilinea oryzae, rice ragged stunt virus, infectious pancreatic necrosis virus, gomvoro disease virus, drosophila melanogaster virus X, saccharomyces cerevisiae virus L-A, giardia lamblia virus, Leishmania RNA virus 1-1, penicillium chrysogenum virus, (Atkinsonella hypoxylon virus), white clover cryptic virus 1, white clover cryptic virus 2, the huge pulse-phase of cryphonectria hypovirus 1-EP713 and lettuce closes virus (Lettuce big-vein associated virus).

In some embodiments, the described viral RNA genome virus RNA of this order virus of encoding.In some embodiments, described virus is to be selected from down the Viraceae of group or a member in the subfamily: Paramyxoviridae (Paramyxoviridae), Pneumovirinae (Pneumovirinae), Rhabdoviridae (Rhabdoviridae), Filoviridae (Filoviridae), Bornaviridae (Bornaviridae), orthomyxoviridae family (Orthomyxoviridae), Bunyaviridae (Bunyaviridae) and Arenaviridae (Arenaviridae).

In some embodiments, described virus is a member that is selected from down in the Tobamovirus of organizing: Respirovirus (Respirovirus), Morbillivirus (Morbillivirus), Rubulavirus (Rubulavirus), (Henipavirus), (Avulavirus), Pneumovirus (Pneumovirus), (Metapneumovirus), Vesiculovirus (Vesiculovirus), lyssavirus (Lyssavirus), Ephemerovirus (Ephemerovirus), Cytorhabdovirus (Cytorhabdovirus), Nucleorhabdovirus (Nucleorhabdovirus), (Novirhabdovirus), (Marburgvirus), (Ebolavirus), Bornavirus (Bornavirus), influenza A virus, Influenza B virus, influenza virus C, Thogotovirus (Thogotovirus), (Isavirus), (Orthobunyavirus), Hantavirus (Hantavirus), Nairovirus (Nairovirus), Phlebovirus (Phlebovirus), (Tospovirus), arenavirus genus (Arenavirus), Ophiovirus (Ophiovirus), oryza virus 3 belongs to (Tenuivirus) or (Deltavirus).

In some embodiments, described virus is selected from down group: Sendai virus, Measles virus, mumps virus, (Hendra virus), Avian pneumo-encephalitis virus, the human respiratory syncytial virus, avian pneumovirus, vesicular stomatitis Indiana virus, rabies virus, bovine ephemeral fever virus, lettuce necrotic yellows virus, potato yellow dwarf virus, infectious haematopoietic necrosis virus, the Lake Victoria Marburg virus, Zaire Ebola virus, berna disease virus, influenza A virus, Influenza B virus, influenza virus C, thogoto virus, infectivity salmon anemia virus (infectious salmon anemia virus), bunyavirus, Hantaan virus, dugbe virus, Rift valley fever virus, tomato spotted wilf virus, lymphocytic choriomeningitis virus, citrus psorsis virus, tenuivirus and hepatitis D.In an embodiment, described virus is influenza A virus or Influenza B virus.In another embodiment, described virus is attenuated influenza virus, acclimatization to cold influenza virus, temperature sensitive influenza virus or the virus with arbitrary combination of these desired characteristic.In one embodiment, described influenza virus comprises the virus of influenza B/Ann Arbor/1/66 strain, for example acclimatization to cold of B/Ann Arbor/1/66, temperature sensitive, attenuation strain.In another embodiment, described influenza virus comprises the virus of influenza A/Ann Arbor/6/60 strain, for example acclimatization to cold of A/Ann Arbor/6/60, temperature sensitive, attenuation strain.

In some embodiments, described virus is to be selected from down the Viraceae of group or a member in the subfamily: Leviviridae (Leviviridae), (Dicistroviridae), pico+ribonucleic acid+virus section (Picornaviridae), Sequiviridae (Sequiviridae), Comoviridae (Comoviridae), (Potyviridae), Caliciviridae (Caliciviridae), Astroviridae (Astroviridae), Nodaviridae (Nodaviridae), (Tetraviridae), Tombusviridae (Tombusviridae), Coronaviridae (Coronaviridae), Arteriviridae (Arteriviridae), (Roniviridae), Alphaherpesvirinae (Togaviridae), flaviviridae (Flaviviridae), Bromoviridae (Bromoviridae), Closteroviridae (Closteroviridae), Barnaviridae (Barnaviridae), (Luteoviridae), (Tymoviridae) and (Flexiviridae).

In some embodiments, described virus is a member that is selected from down in the Tobamovirus of organizing: levivirus belongs to (Levivirus); Allolevivirus (Allolevivirus); (Cripavirus); (Iflavirus); enterovirus genus (Enterovirus); Rhinovirus (Rhinovirus); hepatovirus (Hepatovirus); cardiovirus (Cardiovirus); Hostis (Aphthovirus); (Parechovirus); Sequivirus (Sequivirus); Waikavirus (Waikavirus); Comovirus (Comovirus); broad bean wilt virus belongs to (Fabavirus); nematode passes polyhedrosis virus and belongs to (Nepovirus); potato virus belongs to (Potyvirus); ryegrass mosaic virus belongs to (Rymovirus); bymovirus (Bymovirus); Macluravirus (Macluravirus); (Ipomovirus); (Tritimovirus); (Vesivirus); Lagovirus (Lagovirus); the Norwalk viroid belongs to (Norovirus); (Sapovirus); (Hepevirus); (Mamastrovirus); (Avastrovirus); Alphanodavirus (Alphanodavirus); Betanodavirus (Betanodavirus); Betatetravirus (Betatetravirus); Omegatetravirus (Omegatetravirus); Tombusvirus (Tombusvirus); Carmovirus (Carmovirus); Necrovirus (Necrovirus); Dianthovirus (Dianthovirus); (Machlomovirus); (Avenavirus); (Aureusvirus); Panicovirus (Panicovirus); coronavirus belongs to (Coronavirus); Orbivirus (Torovirus); Arterivirus (Arterivirus); (Okavirus); Alphavirus (Alphavirus); rubella virus genus (Rubivirus); Flavivirus (Flavivirus); pestivirus (Pestivirus); Hepacivirus (Hepacivirus); Alfamovirus (Alfamovirus); isometrical variable Tobamovirus (Ilarvirus); bromovirus (Bromovirus); Cucumovirus (Cucumovirus); Oleavirus (Oleavirus); long linear Tobamovirus (Closterovirus); (Crinivirus); (Ampelovirus); Barnavirus (Barnavirus); barly yellow dwarf virus belongs to (Luteovirus); Polerovirus (Polerovirus); the prominent mosaic virus of Peas ear belongs to (Enamovirus); Tobamovirus (Tobamovirus); Tobravirus (Tobravirus); Hordeivirus (Hordeivirus); furovirus belongs to (Furovirus); (Pomovirus); (Pecluvirus); benyvirus (Benyvirus); (Idaeovirus); Sobemovirus (Sobemovirus); Umbravirus (Umbravirus); Tymovirus (Tymovirus); Marafivirus (Marafivirus); (Maculavirus); Allexivirus (Allexivirus); (Manadrivirus); carnation latent carl belongs to (Carlavirus); capillovirus belongs to (Capillovirus); (Foveavirus); potexvirus (Potexvirus); Trichovirus (Trichovirus); (Vitivirus) and the Ourmia melon viruses belong to (Ourmiavirus).

In some embodiments, described virus is selected from down group: enterobacteria phage MS2; (enterobacteria phage Q β; cricket paralysis virus; infectivity flacherie virus; poliovirus; ERC group virus A; hepatitis A virus; encephalomyocarditis virus; foot and mouth disease virus; the human parechovirus; parsnip yellow fleck virus; rice tungro spherical virus; cowpea mosaic virus; broad bean wilt virus 1; nepovirus; potato virus Y; ryegrass mosaic virus; barley yellow mosaic virus; maclura mosaic virus; sweet potato mild mottle virus; Wheat streak mosaic virus; swine vesicular exanthema virus; rabbit hemorrhagic disease virus; norwalk virus; Sapporo virus; hepatitis E virus; people's Astrovirus; the turkey Astrovirus; nodamura virus; (Striped jack nervous necrosis virus); nudaurelia cyntherea capensis; nudaurelia capensis; tomato bushy stunt virus; carnation mottle virus; tobacco necrosis virus A; carnation ringspot virus; corn chlorotic mottle poison; oat moves back green dysplasia virus (Oat chlorotic stuntvirus); (Pothos latent virus); panicum mosaic virus; infectious bronchitis virus; the horse Orbivirus; equine arteritis virus; (Gill-associated virus); the sindbis virus; rubella virus; yellow fever virus; bovine viral diarrhea virus; hepatitis C virus; alfalfa mosaic virus; annulus orae; brome mosaic virus; CMV; Fructus oleae europaeae cryptovirus 2; (Beet ellows virus); lettuce infectivity flavivirus; grape leaf roll correlated virus 3; (Mushroom bacilliform virus); barly yellow dwarf virus-PAV; corium solani; pea enation mosaic virus-1; tobacco mosaic virus (TMV); Tobacco rattle virus; barley stripe mosaic virus; marmor tritici; potato mop-top virus; peanut clump virus; beet necrotic yellow vein virus; raspberry bushy dwarf virus; SBMV; Carrot mottle virus; turnip yellow mosaic virus; maize rayado fino virus; (Grapevine fleck virus); green onion X virus; India's oranges and tangerines ring spot virus (Indian citrus ringspot virus); carnation latent carl; apple stem grooving virus; apple stem pitting virus; potato virus X; apple chlorotic leaf spot virus; grape vine virus of A and Ourmia melon viruses.

In some embodiments, described virus is the member of Narnaviridae.

In some embodiments, described virus is a member that is selected from down in the Tobamovirus of organizing: Narnavirus (Narnavirus) and Mitovirus (Mitovirus).

In some embodiments, described virus is selected from

In some embodiments, described virus is to be selected from down the Viraceae of group or a member in the subfamily: yeast saccharomyces cerevisiae 20SRNA 20S RNA and cryphonectria parasitica mitovirus-1 NB631.

6. embodiment

1. host cell that contains the nucleic acid of proteins encoded enzyme or pepsinogen, wherein, the nucleic acid of described proteins encoded enzyme or pepsinogen is integrated into the genome of host cell, and under wherein said proteolytic enzyme or the pepsinogen normal circumstances not at described cell inner expression.

2. as enforcement mode 1 described host cell, wherein, described cell expressing proteolytic enzyme.

3. as enforcement mode 2 described host cells, wherein, described proteolytic enzyme is serine protease.

4. as enforcement mode 3 described host cells, wherein, described serine protease is a S1 family protein enzyme.

5. as enforcement mode 4 described host cells, wherein, described proteolytic enzyme is trypsinase.

6. as enforcement mode 2 described host cells, wherein, described serine protease is the bacterium subtilisin.

7. as enforcement mode 6 described host cells, wherein, described proteolytic enzyme is the SPRT from streptomyces griseus.

8. as enforcement mode 2 described host cells, wherein, described proteolytic enzyme is the listed proteolytic enzyme of table 1.

9. as enforcement mode 1 described host cell, wherein, described proteolytic enzyme is pepsinogen.

10. as enforcement mode 9 described host cells, wherein, described pepsinogen is from the trypsinogen of streptomyces griseus or preceding-SPRT pepsinogen.

11. as each described host cell in the above-mentioned embodiment, wherein, the expression of described proteolytic enzyme or pepsinogen is controlled by inducible promoter.

12. as enforcement mode 11 described host cells, wherein, described inducible promoter is induced by Interferon, rabbit or interferon-induced downstream signaling molecule.

13. as enforcement mode 11 described host cells, wherein, described inducible promoter is induced by the expression system of tsiklomitsin-adjusting.

14. as implementing each described host cell among the mode 1-10, wherein, the expression of described proteolytic enzyme or pepsinogen is controlled by the constitutive activity promotor.

15. as each described host cell in the above-mentioned embodiment, wherein, described nucleic acid comprises and instructs proteolytic enzyme or pepsinogen excretory secretion signal.

16. as each described host cell in the above-mentioned embodiment, wherein, described cell is a mammalian cell.

17. as enforcement mode 16 described host cells, wherein, described cell is a canine cells.

18. as enforcement mode 17 described host cells, wherein, described cell is a mdck cell.

19. as enforcement mode 16 described host cells, wherein, described cell is a primate cell.

20. as enforcement mode 19 described host cells, wherein, described cell is African grivet or human cell.

21. as implementing each described host cell among the mode 1-15, wherein, described cell is birds (avian) cells.

22. as enforcement mode 21 described host cells, wherein, described cell is a chicken cell.

23. as each described host cell in the above-mentioned embodiment, wherein, described cell is without adapting to suspension growth.

24. method of producing influenza virus, described method comprises: with each described host cell in influenza infection such as the above-mentioned embodiment, under the condition that allows this influenza virus to duplicate, cultivate this cell, and collect influenza virus from cell culture.

25. method of producing influenza virus, described method comprises: with the genomic nucleic acid transfection of encoding stream sense as implementing each described host cell among the mode 1-23, under the condition that allows this influenza virus to duplicate, cultivate this cell, and collect influenza virus from cell culture.

26. as enforcement mode 24 or 25 described methods, wherein, described host cell expression pepsinogen, and wherein said method also is included in and adds exogenous protease in the substratum.

27. as enforcement mode 26 described methods, wherein, the maximum of described exogenous protease is added the about 0.1 μ g/ml of concentration.

28. as enforcement mode 26 described methods, wherein, described exogenous protease is a trypsinase.

29. the method for a replicating influenza virus when the trypsinase that does not exist external source to add, described method comprises: use the influenza infection host cell, under the condition that allows influenza virus to duplicate, cultivate this cell, and from cell culture collection influenza virus, the condition that wherein said permission influenza virus is duplicated does not comprise the trypsinase that external source is added.

30. the method for a replicating influenza virus when the trypsinase that does not exist external source to add, described method comprises: with the genomic nucleic acid transfection host cell of encoding stream sense, under the condition that allows influenza virus to duplicate, cultivate this cell, and from cell culture collection influenza virus, the condition that wherein said permission influenza virus is duplicated does not comprise the trypsinase that external source is added.

31., wherein, can't help the zymoprotein enzyme or the pepsinogen of described cell expressing under the described host cell expression normal circumstances as enforcement mode 29 or 30 described methods.

32. as enforcement mode 29 or 30 described methods, wherein, described host cell is a mammalian cell.

33. as enforcement mode 29 or 30 described methods, wherein, described host cell is the birds cells.

34. as enforcement mode 32 described methods, wherein, described host cell is a primate cell, canine cells, hamster cell, mouse cell, or rat cell.

35. as enforcement mode 32 described methods, wherein, described host cell is a mdck cell.

36. as enforcement mode 34 described methods, wherein, described host cell is the Vero cell.

37. as enforcement mode 33 described methods, wherein, described host cell is a chicken cell.

38. a raising is grown in the method that the influenza virus in the cell culture is tired, described method is included in and cultivates described influenza virus in the cell culture, cytotostatic expressing protein enzyme or pepsinogen in the wherein said cell culture, this proteolytic enzyme or pepsinogen i) to described cell allos, ii) excise the hemagglutinin of described influenza virus, thereby, with respect in the cell of not expressing heterologous proteolytic enzyme or pepsinogen, cultivating tiring that influenza virus obtains, improved tiring of the influenza virus that is grown in the described cell culture.

39. as enforcement mode 38 described methods, wherein, described proteolytic enzyme of described cell constitutive expression or pepsinogen.

40. as enforcement mode 38 described methods, wherein, described cell induction type is expressed described proteolytic enzyme or pepsinogen.

41. as enforcement mode 38,39 or 40 described methods, wherein, described cell expressing proteolytic enzyme.

42. as enforcement mode 38,39 or 40 described methods, wherein, described cell expressing pepsinogen.

43. as enforcement mode 41 described methods, wherein, described proteolytic enzyme is trypsinase.

44. as enforcement mode 41 described methods, wherein, described proteolytic enzyme is the SPRT proteolytic enzyme of streptomyces griseus.

45. as enforcement mode 42 described methods, wherein, described pepsinogen is trypsinogen or the preceding-SPRT pepsinogen of streptomyces griseus.

46. method of in can supporting the host cell that influenza virus is duplicated, producing heterologous protease or pepsinogen, described method is included in cultivates the cell that comprises under the normal circumstances not at the coding nucleic acid of the proteolytic enzyme of this cell inner expression or pepsinogen under the condition that allows described proteolytic enzyme or pepsinogen to express, thereby produces this proteolytic enzyme or pepsinogen in described cell.

47. as enforcement mode 46 described methods, wherein, described host cell stably express proteolytic enzyme.

48. as enforcement mode 46 described methods, wherein, described host cell stably express pepsinogen.

49. as enforcement mode 46,47 or 48 described methods, wherein, described host cell is secreted into described proteolytic enzyme or pepsinogen in the described cell culture medium.

50. as enforcement mode 46,47,48 or 49 described methods, wherein, the about 0.1ng of described host cell expression is to about 10 μ g proteolytic enzyme or pepsinogen/milliliter host cell culture.

51. as enforcement mode 46,47,48,49 or 50 described methods, wherein, the proteolytic enzyme of described cell expressing or the amount of pepsinogen are enough to improve tiring of the virus that is grown in the host cell culture of expressing this proteolytic enzyme or pepsinogen.

52. as enforcement mode 46,47,48,49,50 or 51 described methods, wherein, the expression of described proteolytic enzyme or pepsinogen is an induction type.

53. as enforcement mode 46,47,48,49,50 or 51 described methods, wherein, the expression of described proteolytic enzyme or pepsinogen is a composing type.

54. a coding and SEQ ID NO:1 are at least about the isolating nucleic acid of 90% identical nucleotide sequence.

55. as enforcement mode 54 described nucleic acid, wherein, nucleotide sequence shown in the described nucleic acid encoding SEQ ID NO.:1.

56. one kind comprises amino acid whose isolated polypeptide shown in the SEQ ID NO:2.

57. the isolating nucleic acid of the embodiment 56 described polypeptide of encoding.

58. one kind comprises embodiment 54,55 or 57 described expression of nucleic acids carriers.

59. isolated cell that comprises embodiment 58 described expression vectors.

60. method of making the host cell of stably express proteolytic enzyme or pepsinogen, described method comprises: the nucleic acid of proteins encoded enzyme or pepsinogen is introduced host cell, not at this host cell inner expression, and separating stable is expressed the host cell of this proteolytic enzyme or pepsinogen under wherein said proteolytic enzyme or the pepsinogen normal circumstances.

61. method that makes adherent host cell suspension growth, described method comprises: the nucleic acid of introducing proteins encoded enzyme or pepsinogen, the nucleic acid of wherein said proteins encoded enzyme or pepsinogen is integrated into the genome of host cell, and under wherein said proteolytic enzyme or the pepsinogen normal circumstances not at this cell inner expression.

62. as enforcement mode 60 or 61 described methods, wherein, described host cell stably express proteolytic enzyme.

63. as enforcement mode 60 or 61 described methods, wherein, described host cell stably express pepsinogen.

64. as enforcement mode 60,61,62 or 63 described methods, wherein, described host cell is secreted into described proteolytic enzyme or pepsinogen in the described cell culture medium.

65. as enforcement mode 60,61,62,63 or 64 described methods, wherein, the about 0.1ng of described host cell expression is to about 50 μ g proteolytic enzyme or pepsinogen/milliliter host cell culture.

66. as enforcement mode 60,61,62,63,64 or 65 described methods, wherein, the proteolytic enzyme of described cell expressing or the amount of pepsinogen are enough to improve tiring of the virus that is grown in the host cell culture of expressing this proteolytic enzyme or pepsinogen.

67. as enforcement mode 60,61,62,63,64,65 or 66 described methods, wherein, the expression of described proteolytic enzyme or pepsinogen is an induction type.

68. as enforcement mode 60,61,62,63,64,65 or 66 described methods, wherein, the expression of described proteolytic enzyme or pepsinogen is a composing type.

69. as enforcement mode 60,61,62,63,64,65,66,67 or 68 described methods, wherein, described host cell is a mammalian cell.

70. as enforcement mode 60,61,62,63,64,65,66,67 or 68 described methods, wherein, described host cell is the birds cells.

71. as enforcement mode 69 described methods, wherein, described host cell is primate cell, canine cells, hamster cell, mouse cell or rat cell.

72. as enforcement mode 71 described methods, wherein, described host cell is a mdck cell.

73. as enforcement mode 71 described methods, wherein, described host cell is the Vero cell.

74. as enforcement mode 70 described methods, wherein, described host cell is a chicken cell.

75. as enforcement mode 60 described methods, wherein, described host cell is a bacterial cell.

76. as implementing each described method among mode 60-62 or the 64-75, wherein, described proteolytic enzyme is trypsinase.

77. as implementing each described method among mode 60-62 or the 64-75, wherein, described proteolytic enzyme is the SPRT proteolytic enzyme of streptomyces griseus.

78. as implementing each described method among mode 60-61 or the 63-75, wherein, described pepsinogen is a trypsinogen.

7. embodiment

The following examples just are used to illustrate the present invention, and are not meant to limit the present invention in any manner.

7.1 embodiment 1: in mdck cell, cultivate strains of influenza viruses

Present embodiment has been described the feature of the several clones that are used to cultivate influenza virus.Several different clone and the primary cell that are used to prepare first type and B-mode these two kinds of laboratory adaptability strains of influenza viruses have been estimated, comprising MRC-5, WI-38, FRhL-2, PerC6,293, NIH 3T3, CEF, CEK, DF-1, Vero and MDCK.Though the cell of many types can both be supported the duplicating of strains of influenza viruses of some acclimatization to cold, has only MDCK can produce first type and B virus that height is tired consistently limitedly.Also detected the ability that mdck cell is supported to be very popular potential vaccine ca A/Vietnam/1203/2004 duplicated in addition.CaA/Vietnam/1203/2004 with low infection multiplicity infects mdck cell, and the virus in the quantitative supernatant of metainfective different time points.Infected back 48 hours, tiring of ca A/Vietnam/1203/2004 reaches about 8log ₁₀TCID ₅₀/ mL kept stable in ensuing 3 to 4 days.Referring to Fig. 1.

In this test, at the substratum that contains 10% U.S.'s foetal calf serum or suitable serum free medium (for example, SFMV100) derive from the mdck cell (accession number No.CCL-34) of ATCC in limited number of times amplification, be used for preliminary The Characteristics to prepare preceding-chief cell (pre-master cell) stoste.Description to suitable serum free medium sees the U.S. Provisional Application No.60/638 that submitted on December 23rd, 2004, the U.S. Provisional Application No.60/641 that on January 5th, 166 and 2005 submitted to, 139.Cell can well be grown in two types substratum, and two kinds of cell stostes are all supported duplicating of the acclimatization to cold vaccine strain and the strain of being very popular, respectively as following table 2 and shown in Figure 1.

Table 2

The reproductivity of the strains of influenza viruses of acclimatization to cold in the mdck cell that contains serum and serum-free culture relatively.

7.2 the tumorigenicity of embodiment 2:MDCK clone

Before two kinds of MDCK-and chief cell stoste cultivates in substratum that contains serum and serum free medium respectively, utilizes its potential tumorigenicity of nude mouse model evaluation, and its residing stage representative has passed the cell in 5 generations, estimates that then cell can be used for vaccine production.In order to estimate tumorigenicity, 10 ⁷Cell is subcutaneously injected in the experimental group that comprises 10 mouse, puts to death animal and inspection after 84 days.There are 6 to observe tumour formation in 10 animals of the cell inoculation that in serum free medium, goes down to posterity.On the contrary, the animal of the cell inoculation that goes down to posterity in the substratum that contains 10% foetal calf serum does not all observe tumour formation; Although observed some fibrosarcoma at inoculation position, as shown in table 3, the cell that goes down to posterity in serum is a non-tumorigenic.

Table 3

The tumorigenicity and the nuclear cytology of the mdck cell that in two kinds of different substratum, goes down to posterity

^*TP ₅₀: in 50% animal body, induce the needed cell count of tumour

ND: do not do

As shown in table 3, to the 4th generation of chief cell stoste before in substratum separately, cultivate two kinds and the 20th generation cell carry out karyotyping.It is 78 Metaphase Chromosome that the non-tumorigenic cell that goes down to posterity in 10%FCS comprises median, and it is relatively limited to contain the chromosomal cell distribution of other numbers (70 to 82).And the contained Metaphase Chromosome median of the cell that goes down to posterity in serum free medium also is 78, but obviously has more cell to contain the non-multiple chromosome number of from 53 to 82 Metaphase Chromosomes of scope.In both cases, going down to posterity afterwards, nuclear cytology does not all change.

7.3 embodiment 3: mdck cell is adapted in serum free medium, grow

The mdck cell that derives from ATCC goes down to posterity containing in the substratum of the FBS of gammairradiation.But these cells are at the limited number of times that goes down to posterity in the serum-free culture based formulation of selection sustenticular cell storehouse preparation then.Serum free medium has description in U.S. Provisional Application number 60/638,166 and 60/641,139.These additional going down to posterity are carried out under 37 ℃ or 33 ℃.In three kinds of substratum of additive that comprises plant origin rather than serum, the go down to posterity caryogram similar (data not shown) of the mdck cell that goes down to posterity in its caryogram of cell that is produced and the substratum that is containing FCS of mdck cell.

7.4 the clone of embodiment 4:MDCK cell

Carry out the biology clone to guarantee to obtain originating from the cell of a genetic background by the limiting dilution pair cell.According to various phenotypic characteristic screening and cloning, comprising doubling time and relative tumorigenicity and viral production.In the Prima Facie Evidence of notion test, in containing the substratum of FCS, obtain 54 MDCK clones.These clones are gone down to posterity, infect each clone with the ca A/New Caledonia/20/99 that hangs down infection multiplicity.Infect the back and took out supernatant in several days, and pass through TCID ₅₀Measure the virus quantity in the supernatant.Have only the minority clone can produce higher relatively virus of tiring, be higher than non-clone's the virus titer that parental cell produced.Clone with good biology and physiology characteristic is used to set up master cell bank (MCB), and is as described below.

7.5 embodiment 5: the detection of master cell bank and sign

Complete detection MCB is to guarantee exogenous factor can not occur.For example, utilize among several PCR one or more and/or at the test of the antibodies specific of existing virokine, shown in following table 4.

Table 4

The detection scheme of MCB

7.6 embodiment 6: clinical preceding evaluation of the influenza virus in cell culture source

Present embodiment has been described the evaluation of preparation from the strains of influenza viruses of cell culture and egg, and has compared two viruses that system produced.Generally speaking, influenza virus is suitable for use as people's vaccine, and has the biological characteristics that the virus of making is applicable to this purposes.In this embodiment, influenza virus is acclimatization to cold (ca; Have the ability of effectively duplicating at a lower temperature), temperature sensitive (ts; When external temperature is higher, duplicate limited) and (att of attenuation; In ferret (ferrets) lung tissue, detect less than duplicating), be called as ca ts att strain in this article.Relatively comprise: the biological chemistry of viral product, antigenicity and genetics evaluation (order-checking); Biology and the biochemical characteristics of virus after people's time multiplexed cell system; Duplicating in (permissive) of susceptible animal model; And the immunogenicity in the animal model of susceptible.

7.6.1 genetics, biological chemistry and antigenic comparability

The ca ts att strain of A/H1N1, A/H5N1, A/H3N2 and B virus can be duplicated with relative higher tiring in mdck cell.In addition, these ca ts att strains of going down to posterity in mdck cell can not change its genome sequence yet.Three ca ts att strain, ca A/Sydney/05/97, ca A/Beijing/262/95 and ca B/Ann Arbor/1/94 go down to posterity in mdck cell once or twice, measure the sequence of the complete coding region of all 6 internal gene, and compare with parent material.The result does not detect Nucleotide and changes, and proves this genetics composition that goes down to posterity and can not change these virus strain that is undertaken by this initiator (substrate).His Sequence Identification of the enterprising Xingqi of different vaccine strains that in mdck cell, prepares, the conditional expectation of vaccine production can be simulated preparation process, comprising substratum form, input dosage (moi), incubation temperature and harvest time.According to these preliminary datas, estimate that the genome sequence of the virus of MDCK preparation does not change.

The genomic genetics feature in back is stable owing to go down to posterity in mdck cell, therefore estimates to be difficult to distinguish in egg or the biological character of the vaccine for preparing in mdck cell.But, the elementary viral product that obtains from cell culture is compared still some subtle difference of possibility with the product of egg preparation, the relevant aspect of posttranslational modification that particularly comprises HA and NA with viral protein, perhaps with viromembrane in lipid form relevant aspect; The two all may change the whole physical property of virus particle.Show with the preliminary clinical preceding data relevant cell culture preparation and on this important parameters, not detect difference with the antigenicity of the vaccine egg preparation.The egg stoste of several vaccine strains goes down to posterity by mdck cell, utilizes reference antisera by measuring the antigenicity that HAI tires to determine two kinds of products.As shown in table 5, all HAI tire in each other 2 times, and the vaccine that time multiplexed cell system is described is compared antigenicity with the material in egg source and do not changed.

Table 5

HAI with the interior virus strain for preparing of mdck cell in the egg tires

7.7 embodiment 7: the HE infection of cultivation

In some embodiments, in order to estimate biological chemistry, biology and the structure homogeny of vaccine after in human archeocyte, duplicating of MDCK and egg preparation, vaccine can go down to posterity 1 time in relevant diploid human archeocyte, as normal people's bronchial epithelial cell (NHBE).This single that can be used for simulating in human respiratory that goes down to posterity infects incident, thereby can carry out the comparison of progeny virus, and described progeny virus finally is responsible for exciting effective immune response.These materials can be used for measuring the hemagglutinin (combination and fusion) and the neuraminic acid enzymic activity of vaccine, and other biological chemistry and structural research, comprising the ratio and the viral genome equivalent of Electronic Speculum, infectious particle and total particle.In a word, these relatively can be used for proving the vaccine in cell source and the same effective and safety of vaccine of egg preparation.The result of analysis and research is summarised in the table 6.

Table 6

The preclinical study that compares the vaccine of cell and egg preparation

In the body (ferret)	External ＊
		The duplicating dynamics of reproduction range in the upper respiratory tract in attenuation/the be replicated in upper respiratory tract	Virus is in conjunction with hemagglutination titer and different sialic combinations
Immunogenicity cross reaction kinetics	The infectious particle of physical property EM morphology: total particle (genome)
		The infection performance detects duplicates the required required dosage of dosage generation antibody response	Fusion-activity optimum pH optimal temperature
	Genome sequence
			The neuraminic acid enzymic activity

^*Compare primary product and the product that in human archeocyte, goes down to posterity once

7.8 embodiment 8: animal model before clinical

Ferret is attenuation characteristic and the immunogenic effective animal model that is used to estimate Gripovax and component vaccine strain.From the performance of the strains of influenza viruses in the cell source of MCB preparation and the identical virus strain comparison of egg preparation.(head-to-head) compares these materials head to head in (controlled) test that contrast is arranged, thereby can guarantee the comparability of these viral product high-levelly.

In order to estimate the ability that two kinds of vaccine infection ferrets are admitted in other words in ferret, the viral prepared product of slight anesthetized animal and intranasal vaccination cell or egg preparation.Wash the nose material in postvaccinal several time points collections, utilize a kind of method in the existing several method to measure virus quantity to estimate the duplicating dynamics and the scope of virus in the animal upper respiratory tract.Test is carried out in range of doses, comprises a plurality of virus strain and different trivalent mixtures, prepares strain prepares strain with respect to egg infectivity relatively to conclude cell culture.These tests equally also can be used for estimating the immunogenicity of influenza virus, and the ability that this specific character and viral promoter infect has inner link.Gather the blood sample of animal and collect nasal wash in postvaccinal different time points (week); These samples are used to estimate serum antibody and IgA reacts in the nose that produces to infecting.These data comprise the reaction of infectivity, serum antibody and mucoantibody, maximum value (culmination) will be used for comparison and estimate the infectivity relatively of vaccine of the relative egg preparation of vaccine of cell preparation.Estimate that most probable result cell preparation has similar infectivity and immunogenicity with vaccine strain egg preparation.If the vaccine in cell source is higher or immunogenicity is stronger than the product infectivity in egg source, so further tests and estimate the possibility of employing than low dosage.

Many immunogenicities and replicated test can carry out the vaccine with the cell culture source of estimating single unit human dosage on the ferret model.Infect with the catsatt virus strain and can in the ferret body, excite powerful usually and antibody response fast.In addition, measured each catsatt virus strain according to a conventional method, the result shows that virus can express attenuation (att) phenotype, copies to higher relatively tiring in nasopharynx, and the levels of replication in these animal lungs then can't detect.Also estimated the influence of in cell culture, cultivating to these biological natures.But it also is impossible that any difference all is observed, because the att phenotype is an integral part of the genetic composition of these virus strain.Giving growth kinetics and the cross reaction of these animal single human dosage with these virus strain of postevaluation.This can inspire and can produce the serum antibody of cross reaction with a plurality of virus strain in the genetic pedigree; The vaccine of estimating the cell source also has same ability.

These similarity evaluations should be able to make us that the potential source biomolecule chemistry and/or the biophysics difference of protovirus product are had more deep understanding, these are estimated has also proved the influence of these acquired differences to catsatt virus strain characteristic, and wherein these characteristics of virus strain are by going down to posterity first in human archeocyte or animal experiment mensuration.According to the sequence information of up to the present grasping, estimate can not exert an influence to the catsatt virus strain because of the immunogenicity characteristic that preparation on mdck cell obtains.

Much more ferret is used for influenza virus as animal model report, and ferret is used to estimate the attenuation phenotype and the immunogenicity of catsatt virus strain usually.In general, the animal in age in 8-10 week is used to estimate the attenuation characteristic; Common test design is that each experimental group or control group comprise n=3-5 animal.Utilize 8 weeks to 6 month big animals to carry out immunogenicity research, each experimental group or control group need n=3-5 animal usually.These number of animals can provide enough information be used for statistics validity between two groups relatively or observe the comparison of importance.In most of processs of the test, may can observe influenza sample symptom, but very not definite.Ferret can not show the symptom of appetite or weight loss, runny nose or discharge of eye; The symptom of observing the influenza virus-like disease is the necessary component of test, and it is inappropriate being intervened as giving anodyne.If uncomfortable other signals obviously descend as stomatocace or body weight, with cure mainly the animal doctor and confer after can suitably handle animal.

7.9 embodiment 9: the foundation of main viral seed (MVS)

At present, the influenza virus vaccine strain is to prepare by separating and screen progeny virus with wild-type virus and first type or B-mode MDV cotransfection bird cell and according to desired 6:2 gene cluster.This process need virus goes down to posterity several times in bird cell culture and/or SPF egg.Recently, plasmid rescue is used to prepare the influenza virus prepared product.In this process, come from through the Vero of the cell bank of duplicate detection and characterized (cercopithecus aethiops) cell usefulness, for example, 8 DNA plasmids, electroporation, each plasmid comprise the cDNA copy of a sections in 8 Influenza Virus RNA sections.Behind the electroporation several days, just influenza virus has been arranged in the supernatant of these electroporation of cells.Then supernatant is inoculated in the SPF egg with amplification and biological cloning vaccine strain.These two processes can produce vaccine strain, and these vaccine strains can be inoculated into and produce MVS in the SPF egg.Though plasmid rescue technique has multiple advantage, comprising more regularly, hereditary property gene segment and be subjected to the exogenous factor contamination of heavy of wild-type strain isolated littler more accurately, but the different MVS of these two kinds of method preparations are nondescript each other, all can be used for starting large batch of production of vaccine.

The final amplification of vaccine strain can be carried out on the cell in source, mdck cell storehouse.This final amplification utilize mdck cell the small-scale culture (＜20L) just can finish.Collect, concentrate and come from the supernatant of these cells and carry out characterized/detections to prepare MVS.

7.10 embodiment 10: with the mdck cell proteolysis activation infectious virus of expressing heterologous pepsinogen

Following examples have been described the structure of the mdck cell system of constitutive expression pig trypsinogen.

At first, the gene clone of coding pig trypsinogen is gone into retroviral vector pLNHX (the clone scientific ﹠ technical corporation of California mountain scene (Clonetech Inc., Mountain View, CA)).For carrying out above-mentioned clone, the recombinant nucleotide sequence of composite coding trypsinogen, its sequence is shown in Figure 11 (SEQ ID NO.:3), and it is cloned into shuttle vectors.From shuttle vectors, digest the trypsinase protogene with Bgl II then and be cloned into the Bgl II site of pLNHX polylinker.The carrier that so makes is called as pTGEN in the text.

Adopt routine techniques that pTGEN is transfected into two kinds of different package cell line-Ampho 293 and GP2 then.Infect and adopt routine techniques cracking packing cell after 48 hours so that separate retrovirus that contains pTGEN (being called vTGEN in the literary composition) or the luciferase expression carrier (being called vLLRN in the literary composition) that is used as positive control.Infecting mdck cell with vLLRN shows, the vLLRN that in the GP2 cell, makes can: 1) luciferase genes is introduced the MDCK genome, this is owing to can detect luciferase activity (Fig. 2) and 2 in back 48 hours in mdck cell in infection) concentration proportional (Fig. 3) of the amount of the luciferase that mdck cell is expressed when infecting back 48 hours and the vLLRN that is used to infect mdck cell.Therefore, this positive control confirms that the pLNHX system can be used for heterologous gene transfection mdck cell, and this gene is expressed by mdck cell.

Adopt following process to identify by the clone of pTGEN stable transfection.In growth medium, add G418 after 48 hours to select resistance MDCK clone with vTGEN, vLLRN or vLNHX (the only negative control of carrier) infection.After about 3 weeks, separate each MDCK clone and with it as homogenic culture proliferation.

Expression from the MDCK of stable conversion clone's luciferase is shown in Fig. 4.As seen from Figure 4, this process 2 different mixtures having obtained 12 different independent clonings and constituted by 3 clones with the high level expression luciferase.These 12 independent clonings produce the active luciferase of about 10-300ng/1000 cells.Therefore, these data acknowledgements, can the obtaining from the pLNHX carrier system of stable conversion with the genetically modified MDCK of high level expression clone.

The summary of the independent clone that the transfection test obtains and clone's mixture sees Fig. 5.As shown in Figure 5, from the retroviral particle of Ampho293 cell and GP2 cell, making, isolate 18 MDCK clones that express trypsinogen respectively.

7.11 embodiment 11: infection and the growth of influenza virus in the mdck cell of the expression trypsinogen of cultivating

This embodiment has described two kinds of different influenza strains infection and growths in the mdck cell of expression trypsinogen when existing and not having external source trypsinase.

Two kinds of abilities of expressing the MDCK clone of trypsinogen (the particle transfection mdck cell manufacturing that makes in a kind of Ampho of being used in 293 cells, another kind are used in the particle transfection mdck cell manufacturing that makes in the GP2 cell) as virus host of following evaluation.0 constantly with ca A/Ann Arbor/6/60 MDV-A or ca A/NewCaladonia/20/90 strain with the cell of infection multiplicity 0.01 (being 1 influenza virus of per 100 cells) infection from suitable clone.Will be with pLLRN or pLNHX cells transfected with comparing.When infection back 12 hours, 24 hours, 48 hours, 72 hours, 96 hours and 120 hours, estimate the virus titer in the culture.

In first group of test, adding external source pig trypsinase to final concentration in infection in the time of back 24 hours in cell culture is 0.1 μ g/ml (solid circles).In another group test, maintain 1 μ g/ml up to the tryptic concentration of external source pig the 5th day (infecting back 120 hours) culture from the 1st day (infecting back 24 hours).In the end in the battery of tests, do not add external source trypsinase.

Test-results is shown in Fig. 6 A-6C.Fig. 6 A has shown with the mdck cell of MDV-A (going up a left side) or ca A/NC (going up right) simulated infection and with the influenza infection result of the mdck cell of the expression luciferase of MDV-A (bottom left) or ca A/NC (bottom right) infection.As expected, when adding 1 μ g/ml external source trypsinase at 1-5 days, the overall productivity of virus all significantly big (hollow square frame) in all tests, the output when not adding external source trypsinase minimum (hollow triangle).Add 0.1 μ g/ml external source trypsinase at the 1st day and do not significantly improve viral yield (solid circles).

Fig. 6 B-6C has shown the influenza infection result of the mdck cell of two kinds of different expression trypsinogens that infect with MDV-A (left side) or ca A/NC (right side).The MDCK clone who is used for testing shown in Fig. 6 B is used in the virion transfection that Ampho 293 cells make to make, and the MDCK clone who is used for testing shown in Fig. 6 C is used in the virion transfection that the GP2 cell makes to make.

Shown in Fig. 6 B-6C, virus titer significantly raises when the cell expressing trypsinogen that infects.In all situations, the virus titer that obtains when not having external source trypsinase (hollow triangle) is lower than tire (the hollow square frame) that obtains when having 1 μ g/ml trypsinase, but greater than the virus titer that contrasts.Yet, (for example, the last left side of Fig. 6 B, the last right side and bottom right in the certain situation; A last left side and the bottom right of Fig. 6 C), added a small amount of trypsin 0.1 μ g/ml at the 1st day) (solid circles) make the gained virus titer be increased to the suitable level of observed virus titer when having external source trypsinase.Do not wish to be confined to any particular theory or mechanism of action, it is believed that adding external source trypsinase is enough to excise the propetide of trypsinogen, thereby proenzyme is activated into its complete activated form.Therefore, " just exempt from " culture with a small amount of peptase and can activate the proenzyme that cells in culture produces, thereby improve whole viral yield.

7.12 embodiment 12: the expression of the trypsinogen of mark in mdck cell

This embodiment has described the trypsinogen of making and detecting with 6 propylhomoserin residue markers in mdck cell.

Be to identify the clone of high level expression trypsinogen, mdck cell is gone in the gene transfection of the trypsinogen fusion rotein of coding 6xHis-mark, thereby made another group clone.In brief, the recombinant nucleotide sequence with coding pig trypsinogen shown in Figure 11 is cloned into pDEST ^TMThe polylinker of 26 (California Ka Ersibaide because of dimension Qu Gen company) also is transfected into mdck cell with the gained carrier.PDEST ^TM26 plasmid-encoded 6xHis labels near this polylinker are so from pDEST ^TM26 express the pig trypsinogen that artificial trypsinogen gene produces 6x His-mark.With transfected cell cultures 48 hours, when having G418, cultivate about 3 weeks afterwards then to select transfectant.Isolate 16 G418-resistance clones then and make it as single clonal expansion.

Then by the trypsinogen of western trace assessment by these 16 clonal expressions.In brief, each clone was cultivated 1-2 days, centrifugal collecting cell then, with the 1x phosphate-buffered saline with cell washing 1 time, then at 200 μ l1x protein cleavage damping fluids (handkerchief agate coffee (the Promega Corp. of company of state of Wisconsin Madison; Lysing cell Madison, WI)), thus from cell isolated protein.Afterwards, each protein cleavage liquid of 10 μ l is loaded into carries out the Western engram analysis on 12% denaturing polyacrylamide gel.With 1: 10,000 dilution be specific to tryptic rabbit antitrypsin polyclonal antibody (Chemicon AB1823; (the Chemicon International of Kai Miken international corporation that Micoud draws is carried in the California; Temecula, CA)) detect the trypsinogen of 6xHis-mark.Added 1: 3, the goat antirabbit HRP link coupled second antibody of 000 dilution, washed twice, and add substrate ((the Healthcare Bio-Sciences Corp. of GE health care biotechnology company of New Jersey Piscataway that adds ECL; Piscataway, NJ)) make the protein video picture.The Western trace the results are shown in Fig. 7 A and 7B.As shown in these figures, but 12 trypsinogens that produce the 6xHis-mark of detection limits are arranged among 16 G418 resistance MDCK clone.

7.13 embodiment 13: trypsinase is expressed in mdck cell under inducible promoter control

This embodiment has described under inducible promoter control and has expressed the structure that tryptic mdck cell is.

In cell culture, provide tryptic another kind of method for detecting in the proper timing behind the virus infection, the following inducible promoter control tryptic mdck cell of expression down that is manufactured on.At first, use pcDNA ^TM6/TR (California Ka Ersibaide because of dimension Qu Gen company) transfection mdck cell system also cultivates when having blasticidin to select transfectant.Separate antibiotic-resistant clone then and from single clonal expansion.

Then, detect tsiklomitsin (tet) aporepressor that integrative vector is expressed among the single clone with positive control carrier pDEST30Luc (California Ka Ersibaide because of dimension Qu Gen company).Thereby the purpose of this test is to identify that expressing capacity tet aporepressor has low-down luciferase expression and have the MDCK of high expression level to clone when having dox when not having doxycycline (dox).For achieving the above object, will treat that respectively about 20,000 cell inoculations of screening and cloning are in each hole of 6 hole microtiter plates.Adopt routine techniques with each sample of pDEST30Luc transient transfection, in 3 holes, added dox at the 1st day after the transfection.The 2nd day harvested cell after the transfection, 20 μ l supernatant liquors are adopted in each luciferase test.

12 clones' luciferase test-results is shown in Fig. 8.As shown in Figure 8, observe clone 3 and when existing and do not have doxycycline, demonstrate high reactivity difference.By 36 MDCK-Trex clones of instantaneous luciferase experiment sieving.Selecting these two clones of MDCK/R3 and MDCK/R7 to make the tryptic mdck cell of inducible expression (induces multiple to be respectively :+DC/-DC=13.1 and 14.0).With pT-Rex-DEST30/ luciferase (Figure 13) transfection MDCK/R3 or R7 in contrast.Isolate 29 antibiotics resistances clone R3/Luc and R7/Luc and breed to be used to be with or without the test of doxycycline (DC) inductive luciferase.After the chemical induction (+DC/-DC=70 and 56), the difference of R3 is up to 70 times, and R7 is up to 56 times.Our data show that R3/Luc and R7/Luc clone to stablize and carry luciferase genes, so the expression of luciferase can be induced by DC.

For making the tryptic mdck cell of inducible expression, with pT-Rex-DEST30/ trypsin Figure 13) transfection MDCK/R3 or R7, and cultivate 2-3 week with MDCK growth medium+blasticidin/G418 and clone to select antibiotics resistance.Isolating 70 expresses tryptic clones and breeds to carry out the protease activity test.Separate these cloned genes group DNA and detect trypsase gene by PCR.Do not find any DNA nucleosides sudden change by dna sequence analysis.

Then, study by growth curve, as the described growth curves research of embodiment 11, estimate the growth of the influenza virus that the clone that demonstrates maximum activity difference when existing and do not have doxycycline cultivates in the cell that is evaluated at the inducible expression trypsinogen and tire.About 15 are cloned in and have high trypsinase when DC induces and express and have low trypsinase express when not having DC.For example, clone R3/6U7 has the baseline trypsinase of about 10ng/ml when not having DC expresses, and this value rises to about 2.5 μ g/ml (inducing multiple about 250) when inducing with 3 μ g/ml DC.For the duplicating of most of influenza vaccines strains, tryptic working concentration is about 1.0 μ g/ml usually.Induce and control in these cells tryptic expression level with different DC concentration to produce influenza vaccines.

The tryptic MDCK of many inducible expressions clone allow to can be used for to prepare vaccine the influenza strain infection and duplicate.Parent's mdck cell only is less than 10% cell suspension growth usually as the attached cell growth.Yet the tryptic MDCK of inducible expression clones less adherent, 30% the cell suspension growth of having an appointment.It is alive observing most of suspension cells (＞90%) by the trypan blue staining analysis.By regulating endogenous tryptic expression, can improve the per-cent of suspension cell.Thereby employing suspension cell manufacturing influenza vaccines can get rid of the demand reduction expense to carrier.

7.14 embodiment 14: the bacterial serine proteolytic enzyme of clone streptomyces griseus

This embodiment has described the serine protease from the streptomyces griseus clone.

For realizing above-mentioned clone, adopt routine techniques with a primer to (Spr T forward and reverse primer) from the increase encoding sequence of SPRT proteolytic enzyme of genomic dna.The right nucleotides sequence of this primer is shown in Figure 12.The streptomyces griseus bacterial strain (accession number 23915) that is preserved in ATCC is used as the source of genomic dna.Adopt routine techniques that pDEST is gone in the sprT gene clone then ^TM14, and adopt routine techniques to identify the nucleotide sequence of sprT gene.

The nucleotides sequence of sprT gene is shown in Fig. 9.

Though for the purpose of clarifying and understanding is described the present invention on some details in the above, but clearly, those skilled in the art can make various modifications in form and details by reading the disclosure, and is only just passable otherwise depart from true scope of the present invention.For example, but above-mentioned all technology and equipment arbitrary combination use.For all purposes, the all complete this paper of including in of all publications that the application quoted, patent, patent application or other documents as a reference, this just as each publication, patent, patent application and/or other documents all by clear and definite and included in this paper independently as a reference.In addition, for all purposes, following U.S. Provisional Patent Application is included this paper by reference in full in: the U.S.60/793 that on April 19th, 2006 submitted to, 522; The U.S.60/793 that on April 19th, 2006 submitted to, 525; The U.S.60/702 that on July 22nd, 2005 submitted to, 006; The U.S.60/699 that on July 15th, 2005 submitted to, 556; The U.S.60/699 that on July 15th, 2005 submitted to, 555; The U.S.60/692 that on June 21st, 2005 submitted to, 965; With the U.S.60/692 that submitted on June 21st, 2005,978.

Sequence table

＜110〉Medimmune Vaccines Inc (MedImmune Vaccines, Inc)

G. Du Ke (Duke, Gregory)

G. the bank treasured (Kemble, George)

J. poplar (Young, James)

Mo Chengjun (Mo, Chengjun)

N. Ha Zharui (Hazari, Nisha)

＜120〉method and composition of expressing heterologous proteolytic enzyme

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<151>2005-07-22

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<151>2005-06-21

<150>60/699,556

<151>2005-07-15

<150>60/692,965

<151>2005-06-21

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<170>PatentIn version 3.3

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＜213〉streptomyces griseus (Streptomyces griseus)

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Met Lys Gln Leu Leu Arg Ala Leu Gln Arg Cys Ser Val Val Val Ala

1 5 10 15

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

20 25 30

Ala Pro Asn Pro Val Val Gly Gly Thr Arg Ala Ala Gln Gly Glu Phe

35 40 45

Pro Phe Met Val Arg Leu Ser Met Gly Cys Gly Gly Ala Leu Tyr Ala

50 55 60

Lys Asp Ile Val Leu Thr Ala Ala His Cys Val Asn Gly Ser Gly Asn

65 70 75 80

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

85 90 95

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

100 105 110

Asn Gly Thr Gly Lys Asp Trp Ala Leu Ile Lys Leu Ala Gln Pro Ile

115 120 125

Asn Gln Pro Thr Leu Lys Ile Ala Thr Thr Thr Ala Tyr Asn Gln Gly

130 135 140

Thr Phe Thr Val Ala Gly Trp Gly Ala Asn Arg Glu Gly Gly Ser Gln

145 150 155 160

Gln Arg Tyr Leu Leu Lys Ala Asn Val Pro Phe Val Ser Asp Ala Ala

165 170 175

Cys Arg Ser Ala Tyr Gly Asn Glu Leu Val Ala Asn Glu Glu Ile Cys

180 185 190

Ala Gly Tyr Pro Asp Thr Gly Gly Val Asp Thr Cys Gln Gly Asp Ser

195 200 205

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

210 215 220

Gly Ile Val Ser Trp Gly Tyr Gly Cys Ala Arg Pro Gly Tyr Pro Gly

225 230 235 240

Val Tyr Thr Glu Val Ser Thr Phe Ala Ser Ala Ile Ala Ser Ala Ala

245 250 255

Arg Thr Leu

<210>3

<211>753

<212>DNA

＜213〉wild boar (Sus scrofa)

<400>3

agatctatga aggctaaatt gctggtgctc ctgtatgctt ttgttgctgg attccctaca 60

gacgacgacg acaaaatcgt cggtggctat acttgtgccg ctaacagcat tccatatcaa 120

gtgagtctca atagtggttc tcacttctgc ggtggctcac tgatcaatag ccagtgggtc 180

gtgtccgcag cccattgtta caaaagccgg atccaggtgc ggcttggcga acataatatc 240

gacgttcttg agggcaacga gcagtttatc aacgctgcta agattatcac ccatcctaat 300

ttcaatggca acaccctgga taatgatatt atgctcatca aacttagcag ccctgccacc 360

ctcaatagtc gggttgcgac agtctccctg cctcgctcct gcgccgctgc cggtacggaa 420

tgtttgatct ccgggtgggg aaacacaaaa tcttctgggt cttcttatcc ttccctgctg 480

cagtgcctga aagccccagt gctgtctgat tcttcttgta agagttccta cccaggtcag 540

atcacgggca acatgatttg cgtcgggttt cttgagggcg gcaaggattc ttgtcagggt 600

gattccggag gacctgttgt ctgcaatggc cagttgcagg gaatcgtgag ttggggatac 660

ggttgcgccc agaagaacaa gcccggagtg tatacgaagg tatgcaacta tgttaactgg 720

atacagcaga ccatcgctgc caactgaaga tct 753

<210>4

<211>24

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic primer

<400>4

ccccacgaaa gaaggcaatc cgtg 24

<210>5

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic primer

<400>5

gggtgccggt acgtgccgtc ag 22

Claims (22)

1. host cell that contains the nucleic acid of proteins encoded enzyme or pepsinogen, wherein, the nucleic acid of described proteins encoded enzyme or pepsinogen is integrated into the genome of host cell, and under wherein said proteolytic enzyme or the pepsinogen normal circumstances not at described cell inner expression.

2. host cell as claimed in claim 1 is characterized in that, described cell expressing proteolytic enzyme.

3. host cell as claimed in claim 2 is characterized in that described proteolytic enzyme is serine protease.

4. host cell as claimed in claim 1 is characterized in that described proteolytic enzyme is pepsinogen.

5. host cell as claimed in claim 1 is characterized in that the expression of described proteolytic enzyme or pepsinogen is controlled by inducible promoter.

6. host cell as claimed in claim 1 is characterized in that, the expression of described proteolytic enzyme or pepsinogen is controlled by the constitutive activity promotor.

7. host cell as claimed in claim 1 is characterized in that, described nucleic acid comprises and instructs proteolytic enzyme or pepsinogen excretory secretion signal.

8. host cell as claimed in claim 1 is characterized in that described cell is a mammalian cell.

9. host cell as claimed in claim 8 is characterized in that, described mammalian cell is human cell, primate cell, canine cells, hamster cell, mouse cell or rat cell.

10. host cell as claimed in claim 1 is characterized in that, described cell is the birds cells.

11. host cell as claimed in claim 1 is characterized in that, described host cell is without adapting to suspension growth.

12. a method of producing influenza virus, described method comprises:

A) with the described host cell of influenza infection claim 1 or with the described host cell of the genomic nucleic acid transfection of encoding stream sense claim 1 so that the influenza genome is introduced described host cell;

B) culturing cell under the condition that allows influenza virus to duplicate; With

C) collect influenza virus from cell culture.

13. method as claimed in claim 12 is characterized in that, described host cell expression pepsinogen, and wherein said method also is included in and adds exogenous protease in the substratum.

14. method as claimed in claim 13 is characterized in that, the maximum of described exogenous protease is added the about 0.1 μ g/ml of concentration.

15. the method for a replicating influenza virus when the proteolytic enzyme that does not exist external source to add, described method comprises:

A) with the described host cell of influenza infection claim 1 or with the described host cell of the genomic nucleic acid transfection of encoding stream sense claim 1 so that the influenza genome is introduced described host cell;

B) culturing cell under the condition that allows influenza virus to duplicate, wherein said condition does not comprise the proteolytic enzyme that external source is added; With

C) collect influenza virus from cell culture.

16. a raising is grown in the method that the influenza virus in the cell culture is tired, described method is included in and cultivates described influenza virus in the cell culture, cytotostatic expressing protein enzyme or pepsinogen in the wherein said cell culture, this proteolytic enzyme or pepsinogen i) to described cell allos, ii) excise the hemagglutinin of described influenza virus, thereby, with respect in the cell of not expressing heterologous proteolytic enzyme or pepsinogen, cultivating tiring that influenza virus obtains, improved tiring of the influenza virus that is grown in the described cell culture.

17. method of in can supporting the host cell that influenza virus is duplicated, producing heterologous protease or pepsinogen, described method is included in cultivates the cell that comprises under the normal circumstances not at the coding nucleic acid of the proteolytic enzyme of this cell inner expression or pepsinogen under the condition that allows described proteolytic enzyme or pepsinogen to express, thereby produces this proteolytic enzyme or pepsinogen in described cell.

18. method as claimed in claim 17 is characterized in that, the proteolytic enzyme of described cell expressing or the amount of pepsinogen are enough to improve tiring of the virus that is grown in the host cell culture of expressing this proteolytic enzyme or pepsinogen.

19. isolating nucleic acid that comprises SEQ ID NO.:1.

20. one kind comprises amino acid whose isolated polypeptide shown in the SEQ ID NO.:2.

21. one kind comprises the described expression of nucleic acids carrier of claim 19.

22. isolated cell that comprises the described expression vector of claim 21.

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