CN101230334A

CN101230334A - Herpes simplex virus and recombinant virus as well as host cell and medicinal combination thereof

Info

Publication number: CN101230334A
Application number: CNA2007100629390A
Authority: CN
Inventors: 李小鹏
Original assignee: BEIJING ORIENGENE BIOTECHNOLOGY Ltd
Current assignee: BEIJING ORIENGENE BIOTECHNOLOGY Ltd
Priority date: 2007-01-22
Filing date: 2007-01-22
Publication date: 2008-07-30
Anticipated expiration: 2027-01-22
Also published as: CN101230334B

Abstract

The invention provides a herpes simplex virus which is separated from yellow race. The virus has high infection rate on the specificity of the host cells of yellow race, has high reproduction speed; so that can be directly used for infecting and dissolve the tumour cells of yellow race, and can also be used as a carrier to carry gene medicine into the cells of yellow race to conduct gene treatment.

Description

Hsv and recombinant virus and host cell and pharmaceutical composition thereof

Technical field

The invention relates to a kind of virus, with this virus as the recombinant virus of carrier, comprise the host cell of described virus and/or described recombinant virus, and the pharmaceutical composition that comprises described virus and/or described recombinant virus.Specifically, the invention relates to a kind of hsv, with this virus as the recombinant virus of carrier, comprise the host cell of described virus and/or described recombinant virus, and the pharmaceutical composition that comprises described virus and/or described recombinant virus.

Background technology

Hsv (Herpes simplex virus, HSV) spherical in shape, be made up of core, capsid, tunicle (Tegument) and cyst membrane by intact virus.Nucleoid contains double-stranded DNA, is wound in the fibril spool.Capsid is the icosahedron symmetry, and diameter is about 100 nanometers, is made up of 162 shell particulates.The outer tunicle by gage distortion of capsid covers.The virus outermost layer is typical double-layer of lipoid cyst membrane.The viral diameter that comprises cyst membrane is the 150-200 nanometer.Viral glycoprotein B (gB), viral glycoprotein C (gC), viral glycoprotein D (gD), viral glycoprotein G (gG) and viral glycoprotein M (gM) are contained in the cyst membrane surface.Hsv comprise the I herpes simplex virus type (Herpes Simplex Virus Type I, HSV-1) and the II herpes simplex virus type.This virus is relevant with the three-dimensional arrangement of hsv membrane glycoprotein to the infection ability of host cell, and promptly can the three-dimensional arrangement of membrane glycoprotein have determined virus enter host cell, and the virus titer that can enter host cell.

Because hsv can be with the human cell as host cell, and human cell's type that can infect is wide, the propagation that meets accident can use the prevention of herpes medicine, burst times and latent period all unconformability host cell gene group to insert sudden change danger little, therefore, hsv has been used for the gene therapy of tumour or nervous system degenerative disease.For example, I herpes simplex virus type F strain HSV1716 is used for the treatment of cerebral glioma (referring to " HSV1716 injection into the brain adjacent to tumourfollowing surgical resection of high-grade glioma:safety data and long-termsurvival ", people such as S.Harrow, Gene Therapy, 11,1648-1658,2004); I herpes simplex virus type 17+ strain infected tumor cell is treated cancer by this viral lytic replication dissolving tumour cell.Yet, the I herpes simplex virus type genome that infects different ethnic groups has visibly different restriction enzyme resolution model, promptly there is restriction fragment length polymorphism (restriction fragment lengthpolymorphism, RFLP) (referring to " Quantitative analysis of genomic polymorphismof herpes simplex virus type 1 strains from six countries:studies of molecularevolution and molecular epidemiology of the virus ", people such as H.Sakaoka, Journal ofGeneral Virology, Vol 75,513-527,1994), herpes simplex virus 1 7+ strain separates from the white race, therefore, when the I herpes simplex virus type 17+ strain of infecting the white race is used for that the anthous carried out gene therapy, exist viral infection rate low, in host cell, duplicate the slow shortcoming of rate of propagation.

Summary of the invention

First purpose of the present invention be the hsv that overcomes prior art when being used for the anthous's gene therapy viral infection rate low, duplicate the slow shortcoming of rate of propagation at host cell, a kind of hsv is provided, when this virus was used for anthous's gene therapy, the viral infection rate height, to duplicate rate of propagation in host cell fast.

Second purpose of the present invention provides with the recombinant virus of above-mentioned virus as carrier.

The 3rd purpose of the present invention provides the host cell that comprises above-mentioned virus and/or above-mentioned recombinant virus.

The 4th purpose of the present invention provides a kind of pharmaceutical composition that comprises above-mentioned virus and/or above-mentioned recombinant virus.

When the present inventor is used for that at the I herpes simplex virus type 17+ strain of infecting the white race anthous carried out gene therapy, exist viral infection rate low, in host cell, duplicate the slow shortcoming of rate of propagation, from Chinese's bicker bleb of growing up, isolate hsv (classification called after herpes simplex virus I-type, the Latin formal name used at school is Herpes Simplex Virus Type I, in preservation on June 14 in 2006 to China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) that is positioned at the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, deposit number is CGMCC No.1736), and this virus carried out genome complete sequence determination and analysis, determined the difference that this clinical separation strain and I herpes simplex virus type 17+ strain exist aspect the gene of coding envelope protein.

The invention provides a kind of hsv, described virus has envelope protein, wherein, described envelope protein comprises a kind of aminoacid sequence that promotes virus infection host cell function that has, described aminoacid sequence is selected from the aminoacid sequence shown in the SEQ ID:NO.1, aminoacid sequence shown in the SEQ ID:NO.3, aminoacid sequence shown in the SEQ ID:NO.5, aminoacid sequence shown in the SEQ ID:NO.7, aminoacid sequence shown in the SEQ ID:NO.9, and to described SEQ ID:NO.1, SEQ ID:NO.3, SEQ ID:NO.5, aminoacid sequence shown in SEQ ID:NO.7 or the SEQ ID:NO.9 carries out one or several aminoacid replacement, add or aminoacid sequence that disappearance and the promotion virus infection host cell function that obtains are constant in one or more.

The present invention also provides a kind of hsv, and described virus has the gene of coding envelope protein, and wherein, described envelope protein is an envelope protein of the present invention.

The present invention also provides a kind of recombinant virus, and described recombinant virus comprises that as the virus of carrier and goal gene wherein, described virus as carrier is hsv of the present invention.

The present invention also provides a kind of infection virulent host cell, and wherein, described virus is above-mentioned virus and/or above-mentioned recombinant virus.

The present invention also provides a kind of pharmaceutical composition, and wherein, described pharmaceutical composition comprises above-mentioned virus and/or above-mentioned recombinant virus.

The invention provides a kind of separation from xanthous hsv, this virus is for the specificity infection rate height of anthous's host cell, reproduction speed is fast, therefore can be directly used in and infect and dissolve xanthous tumour cell, also can be used as carrier and carry genomic medicine and to anthous's cell, carry out gene therapy.Compare with existing I herpes simplex virus type 17+ strain in addition, when virus of the present invention is used for other ethnic groups pilosity tumour cells, stronger to the dissolving power of tumour cell.

The classification called after herpes simplex virus I-type of the hsv that the present invention relates to, the Latin formal name used at school is Herpes Simplex Virus Type I, to China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) that is positioned at the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, deposit number was CGMCC No.1736 in preservation on June 14 in 2006.

Description of drawings

Fig. 1 is the electron micrograph (amplifying 52000 times) of virus of the present invention;

Fig. 2 is the growing state optical microscope photograph (amplifying 400 times) that normal human mammary cancerous cell line MCF-7 cultivated 96 hours;

Fig. 3 is for cultivating the MCF-7 cell after 48 hours, is to cultivate 48 hours growing state optical microscope photograph (amplifying 400 times) again after the 0.1 transfection virus of the present invention by the ratio (MOI) of virus activity particle/cell count;

Fig. 4 is that virus of the present invention is to the inhibiting drug effect photo of human colon adenocarcinoma HT-29 transplanted tumor in nude mice;

Fig. 5 is that virus of the present invention is to the inhibiting curve of adenocarcinoma of colon HT-29 transplanted tumor in nude mice.

Embodiment

Described envelope protein is generally glycoprotein, relevant with virus intrusion host cell, for example viral glycoprotein B (gB), viral glycoprotein C (gC) and viral glycoprotein D (gD) all with adsorb/to penetrate host cell relevant, they also can induce host cell to merge, and the effect that induces the host cell neutralizing antibody is all arranged; Viral glycoprotein B (gB), viral glycoprotein C (gC), viral glycoprotein D (gD), viral glycoprotein G (gG) and viral glycoprotein M (gM) can both induce cytotoxicity.Because hsv envelope protein of the present invention, the viral glycoprotein B shown in the SEQ ID:NO.1 for example, viral glycoprotein C shown in the SEQ ID:NO.3, viral glycoprotein D shown in the SEQ ID:NO.5, among the viral glycoprotein M shown in viral glycoprotein G shown in the SEQ ID:NO.7 and the SEQ ID:NO.9 one or more, compare with wild-type herpes simplex virus 1 7+ variation has taken place, the corresponding target of the three-dimensional arrangement of envelope protein and host cell (for example cell surface receptor) is more identical, therefore especially the virus of anthous's host cell is more to enter host cell, thereby viral infection rate is obviously improved.

20 seed amino acid residues of constitutive protein matter can be divided into four classes according to side chain polarity: 1, nonpolar amino acid: L-Ala (Ala), Xie Ansuan (Val), leucine (Leu), Isoleucine (Ile), methionine(Met) (Met), phenylalanine (Phe), tryptophane (Trp) and proline(Pro) (Pro); 2, the uncharged amino acid of polarity: glycine (Gly), Serine (Ser), Threonine (Thr), halfcystine (Cys), aspartic acid (Asn), glutamine (Gln) and tyrosine (Tyr); 3, positively charged amino acid: arginine (Arg), Methionin (Lys) and Histidine (His); 4, electronegative amino acid: aspartic acid (Asp) and L-glutamic acid (Glu) (referring to " biological chemistry " (second edition) first volume, Shen is with, Wang Jingyan, 82-83 page or leaf, Higher Education Publishing House, December nineteen ninety).If belonging to the amino-acid residue of a classification in the protein together replaces, for example replace Lys or replace Ile by Leu by Arg, described residue role (such as positive charge being provided or forming the effect of hydrophobic capsule bag constructions) in protein domain does not change, therefore can't exert an influence to proteinic three-dimensional arrangement, therefore still can realize proteic function.For example, as well known to those skilled in the art, Ala and Ser, Val and Ile, Asp and Glu, Ser and Thr, Ala and Gly, Ala and Thr, Ser and Asn, Ala and Val, Ser and Gly, Tyr and Phe, Ala and pro, Lys and Arg, Asp and Asn, Leu and Ile, Leu and Val, Ala and Glu and Asp and Gly, replace mutually between any two, can not influence proteic three-dimensional arrangement and function.The described amino-acid residue replacement that belongs to a classification together can occur on any one amino acid residue position of above-mentioned envelope protein.On the contrary, different classes of amino-acid residue replaces, and proteic structure is changed, and difference appears in function.For example compare with herpes simplex virus 1 7+, different classes of replacement has taken place the base of the membrane glycoprotein of hsv of the present invention, and (the 79th amino acids residue as herpes simplex virus 1 7+ viral glycoprotein B is a Methionin, and the 79th amino acids residue of the viral glycoprotein B of hsv of the present invention is a Threonine), result's hsv of the present invention is the easy infection anthous more.

Aminoacid sequence shown in the described SEQ ID:NO.1 is carried out one or several aminoacid replacement and the constant aminoacid sequence of promotion virus infection host cell function that obtains can be selected from the 79th aminoacid sequence for Serine, halfcystine, l-asparagine, glutamine, tyrosine or glycine among the SEQ ID:NO.1.To the aminoacid sequence shown in the described SEQ ID:NO.3 carry out one or several aminoacid replacement and the constant aminoacid sequence of the promotion virus infection host cell function that obtains can among the SEQ ID:NO.3 the 201st among the aminoacid sequence of L-glutamic acid and/or the SEQ ID:NO.3 the 295th be Methionin or arginic aminoacid sequence.To the aminoacid sequence shown in the described SEQ ID:NO.5 carry out one or several aminoacid replacement and the constant aminoacid sequence of the promotion virus infection host cell function that obtains can among the SEQ ID:NO.5 the 369th be Methionin or arginic aminoacid sequence.Aminoacid sequence shown in the described SEQ ID:NO.7 is carried out one or several aminoacid replacement and the constant aminoacid sequence of promotion virus infection host cell function that obtains can be selected among the SEQID:NO.7 the 3rd and is L-Ala, Xie Ansuan, leucine, Isoleucine, methionine(Met), the aminoacid sequence of phenylalanine or tryptophane, the 86th is the aminoacid sequence of aspartic acid among the SEQ ID:NO.7, the 116th is Serine among the SEQ ID:NO.7, Threonine, halfcystine, l-asparagine, the aminoacid sequence of glutamine or tyrosine, the 118th is the aminoacid sequence of aspartic acid among the SEQ ID:NO.7, the 130th is L-Ala among the SEQ ID:NO.7, Xie Ansuan, leucine, Isoleucine, methionine(Met), the aminoacid sequence of phenylalanine or tryptophane, the 151st is glycine among the SEQ ID:NO.7, Threonine, halfcystine, l-asparagine, the aminoacid sequence of glutamine or tyrosine, the 153rd is the aminoacid sequence of aspartic acid among the SEQ ID:NO.7, the 162nd is glycine among the SEQ ID:NO.7, Serine, Threonine, halfcystine, the aminoacid sequence of l-asparagine or tyrosine, the 164th is L-Ala among the SEQ ID:NO.7, Xie Ansuan, leucine, Isoleucine, methionine(Met), the aminoacid sequence of phenylalanine or tryptophane, the 208th is Xie Ansuan among the SEQ ID:NO.7, leucine, Isoleucine, methionine(Met), the aminoacid sequence of phenylalanine or tryptophane and the 236th are L-Ala, Xie Ansuan, leucine, Isoleucine, methionine(Met), in the aminoacid sequence of phenylalanine or tryptophane one or more.To the aminoacid sequence shown in the described SEQ ID:NO.9 carry out one or several aminoacid replacement and the constant aminoacid sequence of the promotion virus infection host cell function that obtains can among the SEQ ID:NO.9 the 440th be the 446th aminoacid sequence among the aminoacid sequence of L-Ala, Xie Ansuan, leucine, Isoleucine, methionine(Met), phenylalanine or tryptophane and/or the SEQ ID:NO.9 for glycine, Serine, Threonine, halfcystine, glutamine or tyrosine.

Aminoacid sequence of the present invention is preferably selected from one or more in the aminoacid sequence shown in aminoacid sequence shown in the aminoacid sequence shown in the aminoacid sequence shown in the aminoacid sequence shown in the SEQ ID:NO.1, the SEQID:NO.3, the SEQ ID:NO.5, the SEQ ID:NO.7 and the SEQ ID:NO.9.More preferably described aminoacid sequence is the aminoacid sequence shown in aminoacid sequence shown in the aminoacid sequence shown in the aminoacid sequence shown in the aminoacid sequence shown in the SEQ ID:NO.1, the SEQ ID:NO.3, the SEQ ID:NO.5, the SEQ ID:NO.7 and the SEQ ID:NO.9.

Known in this field, in 20 kinds of different amino acid of constitutive protein matter, except that Met (ATG) or Trp (TGG) were respectively single password coding, other 18 seed amino acids were respectively by 2-6 codon encode (Sambrook etc., molecular cloning, press of cold spring harbor laboratory, New York, the U.S., second edition, 1989, see 950 pages of appendix D).Promptly because the degeneracy of genetic codon determines more than one mostly of an amino acid whose codon, and the displacement of the 3rd Nucleotide often can not change amino acid whose composition in the triplet codon, the nucleotide sequence of the same protein of therefore encoding can be different.Those skilled in the art are according to known password sublist, from aminoacid sequence disclosed by the invention, and the constant aminoacid sequence of promotion virus infection host cell function that obtains by described aminoacid sequence, can derive their nucleotide sequence of to encode fully, nucleotide sequence as described in obtaining by biological method (as PCR method, mutation method) or chemical synthesis process, be applied in the middle of recombinant technology and the gene therapy, so this partial nucleotide sequence all should be included in the scope of the present invention.On the contrary, utilize dna sequence dna disclosed herein, also can be by means commonly known in the art, for example method (molecular cloning, the press of cold spring harbor laboratory of Sambrook etc., New York, the U.S., second edition, 1989) carry out, by revising nucleotide sequence provided by the invention, obtain and the consistent aminoacid sequence of envelope protein function of the present invention.

The gene of preferred described coding envelope protein comprises that a kind of coding has the nucleotide sequence of the aminoacid sequence that promotes virus infection host cell function, described nucleotide sequence is selected from the nucleotide sequence shown in the SEQ ID:NO.2, nucleotide sequence shown in the SEQ ID:NO.4, nucleotide sequence shown in the SEQ ID:NO.6, nucleotide sequence shown in the SEQ ID:NO.8, nucleotide sequence shown in the SEQ ID:NO.10, and to described SEQ ID:NO.2, SEQ ID:NO.4, SEQ ID:NO.6, nucleotide sequence shown in SEQ ID:NO.8 or the SEQ ID:NO.10 carries out that one or several Nucleotide replaces and in the constant nucleotide sequence of the proteins encoded that obtains one or more.Wherein, the nucleotide sequence shown in the described SEQ ID:NO.2 is carried out that one or several Nucleotide replaces and the constant nucleotide sequence of proteins encoded that obtains can be selected among the SEQ ID:NO.2 the 144th and is the nucleotide sequence of cytosine(Cyt) (C), the 183rd is VITAMIN B4 (A) among the SEQ ID:NO.2, the nucleotide sequence of guanine (G) or thymus pyrimidine (T), the 186th is A among the SEQ ID:NO.2, the nucleotide sequence of G or T, the 210th is the nucleotide sequence of A among the SEQ ID:NO.2, the 219th is the nucleotide sequence of G among the SEQ ID:NO.2, the 237th is A among the SEQ ID:NO.2, the nucleotide sequence of T or C, the 865th is the nucleotide sequence of T among the SEQ ID:NO.2, the 867th is A among the SEQ ID:NO.2, the nucleotide sequence of T or C, the 1161st is A among the SEQ ID:NO.2, the nucleotide sequence of T or G, the 1410th is T among the SEQ ID:NO.2, the nucleotide sequence of G or C, the 1513rd is the nucleotide sequence of A among the SEQ ID:NO.2, the 1515th is A among the SEQ ID:NO.2, the nucleotide sequence of T or C, the 2095th is T among the SEQ ID:NO.2, the nucleotide sequence of G or C, the 2097th is T among the SEQ ID:NO.2, the nucleotide sequence of G or C, the 2196th is the nucleotide sequence of T among the SEQID:NO.2, the 2530th is the nucleotide sequence of T among the SEQ ID:NO.2, the 2532nd is A among the SEQ ID:NO.2, the 2562nd is A among the nucleotide sequence of T or C and the SEQ ID:NO.2, in the nucleotide sequence of T or C one or more.Nucleotide sequence shown in the described SEQ ID:NO.4 is carried out that one or several Nucleotide replaces and the constant nucleotide sequence of proteins encoded that obtains can be selected among the SEQ ID:NO.4 the 120th and is G, the nucleotide sequence of T or C, the 177th is the nucleotide sequence of G among the SEQ ID:NO.4, the 267th is the nucleotide sequence of G among the SEQ ID:NO.4, the 597th is the nucleotide sequence of T among the SEQ ID:NO.4, the 865th is the nucleotide sequence of A among the SEQ ID:NO.4, the 867th is T among the SEQ ID:NO.4, the nucleotide sequence of G or C, the 1059th is A among the SEQ ID:NO.4, the nucleotide sequence of T or C, the 1149th is T among the SEQ ID:NO.4, the 1368th is T among the nucleotide sequence of G or C and the SEQ ID:NO.4, in the nucleotide sequence of the nucleotide sequence of G or C one or more.Nucleotide sequence shown in the described SEQ ID:NO.6 is carried out that one or several Nucleotide replaces and the constant nucleotide sequence of proteins encoded that obtains can be selected among the SEQ ID:NO.6 the 75th and is A, the nucleotide sequence of T or G, the 90th is A among the SEQ ID:NO.6, the nucleotide sequence of T or C, the 822nd is A among the SEQ ID:NO.6, the nucleotide sequence of G or C, the 870th is the nucleotide sequence of G among the SEQ ID:NO.6, the 948th is A among the SEQ ID:NO.6, the nucleotide sequence of T or C, the 963rd is the nucleotide sequence of A or T among the SEQ ID:NO.6, the 1095th is the nucleotide sequence of T among the SEQ ID:NO.6, the 1099th is the nucleotide sequence of A among the SEQ ID:NO.6, the 1101st is A among the SEQ ID:NO.6, the nucleotide sequence of T or G, among the SEQ ID:NO.6 the 1105th be among the nucleotide sequence of A and the SEQ ID:NO.6 the 1107th be A, in the nucleotide sequence of T or C one or more.Nucleotide sequence shown in the described SEQ ID:NO.8 is carried out that one or several Nucleotide replaces and the constant nucleotide sequence of proteins encoded that obtains can be selected among the SEQ ID:NO.8 the 9th and is A, the nucleotide sequence of T or C, the 108th is the nucleotide sequence of A among the SEQ ID:NO.8, the 228th is the nucleotide sequence of A or T among the SEQ ID:NO.8, the 258th is the nucleotide sequence of A among the SEQ ID:NO.8, the 261st is A among the SEQID:NO.8, the nucleotide sequence of T or C, the 270th is the nucleotide sequence of C among the SEQ ID:NO.8, the 327th is A among the SEQ ID:NO.8, the nucleotide sequence of G or C, the 336th is A among the SEQ ID:NO.8, the nucleotide sequence of T or G, the 345th is A among the SEQ ID:NO.8, the nucleotide sequence of T or G, the 348th is A among the SEQ ID:NO.8, the nucleotide sequence of T or C, the 351st is the nucleotide sequence of C among the SEQ ID:NO.8, the 354th is the nucleotide sequence of T among the SEQ ID:NO.8, the 390th is A among the SEQ ID:NO.8, the nucleotide sequence of T or G, the 396th is A among the SEQ ID:NO.8, the nucleotide sequence of G or C, the 430th is the nucleotide sequence of A among the SEQ ID:NO.8, the 432nd is T among the SEQ ID:NO.8, the nucleotide sequence of G or C, the 453rd is the nucleotide sequence of C among the SEQ ID:NO.8, the 459th is the nucleotide sequence of A among the SEQ ID:NO.8, the 472nd is the nucleotide sequence of A among the SEQ ID:NO.8, the 474th is T among the SEQ ID:NO.8, the nucleotide sequence of G or C, the 486th is the nucleotide sequence of G among the SEQ ID:NO.8, the 492nd is A among the SEQ ID:NO.8, the nucleotide sequence of T or G, the 501st is A among the SEQ ID:NO.8, the nucleotide sequence of T or C, the 624th is A among the SEQ ID:NO.8, the 708th is A among the nucleotide sequence of G or C and the SEQ ID:NO.8, in the nucleotide sequence of T or G one or more.Nucleotide sequence shown in the described SEQ ID:NO.10 is carried out that one or several Nucleotide replaces and the constant nucleotide sequence of proteins encoded that obtains can be selected among the SEQ ID:NO.10 the 27th and is A, the nucleotide sequence of T or G, the 136th is the nucleotide sequence of T among the SEQ ID:NO.10, the 138th is A among the SEQ ID:NO.10, the nucleotide sequence of G or C, the 315th is A among the SEQ ID:NO.10, the nucleotide sequence of T or C, the 489th is the nucleotide sequence of G among the SEQ ID:NO.10, the 609th is A among the SEQ ID:NO.10, the nucleotide sequence of T or C, the 618th is A among the SEQ ID:NO.10, the nucleotide sequence of G or C, the 643rd is the nucleotide sequence of A among the SEQ ID:NO.10, the 645th is T among the SEQ ID:NO.10, the nucleotide sequence of G or C, the 728th is T among the SEQ ID:NO.10, the nucleotide sequence of G or C, the 762nd is the nucleotide sequence of T or C among the SEQ ID:NO.10, the 784th is the nucleotide sequence of T among the SEQ ID:NO.10, the 786th is A among the SEQ ID:NO.10, the nucleotide sequence of G or C, the 954th is A among the SEQ ID:NO.10, the nucleotide sequence of T or G, the 966th is A among the SEQ ID:NO.10, the nucleotide sequence of G or C, the 1320th is A among the SEQ ID:NO.10, the nucleotide sequence of T or G, among the SEQ ID:NO.10 the 1338th be among the nucleotide sequence of T and the SEQ ID:NO.10 the 1344th be in the nucleotide sequence of A one or more.

The immediate early gene of hsv (immediate early phase, IE) refer to that virus enters the gene that host cell just utilizes host cell expression (generally in 1 hour) immediately, because hsv is also relevant with the expression abundance of infected cell polypeptides in host cell of this viral immediate early gene coding to the infection ability of host cell, the abundance that the immediate early gene of infected cell polypeptides of promptly encoding is expressed in host cell is high more, and then the rate of propagation of virus in host cell is fast more.Therefore more preferably described virus also comprises a kind of aminoacid sequence that promotes the virus multiplication function that has, the infected cell polypeptides that is a kind of immediate early gene coding is expressed the higher aminoacid sequence of abundance, described aminoacid sequence is selected from the aminoacid sequence shown in the aminoacid sequence shown in the SEQ ID:NO.11, the SEQ ID:NO.13, and the aminoacid sequence shown in described SEQ ID:NO.11 or the SEQ ID:NO.13 is carried out one or several aminoacid replacement, interpolation or disappearance and in the constant aminoacid sequence of the promotion virus multiplication function that obtains one or more.

Described SEQ ID:NO.11 is carried out one or several aminoacid replacement and the constant aminoacid sequence of promotion virus multiplication function that obtains can be selected among the SEQ ID:NO.11 the 34th and is Xie Ansuan, leucine, Isoleucine, methionine(Met), phenylalanine, the aminoacid sequence of tryptophane or proline(Pro), the 102nd is Serine among the SEQ ID:NO.11, glycine, halfcystine, l-asparagine, the aminoacid sequence of glutamine or tyrosine, the 281st is Xie Ansuan among the SEQ ID:NO.11, leucine, Isoleucine, methionine(Met), phenylalanine, the aminoacid sequence of tryptophane or proline(Pro), among the SEQ ID:NO.11 the 351st be among the aminoacid sequence of arginine or Histidine and the SEQ ID:NO.11 the 360th be in the aminoacid sequence of Methionin or Histidine one or more.Described SEQ ID:NO.13 is carried out one or several aminoacid replacement and the constant aminoacid sequence of promotion virus multiplication function that obtains can be selected among the SEQ ID:NO.13 the 36th and is Xie Ansuan, leucine, Isoleucine, methionine(Met), phenylalanine, the aminoacid sequence of tryptophane or proline(Pro), the 178th is Methionin or arginic aminoacid sequence among the SEQ ID:NO.13, the 179th is the aminoacid sequence of Methionin or Histidine among the SEQ ID:NO.13, the 180th is the aminoacid sequence of Methionin or Histidine among the SEQ ID:NO.13, the 181st is Serine among the SEQ ID:NO.3, Threonine, halfcystine, l-asparagine, the aminoacid sequence of glutamine or tyrosine, the 206th is L-Ala among the SEQ ID:NO.13, Xie Ansuan, leucine, Isoleucine, methionine(Met), the aminoacid sequence of phenylalanine or tryptophane, the 383rd is Xie Ansuan among the SEQ ID:NO.13, leucine, Isoleucine, methionine(Met), phenylalanine, in the aminoacid sequence of tryptophane or proline(Pro) one or more.

Further preferred described virus also comprises a kind of aminoacid sequence that promotes the virus multiplication function that has, and described aminoacid sequence is the aminoacid sequence shown in aminoacid sequence shown in the SEQ ID:NO.11 and/or the SEQ ID:NO.13.

In the gene of described coding envelope protein, described envelope protein more preferably is selected from one or more in the envelope protein of the aminoacid sequence shown in aminoacid sequence shown in the aminoacid sequence shown in the aminoacid sequence shown in the aminoacid sequence shown in the SEQ ID:NO.1, the SEQ ID:NO.3, the SEQ ID:NO.5, the SEQ ID:NO.7 and the SEQ ID:NO.9.

Further preferred described nucleotide sequence is selected from one or more in the nucleotide sequence shown in nucleotide sequence shown in the nucleotide sequence shown in the nucleotide sequence shown in the nucleotide sequence shown in the SEQ ID:NO.2, the SEQID:NO.4, the SEQ ID:NO.6, the SEQ ID:NO.8 and the SEQ ID:NO.10.Further preferred described nucleotides sequence is classified the nucleotide sequence shown in nucleotide sequence shown in the nucleotide sequence shown in the nucleotide sequence shown in the nucleotide sequence shown in the SEQ ID:NO.2, the SEQID:NO.4, the SEQ ID:NO.6, the SEQ ID:NO.8 and the SEQ ID:NO.10 as.

More preferably described virus comprises that also a kind of coding has the nucleotide sequence of the aminoacid sequence that promotes the virus multiplication function, described nucleotide sequence is selected from the nucleotide sequence shown in the nucleotide sequence shown in the SEQ ID:NO.12, the SEQ ID:NO.14, and the nucleotide sequence shown in described SEQ ID:NO.12 or the SEQ ID:NO.14 is carried out that one or several Nucleotide replaces and in the constant nucleotide sequence of the proteins encoded that obtains one or more.

Wherein, the nucleotide sequence shown in the described SEQ ID:NO.12 is carried out that one or several Nucleotide replaces and the constant nucleotide sequence of proteins encoded that obtains can be selected among the SEQ ID:NO.12 the 102nd and is VITAMIN B4 (A), the nucleotide sequence of thymus pyrimidine (T) or guanine (G), the 306th is A among the SEQ ID:NO.12, the nucleotide sequence of T or cytosine(Cyt) (C), the 570th is the nucleotide sequence of G among the SEQ ID:NO.12, the 612nd is T among the SEQ ID:NO.12, the nucleotide sequence of G or C, the 652nd is the nucleotide sequence of T among the SEQ ID:NO.12, the 654th is A among the SEQ ID:NO.12, the nucleotide sequence of T or G, the 729th is A among the SEQ ID:NO.12, the nucleotide sequence of G or C, the 843rd is A among the SEQ ID:NO.12, the nucleotide sequence of T or G, among the SEQ ID:NO.12 the 1053rd be among the nucleotide sequence of G and the SEQ ID:NO.12 the 1080th be A, in the nucleotide sequence of T or C one or more.Nucleotide sequence shown in the described SEQID:NO.14 is carried out that one or several Nucleotide replaces and the constant nucleotide sequence of proteins encoded that obtains can be selected among the SEQ ID:NO.14 the 108th and is T, the nucleotide sequence of G or C, the 465th is A among the SEQ ID:NO.14, the nucleotide sequence of T or G, the 519th is A among the SEQ ID:NO.14, the nucleotide sequence of T or C, the 534th is the nucleotide sequence of T among the SEQ ID:NO.14, the 535th is the nucleotide sequence of A among the SEQ ID:NO.14, the 537th is A among the SEQ ID:NO.14, the nucleotide sequence of T or C, the 540th is A among the SEQ ID:NO.14, the nucleotide sequence of T or G, the 588th is T among the SEQ ID:NO.14, the nucleotide sequence of G or C, the 618th is A among the SEQ ID:NO.14, the 1149th is A among the nucleotide sequence of T or C and the SEQ ID:NO.14, in the nucleotide sequence of the nucleotide sequence of T or G one or more.

Further preferred described virus comprises that also a kind of coding has the nucleotide sequence of the aminoacid sequence that promotes the virus multiplication function, and described nucleotides sequence is classified the nucleotide sequence shown in nucleotide sequence shown in the SEQ ID:NO.12 and/or the SEQ ID:NO.14 as.

Most preferably the nucleotide sequence of the I herpes simplex virus type 17+ of ID:NC 001806 has 99.5% homology among the nucleotide sequence of described virus and the NCBI, and the difference of the two is as shown in table 1." variation position " in the table 1 is the base numbering of complete sequence Nucleotide of the I herpes simplex virus type 17+ of ID:NC_001806.

Table 1

The variation position	2351	2352-2353	2538	5730	6067	6385	7412	7604	9663	9980	10085
The variation position	2351	2352-2353	2538	5730	6067	6385	7412	7604	9663	9980	10085	17+	G	CT	C	G	G	C	C	C	C	T	G
CL-1	A	TG	T	A	Disappearance	Disappearance	A	G	T	C	Disappearance	17+	G	CT	C	G	G	C	C	C	C	T	G

Continuous table 1

The variation position	Between 10109 and 10110	10431	10508	10656	10723	10861	11123	11142	11195
The variation position	Between 10109 and 10110	10431	10508	10656	10723	10861	11123	11142	11195	17+	-	A	T	C	G	G	G	A	T
CL-1	Add G	C	C	T	A	T	T	G	C	17+	-	A	T	C	G	G	G	A	T

Continuous table 1

The variation position	111680-111681	11684-11685	11774-11775	11782	11945	11952	12168	12305	13065
The variation position	111680-111681	11684-11685	11774-11775	11782	11945	11952	12168	12305	13065	17+	CC	GC	--	T	C	A	G	A	C
CL-1	AT	Disappearance	Add AG	G	T	G	T	G	A	17+	CC	GC	--	T	C	A	G	A	C

Continuous table 1

The variation position	13331	13353	13787	13995	14520	14934	15209	15229	15873	15908	16094
The variation position	13331	13353	13787	13995	14520	14934	15209	15229	15873	15908	16094	17+	T	G	A	C	G	C	G	C	G	T	T
CL-1	C	A	C	T	A	T	C	G	A	C	C	17+	T	G	A	C	G	C	G	C	G	T	T

Continuous table 1

The variation position	16148	16218	16442	16694	17098	17105	17174	17442	17540	17667	17737
The variation position	16148	16218	16442	16694	17098	17105	17174	17442	17540	17667	17737	17+	A	G	A	T	A	A	T	T	C	C	C
CL-1	G	T	C	C	G	G	C	C	A	A	T	17+	A	G	A	T	A	A	T	T	C	C	C

Continuous table 1

The variation position	17775	17814	17847	17916	17929	18109	18114	18118	18136	18145	18148
The variation position	17775	17814	17847	17916	17929	18109	18114	18118	18136	18145	18148	CL-1	T	T	A	T	G	T	A	C	A	G	T
17+	C	G	G	G	T	G	G	G	G	Disappearance	A	CL-1	T	T	A	T	G	T	A	C	A	G	T

Continuous table 1

The variation position	18284	18458	18515	18580	18646	18722	18850	18852	19081	19253
The variation position	18284	18458	18515	18580	18646	18722	18850	18852	19081	19253	CL-1	T	A	A	C	C	A	G	C	C	A
17+	G	G	G	A	A	G	C	G	T	G	CL-1	T	A	A	C	C	A	G	C	C	A

Continuous table 1

The variation position	Between 20574 and 20575	20856	20967	21093	21237	21345	21525	22011	22014	22422
The variation position	Between 20574 and 20575	20856	20967	21093	21237	21345	21525	22011	22014	22422	CL-1	-	T	A	A	C	A	C	G	C	G
17+	G	C	G	G	T	G	T	A	A	C	CL-1	-	T	A	A	C	A	C	G	C	G

Continuous table 1

The variation position	22452	22651	22905	23232	23343	23519	23694	23813	23823	23850	23933
The variation position	22452	22651	22905	23232	23343	23519	23694	23813	23823	23850	23933	CL-1	T	G	C	C	T	C	A	C	T	A	A
17+	C	A	T	T	C	T	G	T	C	C	T	CL-1	T	G	C	C	T	C	A	C	T	A	A

Continuous table 1

The variation position	23967	23989	24159	24171	24524	24541	24549	24719-24720	24726	Between 24761 and 24762
The variation position	23967	23989	24159	24171	24524	24541	24549	24719-24720	24726	Between 24761 and 24762	CL-1	A	C	C	T	C	A	G	TT	A	-
17+	C	A	T	C	A	G	T	CC	C	A	CL-1	A	C	C	T	C	A	G	TT	A	-

Continuous table 1

The variation position	24790	24864	24895	24932	24991	25116	26049	26316	26349	26715	27256
The variation position	24790	24864	24895	24932	24991	25116	26049	26316	26349	26715	27256	CL-1	C	A	C	A	A	C	T	G	T	C	G
17+	T	G	T	C	G	T	C	A	G	T	A	CL-1	C	A	C	A	A	C	T	G	T	C	G

Continuous table 1

The variation position	27343	27346	27811	27911	28015	28132	28217	28336	28387	28401	28938
The variation position	27343	27346	27811	27911	28015	28132	28217	28336	28387	28401	28938	CL-1	A	A	A	G	G	A	T	C	T	T	A
17+	G	C	G	A	A	G	C	A	G	C	C	CL-1	A	A	A	G	G	A	T	C	T	T	A

Continuous table 1

The variation position	29125	29203	29367	29446	29521	29824	30328	30856	30888	31199	31272
The variation position	29125	29203	29367	29446	29521	29824	30328	30856	30888	31199	31272	CL-1	A	G	A	C	T	A	A	G	T	G	T
17+	C	T	C	T	C	G	G	T	Disappearance	A	G	CL-1	A	G	A	C	T	A	A	G	T	G	T

Continuous table 1

The variation position	31315	31397	31942	31994	32147	32460	32477	32508	32534	32554	32624
The variation position	31315	31397	31942	31994	32147	32460	32477	32508	32534	32554	32624	CL-1	T	A	G	A	G	C	T	A	T	A	G
17+	G	C	A	C	T	A	C	G	G	G	T	CL-1	T	A	G	A	G	C	T	A	T	A	G

Continuous table 1

The variation position	32663	32666	32738	33145	33230	Between 33437 and 33438	33453	33469	33634	33816
The variation position	32663	32666	32738	33145	33230	Between 33437 and 33438	33453	33469	33634	33816	CL-1	A	G	C	T	A	-	A	A	C	C
17+	C	T	T	C	C	C	C	Disappearance	T	A	CL-1	A	G	C	T	A	-	A	A	C	C

Continuous table 1

The variation position	33865	33999	34215	34296	34416	34449	34596	34650	34683	34725	34797
The variation position	33865	33999	34215	34296	34416	34449	34596	34650	34683	34725	34797	CL-1	C	A	T	A	C	G	C	G	G	C	C
17+	A	C	C	G	A	C	T	A	T	T	T	CL-1	C	A	T	A	C	G	C	G	G	C	C

Continuous table 1

The variation position	34811	34863	Between 34880 and 34881	34916	35018	35047	Between 35068 and 35069	35076	35238	35267
The variation position	34811	34863	Between 34880 and 34881	34916	35018	35047	Between 35068 and 35069	35076	35238	35267	CL-1	A	C	--	A	G	G	-	A	G	C
17+	C	T	GG	G	A	T	C	C	A	T	CL-1	A	C	--	A	G	G	-	A	G	C

Continuous table 1

The variation position	35331	35463	36121	36129	36222	36228	36253	36266	36336	36494	36613
The variation position	35331	35463	36121	36129	36222	36228	36253	36266	36336	36494	36613	CL-1	G	G	A	A	T	C	A	C	G	G	T
17+	T	A	G	G	G	A	G	T	Disappearance	A	C	CL-1	G	G	A	A	T	C	A	C	G	G	T

Continuous table 1

The variation position	37115	37236	37280	38141	38225	38231	38312	38390	38516	38672	40069
The variation position	37115	37236	37280	38141	38225	38231	38312	38390	38516	38672	40069	CL-1	T	C	A	G	C	T	G	T	T	T	T
17+	G	G	G	A	T	C	A	C	G	G	C	CL-1	T	C	A	G	C	T	G	T	T	T	T

Continuous table 1

The variation position	40919	40942	40967	41401	41425	41682	41885	42054	42249	42677	42978
The variation position	40919	40942	40967	41401	41425	41682	41885	42054	42249	42677	42978	CL-1	A	T	T	T	A	A	G	C	G	G	A
17+	G	C	G	C	G	G	A	T	C	A	C	CL-1	A	T	T	T	A	A	G	C	G	G	A

Continuous table 1

The variation position	43402	43678	43697	43705	43744	Between 43744 and 43745	43752	43857	44289	44478
The variation position	43402	43678	43697	43705	43744	Between 43744 and 43745	43752	43857	44289	44478	CL-1	A	C	G	C	A	--	A	C	C	G
17+	G	A	A	T	G	GG	G	A	T	A	CL-1	A	C	G	C	A	--	A	C	C	G

Continuous table 1

The variation position	44781	45043	45123	46023	46049	46649	46828	46864	47004	47080	47235
The variation position	44781	45043	45123	46023	46049	46649	46828	46864	47004	47080	47235	CL-1	C	A	G	A	A	C	G	G	T	G	A
17+	A	G	A	G	G	T	A	A	C	A	G	CL-1	C	A	G	A	A	C	G	G	T	G	A

Continuous table 1

The variation position	47251	47526	47626	47729	47738	47904	48303	48304	48402	48406	48445
The variation position	47251	47526	47626	47729	47738	47904	48303	48304	48402	48406	48445	CL-1	T	G	A	C	A	G	G	A	A	C	G
17+	C	A	G	T	G	A	T	G	C	T	A	CL-1	T	G	A	C	A	G	G	A	A	C	G

Continuous table 1

The variation position	48701	48706	Between 48719 and 48720	48785	48970	49414	49444	49457	49522	49555	49598
The variation position	48701	48706	Between 48719 and 48720	48785	48970	49414	49444	49457	49522	49555	49598	CL-1	T	T	-	G	G	G	C	T	A	T	T
17+	G	C	C	T	A	T	A	G	C	C	G	CL-1	T	T	-	G	G	G	C	T	A	T	T

Continuous table 1

The variation position	49819	50353	50588	50762-50774	51397	52281	52326	52518	52521-52822
The variation position	49819	50353	50588	50762-50774	51397	52281	52326	52518	52521-52822	CL-1	C	C	G	TTTTTTTTTTTTT	A	A	G	A	TT
17+	T	T	A	Disappearance	C	G	A	G	Disappearance	CL-1	C	C	G	TTTTTTTTTTTTT	A	A	G	A	TT

Continuous table 1

The variation position	52833	52870	52889	Between 52897 and 52898	52915	52960	52976-52977	52985	53009-53010
The variation position	52833	52870	52889	Between 52897 and 52898	52915	52960	52976-52977	52985	53009-53010	CL-1	G	T	T	--	A	G	AT	T	CA
17+	C	C	G	GG	G	A	Disappearance	C	Disappearance	CL-1	G	T	T	--	A	G	AT	T	CA

Continuous table 1

The variation position	53029	53033-53034	53046	53067	53091	53245	53277	53611	53710	54294	54397
The variation position	53029	53033-53034	53046	53067	53091	53245	53277	53611	53710	54294	54397	CL-1	A	AA	G	A	G	C	T	G	T	G	T
17+	C	Disappearance	A	G	A	A	G	A	C	T	G	CL-1	A	AA	G	A	G	C	T	G	T	G	T

Continuous table 1

The variation position	54646	54942	55571	55589	55598	55623	55626	55663	55895	56027	56684
The variation position	54646	54942	55571	55589	55598	55623	55626	55663	55895	56027	56684	CL-1	G	G	G	T	C	C	G	A	G	T	T
17+	A	A	T	C	T	G	C	G	T	C	C	CL-1	G	G	G	T	C	C	G	A	G	T	T

Continuous table 1

The variation position	57074	58332	58952	59105	59279	59324	59528	60005	60023	60071	60389
The variation position	57074	58332	58952	59105	59279	59324	59528	60005	60023	60071	60389	CL-1	A	T	C	T	C	G	A	G	A	T	T
17+	G	C	T	C	A	A	G	A	G	C	C	CL-1	A	T	C	T	C	G	A	G	A	T	T

Continuous table 1

The variation position	60396	60428	60497	60563	60569	60642	60806	60890	60999	61023	61061
The variation position	60396	60428	60497	60563	60569	60642	60806	60890	60999	61023	61061	CL-1	A	A	G	C	G	C	T	A	T	C	G
17+	G	G	T	T	A	G	C	G	G	A	A	CL-1	A	A	G	C	G	C	T	A	T	C	G

Continuous table 1

The variation position	61150	61175	61205	61388	61394	61475	61721	61805	61936	61949	62071-62072
The variation position	61150	61175	61205	61388	61394	61475	61721	61805	61936	61949	62071-62072	CL-1	G	A	A	A	A	A	G	T	C	C	CC
17+	C	G	G	G	G	G	A	G	A	T	TT	CL-1	G	A	A	A	A	A	G	T	C	C	CC

Continuous table 1

The variation position	62088	62126	62163-62167	62174	62187	62191	62220	Between 62238 and 62239	Between 62253 and 62254
The variation position	62088	62126	62163-62167	62174	62187	62191	62220	Between 62238 and 62239	Between 62253 and 62254	CL-1	A	G	GGGGG	A	A	G	A	--	-
17+	G	A	Disappearance	C	Disappearance	T	C	CC	C	CL-1	A	G	GGGGG	A	A	G	A	--	-

Continuous table 1

The variation position	62271	62276	62329	62342	62759	62784	62786	62788	62793	63273	63459
The variation position	62271	62276	62329	62342	62759	62784	62786	62788	62793	63273	63459	CL-1	A	G	C	A	G	C	C	C	C	T	T
17+	G	Disappearance	T	G	T	A	A	T	Disappearance	C	C	CL-1	A	G	C	A	G	C	C	C	C	T	T

Continuous table 1

The variation position	63654	63811	63812	64897	65493	65536	65537	65772	65808	65835	66445
The variation position	63654	63811	63812	64897	65493	65536	65537	65772	65808	65835	66445	CL-1	C	C	G	C	A	A	C	A	A	A	G
17+	T	G	C	A	C	G	T	C	G	C	A	CL-1	C	C	G	C	A	A	C	A	A	A	G

Continuous table 1

The variation position	73198	73761	80715	80827	80839	80857	80890	80942	81165	81216	81408
The variation position	73198	73761	80715	80827	80839	80857	80890	80942	81165	81216	81408	CL-1	G	G	G	T	A	T	G	C	T	A	A
17+	T	A	T	C	G	C	A	A	C	G	G	CL-1	G	G	G	T	A	T	G	C	T	A	A

Continuous table 1

The variation position	81417	81716	81834	81870	82065	82101	82215	82575	82694	82695	82766
The variation position	81417	81716	81834	81870	82065	82101	82215	82575	82694	82695	82766	CL-1	A	T	G	A	A	G	A	C	G	A	C
17+	G	C	T	G	G	A	G	T	A	C	T	CL-1	A	T	G	A	A	G	A	C	G	A	C

Continuous table 1

The variation position	83172	83175	83643	83856	83857	Between 84505 and 84506	84535	84611	84629	84685
The variation position	83172	83175	83643	83856	83857	Between 84505 and 84506	84535	84611	84629	84685	CL-1	C	T	C	T	G	----	T	C	G	T
17+	G	C	A	G	T	CTCCC	G	A	A	G	CL-1	C	T	C	T	G	----	T	C	G	T

Continuous table 1

The variation position	84920	85091	85340	85409	85892	85942	86018	86144	86256	Between 86334 and 86335	86469
The variation position	84920	85091	85340	85409	85892	85942	86018	86144	86256	Between 86334 and 86335	86469	CL-1	C	C	T	G	C	A	A	C	T	-	C
17+	T	A	C	A	T	C	G	T	C	C	A	CL-1	C	C	T	G	C	A	A	C	T	-	C

Continuous table 1

The variation position	86573	86663	86703	86817	86883	86884	86943	86997	87060	87353	87538
The variation position	86573	86663	86703	86817	86883	86884	86943	86997	87060	87353	87538	CL-1	G	G	A	T	A	G	C	C	C	T	T
17+	A	A	C	G	C	A	T	A	G	C	G	CL-1	G	G	A	T	A	G	C	C	C	T	T

Continuous table 1

The variation position	87548	87705	87732	87816	87975	88146	88881	89852	89923	89930	89980
The variation position	87548	87705	87732	87816	87975	88146	88881	89852	89923	89930	89980	CL-1	A	A	T	C	C	A	T	C	G	C	A
17+	C	G	C	T	T	G	C	T	T	T	T	CL-1	A	A	T	C	C	A	T	C	G	C	A

Continuous table 1

The variation position	89986	89997	90353	90392	90432	90455	90479	90626	90961	91107	91179
The variation position	89986	89997	90353	90392	90432	90455	90479	90626	90961	91107	91179	CL-1	G	C	T	C	C	T	C	T	G	A	T
17+	A	A	C	T	T	C	T	C	T	C	C	CL-1	G	C	T	C	C	T	C	T	G	A	T

Continuous table 1

The variation position	91244	91495	91498	92657	92964	93140	93141	93783	94133	94158	94253
The variation position	91244	91495	91498	92657	92964	93140	93141	93783	94133	94158	94253	CL-1	G	C	C	G	A	C	A	A	T	A	C
17+	A	G	T	A	G	G	C	C	C	G	T	CL-1	G	C	C	G	A	C	A	A	T	A	C

Continuous table 1

The variation position	94255	94257	94296	94336	94605	94636	94693	94709	94764	94771	95190
The variation position	94255	94257	94296	94336	94605	94636	94693	94709	94764	94771	95190	CL-1	T	C	A	T	A	G	G	T	C	C	T
17+	C	T	C	G	G	Disappearance	A	Disappearance	T	Disappearance	C	CL-1	T	C	A	T	A	G	G	T	C	C	T

Continuous table 1

The variation position	95196	95325	95365	95418	95427	95451	95458	95459	95473	95483	95513
The variation position	95196	95325	95365	95418	95427	95451	95458	95459	95473	95483	95513	CL-1	G	T	T	C	G	C	G	T	G	A	A
17+	A	C	C	T	T	T	A	C	C	G	T	CL-1	G	T	T	C	G	C	G	T	G	A	A

Continuous table 1

The variation position	95514	95646	96020	Between 96178 and 96179	96190	96261	96264	96444	96501	96589
The variation position	95514	95646	96020	Between 96178 and 96179	96190	96261	96264	96444	96501	96589	CL-1	C	C	C	--	C	T	G	A	A	C
17+	T	T	T	GC	T	Disappearance	Disappearance	G	G	Disappearance	CL-1	C	C	C	--	C	T	G	A	A	C

Continuous table 1

The variation position	96919	97191	97381	97471-97473	97690	97865	97893	97903	Between 97910 and 97911
The variation position	96919	97191	97381	97471-97473	97690	97865	97893	97903	Between 97910 and 97911	CL-1	G	A	C	GCA	G	G	G	T	-
17+	A	C	T	CAT	A	A	T	G	C	CL-1	G	A	C	GCA	G	G	G	T	-

Continuous table 1

The variation position	97984	98639	99122	Between 99218 and 99219	99347	99427	99460	99511	99522-99524
The variation position	97984	98639	99122	Between 99218 and 99219	99347	99427	99460	99511	99522-99524	CL-1	C	C	G	---	A	C	C	G	GGC
17+	A	T	C	TCG	C	T	T	T	Disappearance	CL-1	C	C	G	---	A	C	C	G	GGC

Continuous table 1

The variation position	99550	99640	99642	99646	99735	99941	99992	100217	100600	101222
The variation position	99550	99640	99642	99646	99735	99941	99992	100217	100600	101222	CL-1	T	G	A	C	C	A	C	T	C	A
17+	G	T	G	T	T	G	G	C	A	G	CL-1	T	G	A	C	C	A	C	T	C	A

Continuous table 1

The variation position	101238	102054	102177	102195	102210	102290	102646	102811	103220
The variation position	101238	102054	102177	102195	102210	102290	102646	102811	103220	CL-1	C	A	A	C	T	T	T	C	A
17+	A	G	C	A	C	C	G	T	G	CL-1	C	A	A	C	T	T	T	C	A

Continuous table 1

The variation position	Between 103273 and 103274	103532	103572	103732	104418	104683	104724	104776	104820
The variation position	Between 103273 and 103274	103532	103572	103732	104418	104683	104724	104776	104820	CL-1	-	T	G	A	G	C	C	G	A
17+	A	Disappearance	T	C	A	A	T	T	G	CL-1	-	T	G	A	G	C	C	G	A

Continuous table 1

The variation position	104832	104938	104989	105057	105194	105203	105293	105411	105502	106256
The variation position	104832	104938	104989	105057	105194	105203	105293	105411	105502	106256	CL-1	A	C	A	C	A	T	C	A	T	G
17+	G	T	G	T	Disappearance	C	A	C	Disappearance	T	CL-1	A	C	A	C	A	T	C	A	T	G

Continuous table 1

The variation position	106263	106859	106927	107025	107181	107253	107278	107286	107510	107240
The variation position	106263	106859	106927	107025	107181	107253	107278	107286	107510	107240	CL-1	C	C	T	C	C	T	T	A	C	A
17+	A	T	C	G	T	C	G	C	A	C	CL-1	C	C	T	C	C	T	T	A	C	A

Continuous table 1

The variation position	107554	107988	108126	108146	108152	108187	108212	108264	108499	108509
The variation position	107554	107988	108126	108146	108152	108187	108212	108264	108499	108509	CL-1	C	T	C	T	G	T	C	G	G	G
17+	A	C	T	C	T	G	A	A	T	C	CL-1	C	T	C	T	G	T	C	G	G	G

Continuous table 1

The variation position	108587	108754	108909	109695	109705	109908	109927	110128	110524	110563
The variation position	108587	108754	108909	109695	109705	109908	109927	110128	110524	110563	CL-1	G	G	A	C	G	A	C	T	T	C
17+	A	A	G	T	T	G	T	C	C	T	CL-1	G	G	A	C	G	A	C	T	T	C

Continuous table 1

The variation position	110599	110604	110606	110611	110842-110844	111237	112978	113225	113239
The variation position	110599	110604	110606	110611	110842-110844	111237	112978	113225	113239	CL-1	A	A	A	G	AAA	G	A	A	G
17+	G	G	C	A	GGG	Disappearance	C	G	T	CL-1	A	A	A	G	AAA	G	A	A	G

Continuous table 1

The variation position	Between 113276 and 113277	113328	113443	113474	113856	114214	114268	114282	114284
The variation position	Between 113276 and 113277	113328	113443	113474	113856	114214	114268	114282	114284	CL-1	-	T	T	T	C	C	G	A	C
17+	G	C	G	C	A	T	T	C	G	CL-1	-	T	T	T	C	C	G	A	C

Continuous table 1

The variation position	114288	114289	114337	114366	114896	115493	118977	132759	132963	133229
The variation position	114288	114289	114337	114366	114896	115493	118977	132759	132963	133229	CL-1	G	C	A	C	G	A	G	G	A	A
17+	C	G	C	A	A	G	T	T	G	C	CL-1	G	C	A	C	G	A	G	G	A	A

Continuous table 1

The variation position	133271	133312	133387	Between 133410 and 133411	133501	133711	133739	133943	133953
The variation position	133271	133312	133387	Between 133410 and 133411	133501	133711	133739	133943	133953	CL-1	A	T	C	-	G	A	G	T	A
17+	Disappearance	Disappearance	T	C	A	G	A	C	C	CL-1	A	T	C	-	G	A	G	T	A

Continuous table 1

The variation position	Between 134041 and 134042	134072	134077	134411	134418	135132	135155	135232	135258
The variation position	Between 134041 and 134042	134072	134077	134411	134418	135132	135155	135232	135258	CL-1	-	C	C	T	T	T	A	A	T
17+	G	A	-	G	C	G	G	G	Disappearance	CL-1	-	C	C	T	T	T	A	A	T

Continuous table 1

The variation position	Between 135279 and 135280	135307	135383	135392	135868	135958	136066	136321	136693
The variation position	Between 135279 and 135280	135307	135383	135392	135868	135958	136066	136321	136693	CL-1	-	A	A	C	C	C	G	C	G
17+	G	G	C	A	T	G	A	G	T	CL-1	-	A	A	C	C	C	G	C	G

Continuous table 1

The variation position	136705	136736	136769	136869	136989	137018-137020	137031	137088	137095
The variation position	136705	136736	136769	136869	136989	137018-137020	137031	137088	137095	CL-1	C	C	C	G	C	AGG	T	T	G
17+	Disappearance	T	A	A	T	Disappearance	C	C	T	CL-1	C	C	C	G	C	AGG	T	T	G

Continuous table 1

The variation position	137106	137108	137112	137114	137149	137156	137193	137213	137219	137235
The variation position	137106	137108	137112	137114	137149	137156	137193	137213	137219	137235	CL-1	C	G	T	A	C	G	A	G	A	A
17+	T	A	C	T	T	A	C	T	G	C	CL-1	C	G	T	A	C	G	A	G	A	A

Continuous table 1

The variation position	137246	137251	137262	137384	137467	Between 137495 and 137496	137498	137512	137516
The variation position	137246	137251	137262	137384	137467	Between 137495 and 137496	137498	137512	137516	CL-1	A	C	G	T	C	-	T	C	T
17+	G	A	A	G	T	G	G	T	G	CL-1	A	C	G	T	C	-	T	C	T

Continuous table 1

The variation position	137539	137542	137558	137586	137605	137610	137664	137763	137825	137952
The variation position	137539	137542	137558	137586	137605	137610	137664	137763	137825	137952	CL-1	C	G	G	G	T	G	G	T	T	G
17+	T	T	A	A	C	Disappearance	A	C	G	A	CL-1	C	G	G	G	T	G	G	T	T	G

Continuous table 1

The variation position	138512	138528	139261	139309	139387	139402	139533	139539	139545-139546
The variation position	138512	138528	139261	139309	139387	139402	139533	139539	139545-139546	CL-1	G	C	T	A	G	C	A	G	GG
17+	A	T	G	G	A	A	G	A	AA	CL-1	G	C	T	A	G	C	A	G	GG

Continuous table 1

The variation position	Between 139634 and 139635	139717	139783	Between 139783 and 139784	140021	140072	140175	140207	140214
The variation position	Between 139634 and 139635	139717	139783	Between 139783 and 139784	140021	140072	140175	140207	140214	CL-1	-	C	C	-	T	G	G	G	G
17+	C	T	T	T	G	A	C	A	A	CL-1	-	C	C	-	T	G	G	G	G

Continuous table 1

The variation position	140220	140259	140265	140308	140338	140420	140443	140464	140465	140467
The variation position	140220	140259	140265	140308	140338	140420	140443	140464	140465	140467	CL-1	T	G	A	A	C	C	G	G	C	T
17+	G	T	G	G	T	A	A	A	T	C	CL-1	T	G	A	A	C	C	G	G	C	T

Continuous table 1

The variation position	140470	140472	140529	140544	140652	140724	140748	140904	140924	140947
The variation position	140470	140472	140529	140544	140652	140724	140748	140904	140924	140947	CL-1	A	C	C	C	T	C	A	G	T	T
17+	G	T	T	T	C	T	G	T	C	C	CL-1	A	C	C	C	T	C	A	G	T	T

Continuous table 1

The variation position	140978	140997	141021	141079-141081	141087-141090	141193	141194	141600
The variation position	140978	140997	141021	141079-141081	141087-141090	141193	141194	141600	CL-1	G	G	A	TCC	CCCC	G	A	C
17+	T	A	C	Disappearance	Disappearance	C	C	G	CL-1	G	G	A	TCC	CCCC	G	A	C

Continuous table 1

The variation position	142037	142147	142159	142564	143054	143189	143298	143452	143510	143582
The variation position	142037	142147	142159	142564	143054	143189	143298	143452	143510	143582	CL-1	G	A	T	G	A	T	T	T	G	C
17+	T	C	C	A	G	C	A	Disappearance	T	T	CL-1	G	A	T	G	A	T	T	T	G	C

Continuous table 1

The variation position	143653-143654	143929	143999	144005	144009	144011-144032
The variation position	143653-143654	143929	143999	144005	144009	144011-144032	CL-1	CT	C	C	T	C	TGGGAGATGGGCCCACCGTCCC
17+	Disappearance	T	Disappearance	A	G	Disappearance	CL-1	CT	C	C	T	C	TGGGAGATGGGCCCACCGTCCC

Continuous table 1

The variation position	144047	144085	144184	144276	144278	After 152311
The variation position	144047	144085	144184	144276	144278	After 152311	CL-1	G	G	G	A	A	---
17+	Disappearance	A	A	G	T	GGC	CL-1	G	G	G	A	A	---

Hsv of the present invention not only strengthens aspect the ability that infects anthous's host cell, and when being used for the multiple tumour cells of other ethnic groups, stronger to the dissolving power of tumour cell.

The present invention also provides a kind of recombinant virus, and described recombinant virus comprises that as the virus of carrier and goal gene wherein, described virus as carrier is selected from hsv of the present invention.Hsv of the present invention not only can act on the cracking killing tumor cell separately, but also can be used as carrier by transmitting goal gene and/or cracking killing tumor cell.Hsv can be inserted the external source fragment (" gene therapy progress and the application in cardiovascular disorder thereof " of 40-50 kilobase (kb), people such as Hua Junyi, the angiocardiology progress, 2000, the 21st volume, the 4th phase, 211-214 page or leaf), therefore can carry the goal gene of 0.1-50kb as the hsv of the present invention of carrier, preferably carry the goal gene of 10-40kb.Described goal gene can be the gene of anti-tumor activity, one or more in the gene of the gene of the prodrug activator of for example encoding, the gene of codes for tumor arrestin, Codocyte antiapoptotic factors (pro-apoptotic factors) precursor and the proteic gene of coding immunostimulation.Described immunostimulation albumen can be for self having Cytotoxic albumen, can stimulating/strengthen the albumen of anti-tumor immune response.Certain virus of the present invention also can be used for one or more goal gene are passed in the cell that needs its expression, this cell can be a for example neurocyte of non-tumor cell, described one or more goal gene can be replied or the gene of the polypeptide of relieve chronic pain pain stimulation for coding changes, described polypeptide for example can completely cut off for example albumen, Substance P and other neuropeptide of nerve growth factor, other regulating pain neurotrophic factor, neurotrophic factor sample molecule.Described one or more goal gene also can stimulate injured nerve regrowth in the degenerative disease or prevent the neural further gene of the polypeptide of sex change for coding.

Described goal gene can also comprise regulating and controlling sequence, for example the promotor of described one or more destination gene expressions, terminator and enhanser.Described goal gene can comprise that also marker gene (for example, the gene of coding beta-galactosidase, green fluorescent protein or other fluorescin) or its product regulate the gene of other genetic expression.

Described goal gene can also be mRNA, tRNA or rRNA except that being the DNA, can also comprise the associated retroviral regulating and controlling sequence relevant with transcription sequence, for example transcription termination signal, polyadenylation site and downstream enhancer element usually.

The present invention also provides a kind of infection virulent host cell, and wherein, described virus is hsv of the present invention and/or recombinant virus of the present invention.Those skilled in the art are known, and hsv need be finished in host cell its life history, and therefore going down to posterity of virus bred and activated and need carry out in host cell.And the host cell of hsv of the present invention and/or recombinant virus of the present invention is arranged in Infection in Vitro, also can be used as gene therapy medicine and import in the body.Described host cell can be the various host cells that can cultivate virus of the present invention and/or recombinant virus of the present invention, for example African green monkey kidney cell (Vero cell), former generation rabbit kidney cell, chick-embryo cell, amnion cell, human cervical carcinoma cell (Hela cell) and human embryonic lung diploid fibroblast (WI-38 cell).

The present invention also provides a kind of pharmaceutical composition, and wherein, described pharmaceutical composition comprises hsv of the present invention and/or recombinant virus of the present invention.Hsv of the present invention can be used the cracking killing tumor cell separately; Can also be as the preparing carriers recombinant virus, described recombinant virus uses separately by transmitting goal gene and/or cracking killing tumor cell.Hsv of the present invention and recombinant virus of the present invention also can use simultaneously.Hsv of the present invention, recombinant virus of the present invention and their mixture all can also comprise medicine acceptable carrier or vehicle, use with the form of pharmaceutical composition.

Carrier that described medicine is accepted or vehicle refer to nontoxic solid-state, semi-solid state or liquid weighting agent, thinner, coating material or other pharmaceutical adjuncts, for example, include but not limited to salt solution, buffering saline solution, glucose, water, glycerine, ethanol and composition thereof.Described pharmaceutical composition is suitable in parenteral, hypogloeeis, the brain pond, in the intravaginal, internal rectum, cheek, in the tumour or the epidermis administration.

Parenteral admin comprises that intravenously, intramuscular, intraperitoneal, breastbone are interior, subcutaneous, intra-articular injection and infusion.The pharmaceutical composition that is suitable for parenteral admin comprises aseptic aqueous solution or non-aqueous solution, dispersion liquid, suspension or emulsion, and is used for before facing use in sterile injectable solution or dispersion liquid powder formulated.Suitable water-based or non-aqueous carrier, thinner, solvent or vehicle comprise water, ethanol, glycerine, interior glycol, polyoxyethylene glycol, carboxymethyl cellulose, vegetables oil and injectable organic ester such as ethyl oleate.These compositions can also contain sanitas, wetting agent, emulsifying agent, protective material and dispersion agent adjuvant, for example inositol, sorbyl alcohol and sucrose.Preferred adding osmotic pressure regulator such as carbohydrate, sodium-chlor, Repone K.

The epidermis administration is included on skin, the mucous membrane and in lung and the surperficial administration of eye.Such pharmaceutical composition comprises pulvis, ointment, drops, transdermal patch, Iontophoretic device and inhalation etc.The composition of rectum or vagina administration is preferably suppository, it can be by being mixed with carrier of the present invention and suitable non-irritating excipient or carrier such as theobroma oil, polyoxyethylene glycol or suppository wax, described vehicle or carrier are solid-state in room temperature, be liquid under body temperature, therefore fusing and discharge active compound in rectum or vaginal canal.

Preferred described pharmaceutical composition is an injection liquid, and described injection liquid comprises pharmaceutically acceptable carrier and virus of the present invention and/or recombinant virus, contains 10 in every milliliter of described injection liquid ²-10 ¹⁰Plaque forms number.Described pharmaceutically acceptable carrier is the phosphoric acid buffer of pH value for 4.0-9.0.Described injection liquid also contains protective material and/or osmotic pressure regulator; With described injection liquid is benchmark, and described protectant content is 0.01-30 weight %, and described protective material is selected from one or more in inositol, sorbyl alcohol and the sucrose; It is 200-700 m osmole/kilogram that the content of described osmotic pressure regulator makes the osmotic pressure of described injection liquid, and described osmotic pressure regulator is sodium-chlor and/or Repone K.When using described injection liquid to carry out, the virus quantity scope that gives is 10 ³-10 ¹⁰Plaque forms number (pfu), preferred 10 ⁵-10 ⁸Pfu, more preferably 10 ⁶-10 ⁸Pfu.When injection medicinal compositions of the present invention, the injection consumption can be this area dosage commonly used, 500 microlitres, general 1-200 microlitre, preferred 1-10 microlitre at the most; But, also can use 10 milliliters dosage at the most according to described tumour and inoculation position.Because those skilled in the art can easily determine best route of administration and dosage, so described route of administration and dosage are for reference only.Described dosage can be according to various parameters, especially determine according to the severity and the route of administration of patient's age to be treated, body weight and illness, disease or illness.In addition, be to be injected directly in the tumour for the preferred route of administration of cancer patients.Medicine composition injection of the present invention also can be administered systemically, and also medicine composition injection of the present invention can be expelled to the intravascular administration into tumor feeding.Most preferred route of administration depends on the position and the size of described tumour.

Further specify the present invention below in conjunction with embodiment, the used reagent of the present invention, substratum are the commercial goods unless stated otherwise.

Embodiment 1

The separation of present embodiment explanation hsv of the present invention.

(1) Bing Du collection

The Chinese male of growing up, 37 years old, Han nationality, the feverblister medical history of showing effect repeatedly repeatedly, no other diseases is the clinical manifestation of typical I herpes simplex virus type recurrent infection---and bicker bleb sample bubble occurs on every side and takes a sample two days later.

Get 15 milliliters aseptic centrifuge tube, fill 3 milliliters of aseptic phosphate buffered saline buffers that contain 100 mcg/ml kantlex (PBS), contain the NaCl of 9 grams per liters, the KH of 0.21 grams per liter in the described damping fluid ₂PO ₄And the Na of 0.97 grams per liter ₂HPO ₄.12H ₂O; Flow out with aseptic cotton carrier extruding hydroa herpetiforme to faint yellow secretory product, dip in cotton swab then and get this secretory product, an end that the gained cotton swab is had secretory product is rinsed in above-mentioned PBS damping fluid and was washed for 10 seconds.Take out cotton swab, with the sterilization micro sample adding appliance that the damping fluid pressure-vaccum that gained contains secretory product is even.

(2) Bing Du inoculation, cultivation and purifying

With the DMEM substratum that contains 10% foetal calf serum, on six porocyte culture plates with 5 * 10 ⁵The density of individual cells/well inoculation African green monkey kidney cell (Vero cell), under 37 ℃ at 5%CO ₂Cultivated 24 hours in the incubator, extremely every hole Vero cell covers with 80%.Under gnotobasis, (1) that adds 200 microlitres in every hole of above-mentioned six porocyte culture plates obtains containing the PBS damping fluid (sample and finished inoculation in back 1 hour) of secretory product then.Continuation is at 5%CO ₂Cultivate down after 24 hours for 37 ℃ in the incubator, under gnotobasis, the magnification with 100 times under opticmicroscope is observed the Vero cell, finds viral plaque; Draw one viral plaque 10 microlitres with the sterilization micro sample adding appliance, the liquid that 10 microlitres are drawn is sneaked in the DMEM substratum of 100 microlitres, and with micro sample adding appliance that the DMEM substratum pressure-vaccum that gained contains virus is even.

Under gnotobasis, in covering with every hole of six porocyte culture plates of 80%Vero cell, another one adds the above-mentioned DMEM substratum that obtains containing purified virus, infect this Vero cell.At 5%CO ₂Cultivate the hsv of the present invention that obtains purifying down for 37 ℃ in the incubator.

(3) Bing Du biological property analysis

As shown in Figure 1, the virus of observing (2) purifying under 52000 times of Electronic Speculum has typical hsv form: capsid is the icosahedron symmetry, and diameter is about 100 nanometers.The outer tunicle by gage distortion of capsid covers.The virus outermost layer is typical double-layer of lipoid cyst membrane, and the viral diameter that comprises cyst membrane is 150 to 200 nanometers.

The contriver entrusts the big genome company of Hangzhou China also the virus of (2) purifying to be carried out genome sequencing in addition, sequencing result is shown in above-mentioned table 1, and the sequence homology that is numbered the I herpes simplex virus type 17+ of ID:NC_001806 among gained virus and the NCBI is 99.5%.The present inventor analyzes the whole genome sequence that obtains, obtain ID:NO.2 as SEQ, SEQ ID:NO.4, SEQ ID:NO.6, SEQ ID:NO.8, and the gene nucleotide series of the coding envelope protein shown in the SEQ ID:NO.10, and obtain as SEQ ID:NO.1 (viral glycoprotein B) by described nucleotide sequence, SEQID:NO.3 (viral glycoprotein C), SEQ ID:NO.5 (viral glycoprotein D), SEQ ID:NO.7 (viral glycoprotein G), and the aminoacid sequence of the envelope protein shown in the SEQ ID:NO.9 (viral glycoprotein M).By comparing, find that there is variation in the gene nucleotide series of the purified virus coding envelope protein that (2) obtain with the corresponding gene of ID:NC_001806.Obtained the nucleotide sequence of the immediate early gene of the coding infected cell polypeptides shown in SEQID:NO.12 and SEQ ID:NO.14 in addition, and obtained as SEQ ID:NO.11 (infected cell polypeptides α 22) and SEQ ID:NO.13 (infected cell polypeptides α 27) by described nucleotide sequence.

Embodiment 2

Present embodiment illustrates the dissolving power of hsv of the present invention to the KB cell (CNE-1) of anthous's pilosity.

(1) mensuration of hsv titre

With the DMEM substratum that contains 10% foetal calf serum, on six porocyte culture plates with 5 * 10 ⁵The density of individual cells/well inoculation African green monkey kidney cell (Vero cell) (U.S. ATCC), under 37 ℃ at 5%CO ₂Cultivated in the incubator 24 hours, and covered with 80% standby to every hole Vero cell.

Under the aseptic condition, collect the supernatant nutrient solution that embodiment 1 cultivates the Vero cell culture that hsv of the present invention is arranged, with the DMEM substratum that does not contain serum respectively gradient dilution become 1 * 10 ²Times liquid, 1 * 10 ³Times liquid, 1 * 10 ⁴Times liquid, 1 * 10 ⁵Times liquid, 1 * 10 ⁶Times liquid and 1 * 10 ⁷Times liquid, every kind of each 100 microlitre of diluent.

Under the aseptic condition, discard the DMEM substratum that contains 10% foetal calf serum on the above-mentioned six porocyte culture plates in the cultured Vero cell culture, every then hole adds 1 milliliter of the DMEM substratum that does not contain serum of above-mentioned dilution usefulness, then the viral dilution liquid with above-mentioned six kinds of concentration that make adds in 6 holes every hole 100 microlitres respectively.Virulent Vero cell six well culture plates of transfection place 5%CO ₂Cultivated 1 hour down for 37 ℃ in the incubator, discard the substratum in the hole under the aseptic condition, add 2 milliliters of carboxymethyl cellulose substratum in every hole (with the carboxymethyl cellulose powder with after PBS solution mixes according to weightmeasurement ratio at 2: 125, preserved 12 hours at 4 ℃ of refrigerators, the back fully 121.3 ℃ of sterilizations of dissolving 30 minutes, under the aseptic condition, with by volume 1: 2 mixed getting of carboxymethyl cellulose PBS solution and the DMEM that contains 10%FBS of the aseptic 1.6 bulking value % of gained).Place 5%CO ₂Cultivate after 48 hours down for 37 ℃ in the incubator, discard nutrient solution, with cell rinse 2 times, discard PBS solution, add 2 milliliters of PBS solution that contain the glutaraldehyde (U.S. Sigma) of 0.1 volume %, placed 10 minutes down for 25 ℃ with PBS solution.Once more cell is used PBS solution rinse twice, discarded PBS solution, add the crystal violet solution (preparing as solvent) of 1 milliliter of 0.1 bulking value %, placed 10 minutes down for 25 ℃ with 20 volume % ethanol.With pure water rinse cell twice, dry the water at the six orifice plate back sides with thieving paper, six orifice plates are placed under 100 times of opticmicroscopes observe, the counting plaque.The plaque number that calculates every milliliter of virus stock solution used formation is 5 * 10 ⁷, promptly the titre of virus stock solution used is 5 * 10 ⁷Virus plaque forms units per ml (pfu/ml).

(2) cultivation of KB cell

Under 37 ℃ at 5%CO ₂In the incubator with 6 milliliters of DMEM substratum that contain 10% foetal calf serum, cultivation is seeded in the CNE-1 cell (available from Shanghai cell research institute of the Chinese Academy of Sciences) 48 hours in 25 square centimeters the culturing bottle, is in logarithmic phase with the inverted microscope CNE-1 cell that amplifies 100 times.Under the aseptic condition, add 1 milliliter in 0.25% trypsinase, at 37 ℃ of described CNE-1 cells that are in logarithmic phase of digestion 5 minutes down.With inverted microscope observe the CNE-1 cell in culture medium solution free after, add fresh culture and be diluted to 1 * 10 ⁴The cell suspension of individual/milliliter, microsyringe piping and druming evenly back adds in 96 orifice plates, and every hole adds 100 microlitre cell suspensions, at 37 ℃, 5%CO ₂Continue in the incubator to cultivate 24 hours.

(3) mensuration of Bing Du transfection and KB cell survival rate

With the hsv stoste of the step (1) of known titre, (multiplicity of infection MOI) is the CNE-1 cell of the above-mentioned cultivation of 0.1 transfection with infection multiplicity according to the method for step (1).Wherein control wells adds and the isopyknic 1% calf serum DMEM substratum that contains of virus stock solution used.Control wells CNE-1 cell and transfected CNE-1 cell are all at 37 ℃ of following 5%CO ₂Cultivate in the incubator, and (concrete grammar is documented in " measuring the foundation of the MTT method of cell survival and propagation ", people such as Zheng Yongtang with mtt assay 24 hours, 48 hours and 72 hours after transfection respectively, Journal of Immunology, 1992, the 8th volume, the 4th phase, the 266-269 page or leaf) the mensuration cell survival rate.With model is that the enzyme linked immunological instrument (manufacturing of East China Electronics Co., Ltd pipe factory) of DG-3022 is measured the OD value under 570 nanometers, and cell survival rate is calculated by following formula:

Cell survival rate=(transfection hole absorbance value/control wells absorbance value) * 100%.

The test result of cell survival rate sees Table 2.

Comparative Examples 1

The hsv of this Comparative Examples explanation prior art is to the dissolving power of the KB cell (CNE-1) of anthous's pilosity.

Method according to embodiment 2, KB cell (CNE-1) (available from Shanghai cell research institute of the Chinese Academy of Sciences) with the herpes simplex virus 1 7+ transfection anthous pilosity of identical virus titer, under same condition, cultivate the CNE-1 cell of transfection, and measure the survival rate of CNE-1 cell according to the method for embodiment 2, measurement result sees Table 2.

Table 2

Virus	Comparative Examples 1	Embodiment 2
Virus	Comparative Examples 1	Embodiment 2	24 hours survival rates (%)	69	45
48 hours survival rates (%)	21	6	24 hours survival rates (%)	69	45
48 hours survival rates (%)	21	6	72 hours survival rates (%)	7	0

As can be seen from Table 2, the survival rate of the CNE-1 cell of transfection hsv of the present invention is significantly less than the survival rate of the CNE-1 cell of transfection prior art herpes simplex virus 1 7+, and the CNE-1 cell is the tumour cell of the multiple human nasopharyngeal carcinoma of the typical anthous, virus of the present invention is described, for the herpes simplex virus 1 7+ of prior art, specificity is stronger to the dissolving power of xanthous tumour cell.

Embodiment 3

Present embodiment illustrates the dissolving power of hsv of the present invention to general tumour cell.

Method according to embodiment 2, descend transfection human breast cancer cell MCF-7, human colon cancer cell HT-29 and human lung adenocarcinoma cell A549 (available from No.2 Research Institute of Military Medical Science Institute) in two kinds of different infection multiplicities (MOI=0.1 and MOI=1) respectively with hsv of the present invention, and the survival number of different time points tumour cell after the test transfection, the result is as shown in table 3.

Comparative Examples 2

The herpes simplex virus 1 7+ of this Comparative Examples explanation prior art is to the dissolving power of general tumour cell.

Method according to embodiment 2, descend transfection MCF-7 MCF-7, CCL188 HT-29 and human lung adenocarcinoma cell line A549 in two kinds of different infection multiplicities (MOI=0.1 and MOI=1) respectively with herpes simplex virus 1 7+, and the survival number of different time points tumour cell after the test transfection, the result is as shown in table 3.

Table 3

As can be seen from Table 3, no matter hanging down under the infection multiplicity (MOI=0.1) or the situation of high infection multiplicity (MOI=1), the survival rate of three kinds of tumor cell lines of 24 hours of transfection hsv of the present invention all is significantly less than the survival rate of three kinds of tumor cell lines of transfection prior art herpes simplex virus 1 7+, illustrates that virus of the present invention also is better than the herpes simplex virus 1 7+ of prior art to the dissolving power of general tumour cell.In addition, Fig. 2 has showed that normal human mammary cancerous cell line MCF-7 cultivates 96 hours growing state optical microscope photograph (amplifying 400 times), and cellularstructure is complete, and intensive growth is arranged; Fig. 3 has showed that cultivation MCF-7 cell is after 48 hours, by the ratio (MOI) of virus activity particle/cell count is to cultivate 48 hours growing state optical microscope photograph (amplifying 400 times) again after the 0.1 transfection virus of the present invention, most cellularstructures are destroyed, and cell number significantly reduces.From two photo contrasts as can be seen, hsv of the present invention all has very strong dissolving power to general tumour cell.

Embodiment 4

The pharmaceutical composition that the present embodiment explanation contains virus of the present invention has oncolysis.

(1) preparation of injection liquid

To prepare one group of phosphate buffered saline buffer respectively according to the dosage shown in the table 4, sterilize 60 minutes down for 121 ℃.Under aseptic condition, hsv stoste with the known titre of step (1) of 0.45 micron filtering with microporous membrane embodiment 2, remove cell debris, collect filtrate to aseptic centrifuge tube, 8000 rev/mins centrifugal 1 hour, supernatant discarded is according to the virus titer shown in the table 4, gained virus precipitation is dispersed in the phosphoric acid buffer behind the above-mentioned high-temperature sterilization, promptly gets injection liquid of the present invention.

Table 4

Medicine	Prescription	1	Prescription 2	Prescription 3	Prescription 4	Prescription 5	Prescription 6	Prescription 7
Medicine	Prescription	1	Prescription 2	Prescription 3	Prescription 4	Prescription 5	Prescription 6	Prescription 7	Phosphoric acid buffer (weight %)	99	95	90	85	80	75	70
Inositol (weight %)	1	0	0	10	15	0	10		Phosphoric acid buffer (weight %)	99	95	90	85	80	75	70
Inositol (weight %)	1	0	0	10	15	0	10	Sorbyl alcohol (weight %)	0	5	0	5	0	15	15
Sucrose (weight %)	0	0	10	0	5	10	5	Sorbyl alcohol (weight %)	0	5	0	5	0	15	15
Sucrose (weight %)	0	0	10	0	5	10	5	Osmotic pressure regulator and concentration thereof (mol)	Sodium-chlor 0.9	Repone K 0.5	Sodium-chlor 0.9	Repone K 0.5	Sodium-chlor 0.9	Repone K 0.5	Sodium-chlor 0.9
Osmotic pressure (m osmole/kilogram)	200	300	400	450	500	600	700	Osmotic pressure regulator and concentration thereof (mol)	Sodium-chlor 0.9	Repone K 0.5	Sodium-chlor 0.9	Repone K 0.5	Sodium-chlor 0.9	Repone K 0.5	Sodium-chlor 0.9
Osmotic pressure (m osmole/kilogram)	200	300	400	450	500	600	700	Virus titer (pfu/ml)	10 ⁴	10 ⁵	10 ⁶	10 ⁷	10 ⁸	10 ⁹	10 ¹⁰

In the present embodiment, the negative control medicine is not for containing the ditalimfos acid buffer of virus; The positive control medicine is Cyclophosphamide for injection (CTX), lot number: 050202, and 200 milligrams/of specifications, Hualian Pharmaceutical Co., Ltd., Shanghai produces.

(2) cell levels pharmacodynamic experiment

According to the method for embodiment 2, with the KB cell (CNE-1) of the virus injection liquid inductance yellow colouration ethnic group pilosity of infection multiplicity MOI=1.The result is as shown in table 5.

Table 5

Virus injection liquid	Negative control	Positive control	Prescription	1	Prescription 2	Prescription 3	Prescription 4	Prescription 5	Prescription 6	Prescription 7
Virus injection liquid	Negative control	Positive control	Prescription	1	Prescription 2	Prescription 3	Prescription 4	Prescription 5	Prescription 6	Prescription 7	24 hours survival rates (%)	100	0	70	50	70	80	80	70	70
48 hours survival rates (%)	100	0	15	10	15	20	15	10	10		24 hours survival rates (%)	100	0	70	50	70	80	80	70	70
48 hours survival rates (%)	100	0	15	10	15	20	15	10	10	72 hours survival rates (%)	100	0	2	0	0	0	5	0	0

As can be seen from Table 5, injection liquid of the present invention has significant lethal effect to the CNE-1 cell of anthous's pilosity, positive control drug can be in 24 hours the kill tumor cell, but positive control drug is also very big to Normocellular toxicity.

(3) the restraining effect pharmacodynamic experiment of human colon adenocarcinoma HT-29 transplanted tumor in nude mice growth

Human colon adenocarcinoma HT-29 transplanted tumor in nude mice is available from institute of Materia Medica,Chinese Academy of Medical Sciences, the Balb/c nude mice, and 4-6 age in week, the 16-20 gram, female, available from Chinese Academy of Medical Sciences's Experimental Animal Center.

When the mice with tumor Subcutaneous tumor that goes down to posterity grew to the about 2-3 of diameter centimetre, aseptic condition took out the knurl piece down, according to the ratio of every gram tumor tissues 3-4 ml physiological saline, makes tumour cell homogenate, gives 0.2 milliliter of every mouse right fore armpit subcutaneous vaccination; Perhaps the knurl piece is cut into 1 cubic centimetre tubercle piece, it is subcutaneous to be inoculated into nude mice right side armpit with trochar.

The tumor growth for the treatment of the inoculation of each treated animal is when animal body surface can be observed the lump of 0.6 centimetre of about 0.6 cm x, divide 9 groups (the present invention fill a prescription 7 groups, positive controls, negative control group) at random, 7 every group begin with the virus injection liquid of the present invention of step (1) and positive control medicine, negative control drug administration.In the 1st day, the 4th day and the 7th day intratumor injection, volumetric injection was 100 microlitres.The dosage of virus of the present invention is 2.5 * 10 ⁷Pfu/kg, positive control drug are endoxan, and the dosage of positive control drug is 100mg/kg, and the negative control medicine is and the isopyknic phosphoric acid buffer of virus injection liquid of the present invention.After the last administration, weigh weekly, measure the observed gross tumor volume of animal body surface energy, continue to observe about 2 weeks, the gross tumor volume change curve is seen Fig. 5, and animal is put to death in the neck dislocation then, peels off taking-up knurl piece and weighs, can draw the average knurl piece weight of tumor control rate=100% * virus injection liquid group of the present invention or the positive control drug group/average knurl piece of negative control medicine weight, the results are shown in Table 6.The negative contrast medicine of Fig. 4 (first row), positive control drug (second row), the knurl piece photo that the present invention's virus prescription 2 injection liquids (the third line) are taken out, the dead mouse of blank position.

Table 6

Virus injection liquid	Negative control	Positive control	Prescription	1	Prescription 2	Prescription 3	Prescription 4	Prescription 5	Prescription 6	Prescription 7
Virus injection liquid	Negative control	Positive control	Prescription	1	Prescription 2	Prescription 3	Prescription 4	Prescription 5	Prescription 6	Prescription 7	Average knurl piece weight (gram)	0.74	0.40	0.59	0.51	0.67	0.62	0.58	0.61	0.61
Tumor control rate (%)	-	46	21	32	10	17	22	18	18		Average knurl piece weight (gram)	0.74	0.40	0.59	0.51	0.67	0.62	0.58	0.61	0.61
Tumor control rate (%)	-	46	21	32	10	17	22	18	18	Mortality ratio (%)	0	30	0	0	0	0	0	0	0

From table 6 and Fig. 4, Fig. 5 as can be seen, growth has the obvious suppression effect to human colon adenocarcinoma HT-29 transplanted tumor in nude mice in the present invention, of the present invention group inhibiting rate scope is 28.0%-77.0%, though the inhibiting rate of positive control drug to the restraining effect of tumour also clearly, but the mortality ratio of positive control drug is very high, thereby virus injection liquid of the present invention is safer.

SEQUENCE LISTING

＜110〉Beijing OrienGene Biotechnology Ltd.

＜120〉hsv and recombinant virus and host cell and pharmaceutical composition thereof

<130>I6627OYH

<160>14

<170>PatentIn version 3.4

<210>1

<211>904

<212>PRT

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>1

Met Arg Gln Gly Ala Pro Ala Arg Gly Arg Arg Trp Phe Val Val Trp

1 5 10 15

Ala Leu Leu Gly Leu Thr Leu Gly Val Leu Val Ala Ser Ala Ala Pro

20 25 30

Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala Asn

35 40 45

Gly Gly Pro Ala Thr Pro Ala Pro Pro Ala Pro Gly Pro Ala Pro Thr

50 55 60

Gly Asp Pro Lys Pro Arg Lys Asn Lys Lys Pro Lys Pro Pro Thr Pro

65 70 75 80

Pro Arg Pro Ala Gly Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr

85 90 95

Leu Arg Glu His Leu Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala Asn

100 105 110

Phe Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu

115 120 125

Gln Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu

130 135 140

Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys

145 150 155 160

Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly

165 170 175

His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val

180 185 190

Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg

195 200 205

Ser Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His

210 215 220

Arg Asp Asp His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn Ala Ala

225 230 235 240

Thr Arg Thr Ser Arg Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro

245 250 255

Ser Arg Val Glu Ala Phe His Arg Tyr Gly Thr Thr Val Asn Cys Ile

260 265 270

Val Glu Glu Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val

275 280 285

Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg

290 295 300

Glu Gly Ser His Thr Glu His Thr Ser Tyr Ala Ala Asp Arg Phe Lys

305 310 315 320

Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala

325 330 335

Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val

340 345 350

Ala Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys

355 360 365

Trp Gln Glu Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser Phe

370 375 380

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

385 390 395 400

Glu Tyr Pro Leu Ser Arg Val Asp Leu Gly Asp Cys Ile Gly Lys Asp

405 410 415

Ala Arg Asp Ala Met Asp Arg Ile Phe Ala Arg Arg Tyr Asn Ala Thr

420 425 430

His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu Ala Asn Gly Gly Phe

435 440 445

Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr

450 455 460

Val Arg Glu His Leu Arg Glu Gln Ser Arg Lys Pro Pro Asn Pro Thr

465 470 475 480

Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile Lys

485 490 495

Thr Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His

500 505 510

Ile Gln Arg His Val Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp

515 520 525

Cys Glu Leu Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys

530 535 540

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

545 550 555 560

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

565 570 575

Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile Ser Ser Arg

580 585 590

Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp

595 600 605

Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg

610 615 620

Leu Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His Arg Arg Tyr

625 630 635 640

Phe Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu Glu Tyr Ala Tyr Ser

645 650 655

His Gln Leu Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp

660 665 670

Leu Asn Ile Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val

675 680 685

Tyr Thr Arg His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu

690 695 700

Val Gln Arg Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp

705 710 715 720

Thr Val Ile His Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Gly

725 730 735

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

740 745 750

Val Met Gly Ile Val Gly Gly Val Val Ser Ala Val Ser Gly Val Ser

755 760 765

Ser Phe Met Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val

770 775 780

Leu Ala Gly Leu Ala Ala Ala Phe Phe Ala Phe Arg Tyr Val Met Arg

785 790 795 800

Leu Gln Ser Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu

805 810 815

Leu Lys Asn Pro Thr Asn Pro Asp Ala Ser Gly Glu Gly Glu Glu Gly

820 825 830

Gly Asp Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met Ile Arg

835 840 845

Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys Ala Lys

850 855 860

Lys Lys Gly Thr Ser Ala Leu Leu Ser Ala Lys Val Thr Asp Met Val

865 870 875 880

Met Arg Lys Arg Arg Asn Thr Asn Tyr Thr Gln Val Pro Asn Lys Asp

885 890 895

Gly Asp Ala Asp Glu Asp Asp Leu

900

<210>2

<211>2715

<212>DNA

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>2

atgcgccagg gcgcccccgc gcgggggcgc cggtggttcg tcgtatgggc gctcttgggg 60

ttgacgctgg gggtcctggt ggcgtcggcg gctccgagtt cccccggcac gcctggggtc 120

gcggccgcga cccaggcggc gaatgggggc cctgccactc cggcgccgcc cgcccctggc 180

cccgccccaa cgggggaccc gaaaccgagg aagaacaaaa aaccgaaacc cccaacgccg 240

ccgcgccccg ccggcgacaa cgcgaccgtc gccgcgggcc acgccaccct gcgcgagcac 300

ctgcgggaca tcaaggcgga gaacaccgat gcaaactttt acgtgtgccc accccccacg 360

ggcgccacgg tggtgcagtt cgagcagccg cgccgctgcc cgacccggcc cgagggtcag 420

aactacacgg agggcatcgc ggtggtcttc aaggagaaca tcgccccgta caagttcaag 480

gccaccatgt actacaaaga cgtcaccgtt tcgcaggtgt ggttcggcca ccgctactcc 540

cagtttatgg ggatctttga ggaccgcgcc cccgtcccct tcgaggaggt gatcgacaag 600

atcaacgcca agggggtctg tcggtccacg gccaagtacg tgcgcaacaa cctggagacc 660

accgcgtttc accgggacga ccacgagacc gacatggagc tgaaaccggc caacgccgcg 720

acccgcacga gccggggctg gcacaccacc gacctcaagt acaacccctc gcgggtggag 780

gcgttccacc ggtacgggac gacggtaaac tgcatcgtcg aggaggtgga cgcgcgctcg 840

gtgtacccgt acgacgagtt tgtgctggcg actggcgact ttgtgtacat gtccccgttt 900

tacggctacc gggaggggtc gcacaccgaa cacaccagct acgccgccga ccgcttcaag 960

caggtcgacg gcttctacgc gcgcgacctc accaccaagg cccgggccac ggcgccgacc 1020

acccggaacc tgctcacgac ccccaagttc accgtggcct gggactgggt gccaaagcgc 1080

ccgtcggtct gcaccatgac caagtggcag gaggtggacg agatgctgcg ctccgagtac 1140

ggcggctcct tccgattctc ctccgacgcc atatccacca ccttcaccac caacctgacc 1200

gagtacccgc tctcgcgcgt ggacctgggg gactgcatcg gcaaggacgc ccgcgacgcc 1260

atggaccgca tcttcgcccg caggtacaac gcgacgcaca tcaaggtggg ccagccgcag 1320

tactacctgg ccaatggggg ctttctgatc gcgtaccagc cccttctcag caacacgctc 1380

gcggagctgt acgtgcggga acacctccga gagcagagcc gcaagccccc aaaccccacg 1440

cccccgccgc ccggggccag cgccaacgcg tccgtggagc gcatcaagac cacctcctcc 1500

atcgagttcg cccggctgca gtttacgtac aaccacatac agcgccatgt caacgatatg 1560

ttgggccgcg ttgccatcgc gtggtgcgag ctgcagaatc acgagctgac cctgtggaac 1620

gaggcccgca agctgaaccc caacgccatc gcctcggcca ccgtgggccg gcgggtgagc 1680

gcgcggatgc tcggcgacgt gatggccgtc tccacgtgcg tgccggtcgc cgcggacaac 1740

gtgatcgtcc aaaactcgat gcgcatcagc tcgcggcccg gggcctgcta cagccgcccc 1800

ctggtcagct ttcggtacga agaccagggc ccgttggtcg aggggcagct gggggagaac 1860

aacgagctgc ggctgacgcg cgatgcgatc gagccgtgca ccgtgggaca ccggcgctac 1920

ttcaccttcg gtgggggcta cgtgtacttc gaggagtacg cgtactccca ccagctgagc 1980

cgcgccgaca tcaccaccgt cagcaccttc atcgacctca acatcaccat gctggaggat 2040

cacgagtttg tccccctgga ggtgtacacc cgccacgaga tcaaggacag cggcctactg 2100

gactacacgg aggtccagcg ccgcaaccag ctgcacgacc tgcgcttcgc cgacatcgac 2160

acggtcatcc acgccgacgc caacgccgcc atgttcgcgg gcctgggcgc gttcttcgag 2220

gggatgggcg acctggggcg cgcggtcggc aaggtggtga tgggcatcgt gggcggcgtg 2280

gtatcggccg tgtcgggcgt gtcctccttc atgtccaacc cctttggggc gctggccgtg 2340

ggtctgttgg tcctggccgg cctggcggcg gccttcttcg cctttcgcta cgtcatgcgg 2400

ctgcagagca accccatgaa ggccctgtac ccgctaacca ccaaggagct caagaacccc 2460

accaacccgg acgcgtccgg ggagggcgag gagggcggcg actttgacga ggccaagcta 2520

gccgaggcca gggagatgat acggtacatg gccctggtgt cggccatgga gcgcacggaa 2580

cacaaggcca agaagaaggg cacgagcgcg ctgctcagcg ccaaggtcac cgacatggtc 2640

atgcgcaagc gccgcaacac caactacacc caagttccca acaaagacgg tgacgccgac 2700

gaggacgacc tgtga 2715

<210>3

<211>423

<212>PRT

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>3

Lys Pro Thr Ser Thr Pro Lys Ser Pro Pro Thr Ser Thr Pro Asp Pro

1 5 10 15

Lys Pro Lys Asn Asn Thr Thr Pro Ala Lys Ser Gly Arg Pro Thr Lys

20 25 30

Pro Pro Gly Pro Val Trp Cys Asp Arg Arg Asp Pro Leu Ala Arg Tyr

35 40 45

Gly Ser Arg Val Gln Ile Arg Cys Arg Phe Arg Asn Ser Thr Arg Met

50 55 60

Glu Phe Arg Leu Gln Ile Trp Arg Tyr Ser Met Gly Pro Ser Pro Pro

65 70 75 80

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

85 90 95

Pro Pro Gly Gly Leu Leu Val Tyr Asp Ser Ala Pro Asn Leu Thr Asp

100 105 110

Pro His Val Leu Trp Ala Glu Gly Ala Gly Pro Gly Ala Asp Pro Pro

115 120 125

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

130 135 140

Glu Val Thr Pro Ala Thr Gln Gly Met Tyr Tyr Leu Ala Trp Gly Arg

145 150 155 160

Met Asp Ser Pro His Glu Tyr Gly Thr Trp Val Arg Val Arg Met Phe

165 170 175

Arg Pro Pro Ser Leu Thr Leu Gln Pro His Ala Val Met Glu Gly Gln

180 185 190

Pro Phe Lys Ala Thr Cys Thr Ala Asp Ala Tyr Tyr Pro Arg Asn Pro

195 200 205

Val Glu Phe Val Trp Phe Glu Asp Asp His Gln Val Phe Asn Pro Gly

210 215 220

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

225 230 235 240

Ser Thr Val Thr Ser Glu Ala Val Gly Gly Gln Val Pro Pro Arg Thr

245 250 255

Phe Thr Cys Gln Met Thr Trp His Arg Asp Ser Val Thr Phe Ser Arg

260 265 270

Arg Asn Ala Thr Gly Leu Ala Leu Val Leu Pro Arg Pro Thr Ile Thr

275 280 285

Met Glu Phe Gly Val Arg His Val Val Cys Thr Ala Gly Cys Val Pro

290 295 300

Glu Gly Val Thr Phe Ala Trp Phe Leu Gly Asp Asp Pro Ser Pro Ala

305 310 315 320

Ala Lys Ser Ala Val Thr Ala Gln Glu Ser Cys Asp His Pro Gly Leu

325 330 335

Ala Thr Val Arg Ser Thr Leu Pro Ile Ser Tyr Asp Tyr Ser Glu Tyr

340 345 350

Ile Cys Arg Leu Thr Gly Tyr Pro Ala Gly Ile Pro Val Leu Glu His

355 360 365

His Gly Ser His Gln Pro Pro Pro Arg Asp Pro Thr Glu Arg Gln Val

370 375 380

Ile Glu Ala Ile Glu Trp Val Gly Ile Gly Ile Gly Val Leu Ala Ala

385 390 395 400

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

405 410 415

Ser Arg Gln Arg His Arg Arg

420

<210>4

<211>1537

<212>DNA

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>4

atggccccgg ggcgggtggg ccttgccgtg gtcctgtgga gcctgttgtg gctcggggcg 60

ggggtgtccg ggggctcgga aactgcctcc accgggccca cgatcaccgc gggagcggta 120

acgaacgcga gcgaggcccc cacatcgggg tcccccgggt cagccgccag cccggaagtc 180

acccccacat cgaccccaaa ccccaacaat gtcacacaaa acaaaaccac ccccaccgag 240

ccggccagcc ccccaacaac cccccaagcc cacctccacg cccaaaagcc cccccacgtc 300

cacccccgac cccaaaccca agaacaacac cacccccgcc aagtcgggcc gccccactaa 360

accccccggg cccgtgtggt gcgaccgccg cgacccattg gcccggtacg gctcgcgggt 420

gcagatccga tgccggtttc ggaattccac ccgcatggag ttccgcctcc agatatggcg 480

ttactccatg ggtccgtccc ccccaatcgc tccggctccc gacctagagg aggtcctgac 540

gaacatcacc gccccacccg ggggactcct ggtgtacgac agcgccccca acctgacgga 600

cccccacgtg ctctgggcgg agggggccgg cccgggcgcc gaccctccgt tgtattctgt 660

caccgggccg ctgccgaccc agcggctgat tatcggcgag gtgacgcccg cgacccaggg 720

aatgtattac ttggcctggg gccggatgga cagcccgcac gagtacggga cgtgggtgcg 780

cgtccgcatg ttccgccccc cgtctctgac cctccagccc cacgcggtga tggagggtca 840

gccgttcaag gcgacgtgca cggccgacgc ctactacccg cgtaaccccg tggagtttgt 900

ctggttcgag gacgaccacc aggtgtttaa cccgggccag atcgacacgc agacgcacga 960

gcaccccgac gggttcacca cagtctctac cgtgacctcc gaggctgtcg gcggccaggt 1020

ccccccgcgg accttcacct gccagatgac gtggcaccgc gactccgtga cgttctcgcg 1080

acgcaatgcc accgggctgg ccctggtgct gccgcggcca accatcacca tggaatttgg 1140

ggtccggcat gtggtctgca cggccggctg cgtccccgag ggcgtgacgt ttgcctggtt 1200

cctgggggac gacccctcac cggcggctaa gtcggccgtt acggcccagg agtcgtgcga 1260

ccaccccggg ctggctacgg tccggtccac cctgcccatt tcgtacgact acagcgagta 1320

catctgtcgg ttgaccggat atccggccgg gattcccgtt ctagagcacc acggcagtca 1380

ccagccccca cccagggacc ccaccgagcg gcaggtgatc gaggcgatcg agtgggtggg 1440

gattggaatc ggggttctcg cggcgggggt cctggtcgta acggcaatcg tgtacgtcgt 1500

ccgcacatca cagtcgcggc agcgtcatcg gcggtaa 1537

<210>5

<211>394

<212>PRT

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>5

Met Gly Gly Ala Ala Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val

1 5 10 15

Ile Val Gly Leu His Gly Val Arg Gly Lys Tyr Ala Leu Ala Asp Ala

20 25 30

Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro

35 40 45

Val Leu Asp Gln Leu Thr Asp Pro Pro Gly Val Arg Arg Val Tyr His

50 55 60

Ile Gln Ala Gly Leu Pro Asp Pro Phe Gln Pro Pro Ser Leu Pro Ile

65 70 75 80

Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu

85 90 95

Asn Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Glu Asp

100 105 110

Val Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Trp Phe Arg Met Gly

115 120 125

Gly Asn Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr Glu Cys Ser

130 135 140

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

145 150 155 160

Asn Tyr Tyr Asp Ser Phe Ser Ala Val Ser Glu Asp Asn Leu Gly Phe

165 170 175

Leu Met His Ala Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu

180 185 190

Val Lys Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His

195 200 205

Arg Ala Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro

210 215 220

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

225 230 235 240

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

245 250 255

Ala Val Tyr Ser Leu Lys Ile Ala Gly Trp His Gly Pro Lys Ala Pro

260 265 270

Tyr Thr Ser Thr Leu Leu Pro Pro Glu Leu Ser Glu Thr Pro Asn Ala

275 280 285

Thr Gln Pro Glu Leu Ala Pro Glu Asp Pro Glu Asp Ser Ala Leu Leu

290 295 300

Glu Asp Pro Val Gly Thr Val Ala Pro Gln Ile Pro Pro Asn Trp His

305 310 315 320

Ile Pro Ser Ile Gln Asp Ala Ala Thr Pro Tyr His Pro Pro Ala Thr

325 330 335

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

340 345 350

Ala Ala Leu Val Ile Cys Gly Ile Val Tyr Trp Met His Arg Arg Thr

355 360 365

Arg Lys Ala Pro Lys Arg Ile Arg Leu Pro His Ile Arg Glu Asp Asp

370 375 380

Gln Pro Ser Ser His Gln Pro Leu Phe Tyr

385 390

<210>6

<211>1185

<212>DNA

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>6

atgggggggg ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60

catggggtcc gcggcaaata tgccttggcg gatgcctctc tcaagatggc cgaccccaat 120

cgctttcgcg gcaaagacct tccggtcctg gaccagctga ccgaccctcc gggggtccgg 180

cgcgtgtacc acatccaggc gggcctaccg gacccgttcc agccccccag cctcccgatc 240

acggtttact acgccgtgtt ggagcgcgcc tgccgcagcg tgctcctaaa cgcaccgtcg 300

gaggcccccc agattgtccg cggggcctcc gaagacgtcc ggaaacaacc ctacaacctg 360

accatcgctt ggtttcggat gggaggcaac tgtgctatcc ccatcacggt catggagtac 420

accgaatgct cctacaacaa gtctctgggg gcctgtccca tccgaacgca gccccgctgg 480

aactactatg acagcttcag cgccgtcagc gaggataacc tggggttcct gatgcacgcc 540

cccgcgtttg agaccgccgg cacgtacctg cggctcgtga agataaacga ctggacggag 600

attacacagt ttatcctgga gcaccgagcc aagggctcct gtaagtacgc cctcccgctg 660

cgcatccccc cgtcagcctg cctctccccc caggcctacc agcagggggt gacggtggac 720

agcatcggga tgctgccccg cttcatcccc gagaaccagc gcaccgtcgc cgtatacagc 780

ttgaagatcg ccgggtggca cgggcccaag gccccataca ctagcaccct gctgcccccg 840

gagctgtccg agacccccaa cgccacgcaa ccagaactcg ccccggaaga ccccgaggat 900

tcggccctct tggaggaccc cgtggggacg gtggcgccgc aaatcccgcc aaactggcac 960

atcccgtcgat ccaggacgc cgcgacgcct taccatcccc cggccacccc gaacaacatg 1020

ggcctgatcg ccggcgcggt gggcggcagt ctcctggcag ccctggtcat ttgcggaatt 1080

gtgtactgga tgcaccgccg cactcggaaa gccccaaagc gcatacgcct cccccacatc 1140

cgggaagacg accagccgtc ctcgcaccag cccttgtttt actag 1185

<210>7

<211>239

<212>PRT

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>7

Met Ser Pro Gly Ala Met Arg Ala Val Val Pro Ile Ile Pro Phe Leu

1 5 10 15

Leu Val Leu Val Gly Val Ser Gly Val Pro Thr Asn Val Ser Ser Thr

20 25 30

Thr Gln Pro Gln Leu Gln Thr Thr Gly Arg Pro Ser His Glu Ala Pro

35 40 45

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

50 55 60

Thr Thr Pro Asp His Thr Pro Pro Met Pro Ser Ile Gly Leu Glu Glu

65 70 75 80

Glu Glu Glu Glu Glu Glu Gly Ala Gly Asp Gly Glu His Leu Glu Gly

85 90 95

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

100 105 110

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

115 120 125

Pro Pro Pro Gly Pro Asn Asn Ser Pro Ala Arg Pro Glu Thr Ser Arg

130 135 140

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

145 150 155 160

Thr Gln Leu Pro Ser Lys Gly Arg Pro Leu Val Pro Thr Pro Gln His

165 170 175

Thr Pro Leu Phe Ser Phe Leu Thr Ala Ser Pro Ala Leu Asp Thr Leu

180 185 190

Phe Val Val Ser Thr Val Ile His Thr Leu Ser Phe Leu Cys Ile Val

195 200 205

Ala Met Ala Thr His Leu Cys Gly Gly Trp Ser Arg Arg Gly Arg Arg

210 215 220

Thr His Pro Ser Val Arg Tyr Val Cys Leu Pro Pro Glu Arg Gly

225 230 235

<210>8

<211>720

<212>DNA

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>8

atgtcgccgg gcgccatgcg tgccgttgtt cccattatcc cattcctttt ggttcttgtc 60

ggtgtatcgg gggttcccac caacgtctcc tccaccaccc aaccccagct ccagaccacc 120

ggtcgtccct cgcatgaagc ccccaacatg acccagaccg gcaccaccga ctctcccacc 180

gccatcagcc ttaccacgcc cgaccacaca ccccccatgc caagtatcgg actggaggag 240

gaggaagagg aggaggaggg ggccggggat ggcgaacatc ttgagggggg agatgggacc 300

cgtgacaccc taccccagtc cccgggtcca gccgtcccgt tggccgggga tgacgagaag 360

gacaaaccca accgtcccgt agtcccaccc cccggtccca acaactcccc cgcgcgcccc 420

gagaccagtc gaccgaagac accccccacc agtatcgagc cgctggcaac tcgacccacg 480

acccaactcc cctcaaaggg gcgacccttg gttccgacgc ctcaacatac cccgctgttc 540

tcgttcctca ctgcctcccc cgccctggac accctcttcg tcgtcagcac cgtcatccac 600

accttatcgt ttttgtgtat tgttgcgatg gcgacacacc tgtgtggcgg ttggtccaga 660

cgcgggcgac gcacacaccc tagcgtgcgtt acgtgtgcc tgccgcccga acgcgggtag 720

<210>9

<211>473

<212>PRT

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>9

Met Gly Arg Pro Ala Pro Arg Gly Ser Pro Asp Ser Ala Pro Pro Thr

1 5 10 15

Lys Gly Met Thr Gly Ala Arg Thr Ala Trp Trp Val Trp Cys Val Gln

20 25 30

Val Ala Thr Phe Val Val Ser Ala Val Cys Val Thr Gly Leu Leu Val

35 40 45

Leu Ala Ser Val Phe Arg Ala Arg Phe Pro Cys Phe Tyr Ala Thr Ala

50 55 60

Ser Ser Tyr Ala Gly Val Asn Ser Thr Ala Glu Val Arg Gly Gly Val

65 70 75 80

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

85 90 95

Ile Thr Ala Val Leu Leu Leu Ala Ala Ala Val Tyr Ala Val Val Gly

100 105 110

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

115 120 125

Ala Ala Ala Arg Met Ala Met Pro His Ala Thr Leu Ile Ala Gly Asn

130 135 140

Val Cys Ser Trp Leu Leu Gln Ile Thr Val Leu Leu Leu Ala His Arg

145 150 155 160

Ile Ser Gln Leu Ala His Leu Val Tyr Val Leu His Phe Ala Cys Leu

165 170 175

Val Tyr Phe Ala Ala His Phe Cys Thr Arg Gly Val Leu Ser Gly Thr

180 185 190

Tyr Leu Arg Gln Val His Gly Leu Met Glu Pro Ala Pro Thr His His

195 200 205

Arg Val Val Gly Pro Ala Arg Ala Val Leu Thr Asn Ala Leu Leu Leu

210 215 220

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

225 230 235 240

Ala Ala Tyr Asn Phe Asn Phe Ser Ala Pro Gly Met Leu Ile Cys Leu

245 250 255

Thr Val Leu Phe Ala Leu Leu Val Val Ser Leu Leu Leu Val Val Glu

260 265 270

Gly Val Leu Cys His Tyr Val Arg Val Leu Val Gly Pro His Leu Gly

275 280 285

Ala Val Ala Ala Thr Gly Ile Val Gly Leu Ala Cys Glu His Tyr Tyr

290 295 300

Thr Asn Gly Tyr Tyr Val Val Glu Thr Gln Trp Pro Gly Ala Gln Thr

305 310 315 320

Gly Val Arg Val Ala Leu Ala Leu Val Ala Ala Phe Ala Leu Gly Met

325 330 335

Ala Val Leu Arg Cys Thr Arg Ala Tyr Leu Tyr His Arg Arg His His

340 345 350

Thr Lys Phe Phe Met Arg Met Arg Asp Thr Arg His Arg Ala His Ser

355 360 365

Ala Leu Lys Arg Val Arg Ser Ser Met Arg Gly Ser Arg Asp Gly Arg

370 375 380

His Arg Pro Ala Pro Gly Ser Pro Pro Gly Ile Pro Glu Tyr Ala Glu

385 390 395 400

Asp Pro Tyr Ala Ile Ser Tyr Gly Gly Gln Leu Asp Arg Tyr Gly Asp

405 410 415

Ser Asp Gly Glu Pro Ile Tyr Asp Glu Val Ala Asp Asp Gln Thr Asp

420 425 430

Val Leu Tyr Ala Lys Ile Gln Pro Pro Arg Hi s Leu Pro Asn Asp Glu

435 440 445

Pro Ile Tyr Asp Thr Val Gly Gly Tyr Asp Pro Glu Pro Ala Glu Asp

450 455 460

Pro Val Tyr Ser Thr Val Arg Arg Trp

465 470

<210>10

<211>1422

<212>DNA

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>10

atgggacgcc cggcccccag aggatccccc gactccgcgc cccccacgaa aggcatgacc 60

ggggcgcgga cggcgtggtg ggtctggtgt gtgcaggtgg cgacgtttgt ggtctctgcg 120

gtctgcgtca cggggcttct cgtcctggcc tctgtgttcc gggcacggtt tccctgcttt 180

tacgccacgg cgagctctta tgccggggtg aactccacgg ccgaggtgcg cgggggtgta 240

gccgtgcccc tcaggttgga cacgcagagc cttgtgggca cttatgtaat cacggccgtg 300

ttgttgttgg ccgcggccgt gtatgccgtg gtcggcgccg tgacctcccg ctacgaccgc 360

gccctggacg cgggccgccg tctggctgcg gcccgcatgg ccatgccgca cgccacgctg 420

atcgccggaa acgtctgctc ttggttgctg cagatcaccg tcctgttgct ggcccatcgc 480

atcagccaac tggcccacct ggtttacgtc ctgcactttg cgtgtctggt gtattttgcg 540

gcccattttt gcaccagggg ggtcctgagc gggacgtatc tgcgtcaggt gcacggcctg 600

atggagccgg ccccgactca tcatcgcgtc gtcggcccgg ctcgagccgt gctgacaaac 660

gccttgctgt tgggcgtctt cctgtgcacg gccgacgccg cggtatccct gaataccatc 720

gccgcgtaca actttaattt ttcggccccg ggcatgctca tatgcctgac cgtgctgttc 780

gcccttctcg tcgtatcgct gttgttggtg gtcgaggggg tgttgtgtca ctacgtgcgc 840

gtgttggtgg gcccccacct gggggccgtg gccgccacgg gcatcgtcgg cctggcctgc 900

gagcactatt acaccaacgg ctactacgtt gtggagacgc agtggccggg ggcccagacg 960

ggagttcgcg tcgccctcgc cctggtcgcc gcctttgccc tcggcatggc cgtgctccgc 1020

tgcacccgcg cctatctgta tcacaggcgg caccacacca aattttttat gcgcatgcgc 1080

gacacgcgac accgcgcaca ttccgccctc aagcgcgtac gcagttccat gcgcggatcg 1140

cgagacggcc gccacaggcc cgcacccggc agcccgcccg ggattcccga atatgcggaa 1200

gacccctacg cgatctcata cggcggccag ctcgaccggt acggagattc cgacggggag 1260

ccgatttacg acgaggtggc ggacgaccaa accgacgtat tgtacgccaa gatacaaccc 1320

ccgcggcacc tgcccaacga cgagcccatc tatgacaccg ttggggggta cgaccccgag 1380

cccgccgagg accccgtgta cagcaccgtc cgccgttggt ag 1422

<210>11

<211>420

<212>PRT

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>11

Met Ala Asp Ile Ser Pro Gly Ala Phe Ala Pro Cys Val Lys Ala Arg

1 5 10 15

Arg Pro Ala Leu Arg Ser Pro Pro Leu Gly Thr Arg Lys Arg Lys Arg

20 25 30

Pro Ala Arg Pro Leu Ser Ser Glu Ser Glu Val Glu Ser Asp Thr Ala

35 40 45

Leu Glu Ser Glu Val Glu Ser Glu Thr Ala Ser Asp Ser Thr Glu Ser

50 55 60

Gly Asp Gln Asp Glu Ala Pro Arg Ile Gly Gly Arg Arg Ala Pro Arg

65 70 75 80

Arg Leu Gly Gly Arg Phe Phe Leu Asp Met Ser Ala Glu Ser Thr Thr

85 90 95

Gly Thr Glu Thr Asp Thr Ser Val Ser Asp Asp Pro Asp Asp Thr Ser

100 105 110

Asp Trp Ser Tyr Asp Asp Ile Pro Pro Arg Pro Lys Arg Ala Arg Val

115 120 125

Asn Leu Arg Leu Thr Ser Ser Pro Asp Arg Arg Asp Gly Val Ile Phe

130 135 140

Pro Lys Met Gly Arg Val Arg Ser Thr Arg Glu Thr Gln Pro Arg Ala

145 150 155 160

Pro Thr Pro Ser Ala Pro Ser Pro Asn Ala Met Leu Arg Arg Ser Val

165 170 175

Arg Gln Ala Gln Arg Arg Ser Ser Ala Arg Trp Thr Pro Glu Leu Gly

180 185 190

Tyr Met Arg Gln Cys Ile Asn Gln Leu Phe Arg Val Leu Arg Val Ala

195 200 205

Arg Asp Pro His Gly Ser Ala Asn Arg Leu Arg His Leu Ile Arg Asp

210 215 220

Cys Tyr Leu Met Gly Tyr Cys Arg Ala Arg Leu Ala Pro Arg Thr Trp

225 230 235 240

Cys Arg Leu Leu Gln Val Ser Gly Gly Thr Trp Gly Met His Leu Arg

245 250 255

Asn Thr Ile Arg Glu Val Glu Ala Arg Phe Asp Ala Thr Ala Glu Pro

260 265 270

Val Cys Lys Leu Pro Cys Leu Glu Ala Arg Arg Tyr Gly Pro Glu Cys

275 280 285

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

290 295 300

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

305 310 315 320

Ala Ser Gln Ser Asp Thr Glu Asp Ala Pro Ser Pro Val Thr Leu Glu

325 330 335

Thr Pro Glu Pro Arg Gly Ser Leu Ala Val Arg Leu Glu Asp Lys Phe

340 345 350

Gly Glu Phe Asp Trp Thr Pro Arg Glu Gly Ser Gln Pro Trp Leu Ser

355 360 365

Ala Val Val Ala Asp Thr Ser Ser Val Glu Arg Pro Gly Pro Ser Asp

370 375 380

Ser Gly Ala Gly Arg Ala Ala Glu Asp Arg Lys Cys Leu Asp Gly Cys

385 390 395 400

Arg Lys Met Arg Phe Ser Thr Ala Cys Pro Tyr Pro Cys Ser Asp Thr

405 410 415

Phe Leu Arg Pro

420

<210>12

<211>1264

<212>DNA

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>12

atggccgaca tttccccagg cgcttttgcg ccttgtgtaa aagcgcggcg tcccgctctc 60

cgatccccgc ccctgggcac gcgcaagcgc aagcgccctg cccgccccct ctcatcggag 120

tctgaggtag aatccgatac agccttggag tctgaggtcg aatccgagac agcatcggat 180

tcgaccgagt ctggggacca ggatgaagcc ccccgcatcg gtggccgtag ggccccccgg 240

aggcttgggg ggcggttttt tctggacatg tcggcggaat ccaccacggg gacggaaacg 300

gatacgtcgg tgtcggacga ccccgacgac acgtccgact ggtcttatga cgacattccc 360

ccacgaccca agcgggcccg ggtaaacctg cggctcacga gctctcccga tcggcgggat 420

ggggttattt ttcctaagat ggggcgggtc cggtctaccc gggaaacgca gccccgggcc 480

cccaccccgt cggccccaag cccaaatgca atgctacggc gctcggtgcg ccaggcccag 540

aggcggagca gcgcacgatg gacccccgaa ctgggctaca tgcgccagtg tatcaatcag 600

ctgtttcggg tacctgcggg tcgcccggga cccccacggc agtgccaacc gctctgcgcc 660

acctgatacg cgactgttac ctgatgggat actgccgagc ccgtctggcc ccgcgcacgt 720

ggtgccgctt gctgcaggtg tccggcggaa ctggggcatg cacctgcgca acaccatacg 780

ggaggtggag gctcgattcg acgccaccgc ggaacccgtg tgcaagcttc cttgtttgga 840

ggccagacgg tacggcccgg agtgtgatct tagtaatctc gagattcatc tcagcgcgac 900

aagcgatgat gaaatctccg atgccaccga tctggaggcc gccggttcgg accacacgct 960

cgcgtcccag tccgacacgg aggatgcccc ctcccccgtt acgctggaaa ccccagaacc 1020

ccgcgggtcc ctcgctgtgc gtctggagga taagtttggg gagtttgact ggaccccccg 1080

ggagggctcc cagccctggc tgtctgcggt cgtggccgat accagctccg tggaacgccc 1140

gggcccatcc gattctgggg cgggtcgcgc cgcagaagac cgcaagtgtc tggacggctg 1200

ccggaaaatg cgcttctcca ccgcctgccc ctatccgtgc agcgacacgt ttctccggcc 1260

gtga 1264

<210>13

<211>512

<212>PRT

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>13

Met Ala Thr Asp Ile Asp Met Leu Ile Asp Leu Gly Leu Asp Leu Ser

1 5 10 15

Asp Ser Asp Leu Asp Glu Asp Pro Pro Glu Pro Ala Glu Ser Arg Arg

20 25 30

Asp Asp Leu Ala Ser Asp Ser Ser Gly Glu Cys Ser Ser Ser Asp Glu

35 40 45

Asp Met Glu Asp Pro His Gly Glu Asp Gly Pro Glu Pro Ile Leu Asp

50 55 60

Ala Ala Arg Pro Ala Val Arg Pro Ser Arg Pro Glu Asp Pro Gly Val

65 70 75 80

Pro Ser Thr Gln Thr Pro Arg Pro Thr Glu Arg Gln Gly Pro Asn Asp

85 90 95

Pro Gln Pro Ala Pro His Ser Val Trp Ser Arg Leu Gly Ala Arg Arg

100 105 110

Pro Ser Cys Ser Pro Glu Gln His Gly Gly Lys Val Ala Arg Leu Gln

115 120 125

Pro Pro Pro Thr Lys Ala Gln Pro Ala Arg Gly Gly Arg Arg Gly Arg

130 135 140

Arg Arg Gly Arg Gly Arg Gly Gly Pro Gly Ala Ala Asp Gly Leu Ser

145 150 155 160

Asp Pro Arg Arg Arg Ala Pro Arg Thr Asn Arg Asn Pro Gly Gly Pro

165 170 175

Arg His Arg Gly Gly Trp Thr Asp Gly Pro Gly Ala Pro His Gly Glu

180 185 190

Ala Trp Arg Gly Ser Glu Gln Pro Asp Pro Pro Gly Gly Pro Arg Thr

195 200 205

Arg Gly Val Arg Gln Ala Pro Pro Pro Leu Met Thr Leu Ala Ile Ala

210 215 220

Pro Pro Pro Ala Asp Pro Arg Ala Pro Ala Pro Glu Arg Lys Ala Pro

225 230 235 240

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

245 250 255

Ser Glu Arg Ala Ala Val Asp Arg Ile Ser Glu Ser Phe Gly Arg Ser

260 265 270

Ala Gln Val Met His Asp Pro Phe Gly Gly Gln Pro Phe Pro Ala Ala

275 280 285

Asn Ser Pro Trp Ala Pro Val Leu Ala Gly Gln Gly Gly Pro Phe Asp

290 295 300

Ala Glu Thr Arg Arg Val Ser Trp Glu Thr Leu Val Ala His Gly Pro

305 310 315 320

Ser Leu Tyr Arg Thr Phe Ala Gly Asn Pro Arg Ala Ala Ser Thr Ala

325 330 335

Lys Ala Met Arg Asp Cys Val Leu Arg Gln Glu Asn Phe Ile Glu Ala

340 345 350

Leu Ala Ser Ala Asp Glu Thr Leu Ala Trp Cys Lys Met Cys Ile His

355 360 365

His Asn Leu Pro Leu Arg Pro Gln Asp Pro Ile Ile Gly Thr Ala Ala

370 375 380

Ala Val Leu Asp Asn Leu Ala Thr Arg Leu Arg Pro Phe Leu Gln Cys

385 390 395 400

Tyr Leu Lys Ala Arg Gly Leu Cys Gly Leu Asp Glu Leu Cys Ser Arg

405 410 415

Arg Arg Leu Ala Asp Ile Lys Asp Ile Ala Ser Phe Val Phe Val Ile

420 425 430

Leu Ala Arg Leu Ala Asn Arg Val Glu Arg Gly Val Ala Glu Ile Asp

435 440 445

Tyr Ala Thr Leu Gly Val Gly Val Gly Glu Lys Met His Phe Tyr Leu

450 455 460

Pro Gly Ala Cys Met Ala Gly Leu Ile Glu Ile Leu Asp Thr His Arg

465 470 475 480

Gln Glu Cys Ser Ser Arg Val Cys Glu Leu Thr Ala Ser His Ile Val

485 490 495

Ala Pro Pro Tyr Val Hi s Gly Lys Tyr Phe Tyr Cys Asn Ser Leu Phe

500 505 510

<210>14

<211>1539

<212>DNA

＜213〉herpes simplex virus I-type (Herpes Simplex Virus Type I)

<400>14

atggcgactg acattgatat gctaattgac ctcggcctgg acctctccga cagcgatctg 60

gacgaggacc cccccgagcc ggcggagagc cgccgcgacg acctggcatc ggacagcagc 120

ggggagtgtt cctcgtcgga cgaggacatg gaagaccccc acggagagga cggaccggag 180

ccgatactcg acgccgctcg cccggcggtc cgcccgtctc gtccagaaga ccccggcgta 240

cccagcaccc agacgcctcg tccgacggag cggcagggcc ccaacgatcc tcaaccagcg 300

ccccacagtg tgtggtcgcg cctcggggcc cggcgaccgt cttgctcccc cgagcagcac 360

gggggcaagg tggcccgcct ccaaccccca ccgaccaaag cccagcctgc ccgcggcgga 420

cgccgtgggc gtcgcagggg tcggggtcgc ggtggtcccg gggccgccga tggtttgtcg 480

gacccccgcc ggcgtgcccc cagaaccaat cgcaacccgg ggggaccccg ccaccggggc 540

gggtggacgg acggccccgg cgccccccat ggcgaggcgt ggcgcggaag tgagcagccc 600

gacccacccg gaggcccgcg gacacggggc gtgcgccaag cacccccccc gctaatgacg 660

ctggcgattg cccccccgcc cgcggacccc cgcgccccgg ccccggagcg aaaggcgccc 720

gccgccgaca ccatcgacgc caccacgcgg ttggtcctgc gctccatctc cgagcgcgcg 780

gcggtcgacc gcatcagcga gagctttggc cgcagcgcac aggtcatgca cgaccccttt 840

ggggggcagc cgtttcccgc cgcgaatagc ccctgggccc cggtgctggc gggccaagga 900

gggccctttg acgccgagac cagacgggtc tcctgggaaa ccttggtcgc ccacggcccg 960

agcctctatc gcacttttgc cggcaatcct cgggccgcat cgaccgccaa ggccatgcgc 1020

gactgcgtgc tgcgccaaga aaatttcatc gaggcgctgg cctccgccga cgagacgctg 1080

gcgtggtgca agatgtgcat ccaccacaac ctgccgctgc gcccccagga ccccattatc 1140

gggacggccg cggctgtgct ggataacctc gccacgcgcc tgcggccctt tctccagtgc 1200

tacctgaagg cgcgaggcct gtgcggcctg gacgaactgt gttcgcggcg gcgtctggcg 1260

gacattaagg acattgcatc cttcgtgttt gtcattctgg ccaggctcgc caaccgcgtc 1320

gagcgtggcg tcgcggagat cgactacgcg acccttggtg tcggggtcgg agagaagatg 1380

catttctacc tccccggggc ctgcatggcg ggcctgatcg aaatcctaga cacgcaccgc 1440

caggagtgtt cgagtcgtgt ctgcgagttg acggccagtc acatcgtcgc ccccccgtac 1500

gtgcacggca aatattttta ttgcaactcc ctgttttag 1539

Claims

1. hsv, described virus has envelope protein, it is characterized in that, described envelope protein comprises a kind of aminoacid sequence that promotes virus infection host cell function that has, described aminoacid sequence is selected from the aminoacid sequence shown in the SEQ ID:NO.1, aminoacid sequence shown in the SEQ ID:NO.3, aminoacid sequence shown in the SEQ ID:NO.5, aminoacid sequence shown in the SEQ ID:NO.7, aminoacid sequence shown in the SEQ ID:NO.9, and to described SEQ ID:NO.1, SEQ ID:NO.3, SEQ ID:NO.5, aminoacid sequence shown in SEQ ID:NO.7 or the SEQ ID:NO.9 carries out one or several aminoacid replacement, add or aminoacid sequence that disappearance and the promotion virus infection host cell function that obtains are constant in one or more.

2. virus according to claim 1, wherein, described aminoacid sequence is selected from one or more in the aminoacid sequence shown in aminoacid sequence shown in the aminoacid sequence shown in the aminoacid sequence shown in the aminoacid sequence shown in the SEQ ID:NO.1, the SEQ ID:NO.3, the SEQ ID:NO.5, the SEQ ID:NO.7 and the SEQ ID:NO.9.

3. hsv, described virus have the gene of coding envelope protein, it is characterized in that described envelope protein is the described envelope protein of claim 1.

4. virus according to claim 3, wherein, the gene of described coding envelope protein comprises that a kind of coding has the nucleotide sequence of the aminoacid sequence that promotes virus infection host cell function, described nucleotide sequence is selected from the nucleotide sequence shown in the SEQ ID:NO.2, nucleotide sequence shown in the SEQ ID:NO.4, nucleotide sequence shown in the SEQ ID:NO.6, nucleotide sequence shown in the SEQ ID:NO.8, nucleotide sequence shown in the SEQ ID:NO.10, and to described SEQ ID:NO.2, SEQ ID:NO.4, SEQ ID:NO.6, nucleotide sequence shown in SEQ ID:NO.8 or the SEQ ID:NO.10 carries out that one or several Nucleotide replaces and in the constant nucleotide sequence of the proteins encoded that obtains one or more.

5. virus according to claim 3, wherein, described envelope protein is the described envelope protein of claim 2.

6. according to claim 3 or 5 described viruses, wherein, the gene of described coding envelope protein comprises that a kind of coding has the nucleotide sequence of the aminoacid sequence that promotes virus infection host cell function, and described nucleotide sequence is selected from one or more in the nucleotide sequence shown in nucleotide sequence shown in the nucleotide sequence shown in the nucleotide sequence shown in the nucleotide sequence shown in the SEQ ID:NO.2, the SEQ ID:NO.4, the SEQ ID:NO.6, the SEQ ID:NO.8 and the SEQ ID:NO.10.

7. virus according to claim 3, wherein, the nucleotide sequence of the I herpes simplex virus type 17+ of ID:NC_001806 has 99.5% homology among the nucleotide sequence of described virus and the NCBI.

8. recombinant virus, described recombinant virus comprises as the virus of carrier and goal gene, it is characterized in that, described virus as carrier is any described virus among the claim 1-7.

9. one kind is infected virulent host cell, it is characterized in that described virus is any described virus and/or the described recombinant virus of claim 8 among the claim 1-7.

10. a pharmaceutical composition is characterized in that, described pharmaceutical composition comprises any described virus and/or the described recombinant virus of claim 8 among the claim 1-7.

11. pharmaceutical composition according to claim 10, wherein, described pharmaceutical composition is an injection liquid, described injection liquid comprises any described virus and/or the described recombinant virus of claim 8 among pharmaceutically acceptable carrier and the claim 1-7, contains 10 in every milliliter of described injection liquid ²-10 ¹⁰Plaque forms several described virus and/or recombinant virus.

12. pharmaceutical composition according to claim 11, wherein, described pharmaceutically acceptable carrier is the phosphoric acid buffer of pH value for 4.0-9.0.

13. pharmaceutical composition according to claim 11, wherein, described injection liquid also contains protective material and/or osmotic pressure regulator; With described injection liquid is benchmark, and described protectant content is 0.01-30 weight %, and described protective material is selected from one or more in inositol, sorbyl alcohol and the sucrose; It is 200-700 m osmole/kilogram that the content of described osmotic pressure regulator makes the osmotic pressure of described injection liquid, and described osmotic pressure regulator is sodium-chlor and/or Repone K.