CN1133594A - Hsv-2 UL26 gene, capsid proteins, immunoassays and protease inhibitors - Google Patents

Hsv-2 UL26 gene, capsid proteins, immunoassays and protease inhibitors Download PDF

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CN1133594A
CN1133594A CN94193881A CN94193881A CN1133594A CN 1133594 A CN1133594 A CN 1133594A CN 94193881 A CN94193881 A CN 94193881A CN 94193881 A CN94193881 A CN 94193881A CN 1133594 A CN1133594 A CN 1133594A
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A·G·迪莱拉
C·M·迪布克
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SmithKline Beecham Corp
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Abstract

Essentially pure HSV-2 UL26 gene products and fragments thereof including mature HSV-2 protease and active fragments thereof are disclosed. Essentially pure HSV-2 UL26.5 gene products and fragments thereof including mature HSV-2 capsid protein and functional fragments are disclosed. Isolated nucleic acid molecules comprising all or part of the HSV-2 UL26 gene and/or the HSV-2 UL26.5 gene are disclosed. Expression vectors and host cells comprising such nucleic acid molecules are disclosed. Methods of identifying compounds that inhibit HSV-2 protease activity and methods of identifying compounds that inhibit HSV-2 virion assembly are disclosed. Synthetic HSV-2 substrates are disclosed. Antibodies that selectively bind to HSV-2 protease processed substrates but not unprocessed substrates or unprocessed substrates but not processed substrates are disclosed. Methods of and kits for distinguishing between HSV-1 DNA or protein and HSV-2 DNA or protein and reagents useful in such methods and kits are disclosed.

Description

Herpes simplex virus-2 UL26 gene, capsid protein, immunoassay and proteinase inhibitor
The mutual reference of relevant application
This application is a part continuation application of the common unsettled U.S. Patent Application Serial 08/110,522 of submission on August 20th, 1993, and this application whole contents is hereby incorporated by.Invention field
The present invention relates to the gene of HSV-2 (herpes simplex virus-2) UL26 and HSV-2 UL26.5; Each product of gene that relates to pure basically HSV-2 UL26 and HSV-2 UL26.5; Relate to the composition and the method for producing and utilizing HSV-2 UL26 and HSV-2 UL26.5 dna sequence dna and gene product.Background of invention
The genomic virus particle of two strands that includes that simplexvirus is sealed by about icosahedron is formed.Current, six kinds of people's simplexvirus is split into, and known they are relevant to the impaired state of panimmunity of the disease state that causes death from subclinical infection.The simplexvirus that a kind of people is arranged, herpes simplex virus type 2 is called HSV-2, often is that trafficability characteristic contact is infected and cause genital herpes.Genital herpes recurrence rate scope is between annual 1 to 6 time for the second time.According to estimates, having 1,000 ten thousand to 6,000 ten thousand people that sexual organ HSV-2 takes place in the U.S. infects.Now, also there is not to be used for preventing the vaccine of HSV-2 infection.
Genome composition about HSV-2 is known little.Yet HSV-2 has drawn an important public health problem.Most individuals continue by virus infection but also satisfied fully antiviral agent or vaccine.Need a kind of method of anti-HSV-2 agent and reagent that in this quadrat method, uses identified.Need a kind of method of authenticating compound, this kind compound can be regulated proteic activity of HSV-2 and the viral ability of duplicating and produce the virus particle of multiple infection in the cell of an infection of influence.Also need the method and the other test kit of inspection of difference HSV-2 infection and other herpesvirus infections.Brief summary of the invention
The present invention relates to pure basically HSV-2 UL-26 gene product and its fragment, they comprise HSV-2 proteolytic enzyme precursor protein, sophisticated HSV-2 proteolytic enzyme and active fragments thereof, HSV capsid precursor protein and sophisticated HSV-2 capsid protein.
The present invention relates to pure basically the HSV-2 UL26.5 gene product and the fragment thereof that comprise HSV-2 capsid precursor protein and sophisticated HSV-2 capsid protein.
The present invention relates to the nucleic acid molecule of the isolating HSV-2 of comprising UL-26 gene or its part, the nucleic acid molecule of this group or its part comprises the HSV-2 proteolytic enzyme of encoding mature and nucleic acid molecule and the coding precursor or the sophisticated HSV-2 capsid protein of its active fragments, regulation domain, for example nucleic acid molecule of promoter region or its function fragment.
The present invention relates to comprise HSV-2 UL-26 gene or its a part of expression vector, comprising the nucleotide sequence of encoding mature HSV-2 proteolytic enzyme and its active fragments, and the nucleotide sequence of coding precursor or sophisticated HSV-2 capsid protein or its function fragment.
The present invention relates to contain the host cell of the expression vector that comprises a HSV-2 UL-26 gene or his part, comprising HSV-2 proteolytic enzyme and the nucleotide sequence of his active fragments and the nucleotide sequence of coding precursor or sophisticated HSV-2 capsid protein or his function fragment of encoding mature.
The present invention relates to comprise the nucleic acid molecule that is separated to of HSV-2 UL 26.5 genes or his part, these UL 26.5 genes or his part comprise the HSV-2 capsid protein of encoding mature, the nucleic acid that is separated to of regulatory region (for example promoter region or his fragment) and the nucleotide sequence of coding precursor or sophisticated HSV-2 capsid protein or his function fragment.
The present invention relates to comprise the expression vector of HSV-2 UL 26.5 genes or his part, these UL 26.5 genes or his part comprise the HSV-2 capsid protein of encoding mature or the nucleotide sequence of his segmental nucleotide sequence and coding precursor or sophisticated HSV-2 capsid protein or his function fragment.
The present invention relates to contain the host cell of the expression vector that comprises HSV-2 UL 26.5 genes or his part, these UL 26.5 genes or its part comprise the HSV-2 capsid protein of encoding mature or the nucleotide sequence of his segmental nucleotide sequence and coding precursor or sophisticated HSV-2 capsid protein or his function fragment.
The present invention relates to identify the method for the compound that suppresses the HSV-2 protease activity, being included in a kind of tested compound exists down, contact with HSV-2 proteolytic enzyme or his active fragments detecting substrate with the substrate of a HSV-2 proteolytic enzyme by the level of protease cracking, and with do not have tested compound under the level of enzymolysis compare.
The present invention relates to identify the method for the compound that suppresses the assembling of HSV-2 virus particle, by in the presence of a kind of tested compound with the contacting of HSV-2 capsid protein, detect the level of capsid-capsid associating, and with this level with compare in the level that does not have to take place under the tested compound.
The present invention relates to produce the HSV-2 protease substrate, or recombinant production concludes it is whole UL 26 gene products or the segmental HSV-2 protease substrate of part with the chemosynthesis means.
The present invention relates to antibody, the substrate that he optionally crosses in conjunction with the HSV-2 protease treatment but be not the substrate of not handling, or optionally combine but be not treated substrate with the substrate of treated mistake not.
The present invention relates to distinguish the method between HSV-1 DNA and HSV-2 DNA, comprise amplification HSV-1 DNA but be not that the primer of HSV-2 DNA is with pcr amplified dna and/or with increasing HSV-2 DNA but be not the primer pcr amplified dna of HSV-1 DNA.
HSV-1 DNA but be not the PCR primer of HSV-2 DNA and amplification HSV-2 DNA but be not the PCR primer of HSV-1 DNA the present invention relates to increase.
The present invention relates to test kit in order to difference HSV-1 DNA and HSV-2 DNA, comprise amplification HSV-1 DNA is housed in one but be not HSV-2 DNA the PCR primer and-individual positive control and the definite HSV-1 DNA container of the molecular size sign by primer amplification whether, and/or amplification HSV-2 DNA be housed in one but be not the PCR primer of HSV-1 DNA and positive control and in order to determine the whether container of the molecular size sign by primer amplification of HSV-2 DNA.
The present invention relates to distinguish HSV-1 albumen and the proteic method of HSV-2 and comprise that employing is optionally with HSV-1 albumen but be not the immunoassay of the antibody that combines of HSV-2 albumen and/or utilize optionally with HSV-2 albumen but be not an immunoassay of the antibody that combines of HSV-1 albumen.
The present invention relates to optionally with HSV-1 albumen but be not the antibody that combines of HSV-2 albumen or optionally with HSV-2 albumen but be not the antibody that HSV-1 albumen combines.
The present invention relates in order to difference HSV-1 albumen and the proteic test kit of HSV-2.Said test kit comprises a delivery case that the compartment is arranged with the container of accepting a series of strict regulation, it is equipped with one and comprises optionally with HSV-1 albumen but be not that an antibody and the cover that HSV-2 albumen combines detects first container whether antibody is incorporated into instrument on the HSV-1 albumen, and/or one comprises optionally with HSV-2 albumen but is not that an antibody and the cover that HSV-1 albumen combines detects second container whether antibody is incorporated into the instrument on the HSV-2 albumen.
The present invention relates to HSV-2 protease promoter and/or enhancer element and their application.
The present invention relates to HSV-2 capsid protein promotor and/or enhancer element and their application.The accompanying drawing summary
Fig. 1 illustrates HSV-2 UL 26 groups.Symbol＜〉margin line of expression HSV-2 UL 26 group products.The termination codon of generally acknowledging is at underline.
The margin line of symbol [[]] expression HSV-2 UL 26 group products.
The margin line in two major protein hydrolysis sites of symbol [] expression.
The key that splits is represented with *.
Symbol ‖ represents the promoter region of HSV-2 UL 26.5 genes, and generally acknowledged " a TATA box " is used in underline and represents.
Fig. 2 illustrates under HSV-2 UL 26.5 promotors are regulated, the expression of E.C. 2.3.1.28.Detailed Description Of The Invention
Terminology used here UL 26 groups refer to a dna molecular of the nucleotide sequence that comprises a coding HSV-2 proteolytic enzyme and a kind of HSV-2 capsid protein.UL 26 genes are disclosed among the SEQ ID NO:1 (No. 1, Sequence Identification).The coding region of UL 26 genes is made up of Nucleotide between the 534-2447 of SEQ ID NO:1.One 638 amino acid whose activated protein enzyme precursors of coding during UL 26 genetic expressions (open in SEQ ID NO:1 and SEQ ID NO:2).
As used here, term " activated proteolytic enzyme precursor " refers to the product without UL 26 translations of processing.Activated proteolytic enzyme precursor is an activated HSV-2 proteolytic enzyme.When producing, before the activated proteolytic enzyme position on the proteolytic enzyme of an inside between 247 and 248 amino acid splits the site from shearing.247 amino acid moieties of amino acid still keep protease activity.
Used here, term " sophisticated proteolytic enzyme " refers to 247 amino acid whose albumen of amino acid of producing from shearing of passing through of activated proteolytic enzyme precursor.The aminoacid sequence of maturation protein enzyme is open with the 1-247 amino acid of SEQ ID NO:1 and SEQ ID NO:2.
As used here, term " HSV-2 proteolytic enzyme " meaning is exactly alternately to be expressed as activated proteolytic enzyme precursor, sophisticated proteolytic enzyme or its active fragment.
As used here, term " UL 26.5 " gene refers to a dna molecular that comprises coding HSV-2 capsid protein nucleotide sequence.UL 26.5 genes are internal sequences that are separated to transcribe in UL 26 genes.UL 26.5 genes are open in SEQ ID NO:1, comprise from the coding region of 1461-2447 Nucleotide.When expressing, one 329 amino acid whose capsid precursors of UL 26.5 genes encodings (in SEQ ID NO:1 and SEQ ID NO:2 with 310-638 between amino acid open).
As used here, term " capsid precursor " refers to does not have finished UL 26.5 translation products.When the function of the relevant gene product of the present invention of hope is not by any special mechanism theory constraint, but according to the part document of relevant HSV-1, thinkable is to be cut open on an inner protease cracking site between 613 and 614 amino-acid residues of SEQ ID NO:1 and SEQ ID NO:2 by HSV-2 proteolytic enzyme after the capsid precursor produces.304 amino acid moieties are the capsid proteins that are used for virus assembling and viral DNA bag quilt.The C-terminal processing of UL 26.5 impels the viral DNA bag to be become ripe capsid.The inhibition of this processing incident can cause DNA being packaged into ripe capsid.
As used here, term " sophisticated capsid protein " refers to by HSV-2 proteolytic enzyme by 304 amino acid proteins that cracking produced to the capsid precursor.The aminoacid sequence of ripe capsid protein is disclosed as the 310-613 amino acid of SEQ ID NO:1 and SEQ ID NO:2.
As used here, term " HSV-2 capsid protein " meaning is exactly capsid precursor and ripe capsid protein (conversion mutually).
As used here, term " function fragment ", when being used to modify specific genes or gene product, mean one than gene or gene product total length partly short and it remains with all biological functions with the relevant full-length gene product of full-length gene associated in fact.Determine whether a fragment of a special gene or gene product is a function fragment, only produce a fragment and measure so segmental biological function of generation with nucleic acid decomposition of knowing and Proteolytic technology.
The present invention relates to pure basically HSV-2 proteolytic enzyme, relate to it and form production method and application, relate to the method for nucleic acid molecule and production and this coding of application HSV-2 proteolytic enzyme nucleic acid molecule of coding HSV-2 proteolytic enzyme.The present invention relates to pure basically HSV-2 capsid protein, relate to the method for its composition and production and application HSV-2 capsid protein, relate to the nucleic acid molecule of coding HSV-2 capsid protein, relate to the method for the nucleic acid molecule that produces and use coding HSV-2 capsid protein.The present invention relates to substrate, relate to the authentication method of the compound that suppresses the HSV-2 protease activity as the HSV-2 protease cracking.The authentication method that relates to the compound that suppresses the assembling of HSV-2 capsid, relate to the method that difference contains the sample of HSV-1 DNA and contains the HSV-2 dna sample, relate to the method that difference contains HSV-1 albumen sample and contains HSV-2 albumen sample, and relate in order to finish the reagent of this quadrat method, comprise oligonucleotide and antibody.
Embodiments more of the present invention provide the method that suppresses or regulate HSV-2 protease activity compound in other respects of identifying.Therefore, the invention provides the method for identifying as the compound of anti-HSV-2 agent, because the HSV-2 protease activities is requisite in the viral life cycle.According to the present invention, when having the compound that will measure, HSV-2 proteolytic enzyme contacts with a HSV-2 protease substrate (substrate) and protein decomposing activity is with or without influences to determine to be measured compound.Protein decomposing activity when test-compound has test-compound to the influence of HSV-2 proteolytic enzyme is also available is compared the protein decomposing activity that is observed when not having test-compound and is determined.
Protein decomposing activity refers to the ability that HSV-2 protease hydrolyzed processing substrate becomes product, promptly decomposes a single peptide substrate molecule and becomes two or more peptide molecules (protein,split).In viral life cycle, by such protein cleavage, the proteolytic enzyme precursor is processed to sophisticated proteolytic enzyme, and the capsid precursor is processed to sophisticated capsid.This transfer pair virus particle assembling and viral DNA packing are necessary.The level of protein decomposing activity can be by those have the known several different methods of routine techniques personnel and determine in the art.A kind of method that unprocessed substrate and protein,split are differentiated is provided basically.Therefore, after substrate added, HSV-2 proteolytic enzyme contacted with test-compound, by detecting the finished amount of substrate that do not have that keeps, do not process the consumption of substrate, or the growing amount of protein,split just can be observed the level of protein decomposing activity.
The invention provides pure basically HSV-2 proteolytic enzyme, it is useful in the analysis of identifying the compound of regulating the HSV-2 protease activity.The present invention also provides the method that produces pure basically HSV-2 proteolytic enzyme.The aminoacid sequence of HSV-2 proteolytic enzyme is open in SEQ ID NO:1 and SEQ ID NO:2.As mentioned above, 638 amino acid whose activated protein enzyme precursors are also open in SEQ ID NO:1 and SEQ ID NO:2.Activated proteolytic enzyme precursor is an activated HSV-2 proteolytic enzyme, and it is to process to produce 247 amino acid whose albumen by the automatic cracking of the inner cracking site of a proteolytic enzyme between amino-acid residue 247 and 248 to be called sophisticated proteolytic enzyme.The synthetic method of the peptide by routine or by the information that provides among the SEQ ID NO:1 is provided is used recombinant DNA technology and can be produced pure activated proteolytic enzyme precursor, maturation protein enzyme and its active fragments.The operation of application standard and the parent material that obtains easily, the person just can produce HSV-2 proteolytic enzyme to have the general technology in this area.On the other hand, with the operation of standard and the parent material that obtains easily, the person just can determine a fragment and/or have whether the derivative or the sophisticated proteolytic enzyme of the precursor of protease activity are an activated fragment to have the general technology in this area.
Determine that the assay method that albumen or peptide can special substrates of cracking is open at this.Determine whether a HSV-2 proteolytic enzyme fragment has protein decomposing activity, the person just can finish protease activity determination with the fragment or the derivative replacement proteolytic enzyme identical with SEQ ID NO:2 of proteolytic enzyme as not having under the test compound condition that this narrates then to have the general technology in this area.If fragment or derivative cracking substrate, then he is activated, and promptly this fragment or derivative have protein decomposing activity.Therefore, the person that has the general technology in this area just can determine routinely that fragment of proteolytic enzyme or derivative are an activated fragment or an activated derivative.
The present invention relates to encode HSV-2 proteolytic enzyme nucleotide sequence and relate to the nucleotide sequence of coding HSV-2 capsid protein.UL 26 genes comprise that the nucleotides sequence of a precursor forms of coding HSV-2 proteolytic enzyme and HSV-2 capsid protein is listed among the SEQ ID NO:1 and are disclosed.UL 26 genes comprise that the nucleotide sequence of a coding HSV-2 capsid protein also is disclosed in SEQ ID NO:1.The person that has the general technology in the art, application standard technology and the parent material that obtains easily, utilization comprises openly that here the information of SEQ ID NO:1 can obtain or the nucleic acid molecule of a synthetic coding HSV-2 proteolytic enzyme, or the nucleic acid molecule of a coding HSV-2 capsid protein.And, the information of application standard technology, the parent material that obtains easily and the disclosed here SEQ of comprising ID NO:1, the person that has the general technology just can produce pure basically HSV-2 proteolytic enzyme in the art, include active precursor protein enzyme, maturation protein enzyme or activated HSV-2 proteolytic enzyme fragment.Similarly, the technology of application standard, the information of parent material that obtains easily and the disclosed here SEQ of comprising ID NO:1, in the art the person that has the general technology just can produce pure basically HSV-2 capsid protein comprises the capsid precursor, sophisticated capsid maybe can be assembled into the HSV-2 capsid fragment of function fragment.One has the general technology universe in this area, the technology of application standard and the parent material that obtains easily can utilize the information of the disclosed SEQ of being included in ID NO:1 to utilize given host cell in the expression system to obtain with the codon that produces ideal and white matter product or the nucleic acid molecule of composite coding HSV-2 proteolytic enzyme or HSV-2 capsid protein here.
Those persons that have the general technology utilize different technology and suitable experiment also can produce the nucleic acid molecule of coding HSV-2 proteolytic enzyme or HSV-2 capsid protein in the art.For example, use polymerase chain reaction,PCR (PCR) methodology.Can design primer in order to produce the multiple copied nucleotide sequence of coding HSV-2 proteolytic enzyme or HSV-2 capsid protein.Amplification by viral DNA also may obtain routinely the encoding complete nucleotide sequence of activated proteolytic enzyme precursor.Equally, also can obtain the nucleotide sequence of encoding mature proteolytic enzyme routinely by the amplification of viral DNA.Similarly, by the amplification of viral DNA, a segmental nucleotide sequence of activated HSV-2 proteolytic enzyme routinely also can obtain encoding.In an identical situation, the capsid precursor that also can obtain routinely encoding of the amplification by viral DNA, the complete nucleotide sequence of ripe capsid or its function fragment.Change a kind of method, utilize Restriction Enzyme, the DNA of coding HSV-2 proteolytic enzyme, include active proteolytic enzyme precursor, the maturation protein enzyme, or it has active fragments or HSV-2 capsid protein to comprise the capsid precursor, and ripe capsid or its function fragment also can obtain from viral DNA is cloned into the carrier, uses from disclosed nucleotide sequence designed probe and identifies by the hybridization back.In addition, the nucleic acid molecule of coding HSV-2 proteolytic enzyme or HSV-2 capsid protein also can be with those have the general technology the known technology of person is synthesized in the art.For can be used for optimizing, the codon of HSV-2 proteolytic enzyme or HSV-2 capsid protein coding is elected to be the protein product that HSV-2 proteolytic enzyme or HSV-2 capsid protein master organize the host cell of production.The HSV-2 genome has the G+C Nucleotide of the high amount of being rich in.This point is correct especially to the UL26 gene of coding HSV-2 proteolytic enzyme.Like this feature of high-content G+C in E.coli (intestinal bacteria) in the overexpression gene because codon uses and the increase of phase shift mutation chance has proposed a problem.In effort improvement UL 26 expressed in E.coli, change UL 26 genes and its fragment were to be provided at the preferred codon of expressing the reliable aminoacid sequence that still keeps proteolytic enzyme among the E.coli.The reference that relevant preferred codon uses is: Wada etc., (1992) " the codon use table that from gene library genetic sequence data, is made into ", Nncleic Acid Research, Vol.20 Supplement, the 2111-2118 page or leaf here enrolls with reference.The suitableeest use of codon is well known, and can be applied to the designing nucleic acid molecule according to the present invention, makes it can be expressed the level that an efficient increases in a host who selected.
Person that has the general technology in the art uses technology that everybody knows and can be inserted into such dna molecular as commercial and availablely know in the carrier of doing the carrier that the expression system uses.For example, commercial available resemble psE 420 (Invitrogen, SanDiego, CA) or pET-16 (b) (Novagen, Modison N.I.) can be with in E.coli, producing HSV-2 proteolytic enzyme.(Invitrogen, San Diego CA) for example, also can be used as production at saccharomycetic S.cerevisiae (saccharomyces cerevisiae) to commercial available plasmid pYES2.Commercial available NAXBAC TM(Invitrogen, San Diego CA) for example also can be in order to produce in insect cell for the bacilliform virus expression systems completely.Commercial available plasmid pcDNAI (Invitrogen, San Diego, CA), for example, in cells of mamma animals as also can be used as production in the Chinese hamster ovary cell.Personnel that general technology is arranged in the art use conventional technology and the parent material that obtains easily, can utilize these coml expression vectors and system or other with production HSV-2 proteolytic enzyme or HSV-2 capsid protein.(ask for an interview, for example, Sambrook etc., molecular cloning, a laboratory manual, second edition, cold spring port press (1989), this book here enrolls with reference) therefore.In protokaryon and two systems eucaryon, can prepare desired albumen, produce the series of an albumen form processing.
Be fit to the details of construction of expectation host's expression system, those people in this area know.Brief says, is the production of recombinant protein, and the DNA of coded polypeptide will be connected on the selected good expression vector suitably.DNA can be operationally connected to necessary all regulatory elements of DNA expression in selecting the host.One has those skilled in the art in this area, can prepare reorganization with the technology of knowing and produce the polypeptide expression carrier.
The expression vector that comprises the DNA of coding HSV-2 proteolytic enzyme or HSV-2 capsid protein is used to transform or the transfection suitable host, then this host is cultivated and remains on foreign DNA and express under the condition that takes place.From culture, or by lysing cell, or from suitable and for reclaiming the albumen that the present invention produces in the substratum known to those people this area.Personnel that general technology is arranged in this area use the technology of knowing, and can separate and use the albumen that such expression system produced.
According to one embodiment of present invention, albumen also can be as follows by production and purifying.A dna molecular that comprises the nucleotide sequence of coding HSV-2 proteolytic enzyme or HSV-2 capsid protein can be made into and is included in the encode nucleotide sequence of a plurality of histidine residues of a proteic terminal portions.This dna molecular is incorporated into an expression vector, and this carrier is introduced in the host cell that is fit to.DNA is expressed, and the albumen that is produced comprises terminal histidine residues, and he is known as Histidine tail tag or His-tag here.Collecting this cell puts into the phosphoric acid buffer physiological saline of pH8.5 and remains on ice.With ultrasonic wave cell is split then.Shake the cellular material that splits 30 through ultrasonic wave, and 000xg is centrifugal.Then supernatant liquor is passed through 2 microns membrane filtrations.(for example crossing filterable supernatant liquor with a kind of metal-chelating resin, nitrilotriacetic acid nickel resin be one to one of useful various kinds of resin of such purpose) incubated altogether 2 hours in room temperature,, resin is never separated in the bonded material after 2 hours through this by centrifugal.Clean to remove the albumen that does not have specific combination resin dress post and with 50mM imidazoles liquid then.With the 150mM imidazole buffer end mark histidine protein enzyme is eluted from the Ni post then.Elutriant, in phosphate buffered saline, be further purified by column chromatography with the post of Pharmacia Superdex 75 sizes from post.
Dna molecular can be designed at the tail tag Histidine and comprise a special cracking site between HSV-2 proteolytic enzyme reliably, enables to remove tail tag note Histidine from expressed proteins.The removal of the Histidine of tail tag note also can be done as follows: when tail tag is remembered the Histidine position at HSV-2 proteolytic enzyme N-terminal, and can be with (aspartic acid) 4The Methionin sequence places before tail tag note Histidine back and the HSV-2 sequence.Enteropeptidase is at (aspartic acid) 4Cut specifically after the Methionin sequence and split thereby produce credible HSV-2 proteolytic enzyme.
Except produce these albumen with the recombinant chou technology, also can use automatic peptide synthesizer and produce HSV-2 proteolytic enzyme or HSV-2 capsid protein.Have those people of general technology in this area, such technology is known.
The invention provides the pure basically active substrate of HSV-2 protease cracking that is used for, comprise the synthetic substrate.A HSV-2 protease substrate is a peptide, and the proteolyzing that is caused by HSV-2 proteolytic enzyme is cracked at least two peptides that separate.In certain embodiments, cracked and not between the cracked substrate difference of size to be used to detect proteolytic enzyme be active or do not have active.In certain embodiments, substrate of the present invention be labeled therefore they also can be predicted.In certain embodiments, substrate is to be fixed on the solid phase.In some embodiments of the invention, or substrate or protein,split have activity biologically or chemistry but then do not exist in other part activity, and this point can be used for this and other differences are come.Bioactive example comprise enzymic activity and with the binding ability of specific antibody.
In the UL 26 that natural cracking site is arranged by evaluation, contain two aminoacid sequences.First be LQAS (SEQ ID NO:3) there HSV-2 proteolytic enzyme between A and S the peptide cracking.Second is VNAS (SEQ ID NO:4), and HSV-2 proteolytic enzyme splits peptide between A and S there.Natural or synthetic substrate can be produced any that contains these two cracking sites.Therefore, according to substrate of the present invention general formula is arranged
R 1-SEQ ID NO:3-R 2Or general formula
R 1-SEQ ID NO:4-R 2R in formula 1And R 2Be expressed as hydrogen or one or more amino acid respectively.In some embodiments, substrate is the product of UL 26 genes, and it contains two protease cracking sites; One comprises SEQ ID NO:3 and comprises SEQ ID NO:4 with another.In some embodiments, substrate is UL 26.5 gene products, and it contains a protease cracking site that comprises SEQ ID NO:4.In some embodiments, R 1Preferred 1-20 amino acid is more preferably 1-10 and is preferably 3,4,5,6,7,8 or 9 amino acid.In some embodiments, R 2Can be 1-20 amino acid, be more preferably 1-10, preferably 3,4,5,6,7,8 or 9 amino acid.One has those skilled in the art and can design substrate easily according to above-mentioned general formula in this area.Following each peptide is designed as substrate.
1. comprise inner cracking site SEQ ID NO:3 (LQA *S) each peptide:
AHTYLQA *SEKFK SEQ?ID?NO：5
AGIAGHTYLQA *SEKFK SEQ?ID?NO：6
GIAGHTYLQA *SEKFK SEQ?ID?NO：7
IAGHTYLQA *SEKFK SEQ?ID?NO：8
GHTYLQA *SEKFK SEQ?ID?NO：9
HTYLQA *SEKFKM SEQ?ID?NO：10
HTYLQA *SEKFKMW SEQ?ID?NO：11
HTYLQA *SEKFKMWG SEQ?ID?NO：12
HTYLQA *SEKFKMWGA SEQ?ID?NO：13
HTYLQA *SEKFKMWGAE SEQ?ID?NO：14
2. comprise terminal cracking site SEQ ID NO:4 (VNA *S) each peptide
ALVNA *SSAAHVDVD SEQ ID NO:15 asterisk ( *) represent to split key, HSV-2 proteolytic enzyme just splits chain here.
Substrate also can obtain from the proteoclastic cracking of UL 26 or in UL 26.5 protein products.They can produce or contain its fragment of cracking site by the reorganization of UL 26 genes or UL 26.5 expression of gene; Or with the standard peptide synthesis step of knowing in this area, for example the Merrifield synthesis method relies on the synthetic substrate of organic chemistry synthesizing mean.
One has those skilled in the art in this area, uses HSV-2 proteolytic enzyme and substrate and just can easily design the compound that measuring method removes to identify adjusting HSV-2 protease activity.Refer to comprising HSV-2 proteolytic enzyme, the mensuration of a mixture of substrate and test-compound as used here term " determination test "; Term " controlled trial " refers to comprising HSV-2 proteolytic enzyme and substrate but does not have the mensuration of a mixed solution of test-compound.Determine whether a test-compound can regulate the HSV-2 protease activity, the level of HSV-2 protease activity can be compared with the level of HSV-2 protease activity in a controlled trial in a determination test.
In some embodiments of the invention, when having a test-compound when contacting with HSV-2 proteolytic enzyme, cracked and not between the cracked substrate difference of size be to be used for determining whether substrate cleaved.In some embodiments, adopt HPLC (high pressure liquid chromatography (HPLC)) to finish.The sample and the substrate that contain proteolytic enzyme, for example HTYLQASEKFKNWGAE (SEQ ID NO:14) 37C insulation 4 hours, uses the trifluoroacetic acid termination reaction after the insulation in phosphate buffered saline (PBS).Then reaction solution through a HPLC post, show activity with the split product of peptide.
Among some embodiment of the present invention, when test-compound exists, and whether cleaved with immunoassay detection substrate when contact with HSV-2 proteolytic enzyme.In certain embodiments, the antibody that provides specifically with combine without the cracked substrate but do not combine with the product of protease cracking.Such antibody is called as " substrate-specific antibody " here.In certain embodiments, the antibody that provides combines with HSV-2 protease cracking product specifically, but with without the cracking substrate does not combine.Such antibody is called " product-specific antibody " here.Or with a product, or the antibody that with a substrate reactions but does not all react with the two (being the colony of substrate-specific antibody and product-specific antibody) is called as " non-cross-reacting antibody " here.In some embodiments, antibody is fixed on the solid phase.In certain embodiments, antibody is labeled.
For example, one contains HSV-2 proteolytic enzyme, and the mixed solution of the substrate and the compound that is put to the test is maintained under the appropriate condition and through an adequate time lets alone to take place proteolyzing, unless test-compound influences its effect.Can be added to an inner surface to mixed solution is pasted with in the container of no cross reaction antibody.If no cross reaction antibody is the antibody of substrate specificity, then be retained in the mixed solution any without the cracked substrate will with antibodies.If the substrate mark, container can be by drip washing, and the mark total amount that exists can be predicted.Therefore the level of HSV-2 protease activity just is determined.If non-cross-reacting antibody is product-specific antibody, in mixed solution any HSV-2 proteolytic enzyme product will with antibodies.If substrate is to be marked on the part that discharges as product, container can be by drip washing, and the labelled amount of existence just can be determined.Therefore HSV-2 protease activity level just is determined.
ICP35 antibody (catalog number (Cat.No.): B-118-100; Rivers Park, 9108 GulfordRd.Columbia Maryland) also can be used to measure the cracked substrate.Such antibody is that product is special, it only with combined by the finished capsid protein of HSV-2 protease hydrolysis.
Change a kind of method, replace using the substrate of mark, can be modified the immunoassay of exampleization and make cappelletti packet mode mensuration, just can be determined in method to the special antibody of firmly antigenic compound in bond.Such antibody is known as " compound-specific antibody " here.Container is once more by drip washing, and allows the combining of compound-specific antibody and the alloy that exists with adequate time.Measure the level of compound-specific antibody and just express HSV-2 protease activity level.
In some immunoassay embodiment, the substrate of mark is not used in reaction mixture.After reaction mixture is added to the container that comprises a non-cross-reacting antibody and keep adequate time to be beneficial to non-cross-reacting antibody combining with substrate or with product, again the product of the substrate of mark or mark, add respectively and will make him and also not have mixed liquid in substrate or the non-cross-reacting antibody of product bonded combine.The total amount of measuring labeled substrate or marked product just can show the level of proteolytic cleavage.
In certain embodiments, substrate is labeled, and is released when substrate changes into the protein hydrolysate tense marker.Predict the release of mark, just can represent the HSV-2 protease activity, the means that this available class is known are finished.In certain embodiments, the substrate that is labeled is fixing on solid phase.Because HSV-2 proteolytic enzyme is to its cracking, the mark that is connected on the part substrate is released, and this part substrate is the product of linkage flag not.Before relatively being present in the reaction solution and after the mark level just know that how many marks are released, thereby also know the level of HSV-2 protease activity.Alternatively, measure the total amount of the mark that breaks away from solid phase, also represent the level of HSV-2 protease activity.
In another embodiment, the method that detects the HSV-2 protease activity comprises that fluorescence separates out assay method, and wherein, substrate contains fluorescent mark in the key vicinity of splitting.On such position, mark can not be measured in not cracking substrate.Yet, when substrate by HSV-2 proteolytic enzyme on cracking site when cleaved, fluorophor becomes exposure, fluorescence just becomes can be determined.Therefore, when a test-compound exists substrate with after HSV-2 proteolytic enzyme contacts, can measure the level of proteolytic activity with the detectable fluorescent method of tolerance.
In another embodiment, the method for measuring the HSV-2 protease activity comprises flicker neighbour assay method, and in this method, radiolabeled substrate is incorporated on the solid beads, is excited when the closely adjacent radio-labeled of globule, and is just detectable by scintillation method.When substrate is cleaved, the no longer closely adjacent globule of radio-labeled, so and globule be not excited and can not have detected with scintillation method.Therefore when having a test-compound the bonded substrate with after HSV-2 proteolytic enzyme contacts, can measure exciting the level of globule with scintillation method with the mensuration proteolytic activity.
Except these embodiment, the personnel with general technology can use the whole bag of tricks that the technology of knowing adopts mensuration HSV-2 protease cracking or its disappearance in this area, remove to design the additive method of identifying the compound of regulating the HSV-2 protease activity.
The present invention relates to identify the test kit of the compound of regulating the HSV-2 protease activity.Such test kit includes container separately, comprises HSV-2 proteolytic enzyme in the container, substrate and optional antibody, or other are in order to the reagent that detects the HSV-2 protease activity or be used to distinguish the not reagent of cracked substrate and product.Substrate or antibody also may be the surfaces, inside that is fixed on container.Substrate or antibody also may be to be labeled.
Some embodiments of the present invention, also provide one with the diversity assay method identify to suppress or other regulate the method for the compound of HSV-2 capsid protein assembling.The invention provides the method for discriminating, because the capsid assembling is essential to virus replication and infection as the useful compound of anti-HSV-2 medicament.According to the present invention, HSV-2 UL 26.5 genes (or its part) that the mosaic gene that provides comprises a coding HSV-2 capsid protein are connected on the protein-bonded sequence of coding yeast GAL4 DNA: or HSV-2 UL 26.5 genes (or its part) of coding HSV-2 capsid protein are connected on the coding yeast GAL4 activated protein sequence.The mosaic gene part of coding HSV-2 capsid protein is encoding mature capsid, the also gene of available code capsid precursor protein preferably.Mosaic gene is inserted on saccharomyces cerevisiae (Saccharomyces Cerevisiae) plasmid and this plasmid introduced in the saccharomyces cerevisiae of the IacZ indicator that the GAL4-that contains an integration replys, when mosaic gene on plasmid is expressed, just produce fusion rotein.The fusion rotein each several part that comprises the HSV-2 capsid protein will be bonded to each other and DNA-land and the region of activation of GAL4 be converged under selected condition.When the lacZ indicator of these two very contiguous GAL4 districts and GAL4-reaction interacted, indicator was expressed in that a kind of detectable blue look just is observed under the appropriate condition.If fusion rotein is prevented from combination, these two GAL4 districts are not contiguous the existence each other, so indicator is not activated.Therefore, can't see blue look.
Therefore, this Yeast system provides one to measure the interactional quick and special method that the HSV-2 capsid protein takes place in the virus particle assembling.When having blocking-up or suppressing the interactional compound of HSV-2 capsid protein, express GAL4 district in the fusion rotein that produces by mosaic gene with debond so will not activate lacZ gene in the yeast system.Therefore, the compound that to identify the assembling of inhibition HSV-2 capsid do not occur activating by the yeast lacZ gene that transformed, so compound has antiviral characteristic.
Among some embodiment of the present invention, provide difference sample that contains HSV-1 DNA and the method that contains the sample of HSV-2 DNA, or difference contains HSV-1 protein sample and the method that contains the HSV-2 protein sample.Therefore, the invention provides the diagnostic method whether certain individuality of diagnosis infects HSV-1 and/or HSV-2.Disclosed is to identify the method whether people infects HSV-1 and/or HSV-2, and wherein HSV-1 infects to infect to distinguish with HSV-2 and comes.
According to some embodiments of the present invention, contain the HSV-1 dna sample and contain the HSV-2 dna sample with the round pcr difference.The means that such method provides a difference HSV-1 and HSV-2 to infect, and be considered as a people infected the diagnostic method of HSV type.Design that special primer is beneficial to HSV-1 DNA but be not the amplification of HSV-2 DNA and/or HSV-2DNA but be not the amplification of HSV-1 DNA.Therefore.With such primer by amplification technique and take from for example cell of individual biological sample, serum or tissue samples, particularly sending out the blister district or other can in sightly have virus to distribute the sample that the performance district is got, whether people just can determine DNA in the sample from HSV-1 or HSV-2, and taking a sample, whether individuality infects HSV-1 or HSV-2 just is determined.
The nucleotide sequence of UL26 gene comprises that the nucleotides sequence of coding HSV-2 proteolytic enzyme and HSV-2 capsid protein is listed in SEQ ID NO:1 and is disclosed.The nucleotides sequence of coding HSV-1 proteolytic enzyme and HSV-1 capsid protein is listed among the SEQ ID NO:16 and is disclosed.Design one cover PCR primer amplifies the HSV-2 sequence but does not amplify the HSV-1 sequence.Therefore, the detection of amplification DNA shows that HSV-2 exists.Equally, design one cover PCR primer only amplifies the HSV-1 sequence and does not amplify the HSV-2 sequence.Therefore, the detection of amplification DNA just shows that HSV-1 exists.The best two cover primers that provide be used in the disclosed amplification scheme and with from the material of same sample so that supply with an extra contrast.The contrast of Xuan Zeing comprises positive control that the dna sequence dna that contains can be exaggerated and/or the negative control that can not be amplified by primer in addition.After the amplification scheme is finished, the DNA of amplification be used on the running gel run electrophoretic method can be detected.A dna molecular that amplifies the desired length of product can be provided as the size sign.
The present invention also relates to distinguish DNA that a sample contains whether from the test kit of HSV-1 or HSV-2.Whether test kit of the present invention is useful diagnosing one by one the people to infect on HSV-1 and/or the HSV-2.Test kit contains to include and amplifies HSV-1 DNA but be not that each container of the primer of HSV-2DNA maybe will amplify HSV-2 DNA but is not each container of HSV-1 DNA.Test kit can randomly contain two cover primers and be placed in the container separately, divides other amplification procedure with the different piece of same sample.Test kit also can randomly contain the positive and/or negative control in the container that separates.Test kit can randomly contain can be as the dna molecular of size sign in a container that separates.Dna molecular can be exaggerated into the dna molecular of a desired length with primer.
According to some embodiment of invention, used immunoassay is gone difference to contain the HSV-1 protein sample and is contained the proteic sample of HSV-2.Thereby immunoassay is used to distinguish the also diagnosable people one by one of infection of HSV-1 and HSV-2 holds the type that infects HSV.Such immunoassay is according to the different of UL 26 gene products of HSV-1 and HSV-2 or according to the difference of UL 26.5 gene products of HSV-1 and HSV-2.Immunoassay also can be according to different between proteolytic enzyme and/or capsid protein of HSV-1 and HSV-2.The specific antibody that provides it combine with epi-position on the HSV-1 antigen selectively but not with HSV-2 antigen on exist combine, or it selectively with combine in the epi-position on the HSV-2 antigen but not with HSV-1 antigen on exist combine.For example, the specific antibody that provides it selectively with HSV-2 proteolytic enzyme but be not that HSV-2 proteolytic enzyme combines, or it is selectively with HSV-2 proteolytic enzyme but do not combine with HSV-1 proteolytic enzyme.Similarly, the specific antibody that provides it specifically with the HSV-1 capsid but be not that the HSV-2 capsid combines, or it is selectively with the HSV-2 capsid but be not that the HSV-1 capsid combines.
Therefore, with special antibody, implementation by the antibodies analytical method and the biological sample of taking from the individual be cell for example, serum or tissue sample, particularly sending out the blister district or other viruses that have that can be observed are distributed the sample that the performance district is got, people just can determine whether HSV-1 specific antibody or HSV-2 specific antibody combine with albumen in the sample, thereby the sampling individuality whether infects HSV-1 and/or HSV-2 just is determined.The aminoacid sequence of HSV-2 activated protein enzyme precursor is crossed over 1-638 amino acid in SEQ ID NO:1 and SEQ ID NO:2.The aminoacid sequence of HSV-2 maturation protein enzyme is crossed over 1-247 the amino acid of SEQ ID NO:1 and SEQ ID NO:2.The aminoacid sequence of HSV-2 capsid precursor is crossed over 310-638 amino acid in SEQ ID NO:1 and SEQ IDNO:2.The aminoacid sequence of the ripe capsid of HSV-2 is crossed over the 310-613 amino acid of SEQ ID NO:1 and SEQ ID NO:2.The aminoacid sequence of HSV-1 proteolytic enzyme and capsid is disclosed among the SEQ ID NO:17.The aminoacid sequence of HSV-1 activated protein enzyme precursor is crossed over 1-635 amino acid in SEQ ID NO:17.The aminoacid sequence of HSV-1 maturation protein enzyme is crossed over the 1-247 amino acid of SEQ ID NO:17.The aminoacid sequence of HSV-1 capsid precursor is crossed over 307-635 amino acid in SEQ ID NO:17.The aminoacid sequence of the ripe capsid of HSV-1 is crossed over the 307-610 amino acid of SEQ ID NO:17.
The method and can available widely parent material can producing that those human routines of general technology are arranged in this area combine with HSV-2 proteolytic enzyme specifically but not with HSV-1 proteolytic enzyme bonded antibody.Equally, those people of general technology are arranged in this area, can produce the HSV-2 capsid with the conventional method and the parent material that can obtain widely but be not HSV-1 capsid bonded antibody specifically.Any of these HSV-2 specific antibodies all can detect HSV-2 in the immunoassay of a difference HSV-1 and HSV-2.Equally, those persons that have the general technology are with conventional method and the parent material that can obtain widely in the art, can produce HSV-1 proteolytic enzyme but are not HSV-2 proteolytic enzyme bonded antibody specifically.Similarly, those persons that have the general technology are with conventional method with can obtain parent material widely in the art, can produce not to be the HSV-2 capsid but to HSV-1 capsid bonded antibody specifically.The special antibody of these HSV-1 is used to detect HSV-1 in the immunoassay of a difference HSV-1 and HSV-2.Best is to use from same specimen material and carry out these two immunoassays so that an additional contrast is provided.The contrast of other selections comprises positive control, the peptide that combines with antibody used in immunoassay when it includes, and/or negative control, and it includes not the peptide with antibodies used in immunoassay.Antibody can be labeled, also can use one specifically with HSV specific antibody bonded antibody.One has the people of general technology that the immunoassay kit that the information that provides can easily be produced the reagent that comprises all needs is provided here in this area.
The HSV-1 protease antibody is produced as antibody 45KD by Serotech and can be obtained from biological product scientific company (Bioproducts for Science Inc.) commercial, its catalog number (Cat.No.) is that (P.O.Box 29176 for MCA 406, Indianapolis, IN), it can be used in immunoassay to distinguish HSV-2 from HSV-1.This Serotech antibody is to HSV-1 precursor or ripe capsid protein but be not that HSV-2 precursor or ripe capsid protein combine.Therefore, can finish to determine whether contain HSV-1 or HSV-2 in a sample with an immunoassay of Serotech antibody, thereby determine whether infect HSV-1 or HSV-2 from itself obtaining that people of sample.
The present invention also relates to diagnose certain individuality whether to infect the test kit of HSV-1 or HSV-2.Test kit of the present invention can comprise contain combine with HSV-1 proteolytic enzyme but not with one of HSV-2 bonded antibody container and/or contain combine with HSV-2 proteolytic enzyme but not with a container of HSV-1 proteolytic enzyme bonded antibody.Best is, and test kit comprises two types of antibody deposits in the container separately.The antibody that is used for test kit can be labeled.Test kit contains finishes a kind of other all reagent and materials of using the immunoassay of antibody.Test kit also can randomly contain positive control and/or negative control in the container that separates.Test kit also can randomly contain the means that detect antibody and comprise, for example, a second antibody, he combines with anti--HSV protease antibody specifically.Test kit of the present invention also can comprise contain combine with the HSV-1 capsid but not with container of HSV-2 capsid bonded antibody and/or one contain combine with the HSV-2 capsid but not with a container of HSV-1 capsid bonded antibody.Best is test kit comprises antibody in the container that separates two types.The antibody that is used for test kit can be labeled.Test kit contains finishes a kind of other all reagent and materials of using with the immunoassay of antibody.Test kit also can randomly contain the positive and/or negative control in the container that separates.Test kit also can randomly comprise the means that detect antibody and comprise, for example, a second antibody, he combines with anti--HSV-1 capsid antibody specifically.Test kit also can comprise Serotech antibody.
Another aspect of the present invention relates to HSV-2 protease promoter and/or enhanser and their purposes.The HSV-2 protease promoter can be synthesized or the separated coding that is connected to except that HSV-2 proteolytic enzyme on the proteic encoding sequence.Therefore, the present invention relates to recombinant DNA molecules, which comprises at least the part of nucleotide sequence between SEQ ID NO:1 1-534 Nucleotide, and be operationally connected to a coding proteic nucleotides sequence except that HSV-2 proteolytic enzyme and list.The present invention relates to comprise the cell of dna molecular, this dna molecular includes the part at 1 and 534 nucleotide sequence of SEQ ID NO:1 at least, can be operationally connected to the proteic nucleotides sequence of coding except that HSV-2 proteolytic enzyme and list.
Being applicable on the other hand into biting body of invention cloned, and this clone has the upstream and downstream sequence of HSV-2 UL 26 genes (SEQ ID NO:1) and gene.Therefore, the sequence of connection can be used as the screening of UL 26 promotor regulatory regions and/or promotor enhancement region.
The present invention relates to HSV-2 capsid protein promotor and its purposes on the other hand.HSV-2 capsid protein promotor position is in the 1461 Nucleotide upstreams of SEQ ID NO:1.It can be synthesized or be separated and be connected on the sequence of other proteins encoded except that the HSV-2 capsid protein.Therefore, the present invention relates to recombinant DNA molecules, it comprises the part of the 1461 Nucleotide upstream nucleotide sequences of SEQ ID NO:1 at least, and a nucleotides sequence that can be operationally connected to the proteins encoded except that the HSV-2 capsid protein lists.The present invention relates to cell, the dna molecular that it comprises contains the part of upstream nucleotide sequence of the 1461st Nucleotide of SEQ ID NO:1 at least, and can be operationally connected to that proteic nucleotides sequence of coding lists except that the HSV-2 capsid protein.Nucleotide 1191 to 1461 (SEQ ID NO:1) for example, is connected on the chloramphenicol acetyl transferasegene and shows when transfection enters the VERO cell and have important promoter activity.
EXAMPLE Example 1
Proteolytic activity is to the ripe essential characteristic that has been represented as HSV-2 of virus particle of simplexvirus.HSV-2 proteolytic enzyme is also referred to as HSV-2 UL26, has an appointment 67, the molecular weight (apparent) of 028Da (dalton) and pI (iso-electric point)=6.94.HSV-2 proteolytic enzyme passes through reasonably design and screens the antiviral activity of identifying this active inhibitor and measure these inhibitor in experiment in vitro in the cell of infection with technology biochemistry with molecule.
HSV-2 UL 26 genes are cloned into a NcoI-EcoRI fragment (1938 base pair), and it contains start codon, whole open reading frame, stop codon and 3 '-22 pairs of bases of translation sequences not.Express HSV-2 UL 26 whole length with the pOTS carrier system in intestinal bacteria (E.coli), gene is inserted in the P that strongly and closely regulates that going into phage from the pOTS-207 carrier in carrier 2The downstream of promotor.When the gene of expressing is likely that pair cell is poisonous, proteolytic enzyme for example, the tight adjusting of promotor is important.27KD proteolytic enzyme zone is equivalent to a product from the automatic protein hydrolysate of HSV-2 UL 26 original translation products, and this original translation product is to use the expression vector pET-16 (b) (Novagen, Madison W.I.) of the tight adjusting that contains the T7 promotor to produce in intestinal bacteria.
Each construction of design comprises 6 Histidine codon and (aspartic acid) of position in HSV-2 UL 26 promotor codon fronts 4The Methionin codon, so the expressed proteins Histidine tail that contains a cleavable at the N-end is beneficial at the nickel post and carries out protein purification.Also available other chelate column, Histidine-tail tag note albumen elutes from post by the adding of imidazoles.In addition, it also can be eluted for example to change pH with other method.Also can be to post and technical scheme that purifying protein is useful from for example Qiagen acquisition in commercial available source.
To P 2Promoter vector is incorporated into the recombinant construction thing and is used for expressing and handle/the intestinal bacteria AR120 (nalidixic acid is induced strain) and the intestinal bacteria AR58 (thermal induction strain) of purifying research.To T 7Promoter vector is incorporated into e. coli bl21 to the recombination to construct thing, a strain IPTG (sec.-propyl-β-D thiogalactoside) inducible strain.On the nickel chelate column, be easy to be purified by chromatography albumen.
P27 proteolytic enzyme fragment is great-hearted as its ability shows, and its vigor can be removed last 25 amino acid from comprising the most construction in UL 26.5 coding regions.Embodiment 2
The p27 proteinase gene can be synthesized and make the bacillus coli gene that contains high expression level is the codon of feature, but still remains with the aminoacid sequence of p27 proteolytic enzyme.The synthetic gene is at the upper T in urgent adjusting of expression vector pET-16 (b) 7The downstream of promotor.Along with IPTG (1mM) induces, just express at the intestinal bacteria camber in 27KDa albumen zone.Embodiment 3
Above-mentioned HSV-2 UL 26 genes (NcoI-EcoRI fragment) and p27 protease clone to insect cell expression carrier pVL1392.Then the recombinant construction thing is incorporated in the insect cell from fall army worm.Then for assays for protease enzyme activity prepares the infectious titer mother liquor, the protein production that scales up that continues.Embodiment 4
Branch is enjoyed HSV-1 UL 26 genes of minimum homogeny and the DNA district design oligonucleotides PCR primer of HSV-2 UL 26 groups, with the specificity that guarantees to measure.Be disclosed in two dna sequence dnas among SEQ ID NO:1 and the SEQ ID NO:2 more respectively by Computer Analysis, just can easily observe such zone.In a small amount of identical position, zone between two homologous chromosomess in UL 26.5 zones, the part of the capsid gene of promptly encoding.According to the few nucleotide that provides in relatively at nucleotide sequence that in the Computer Analysis of input, is provided, provide following the primer sequence and to the position of primer.As shown below, it is helpful to designing a system that produces big or small HSV-1 of difference and HSV-2 product to improve analysis.5 '-the PCR primer, (justice-chain-ordering): SEQ ID NO:18 HSV-1:5 '-CCGGTGCCCAATCGTCCGT-3 ', (#864-882) SEQ ID NO:19 HSV-2:5 '-GTCCGTGCGCGTCAAGTCG-3 ', (#1397-1416) 3 '-the PCR primer, (antisense-chain-ordering): SEQ ID NO:20 HSV-1:5 '-TTCCGGCTCCCCCACCTGA-3 ', (#1560-1542) SEQ ID NO:21 HSV-2:5 '-ATTCGGATCCTGGAGGCGA-3 ', (#2470-2452) with the size of these primer sets expectation PCR product:
The HSV-1:696 base pair
The HSV-2:1073 base pair.
Use SEQ ID NO:18 and SEQ ID NO:20 or use SEQ ID NO:19 and SEQ ID NO:21 in HSV-1 analyzes in HSV-2 analyzes, the sample that suspection is contained HSV-1 or HSV-2 DNA uses different pcr amplification methods.If the dna fragmentation of one 696 base pair produces, just show that HSV-DNA exists in sample in HSV-1 analyzes.Detect the segmental existence of 696 base pairs, can be amplified production by being moved on the electrophoresis matrix.The also swimming of leakage of electricity simultaneously on same matrix of a size sign of the DNA of about 696 base pairs.If what produce is the dna fragmentation of one 1083 base pair, then just show the existence that HSV-2 DNA is arranged in the sample in HSV-2 analyzes.For detecting one the 1073 segmental existence of base pair, can be amplified production by the migration on the electrophoresis matrix.The size sign of the DNA of about 1073 base pairs also while electrophoresis on identical matrix.
A kind of test kit is provided, and it comprises that one contains SEQ ID NO:18 that HSV-1 uses in analyzing and the container of SEQ ID NO:20.A kind of test kit is provided, and it comprises that one contains the SEQ ID NO:19 that uses and the container of SEQ ID NO:21 in HSV-2 analyzes.A kind of test kit is provided, and it comprises the two, and container contains SEQ ID NO:18 and the SEQ ID NO:20 in HSV-1 analyzes, and contains SEQID NO:19 and SEQ ID NO:21 in HSV-2 analyzes in another container.Size sign DNA also can randomly be provided.The sign of one 696 base pair size is provided in some test kit.The sign of one 1073 base pair size is provided in some test kit.The sign of one 696 base pair size and the sign of one 1073 base pair size are provided in some test kits.Embodiment 5
The activity of the regional cloning that is included in HSV-2 UL 26.5 promotors of inferring in HSV-2 UL 26 genes with test starting.SEQ ID NO:1's crossed in 256 analyzed base pair districts #1191 to #1147.Cross over the Nucleotide of SEQ ID NO:1 with positive-sense strand #1191 arrive #1209 primer (5 '-AACATGAGCTGCGTGACC-3 ') and cross over SEQ ID NO:1's #1447 to #The antisense strand primer of 1429 Nucleotide (5 '-AAAGAAGAAGAAGAAGAC-3 ') by polymerase chain reaction,PCR dna fragmentation is cloned.By the activity of 256 base pair PCR fragment clonings at upstream test starting of E.C. 2.3.1.28 (CAT) reporter gene of commercial available plasmid pCAT Basic (Promega).Then the construction that produces being incorporated into a suitable cells of mamma animals is as in the Vero cell, with analyzing CAT activity level test promoter activity.This clone lacks endogenous CAT, therefore, is introducing after the promotor construction enters such cell strain, and the active level of CAT is exactly that of HSV-2 UL 26.5 promoter activities directly measures.
The Vero cell is grown in the DMEM+10%FCS that contains gentamycin (10 μ g/m1),, carry out electroporation with the scheme of standard and be incorporated in the 500 ten thousand Vero cells 15 microgram HSV-2 UL 26.5/pCAT constructions.Through behind the electroporation 48 hours, collecting cell was placed in the 100 microlitre 0.25M pH8.0 Tris damping fluids.With freezing repeatedly-melt, centrifugal and supernatant liquor transferred in the new test tube with 15000rpm lysis.Determine total protein concentration with Bio-Rad protein determination dye reagent box (catalog number (Cat.No.) 500-0006).5 microlitre D-Soviet Union [two chloracetyls-1- 14C] paraxin (Amersham, 56mCi/mmol) and 5 microlitre n-butyraldehyde coenzyme As (5mg/ml) be added to that to make its last volume in the sample of a cell extraction supernatant liquor be 100 microlitres, with acetic acid ethyl acetate extract then with tlc to measure the CAT activity.
Except that above-mentioned construction, also can be the CAT reporter gene upstream of 256 base pair HSV-2 UL, 26.5 fragment clonings in containing the pCAT Enhancen carrier of a SV-40 enhanser.This construction also can be tested the CAT activity with above-mentioned identical method in the Vero cell.
Control vector pCAT (containing SV40 promotor and enhanser) can be with the comparison that is HSV-2 UL26.5 promotor ability.
Fig. 2 has summed up four result of experiment.Cylindricality 1 be a negative control and representative be not activated son and enhanser transcribe controlling elements the time the expression of CAT.2, one male contrasts of cylindricality are used the promotor of SV40 and the enhancement factor of SV40 and are ordered about CAT genetic expression.Cylindricality 3 representatives are ordered about the CAT group separately by UL 26.5 promotors and are expressed, and cylindricality 4 representatives CAT group when UL 26.5 promotors and SV40 enhanser combined utilization is expressed.
Basic UL 26.5 expression levels (cylindricality 3) have been set up as long as separate the fragment of appropriate length by get back to Nucleotide 1 upstream from Nucleotide 1191, and this fragment is incorporated into respect to promoter region works in the primary expression construction of position and measure the enhancing CAT ability to express that their surpass basic horizontal, the additional clip that so just can be used in gene order among Fig. 1 is with evaluation UL 26.5 enhancement factors.
Promotor described here when the adjusting that be operatively connected thereunto then to allogeneic gene expression be useful.
Sequence catalogue (1) general information
(i) applicant: Dilella, Anthony G.
Debouck，Christine
(ii) Fa Ming exercise question: new gene
(iii) sequence number: 21
(iv) corresponding address:
(A) addressee: Smithkline Beecham corporate juridical person patent-US
UW2220
(B) street: P.O.Box 1539
(C) city: King of Prussia
(D) state: Pennsylvania
(E) country: USA
(F)ZIP：?19406-0939
(V) computer readable
(A) matrix type: diskette
(B) computer: IBM PC compatibility
(C) operating system: PC-DOS/MS-DOS
(D) software: PatentIn discharges #1.0 file, #1.25, mmd
(vi) the application's data
(A) application number:
(B) date of filling:
(C) classification:
(viii) proxy/business agent's information
(A) name: Jervis, Herbert H.
(B) number of registration: 31,171
(C) reference/summary number: P50188
(ix) telecommunications exchange of information
(A) phone: 215-270-5019
(B) fax: 215-270-5090 (2) SEQ ID NO:1 information
(i) sequence signature:
(A) length: 2472
(B) type: nucleic acid
(C) chain: two strands
(D) topology: linearity
(ii) molecule type: group group DNA
(ix) feature
(A) title/button: CDS
(B) position: 534..2447
(ix) feature
(A) title/button: misc feature
(B) position: 1461,2442
( xi ) ：SEQ ID NO：1GTCGACGAGG CGCGTGGTGG ATATGTCGTC GGGCGCCCGC CAGGCGGCGC TCGTGCGCCT 60CACCGCGCTG GAGCTCATCA ACCGCACCCG CACAAACACC ACCCCTGTGG GGGAGATTAT 120TAACGCCCAC GATGCCTTGG GGATACAATA CGAACAGGGC CTGGGGCTGC TCGCCCAGCA 180GGCACGCATC GGCTTGGCGT CGAACGCCAA GCGATTCGCC ACGTTCAACG TGGGCAGCGA 240CTACGACCTG TTGTACTTTT TGTGTCTCGG GTTCATTCCC CAGTACCTGT CCGTGGCCTA 300GGGAAGGGTG GGGGTGGTGG TGGTGGGGTG TTTTTCTGTT GTTGTTGTTT CTGGTCCGCC 360TGGTCACAAA AGGCACGGCG CCCCGAAACG CGGGCTTTAG TCCCGGCCCG GACGTCGGCG 420GACACACAAC AACGGCGGGC CCCGTGGGTG GGTAAGTTGG TTCGGGGGCA TCGCTGTATT 480CCCTTGCCCG CTTCCACCCC CCCTTCCCGT TTGGTTTGTT TGTGCGGGTG CCC ATG 536
Met
1GCG?TCG?GCG?GAA?ATG?CGC?GAG?CGG?TTG?GAG?GCG?CCT?CTG?CCC?GAC?CGG 584Ala?Ser?Ala?Glu?Met?Arg?Glu?Arg?Leu?Glu?Ala?Pro?Leu?Pro?Asp?Arg
5 10 15GCG?GTG?CCC?ATC?TAC?GTG?GCC?GGG?TTT?TTG?GCC?CTG?TAC?GAC?AGC?GGG 632Ala?Val?Pro?Ile?Tyr?Val?Ala?Gly?Phe?Leu?Ala?Leu?Tyr?Asp?Ser?Gly
20 25 30GAC?CCG?GGC?GAG?CTG?GCC?CTG?GAC?CCA?GAC?ACG?GTG?CGT?GCG?GCC?CTG 680Asp?Pro?Gly?Glu?Leu?Ala?Leu?Asp?Pro?Asp?Thr?Val?Arg?Ala?Ala?Leu
35 40 45CCT?CCG?GAG?AAC?CCC?CTG?CCG?ATC?AAC?GTA?GAC?CAC?CGC?GCT?CGG?TGC 728Pro?Pro?Glu?Asn?Pro?Leu?Pro?Ile?Asn?Val?Asp?His?Arg?Ala?Arg?Cys?50 55 60 65GAG?GTG?GGC?CGG?GTG?CTC?GCC?GTG?GTC?AAC?GAC?CCT?CGG?GGG?CCG?TTT 776Glu?Val?Gly?Arg?Val?Leu?Ala?Val?Val?Asn?Asp?Pro?Arg?Gly?Pro?Phe
70 75 80TTT?GTG?GGG?CTG?ATC?GCG?TGC?GTG?CAG?CTG?GAG?CGC?GTC?CTC?GAG?ACG 824Phe?Val?Gly?Leu?Ile?Ala?Cys?Val?Gln?Leu?Glu?Arg?Val?Leu?Glu?Thr
85 90 95GCC?GCC?AGC?GCC?GCT?ATT?TTT?GAG?CGC?CGC?GGA?CCC?GCG?CTC?TCC?CGG 872Ala?Ala?Ser?Ala?Ala?Ile?Phe?Glu?Arg?Arg?Gly?Pro?Ala?Leu?Ser?Arg
100 105 110GAG?GAG?CGT?CTG?CTG?TAC?CTG?ATC?ACC?AAC?TAC?CTG?CCA?TCG?GTC?TCG 920Glu?Glu?Arg?Leu?Leu?Tyr?Leu?Ile?Thr?Asn?Tyr?Leu?Pro?Ser?Val?Ser
115 120 125CTG?TCC?ACA?AAA?CGC?CGG?GGG?GAC?GAG?GTT?CCG?CCC?GAC?CGC?ACC?CTG 968Leu?Ser?Thr?Lys?Arg?Arg?Gly?Asp?Glu?Val?Pro?Pro?Asp?Arg?Thr?Leu130 135 140 145TTT?GCG?CAC?GTG?GCC?CTG?TGC?GCC?ATC?GGG?CGG?CGC?CTT?GGA?ACC?ATC 1016Phe?Ala?His?Val?Ala?Leu?Cys?Ala?Ile?Gly?Arg?Arg?Leu?Gly?Thr?Ile
150 155 160GTC?ACC?TAC?GAC?ACC?AGC?CTA?GAC?GCG?GCC?ATC?GCT?CCG?TTT?CGC?CAC 1064Val?Thr?Tyr?Asp?Thr?Ser?Leu?Asp?Ala?Ala?Ile?Ala?Pro?Phe?Arg?His
165 170 175CTG?GAC?CCG?GCG?ACG?CGC?GAG?GGG?GTG?CGA?CGC?GAG?GCC?GCC?GAG?GCC 1112Leu?Asp?Pro?Ala?Thr?Arg?Glu?Gly?Val?Arg?Arg?Glu?Ala?Ala?Glu?Ala
180 185 190GAG?CTC?GCG?CTG?GCC?GGG?CGC?ACC?TGG?GCC?CCC?GGC?GTG?GAG?GCG?CTC 1160Glu?Leu?Ala?Leu?Ala?Gly?Arg?Thr?Trp?Ala?Pro?Gly?Val?Glu?Ala?Leu
195 200 205ACA?CAC?ACG?CTG?CTC?TCC?ACC?GCC?GTC?AAC?AAC?ATG?ATG?CTG?CGT?GAC 1208Thr?His?Thr?Leu?Leu?Ser?Thr?Ala?Val?Asn?Asn?Met?Met?Leu?Arg?Asp210 215 220 225CGC?TGG?AGC?CTC?GTG?GCC?GAG?CGG?CGG?CGG?CAG?GCC?GGG?ATC?GCC?GGA 1256Arg?Trp?Ser?Leu?Val?Ala?Glu?Arg?Arg?Arg?Gln?Ala?Gly?Ile?Ala?Gly
230 235 240CAC?ACG?TAC?CTT?CAG?GCG?AGC?GAA?AAA?TTT?AAA?ATA?TGG?GGG?GCG?GAG 1304His?Thr?Tyr?Leu?Gln?Ala?Ser?Glu?Lys?Phe?Lys?Ile?Trp?Gly?Ala?Glu
245 250 255TCT?GCC?CCT?GCG?CCG?GAG?CGT?GGG?TAT?AAA?ACC?GGC?GCC?CCG?GGT?GCC 1352Ser?Ala?Pro?Ala?Pro?Glu?Arg?Gly?Tyr?Lys?Thr?Gly?Ala?Pro?Gly?Ala
260 265 270ATG?GAC?ACA?TCC?CCC?GCC?GCG?AGC?GTT?CCC?GCG?CCG?CAG?GTC?GCC?GTC 1400Met?Asp?Thr?Ser?Pro?Ala?Ala?Ser?Val?Pro?Ala?Pro?Gln?Val?Ala?Val
275 280 285CGT?GCG?CGT?CAA?GTC?GCG?TCG?TCG?TCG?TCT?TCT?TCT?TCT?TCT?TTT?CCG 1448Arg?Ala?Arg?Gln?Val?Ala?Ser?Ser?Ser?Ser?Ser?Ser?Ser?Ser?Phe?Pro290 295 300 305GCA?CCG?GCC?GAT?ATG?AAC?CCC?GTT?TCG?GCA?TCG?GGC?GCC?CCG?GCC?CCT 1496Ala?Pro?Ala?Asp?Met?Asn?Pro?Val?Ser?Ala?Ser?Gly?Ala?Pro?Ala?Pro
310 315 320CCG?CCG?CCC?GGC?GAC?GGG?AGT?TAT?TTG?TGG?ATC?CCC?GCC?TCT?CAT?TAC 1544Pro?Pro?Pro?Gly?Asp?Gly?Ser?Tyr?Leu?Trp?Ile?Pro?Ala?Ser?His?Tyr
325 330 335AAT?CAG?CTC?GTC?ACC?GGG?CAA?TCC?GCG?CCC?CGC?CAC?CCG?CCG?CTG?ACC 1592Asn?Gln?Leu?Val?Thr?Gly?Gln?Ser?Ala?Pro?Arg?His?Pro?Pro?Leu?Thr
340 345 350GCG?TGC?GGC?CTG?CCG?GCC?GCG?GGG?ACG?GTG?GCC?TAC?GGA?CAC?CCC?GGC 1640Ala?Cys?Gly?Leu?Pro?Ala?Ala?Gly?Thr?Val?Ala?Tyr?Gly?His?Pro?Gly
355 360 365GCC?GGC?CCG?TCC?CCG?CAC?TAC?CCG?CCT?CCT?CCC?GCC?CAC?CCG?TAC?CCG 1688Ala?Gly?Pro?Ser?Pro?His?Tyr?Pro?Pro?Pro?Pro?Ala?His?Pro?Tyr?Pro370 375 380 385GGT?ATG?CTG?TTC?GCG?GGC?CCC?AGT?CCC?CTG?GAG?GCC?CAG?ATC?GCC?GCG 1736Gly?Met?Leu?Phe?Ala?Gly?Pro?Ser?Pro?Leu?Glu?Ala?Gln?Ile?Ala?Ala
390 395 400CTG?GTG?GGG?GCC?ATC?GCC?GCC?GAC?CGC?CAG?GCG?GGT?GGG?CTT?CCG?GCG 1784Leu?Val?Gly?Ala?Ile?Ala?Ala?Asp?Arg?Gln?Ala?Gly?Gly?Leu?Pro?Ala
405 410 415GCC?GCC?GGA?GAC?CAC?GGG?ATC?CGG?GGG?TCG?GCG?AAG?CGC?CGC?CGA?CAC 1832Ala?Ala?Gly?Asp?His?Gly?Ile?Arg?Gly?Ser?Ala?Lys?Arg?Arg?Arg?His
420 425 430GAG?GTG?GAG?CAG?CCG?GAG?TAC?GAC?TGC?GGC?CGT?GAC?GAG?CCG?GAC?CGG 1880Glu?Val?Glu?Gln?Pro?Glu?Tyr?Asp?Cys?Gly?Arg?Asp?Glu?Pro?Asp?Arg
435 440 445GAC?TTC?CCG?TAT?TAC?CCG?GGC?GAG?GCC?CGC?CCC?GAG?CCG?CGC?CCG?GTC 1928Asp?Phe?Pro?Tyr?Tyr?Pro?Gly?Glu?Ala?Arg?Pro?Glu?Pro?Arg?Pro?Val450 455 460 465GAC?TCC?CGG?CGC?GCC?GCG?CGC?CAG?GCT?TCC?GGG?CCC?CAC?GAA?ACC?ATC 1976Asp?Ser?Arg?Arg?Ala?Ala?Arg?Gln?Ala?Ser?Gly?Pro?His?Glu?Thr?Ile
470 475 480ACG?GCG?CTG?GTG?GGG?GCG?GTG?ACG?TCC?CTG?CAG?CAG?GAA?CTG?GCG?CAC 2024Thr?Ala?Leu?Val?Gly?Ala?Val?Thr?Ser?Leu?Gln?Gln?Glu?Leu?Ala?His
485 490 495ATG?CGC?GCG?CGT?ACC?CAC?GCC?CCC?TAC?GGG?CCG?TAT?CCG?CCG?GTG?GGG 2072Met?Arg?Ala?Arg?Thr?His?Ala?Pro?Tyr?Gly?Pro?Tyr?Pro?Pro?Val?Gly
500 505 510CCC?TAC?CAC?CAC?CCC?CAC?GCA?GAC?ACG?GAG?ACC?CCC?GCC?CAA?CCA?CCC 2120Pro?Tyr?His?His?Pro?His?Ala?Asp?Thr?Glu?Thr?Pro?Ala?Gln?Pro?Pro
515 520 525CGC?TAC?CCC?GCC?GAG?GCC?GTC?TAT?CTG?CCG?CCG?CCG?CAC?ATC?GCC?CCC 2168Arg?Tyr?Pro?Ala?Glu?Ala?Val?Tyr?Leu?Pro?Pro?Pro?His?Ile?Ala?Pro530 535 540 545CCG?GGG?CCT?CCT?CTA?TCC?GGG?GCG?GTC?CCC?CCA?CCC?TCG?TAT?CCC?CCA 2216Pro?Gly?Pro?Pro?Leu?Ser?Gly?Ala?Val?Pro?Pro?Pro?Ser?Tyr?Pro?Pro
550 555 560GTT?GCG?GTT?ACC?CCC?GGT?CCC?GCT?CCC?CCG?CTA?CAT?CAG?CCC?TCC?CCC 2264Val?Ala?Val?Thr?Pro?Gly?Pro?Ala?Pro?Pro?Leu?His?Gln?Pro?Ser?Pro
565 570 575GCA?CAC?GCC?CAC?CCC?CCT?CCG?CCG?CCG?CCG?GGA?CCC?ACG?CCT?CCC?CCC 2312Ala?His?Ala?His?Pro?Pro?Pro?Pro?Pro?Pro?Gly?Pro?Thr?Pro?Pro?Pro
580 585 590GCC?GCG?AGC?TTA?CCC?CAA?CCC?GAG?GCG?CCC?GGC?GCG?GAG?GCC?GGC?GCC 2360Ala?Ala?Ser?Leu?Pro?Gln?Pro?Glu?Ala?Pro?Gly?Ala?Glu?Ala?Gly?Ala
595 600 605TTA?GTT?AAC?GCC?AGC?AGC?GCG?GCC?CAC?GTG?AAC?GTG?GAC?ACG?GCC?CGG 2408Leu?Val?Asn?Ala?Ser?Ser?Ala?Ala?His?Val?Asn?Val?Asp?Thr?Ala?Arg610 615 620 625GCC?GCC?GAT?CTG?TTT?GTG?TCA?CAG?ATG?ATG?GGG?TCC?CGC?TAACTCGCCT 2457Ala?Ala?Asp?Leu?Phe?Val?Ser?Gln?Met?Met?Gly?Ser?Arg
630 635CCAGGATCCG AATTC, 2472 (2) SEQ ID NO:2 information
(i) sequence signature:
(A) length: 638 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: protein
(xi) sequence description: SEQ ID NO:2Met Ala Ser Ala Glu Met Arg Glu Arg Leu Glu Ala Pro Leu Pro Asp 15 10 15Arg Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser
20 25 30Gly?Asp?Pro?Gly?Glu?Leu?Ala?Leu?Asp?Pro?Asp?Thr?Val?Arg?Ala?Ala
35 40 45Leu?Pro?Pro?Glu?Asn?Pro?Leu?Pro?Ile?Asn?Val?Asp?His?Arg?Ala?Arg
50 55 60Cys?Glu?Val?Gly?Arg?Val?Leu?Ala?Val?Val?Asn?Asp?Pro?Arg?Gly?Pro?65 70 75 80Phe?Phe?Val?Gly?Leu?Ile?Ala?Cys?Val?Gln?Leu?Glu?Arg?Val?Leu?Glu
85 90 95Thr?Ala?Ala?Ser?Ala?Ala?Ile?Phe?Glu?Arg?Arg?Gly?Pro?Ala?Leu?Ser
100 105 110Arg?Glu?Glu?Arg?Leu?Leu?Tyr?Leu?Ile?Thr?Asn?Tyr?Leu?Pro?Ser?Val
115 120 125Ser?Leu?Ser?Thr?Lys?Arg?Arg?Gly?Asp?Glu?Val?Pro?Pro?Asp?Arg?Thr
130 135 140Leu?Phe?Ala?His?Val?Ala?Leu?Cys?Ala?Ile?Gly?Arg?Arg?Leu?Gly?Thr145 150 155 160Ile?Val?Thr?Tyr?Asp?Thr?Ser?Leu?Asp?Ala?Ala?Ile?Ala?Pro?Phe?Arg
165 170 175His?Leu?Asp?Pro?Ala?Thr?Arg?Glu?Gly?Val?Arg?Arg?Glu?Ala?Ala?Glu
180 185 190Ala?Glu?Leu?Ala?Leu?Ala?Gly?Arg?Thr?Trp?Ala?Pro?Gly?Val?Glu?Ala
195 200 205Leu?Thr?His?Thr?Leu?Leu?Ser?Thr?Ala?Val?Asn?Asn?Met?Met?Leu?Arg
210 215 220Asp?Arg?Trp?Ser?Leu?Val?Ala?Glu?Arg?Arg?Arg?Gln?Ala?Gly?Ile?Ala225 230 235 240Gly?Mis?Thr?Tyr?Leu?Gln?Ala?Ser?Glu?Lys?Phe?Lys?Ile?Trp?Gly?Ala
245 250 255Glu?Ser?Ala?Pro?Ala?Pro?Glu?Arg?Gly?Tyr?Lys?Thr?Gly?Ala?Pro?Gly
260 265 270Ala?Met?Asp?Thr?Ser?Pro?Ala?Ala?Ser?Val?Pro?Ala?Pro?Gln?Val?Ala
275 280 285Val?Arg?Ala?Arg?Gln?Val?Ala?Ser?Ser?Ser?Ser?Ser?Ser?Ser?Ser?Phe
290 295 300Pro?Ala?Pro?Ala?Asp?Met?Asn?Pro?Val?Ser?Ala?Ser?Gly?Ala?Pro?Ala305 310 315 320Pro?Pro?Pro?Pro?Gly?Asp?Gly?Ser?Tyr?Leu?Trp?Ile?Pro?Ala?Ser?His
325 330 335Tyr?Asn?Gln?Leu?Val?Thr?Gly?Gln?Ser?Ala?Pro?Arg?His?Pro?Pro?Leu
340 345 350Thr?Ala?Cys?Gly?Leu?Pro?Ala?Ala?Gly?Thr?Val?Ala?Tyr?Gly?His?Pro
355 360 365Gly?Ala?Gly?Pro?Ser?Pro?His?Tyr?Pro?Pro?Pro?Pro?Ala?His?Pro?Tyr
370 375 380Pro?Gly?Met?Leu?Phe?Ala?Gly?Pro?Ser?Pro?Leu?Glu?Ala?Gln?Ile?Ala385 390 395 400Ala?Leu?Val?Gly?Ala?Ile?Ala?Ala?Asp?Arg?Gln?Ala?Gly?Gly?Leu?Pro
405 410 415Ala?Ala?Ala?Gly?Asp?His?Gly?Ile?Arg?Gly?Ser?Ala?Lys?Arg?Arg?Arg
420 425 430His?Glu?Val?Glu?Gln?Pro?Glu?Tyr?Asp?Cys?Gly?Arg?Asp?Glu?Pro?Asp
435 440 445Arg?Asp?Phe?Pro?Tyr?Tyr?Pro?Gly?Glu?Ala?Arg?Pro?Glu?Pro?Arg?Pro
450 455 460Val?Asp?Ser?Arg?Arg?Ala?Ala?Arg?Gln?Ala?Ser?Gly?Pro?His?Glu?Thr465 470 475 480Ile?Thr?Ala?Leu?Val?Gly?Ala?Val?Thr?Ser?Leu?Gln?Gln?Glu?Leu?Ala
485 490 495His?Met?Arg?Ala?Arg?Thr?His?Ala?Pro?Tyr?Gly?Pro?Tyr?Pro?Pro?Val
500 505 510Gly?Pro?Tyr?His?His?Pro?His?Ala?Asp?Thr?Glu?Thr?Pro?Ala?Gln?Pro
515 520 525Pro?Arg?Tyr?Pro?Ala?Glu?Ala?Val?Tyr?Leu?Pro?Pro?Pro?His?Ile?Ala
530 535 540Pro?Pro?Gly?Pro?Pro?Leu?Ser?Gly?Ala?Val?Pro?Pro?Pro?Ser?Tyr?Pro545 550 555 560Pro?Val?Ala?Val?Thr?Pro?Gly?Pro?Ala?Pro?Pro?Leu?His?Gln?Pro?Ser
565 570 575Pro?Ala?His?Ala?His?Pro?Pro?Pro?Pro?Pro?Pro?Gly?Pro?Thr?Pro?Pro
580 585 590Pro?Ala?Ala?Ser?Leu?Pro?Gln?Pro?Glu?Ala?Pro?Gly?Ala?Glu?Ala?Gly
595 600 605Ala?Leu?Val?Asn?Ala?Ser?Ser?Ala?Ala?His?Val?Asn?Val?Asp?Thr?Ala
610 615 620Arg Ala Ala Asp Leu Phe Val Ser Gln Met Met Gly Ser Arg625,630 635 (2) SEQ ID NO:3 information
(i) sequence signature:
(A) length: 4 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:3
Leu?Gl?n?Ala?Ser
1 (2) SEQ ID NO:4 information
(i) sequence signature:
(A) length: 4 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:4
Val?Asn?Ala?Ser
1 (2) SEQ ID NO:5 information
(i) sequence signature:
(A) length: 12 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:5Ala His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys 15 10 (2) SEQ ID NO:6 information
(i) sequence signature:
(A) length: 16 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:6Ala Gly Ile Ala Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys 15 10 15 (2) SEQ ID NO:7 information
(i) sequence signature:
(A) length: fifteen amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:7Gly Ile Ala Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys 15 10 15 (2) SEQ ID NO:8 information
(i) sequence signature:
(A) length: 14 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:8Ile Ala Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys1 5 10 (2) SEQ ID NO:9 information
(i) sequence signature:
(A) length: 12 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:9Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys 15 10 (2) SEQ ID NO:10 information
(i) sequence signature:
(A) length: 12 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:10His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met 15 10 (2) SEQ ID NO:l1 information
(i) sequence signature:
(A) length: 13 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:11His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp 15 10 (2) SEQ ID NO:12 information
(i) sequence signature:
(A) length: 14 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:12His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly 15 10 (2) SEQ ID NO:13 information
(i) sequence signature:
(A) length: fifteen amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:13His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly Ala 15 10 15 (2) SEQ ID NO:14 information
(i) sequence signature:
(A) length: 16 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:14His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly Ala Glu 15 10 15 (2) SEQ ID NO:15 information
(i) sequence signature:
(A) length: 14 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: peptide
(xi) sequence description: SEQ ID NO:15Ala Leu Val Asn Ala Ser Ser Ala Ala His Val Asp Val Asp 15 10 (2) SEQ ID NO:16 information
(i) sequence signature:
(A) length: 1908 base pairs
(B) type: nucleic acid
(C) chain: two strands
(D) topology: linearity
(ii) molecule type: cDNA
(ix) characteristic
(A) title/button: CDS
(B) position: 1....1908
(ix) characteristic:
(A) title/button: misc characteristic
(B) position: 919....1908
(xi) sequence signature: SEQ ID NO:16ATG GCA GCC GAT GCC CCG GGA GAC CGG ATG GAG GAG CCC CTG CCC GAC 48Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp 15 10 l5AGG GCC GTG CCC ATT TAC GTG GCT GGG TTT TTG GCC CTG TAT GAC AGC 96Arg Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser
20 25 30GGG?GAC?TCG?GGC?GAG?TTG?GCA?TTG?GAT?CCG?GAT?ACG?GTG?CGG?GCG?GCC 144Gly?Asp?Ser?Gly?Glu?Leu?Ala?Leu?Asp?Pro?Asp?Thr?Val?Arg?Ala?Ala
35 40 45CTG?CCT?CCG?GAT?AAC?CCA?CTC?CCG?ATT?AAC?GTG?GAC?CAC?CGC?GCT?GGC 192Leu?Pro?Pro?Asp?Asn?Pro?Leu?Pro?Ile?Asn?Val?Asp?His?Arg?Ala?Gly
50 55 60TGC?GAG?GTG?GGG?CGG?GTG?CTG?GCC?GTG?GTC?GAC?GAC?CCC?CGC?GGG?CCG 240Cys?Glu?Val?Gly?Arg?Val?Leu?Ala?Val?Val?Asp?Asp?Pro?Arg?Gly?Pro 65 70 75 80TTT?TTT?GTG?GGG?CTG?ATC?GCC?TGC?GTG?CAG?CTG?GAG?CGC?GTC?CTC?GAG 288Phe?Phe?Val?Gly?Leu?Ile?Ala?Cys?Val?Gln?Leu?Glu?Arg?Val?Leu?Glu
85 90 95ACG?GCC?GCC?AGC?GCT?GCG?ATT?TTC?GAG?CGC?CGC?GGG?CCG?CCG?CTC?TCC 336Thr?Ala?Ala?Ser?Ala?Ala?Ile?Phe?Glu?Arg?Arg?Gly?Pro?Pro?Leu?Ser
100 105 110CGG?GAG?GAG?CGC?CTG?TTG?TAC?CTG?ATC?ACC?AAC?TAC?CTG?CCC?TCG?GTC 384Arg?Glu?Glu?Arg?Leu?Leu?Tyr?Leu?Ile?Thr?Asn?Tyr?Leu?Pro?Ser?Val
115 120 125TCC?CTG?GCC?ACA?AAA?CGC?CTG?GGG?GGC?GAG?GCG?CAC?CCC?GAT?CGC?ACG 432Ser?Leu?Ala?Thr?Lys?Arg?Leu?Gly?Gly?Glu?Ala?His?Pro?Asp?Arg?Thr
130 135 140CTG?TTC?GCG?CAC?GTC?GCG?CTG?TGC?GCG?ATC?GGG?CGG?CGC?CTC?GGC?ACT 480Leu?Phe?Ala?His?Val?Ala?Leu?Cys?Ala?Ile?Gly?Arg?Arg?Leu?Gly?Thr145 150 155 160ATC?GTC?ACC?TAC?GAC?ACC?GGT?CTC?GAC?GCC?GCC?ATC?GCG?CCC?TTT?CGC 528Ile?Val?Thr?Tyr?Asp?Thr?Gly?Leu?Asp?Ala?Ala?Ile?Ala?Pro?Phe?Arg
165 170 175CAC?CTG?TCG?CCG?GCG?TCT?CGC?GAG?GGG?GCG?CGG?CGA?CTG?GCC?GCC?GAG 576His?Leu?Ser?Pro?Ala?Ser?Arg?Glu?Gly?Ala?Arg?Arg?Leu?Ala?Ala?Glu
180 185 l90GCC?GAG?CTC?GCG?CTG?TCC?GGG?CGC?ACC?TGG?GCG?CCC?GGC?GTG?GAG?GCG 624Ala?Glu?Leu?Ala?Leu?Ser?Gly?Arg?Thr?Trp?Ala?Pro?Gly?Val?Glu?Ala
195 200 205CTG?ACC?CAC?ACG?CTG?CTT?TCC?ACC?GCC?GTT?AAC?AAC?ATG?ATG?CTG?CGG 672Leu?Thr?His?Thr?Leu?Leu?Ser?Thr?Ala?Val?Asn?Asn?Met?Met?Leu?Arg
210 215 220GAC?CGC?TGG?AGC?CTG?GTG?GCC?GAG?CGG?CGG?CGG?CAG?GCC?GGG?ATC?GCC 720Asp?Arg?Trp?Ser?Leu?Val?Ala?Giu?Arg?Arg?Arg?Gln?Ala?Gly?Ile?Ala225 230 235 240GGA?CAC?ACC?TAC?CTC?CAG?GCG?AGC?GAA?AAA?TTC?AAA?ATG?TGG?GGG?GCG 768Gly?His?Thr?Tyr?Leu?Gln?Ala?Ser?Glu?Lys?Phe?Lys?Met?Trp?Gly?Ala
245 250 255GAG?CCT?GTT?TCC?GCG?CCG?GCG?CGC?GGG?TAT?AAG?AAC?GGG?GCC?CCG?GAG 816Glu?Pro?Val?Ser?Ala?Pro?Ala?Arg?Gly?Tyr?Lys?Asn?Gly?Ala?Pro?Glu
260 265 270TCC?ACG?GAC?ATA?CCG?CCC?GGC?TCG?ATC?GCT?GCC?GCG?CCG?CAG?GGT?GAC 864Ser?Thr?Asp?Ile?Pro?Pro?Gly?Ser?Ile?Ala?Ala?Ala?Pro?Gln?Gly?Asp
275 280 285CGG?TGC?CCA?ATC?GTC?CGT?CAG?CGC?GGG?GTC?GCC?TTG?TCC?CCG?GTA?CTG 912Arg?Cys?Pro?Ile?Val?Arg?Gln?Arg?Gly?Val?Ala?Leu?Ser?Pro?Val?Leu
290 295 300CCC?CCC?ATG?AAC?CCC?GTT?CCG?ACA?TCG?GGC?ACC?CCG?GCC?CCC?GCG?CCG 960Pro?Pro?Met?Asn?Pro?Val?Pro?Thr?Ser?Gly?Thr?Pro?Ala?Pro?Ala?Pro305 310 315 320CCC?GGC?GAC?GGG?AGC?TAC?CTG?TGG?ATC?CCG?GCC?TCC?CAT?TAC?AAC?CAG 1008Pro?Gly?Asp?Gly?Ser?Tyr?Leu?Trp?Ile?Pro?Ala?Ser?His?Tyr?Asn?Gln
325 330 335CTC?GTC?GCC?GGC?CAT?GCC?GCG?CCC?CAA?CCC?CAG?CCG?CAT?TCC?GCG?TTT 1056Leu?Val?Ala?Gly?His?Ala?Ala?Pro?Gln?Pro?Gln?Pro?His?Ser?Ala?Phe
340 345 350GGT?TTC?CCG?GCT?GCG?GCG?GGG?TCC?GTG?GCC?TAT?GGG?CCT?CAC?GGT?GCG 1104Gly?Phe?Pro?Ala?Ala?Ala?Gly?Ser?Val?Ala?Tyr?Gly?Pro?His?Gly?Ala
355 360 365GGT?CTT?TCC?CAG?CAT?TAC?CCT?CCC?CAC?GTC?GCC?CAT?CAG?TAT?CCC?GGG 1152Gly?Leu?Ser?Gln?His?Tyr?Pro?Pro?His?Val?Ala?His?Gln?Tyr?Pro?Gly
370 375 380GTG?CTG?TTC?TCG?GGA?CCC?AGC?CCA?CTC?GAG?GCG?CAG?ATA?GCC?GCG?TTG 1200Val?Leu?Phe?Ser?Gly?Pro?Ser?Pro?Leu?Glu?Ala?Gln?Ile?Ala?Ala?Leu385 390 395 400GTG?GGG?GCC?ATA?GCC?GCG?GAC?CGC?CAG?GCG?GGC?GGT?CAG?CCG?GCC?GCG 1248Val?Gly?Ala?Ile?Ala?Ala?Asp?Arg?Gln?Ala?Gly?Gly?Gln?Pro?Ala?Ala
405 410 415GGA?GAC?CCT?GGG?GTC?CGG?GGG?TCG?GGA?AAG?CGT?CGC?CGG?TAC?GAG?GCG 1296Gly?Asp?Pro?Gly?Val?Arg?Gly?Ser?Gly?Lys?Arg?Arg?Arg?Tyr?Glu?Ala
420 425 430GGG?CCG?TCG?GAG?TCC?TAC?TGC?GAC?CAG?GAC?GAA?CCG?GAC?GCG?GAC?TAC 1344Gly?Pro?Ser?Glu?Ser?Tyr?Cys?Asp?Gln?Asp?Glu?Pro?Asp?Ala?Asp?Tyr
435 440 445CCG?TAC?TAC?CCC?GGG?GAG?GCT?CGA?GGC?GCG?CCG?CGC?GGG?GTC?GAC?TCC 1392Pro?Tyr?Tyr?Pro?Gly?Glu?Ala?Arg?Gly?Ala?Pro?Arg?Gly?Val?Asp?Ser
450 455 460CGG?CGC?GCG?GCC?CGC?CAT?TCT?CCC?GGG?ACC?AAC?GAG?ACC?ATC?ACG?GCG 1440Arg?Arg?Ala?Ala?Arg?His?Ser?Pro?Gly?Thr?Asn?Glu?Thr?Ile?Thr?Ala465 470 475 480CTG?ATG?GGG?GCG?GTG?ACG?TCT?CTG?CAG?CAG?GAA?CTG?GCG?CAC?ATG?CGG 1488Leu?Met?Gly?Ala?Val?Thr?Ser?Leu?Gln?Gln?Glu?Leu?Ala?His?Met?Arg
485 490 495GCT?CGG?ACC?AGC?GCC?CCC?TAT?GGA?ATG?TAC?ACG?CCG?GTG?GCG?CAC?TAT 1536Ala?Arg?Thr?Ser?Ala?Pro?Tyr?Gly?Met?Tyr?Thr?Pro?Val?Ala?His?Tyr
500 505 510CGC?CCT?CAG?GTG?GGG?GAG?CCG?GAA?CCA?ACA?ACG?ACC?CAC?CCG?GCC?CTT 1584Arg?Pro?Gln?Val?Gly?Glu?Pro?Glu?Pro?Thr?Thr?Thr?His?Pro?Ala?Leu
515 520 525TGT?CCC?CCG?GAG?GCC?GTG?TAT?CGC?CCC?CCA?CCA?CAC?AGC?GCC?CCC?TAC 1632Cys?Pro?Pro?Glu?Ala?Val?Tyr?Arg?Pro?Pro?Pro?His?Ser?Ala?Pro?Tyr
530 535 540GGT?CCT?CCC?CAG?GGT?CCG?GCG?TCC?CAT?GCC?CCC?ACT?CCC?CCG?TAT?GCC 1680Gly?Pro?Pro?Gln?Gly?Pro?Ala?Ser?His?Ala?Pro?Thr?Pro?Pro?Tyr?Ala545 550 555 560CCA?GCT?GCC?TGC?CCG?CCA?GGC?CCG?CCA?CCG?CCC?CCA?TGT?CCT?TCC?ACC 1728Pro?Ala?Ala?Cys?Pro?Pro?Gly?Pro?Pro?Pro?Pro?Pro?Cys?Pro?Ser?Thr
565 570 575CAG?ACG?CGC?GCC?CCT?CTA?CCG?ACG?GAG?CCC?GCG?TTC?CCC?CCC?GCC?GCC 1776Gln?Thr?Arg?Ala?Pro?Leu?Pro?Thr?Glu?Pro?Ala?Phe?Pro?Pro?Ala?Ala
580 585 590ACC?GGA?TCC?CAA?CCG?GAG?GCA?TCC?AAC?GCG?GAG?GCC?GGG?GCC?CTT?GTC 1824Thr?Gly?Ser?Gln?Pro?Glu?Ala?Ser?Asn?Ala?Glu?Ala?Gly?Ala?Leu?Val
595 600 605AAC?GCC?AGC?AGC?GCA?GCA?CAC?GTG?GAC?GTT?GAC?ACG?GCC?CGC?GCC?GCC 1872Asn?Ala?Ser?Ser?Ala?Ala?His?Val?Asp?Val?Asp?Thr?Ala?Arg?Ala?Ala
610 615 620GAT TTG TTC GTC TCT CAG ATG ATG GGG GCC CGC TGA 1908Asp Leu Phe Val Ser Gln Met Met Gly Ala Arg625,630 635 (2) SEQ ID NO:17 information
(i) sequence signature:
(A) length: 635 amino acid
(B) type: amino acid
(C) topology: linearity
(ii) molecule type: amino acid
(xi) sequence description: SEQ ID NO:17Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp 15 10 15Arg Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser
20 25 30Gly?Asp?Ser?Gly?Glu?Leu?Ala?Leu?Asp?Pro?Asp?Thr?Val?Arg?Ala?Ala
35 40 45Leu?Pro?Pro?Asp?Asn?Pro?Leu?Pro?Ile?Asn?Val?Asp?His?Arg?Ala?Gly
50 55 60Cys?Glu?Val?Gly?Arg?Val?Leu?Ala?Val?Val?Asp?Asp?Pro?Arg?Gly?Pro 65 70 75 80Phe?Phe?Val?Gly?Leu?Ile?Ala?Cys?Val?Gln?Leu?Glu?Arg?Val?Leu?Glu
85 90 95Thr?Ala?Ala?Ser?Ala?Ala?Ile?Phe?Glu?Arg?Arg?Gly?Pro?Pro?Leu?Ser
100 105 110Arg?Glu?Glu?Arg?Leu?Leu?Tyr?Leu?Ile?Thr?Asn?Tyr?Leu?Pro?Ser?Val
115 120 125Ser?Leu?Ala?Thr?Lys?Arg?Leu?Gly?Gly?Glu?Ala?His?Pro?Asp?Arg?Thr
130 135 140Leu?Phe?Ala?His?Val?Ala?Leu?Cys?Ala?Ile?Gly?Arg?Arg?Leu?Gly?Thr145 150 155 160Ile?Val?Thr?Tyr?Asp?Thr?Gly?Leu?Asp?Ala?Ala?Ile?Ala?Pro?Phe?Arg
165 170 175His?Leu?Ser?Pro?Ala?Ser?Arg?Glu?Gly?Ala?Arg?Arg?Leu?Ala?Ala?Glu
180 185 190Ala?Glu?Leu?Ala?Leu?Ser?Gly?Arg?Thr?Trp?Ala?Pro?Gly?Val?Glu?Ala
195 200 205Leu?Thr?His?Thr?Leu?Leu?Ser?Thr?Ala?Val?Asn?Asn?Met?Met?Leu?Arg
210 215 220Asp?Arg?Trp?Ser?Leu?Val?Ala?Glu?Arg?Arg?Arg?Gln?Ala?Gly?Ile?Ala225 230 235 240Gly?His?Thr?Tyr?Leu?Gln?Ala?Ser?Glu?Lys?Phe?Lys?Met?Tp?Gly?Ala
245 250 255Glu?Pro?Val?Ser?Ala?Pro?Ala?Arg?Gly?Tyr?Lys?Asn?Gly?Ala?Pro?Glu
260 265 270Ser?Thr?Asp?Ile?Pro?Pro?Gly?Ser?Ile?Ala?Ala?Ala?Pro?Gln?Gly?Asp
275 280 285Arg?Cys?Pro?Ile?Val?Arg?Gln?Arg?Gly?Val?Ala?Leu?Ser?Pro?Val?Leu
290 295 300Pro?Pro?Met?Asn?Pro?Val?Pro?Thr?Ser?Gly?Thr?Pro?Ala?Pro?Ala?Pro305 310 315 320Pro?Gly?Asp?Gly?Ser?Tyr?Leu?Trp?Ile?Pro?Ala?Ser?His?Tyr?Asn?Gln
325 330 335Leu?Val?Ala?Gly?His?Ala?Ala?Pro?Gln?Pro?Gln?Pro?His?Ser?Ala?Phe
340 345 350Gly?Phe?Pro?Ala?Ala?Ala?Gly?Ser?Val?Ala?Tyr?Gly?Pro?His?Gly?Ala
355 360 365Gly?Leu?Ser?Gln?His?Tyr?Pro?Pro?His?Val?Ala?His?Gln?Tyr?Pro?Gly
370 375 380Val?Leu?Phe?Ser?Gly?Pro?Ser?Pro?Leu?Glu?Ala?Gln?Ile?Ala?Ala?Leu385 390 395 400Val?Gly?Ala?Ile?Ala?Ala?Asp?Arg?Gln?Ala?Gly?Gly?Gln?Pro?Ala?Ala
405 410 415Gly?Asp?Pro?Gly?Val?Arg?Gly?Ser?Gly?Lys?Arg?Arg?Arg?Tyr?Glu?Ala
420 425 430Gly?Pro?Ser?Glu?Ser?Tyr?Cys?Asp?Gln?Asp?Glu?Pro?Asp?Ala?Asp?Tyr
435 440 445Pro?Tyr?Tyr?Pro?Gly?Glu?Ala?Arg?Gly?Ala?Pro?Arg?Gly?Val?Asp?Ser
450 455 460Arg?Arg?Ala?Ala?Arg?His?Ser?Pro?Gly?Thr?Asn?Glu?Thr?Ile?Thr?Ala465 470 475 480Leu?Met?Gly?Ala?Val?Thr?Ser?Leu?Gln?Gln?Glu?Leu?Ala?His?Met?Arg
485 490 495Ala?Arg?Thr?Ser?Ala?Pro?Tyr?Gly?Met?Tyr?Thr?Pro?Val?Ala?His?Tyr
500 505 510Arg?Pro?Gln?Val?Gly?Glu?Pro?Glu?Pro?Thr?Thr?Thr?His?Pro?Ala?Leu
515 520 525Cys?Pro?Pro?Glu?Ala?Val?Tyr?Arg?Pro?Pro?Pro?His?Ser?Ala?Pro?Tyr
530 535 540Gly?Pro?Pro?Gln?Gly?Pro?Ala?Ser?Mis?Ala?Pro?Thr?Pro?Pro?Tyr?Ala545 550 555 560Pro?Ala?Ala?Cys?Pro?Pro?Gly?Pro?Pro?Pro?Pro?Pro?Cys?Pro?Ser?Thr
565 570 575Gln?Thr?Arg?Ala?Pro?Leu?Pro?Thr?Glu?Pro?Ala?Phe?Pro?Pro?Ala?Ala
580 585 590Thr?Gly?Ser?Gln?Pro?Glu?Ala?Ser?Asn?Ala?Glu?Ala?Gly?Ala?Leu?Val
595 600 605Asn?Ala?Ser?Ser?Aia?Ala?His?Val?Asp?Val?Asp?Thr?Ala?Arg?Ala?Ala
610 615 620Asp Leu Phe Val Ser Gln Met Met Gly Ala Arg625,630 635 (2) SEQ ID NO:18 information
(i) sequence signature:
(A) length: 19 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ii) molecule type: cDNA
(xi) sequence description: SEQ ID NO:18
CCGGTGCCCA ATCGTCCGT (2) SEQ ID NO:19 says breath
(i) sequence signature:
(A) length: 19 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ii) molecule type: cDNA
(xi) sequence description: SEQ ID NO:19
GTCCGTGCGC GTCAAGTGG (2) SEQ ID NO:20 information
(i) sequence signature:
(A) length: 19 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ii) molecule type: cDNA
(xi) sequence description: SEQ ID NO:20
TTCCGGcTCC CCCACCTGA (2) SEQ ID NO:21 information
(i) sequence signature:
(A) length: 19 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ii) molecule type: cDNA
(xi) sequence description: SEQ ID NO:21
ATTCGGATCC?TGGAGGCGA

Claims (29)

1. pure basically an albumen and function fragment thereof by HSV-2 UL26 genes encoding.
2. pure basically albumen in the claim 1, wherein said albumen is selected from by HSV-2 proteolytic enzyme precursor protein, in the group that the function fragment of sophisticated HSV-2 proteolytic enzyme and said sophisticated HSV-2 proteolytic enzyme is formed.
3. pure basically albumen in the claim 1, wherein said albumen is sophisticated HSV-2 proteolytic enzyme.
4. pure basically albumen or its fragment by HSV-2 UL26.5 coded by said gene.
5. pure basically albumen in the claim 4, wherein said albumen is selected from by HSV-2 capsid precursor protein, in the group that sophisticated HSV-2 capsid protein and its function fragment are formed.
6. pure basically albumen in the claim 1, wherein said albumen is sophisticated HSV-2 capsid protein.
7. the nucleic acid molecule that comprises a HSV-2 UL26 group or its function fragment that is separated to.
8. the nucleic acid molecule that is separated to of claim 7 comprises the nucleotide sequence of a SEQ ID NO:1 or its function fragment.
9. the nucleic acid molecule that is separated to of claim 7 comprises the nucleotide sequence of an encoding mature HSV-2 proteolytic enzyme.
10. the nucleic acid molecule that is separated to of claim 7 comprises a HSV-2 UL26.5 group or its function fragment.
11. the nucleic acid molecule that is separated to of claim 10 comprises the nucleotide sequence of an encoding mature HSV-2 capsid protein.
12. the nucleic acid molecule that is separated to of claim 10 comprises HSV-2 26.5 promotors.
13. expression vector that comprises HSV-2 UL26 gene or its function fragment.
14. the expression vector of claim 13, wherein said UL26 gene are open in SEQ IDNO:1.
15. the expression vector of claim 13, the said fragment of wherein said UL26 gene is selected from a nucleotide sequence by encoding mature HSV-2 proteolytic enzyme, the nucleotide sequence of an encoding mature HSV-2 capsid protein, the nucleotide sequence of a coding HSV-2 UL26.5 gene, the nucleotide sequence and the HSV-2UL26.5 of an encoding mature HSV-2 capsid protein start in molecular group.
16. used the expression vector transformed host cells of claim 13, said host cell can be expressed said UL26 gene or its function fragment.
17. a method of identifying the compound that suppresses the HSV-2 protease activity comprises the following steps:
A) in the presence of a kind of test compound, contact with HSV-2 proteolytic enzyme or his function fragment with a HSV-2 protease substrate:
B) level of the proteolytic cleavage of the said substrate of detection, and
C) (b) level with do not having in the presence of the test compound, the level of proteolytic activity of generation is compared when contacting with HSV-2 proteolytic enzyme or its function fragment with a HSV-2 protease substrate.
18. a method of identifying the compound that suppresses the assembling of HSV-2 virus particle comprises
A) in the presence of a kind of test-compound, the albumen that comprises HSV-2 capsid protein part at least that is present in more than two or two in the test-compound is contacted,
B) detect the level that capsid-capsid is united, and
C) with the level of said capsid-capsid associating with do not having test-compound in the presence of, the level that the capsid that takes place when the Partial Protein that comprises the HSV-2 capsid protein more than two or two at least contacts-capsid is united is compared.
19. a synthetic HSV-2 protease substrate, it has general formula R 1-SEQ IDNO:3-R 2Or R 1-SEQ ID NO:4-R 2
20. synthetic HSV-2 protease substrate is selected from SEQ IDNO:3 in the claim 19; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7:SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14 and SEQID NO:15.
21. an antibody that optionally combines with a crude HSV-2 proteolytic enzyme, wherein said antibody can not combine with finished HSV-2 substrate.
22. a method of distinguishing HSV-1 DNA and HSV-2 DNA comprises the following steps:
A) use amplification HSV-1 DNA but not DNA in the primer amplification sample of HSV-2 DNA, and/or use amplification HSV-2 DNA but not DNA in the primer amplification sample of HSV-1 DNA,
B) existence of detection DNA amplification.
23. one group of PCR primer, comprise the HSV-1 DNA that can only be used to increase but not the nucleotide sequence of HSV-2DNA maybe can only be used to increase HSV-2 DNA but not the nucleotide sequence of HSV-1 DNA.
24. a test kit of distinguishing HSV-1 DNA and HSV-2 DNA comprises a container and a container that contains a DNA size marker molecule that contains one group of PCR primer of claim 23.
25. distinguish HSV-1 albumen and the proteic method of HSV-2, comprise the following steps: for one kind
A) using can be selectively with the HSV-1 protein binding but can not carry out immunoassay with the protein bound antibody of HSV-2, and/or use can be selectively with the HSV-2 protein binding but can not carry out immunoassay with the protein bound antibody of HSV-1; And
B) existence of detection binding antibody.
26. one kind can be selectively with the HSV-2 protein binding but can not with the protein bound antibody of HSV-1 and a kind of can be selectively with the HSV-1 protein binding can not with the protein bound antibody of HSV-2.
27. a difference HSV-1 albumen and the proteic test kit of HSV-2 comprise that the container of an antibody that contains claim 26 and/or one contains that can be selectively can not contain in conjunction with the proteic antibody of HSV-2 and/or one in conjunction with HSV-1 albumen can selectively can not be in conjunction with the container of the proteic antibody of HSV-1 in conjunction with HSV-2 albumen.
28. the HSV-2 protease inhibitor compound of identifying with the method for claim 17.
29. the compound of the inhibition HSV-2 virus particle assembling of identifying with the method for claim 18.
CN94193881A 1993-08-20 1994-08-19 Hsv-2 UL26 gene, capsid proteins, immunoassays and protease inhibitors Pending CN1133594A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US11052293A 1993-08-20 1993-08-20
US08/110522 1993-08-20
US26453794A 1994-06-23 1994-06-23
US08/264537 1994-06-23

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EP (1) EP0714399A4 (en)
JP (1) JPH09503385A (en)
CN (1) CN1133594A (en)
AU (1) AU7568294A (en)
CA (1) CA2169748A1 (en)
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CN107305213A (en) * 2016-04-25 2017-10-31 赵芳 A kind of method and kit for detecting organophosphate and carbamate pesticide
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CN102363805A (en) * 2010-10-15 2012-02-29 成都医学院 Kit and method for screening HSV-1 resisting medicines
CN107305213A (en) * 2016-04-25 2017-10-31 赵芳 A kind of method and kit for detecting organophosphate and carbamate pesticide
CN107305212A (en) * 2016-04-25 2017-10-31 赵芳 The immunological detection method and kit of a kind of organophosphate and carbamate pesticide
CN107305212B (en) * 2016-04-25 2019-08-30 赵芳 A kind of immunological detection method and kit of organophosphate and carbamate pesticide

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EP0714399A1 (en) 1996-06-05
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WO1995006055A1 (en) 1995-03-02
CA2169748A1 (en) 1995-03-02
MXPA94006367A (en) 2004-09-09
AU7568294A (en) 1995-03-21

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