CN100381456C - A segment differential nucleotide sequence of smallpox virus genom - Google Patents

A segment differential nucleotide sequence of smallpox virus genom Download PDF

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CN100381456C
CN100381456C CNB031212603A CN03121260A CN100381456C CN 100381456 C CN100381456 C CN 100381456C CN B031212603 A CNB031212603 A CN B031212603A CN 03121260 A CN03121260 A CN 03121260A CN 100381456 C CN100381456 C CN 100381456C
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variola
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variola virus
nucleotide
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CN1482131A (en
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阮力
王世峰
郑楠
陆柔剑
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National Institue for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention
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National Institue for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention
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Abstract

The present invention belongs to the field of biological techniques and virus detection techniques and provides a section of specific nucleotide sequence U160K of variola virus genomes. The sequence comprises 920 basic groups, widely exists variola virus genomes and has different distribution modes in common type variola viruses and light variola viruses, but the nucleotide homology is larger than 90%; the sequence has no obvious homology with viruses except for variola viruses, is a specific genetic material mark of variola viruses and can be used for the research and the development of the specific detection technique of variola viruses.

Description

The nucleotide sequence of one section variola virus genome specific
Technical field
The present invention relates generally to biotechnology, particularly detection of variola virus and smallpox diagnostic techniques.
Background technology
Smallpox is the deadly infectious disease that is caused by the variola virus that belongs to the Poxviridae orthopoxvirus, can and mucocutaneously propagate in each age groups through respiratory tract.Once popular on a large scale in history, case fatality rate is up to 20%-30%.Because the extensive inoculation of bovine vaccine (antismallpox vaccine) has effectively prevented the popular of smallpox, and has eliminated smallpox in worldwide the seventies in 20th century the most finally.From 1980, the whole world stopped the bovine vaccine inoculation, had at present the colony that in a large number variola virus is not had immunizing power or hypoimmunity among the crowd, and China had 400,000,000 people at least to the crowd that smallpox does not have immunizing power below 22 years old, and had more than 20 hundred million people in the whole world.Like this, in case because factors such as bio-terrorism, laboratory leakage, archaeological excavation cause variola virus to reappear, the diffusion of disease is with very fast.In order to tackle such situation, must when reappearing, variola virus find rapidly also to take emergency measures as early as possible, could at utmost reduce loss.Under such situation, all take much count of variola virus both at home and abroad and detect and day research of blossom disease diagnostic techniques.
Variola virus detects the main technology such as cause of disease negative staining electron microscope, the cultivation of cause of disease chick embryo allantois chorion, virus antigen and antibody test of using at present.What use was recommended by The World Health Organization (WHO) mainly is cause of disease negative staining electron microscope and cause of disease chick embryo allantois chorion inoculation technique.Cause of disease negative staining electron microscope technology can determine accurately whether test sample is poxvirus from morphology, but can not distinguish variola virus and other poxvirus, as monkeypox, vaccinia virus etc.; Though cause of disease chick embryo allantois chorion inoculation method can be distinguished variola virus and other poxvirus to a certain extent from the difference of pock form, length consuming time (2-4 days), complicated operation, result reliability is limited; Virus antigen and antibody test technology all can not clearly be distinguished variola virus and other poxvirus.That is to say that above-mentioned common technology all can not carry out specific detection to variola virus.Present above-mentioned technology only is used for the auxiliary detection and the diagnosis of variola virus and day blossom disease.
Recent years, abroad the someone attempts to utilize the technology of light circulating polymerization polymerase chain reaction (Light Cycler PCR) and oligonucleotide chip (Oligonucleotide microchip) to detect variola virus.But the former still can't distinguish variola virus and other poxvirus, and the latter does not also have substantive progress at present.More than two kinds of methods also all be in the laboratory study stage at present.
Summary of the invention
In sum, also there is not the special detection method of a kind of variola virus fast and accurately at present.This is largely because poxvirus (comprising variola virus) is maximum at present and a most complicated viroid, its proteantigen homology is very high, morphology of virus is very similar again, therefore be difficult to use routine techniques set up can with the special detection method of other other variola virus of poxvirus phase region.All by its genome decision, virus is no exception for all biological proterties.Therefore, the objective of the invention is: from virus genomic otherness, searching can be different from other viral variola virus specific gene group sequence, be that this sequence should all exist in all variola virus, and at other known viruse, particularly with other very near poxvirus of variola virus sibship, as not existing in the viruses such as monkeypox, cowpox, bovine vaccine, for the special detection of variola virus provides the sign gene.
The present invention can realize with principle by the following method: 1. classify the basis as with the variola virus genome sequence of having published at present; 2. has the virus of close sibship with bovine vaccine, monkey pox virus etc. and variola virus and eruption virus genome sequence such as varicella, Measles virus are the comparison object; 3. sequence data is handled in the comparison of applying biological information science means, seeks variola virus genome and above-mentioned virus genomic difference, tentatively determines the variola virus distinguished sequence; 4. the variola virus genome specific sequence is determined in the checking of again all viral nucleotide sequences of the distinguished sequence that obtains and European Molecular Bioglogy Laboratory (EMBL) data-base recording being compared substantially; 5. this distinguished sequence of synthetic variola virus is the template of polymerase chain reaction (PCR) with this sequence, compares with other virus gene genome nucleic acid template, and checking by experiment determines that finally this distinguished sequence is the sign gene of variola virus.
Main contents of the present invention: the present invention (has three: comprise two strain coventional type variola virus with the variola virus genome sequence of publishing at present, India strain India-1967 and Bangladesh strain bangladesh-1975, its Genbank database number of asking for is respectively X69198 and L22579; One strain milk-pox virus, add West Asia strain Garcia-1966, the number of asking for is Y16780) for basic, the virus and the eruption virus genome sequence such as varicella, measles that have close sibship with bovine vaccine, monkey pox virus etc. and variola virus are the comparison object, use the network edition shareware of American National information center (NCBI), carry out the screening of the special sign gene of variola virus.The genome sequence of at first selecting India strain variola virus is as work sequence, and the network edition shareware " in pairs basic local connection is joined lookup tool " (Pairwise BLAST) that uses American National bioinformation center is the homology of the genome sequence of this sequence and above-mentioned comparison object virus relatively one by one.Return the homology zone of two kinds of virus genome sequence of participating in comparison after the comparison, remaining non-homogeneous zone.With above-mentioned comparison object virus compare one by one finish after, to compare the non-homogeneous zone that obtains again at every turn and get common factor, obtain on the variola virus genome and all comparison object viruses not homologous zone, i.e. variola virus genome specific sequence, long 920 Nucleotide of this sequence, called after U160K.Then, this sequence is compared to all viral nucleotide sequences (154,696) of EMBL data-base recording through the network edition shareware FASTA of European Molecular Bioglogy Laboratory (EMBL) again.The result shows that all other viral nucleotide sequences in this sequence and the database beyond the variola virus all do not have significant homology, have promptly further confirmed the specificity of sequence U160K.At last, the variola virus genome sequence delivered of aligned sequences U160K and other two strains again, prove its ubiquity and conservative (sequence homology is greater than 90%) in the variola virus genome, prove that simultaneously U160K is continuous distribution in the coventional type variola virus, and in weak variola virus, distribute for being interrupted.The present invention is according to the U160K sequence, and its homologous sequence of synthetic is contrast as template with the correlated virus, carries out polymerase chain reaction (PCR) experiment, proves that this distinguished sequence can be used as the sign gene of variola virus, is used for the variola virus specific detection.
The invention has the advantages that: the variola virus genome sign gene U160K that provides has very high specificity.It all exists in the three strain variola virus sequences of having delivered, and with viruses such as the higher monkeypox of its affinity, cowpox, hunchbacked acne, bovine vaccine, and do not exist in all other virus gene sequences beyond the variola virus in the European molecular biology database.This section sequence with synthetic is a template, does polymerase chain reaction (PCR) with other poxvirus and proves that relatively this sequence can be used as the sign gene of variola virus, is used for the special gene test of variola virus.Because this sequence is different with arrangement mode in the milk-pox virus genome sequence the coventional type variola virus, the difference that this sign gene also can be used for coventional type variola virus and milk-pox virus detects.
Description of drawings
Fig. 1. use Pairwise BLAST software to compare the result of variola virus genome sequence and other virus genome sequence one by one."-" homologous region, " *" non-homogeneous district (special district), dash area is a unique all special sequence U160K of other viral genome that compares with all participation on the variola virus genome.On the variola virus genome with vaccinia virus Copenhagen pnca gene group homology and nonhomologous zone, on the variola virus genome with vaccinia virus Tiantan strain genome homology and nonhomologous zone, on the variola virus genome with monkey pox virus genome homology and nonhomologous zone, on the variola virus genome with vaccinia virus genome homology and nonhomologous zone, on the variola virus genome with hunchbacked poxvirus genome group homology and nonhomologous zone, on the variola virus genome with mouse pox virus genome homology and nonhomologous zone, on the variola virus genome with avian pox virus gene group homology and nonhomologous zone, on the variola virus genome with sheep pine skin disease virus genes group homology and nonhomologous zone, on the variola virus genome with rabbit fibroma virus genome homology and nonhomologous zone, on the variola virus genome with pig pox virus genome homology and nonhomologous zone, 11. on the variola virus genome with Asia crust monkey sample disease virus genes group homology and nonhomologous zone, on the 12. variola virus genomes with the molluscum contagiosum virus, herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Measles virus, rubella virus genome group homology and nonhomologous zone.Variola virus and this several viral genome homologous zone are seldom very short, do not show corresponding homology symbol on the figure.
Fig. 2. variola virus genome specific sequence U160K.Totally 920 Nucleotide, dash area are two encoding histone frames in the U160K sequence.
Fig. 3. use the specificity of FASTA software certification sequence U160K.When using the E value change curve of FASTA software comparison virus sequence homology, the E value is more little, and the homology degree is high more.Preceding 7 points are represented the E value of the virus of 7 remarkable homologous sequences among the figure, and the E value all is less than 0.01, and point is after this represented other virus sequence E value relatively of EMBL data-base recording, and the E value is higher, does not all have remarkable homology.Preceding 7 some correspondences is that FASTA software (amounts to 154 at all viral nucleotide sequences of EMBL data-base recording among the figure, article 696, find) with the remarkable homologous sequence of U160K, totally 7, be the variola virus sequence, numbering and name are called: 1.EM_VI:VVCGAA X69198.1 Variola virus DNA comple (185578) [f], 2.EM_VI:VVXHOIFOH X67117.1 Variola virus (XhoI-F 25344) [f], 3.EM_VI:VV18341U18341.1 Variola virus Somalia-1 (30869) [f], 4.EM_VI:PXVARCG L22579.1 Variola majorvirus (st 186103) [f], 5.EM_VI:VV18339 U18339.1 Variola virus Garcia-19 (31562) [f], 6.EM_VI:VMVY16780 Y16780.1 variola minor virus c (186986) [f], 7.EM_VI:VMVGENX72086.1 Variola minor virus comp (20259) [f].
Fig. 4. the arranged distribution of variola virus distinguished sequence U160K on three strain variola virus genomes." " be the U160K fragment, this fragment is positioned at India's strain the 160681st to 161600 Nucleotide place, is positioned at Bangladesh's strain the 160713rd to 161680 Nucleotide place, is positioned to add West Asia strain 161745 to 162261 and 162889 to 163261 Nucleotide places.
The plasmid figure of Fig. 5 a. clone synthetic U160K homologous sequence.Use pMD18T commodity plasmid to be carrier, the U160K homologous sequence is inserted between SalI and the XbaI enzyme cutting point.
The sequence-specific agarose electrophoresis result of Fig. 5 b. polymerase chain reaction (PCR) check U160K.1.DL2000DNA molecular weight marker, unit is base pair (bp), 2. bovine vaccine the Temple of Heaven strain special primer PCR on bovine vaccine the Temple of Heaven pnca gene group dna profiling, as positive technology contrast (→), 3. variola virus special primer PCR on synthetic variola virus distinguished sequence U160K template, as the positive indication of variola virus (→), 4. the smallpox special primer is gone up PCR in blank template (pure water), as negative technology contrast, 5. all be smallpox special primer PCR on bovine vaccine the Temple of Heaven pnca gene group dna profiling with 6., as the variola virus negative control.
Table 1. uses other Virus Name and the genome sequence source of Pairwise BLAST software and the comparison of variola virus genome.Select three viroids, 1. the poxvirus of all infected person: comprise vaccinia virus (the Temple of Heaven strain and Copenhagen strain), vaccinia virus, monkey pox virus, molluscum contagiosum virus; 2. other important poxvirus (vertebrates poxvirus, each genus get to two kind for representative): comprise that hunchbacked poxvirus, mouse pox virus, fowlpox virus, the sick virus of sheep pine skin, rabbit fibroma virus, pig pox virus, inferior crust monkey sample are sick viral; 3. infected person and cause the virus of eruption disease: herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus (being herpes virus hominis's 3 types), Measles virus, rubella virus.Select 16 kinds of viruses altogether, wherein except that vaccinia virus was selected two strains, all the other every kind virus was selected a strain, amounted to 17 strain virus.Each virus genome sequence source sees the above table with numbering is detailed.
Embodiment
The present invention is described in detail below in conjunction with accompanying drawing:
Example 1. comparison variola virus genomes and other viral genome are sought the variola virus genome specific sequence
Use Pairwise BLAST software to compare the homology of variola virus genome sequence and other virus (for details see attached table 1) genome sequence one by one.The result shows that other viral genome of having only a zone and all to participate in comparison on the variola virus genome is all special, called after variola virus distinguished sequence U160K (the results are shown in accompanying drawing 1).Long 920 Nucleotide of this sequence contain two encoding histone frames, lay respectively at 5 ' of sequence-end and play 500 Nucleotide of the 207th Nucleotide to the and 713 Nucleotide of the 516th Nucleotide to the (seeing accompanying drawing 2).
Example 2. uses FASTA software further to confirm the specificity of variola virus genome specific sequence U160K
Using FASTA software compares (ending in October, 2002) all viral nucleotide sequences delivered (amounting to 154,696) that write down in variola virus distinguished sequence U160K and the EMBL database.The result shows, except that the nucleotide sequence of variola virus, without any virus and the remarkable homology of U160K sequence (the results are shown in accompanying drawing 3), thereby confirmed the specificity of this sequence.
Example 3. sequence U160K exist in variola virus and are conservative
With sequence U160K and the India's strain of coventional type variola virus and Bangladesh's strain, and milk-pox virus adds this three strains variola virus genome of West Asia strain and compares.The result shows (seeing Fig. 4 for details): this sequence and India's pnca gene group record chain 5 '-161600 Nucleotide zone homologies of the 160681st Nucleotide to the of end and arrangement continuously, and the homology ratio is 100%; With Bangladesh's 161680 Nucleotide zone homologies of pnca gene group record chain 5 '-terminal the 160713rd Nucleotide to the and arrangement continuously, the homology ratio is 94.0%; Arrange with adding 163261 Nucleotide zone homologies of West Asia pnca gene group record chain 5 '-the 161745th Nucleotide to the of end but be to be interrupted, the homology ratio is 94.7%.
The specific experimental evidence of example 4. sequence U160K
Synthetic U160K homologous sequence inserts among the commodity vector plasmid pMD18-T plasmid called after pMD18TSU160K (seeing accompanying drawing 5a) that has the U160K homologous sequence of acquisition.With this plasmid is the variola virus positive template, with the negative contrast of vaccinia virus Tiantan strain genomic deoxyribonucleic acid (DNA), according to the synthetic variola virus special primer of U160K sequence, carries out polymerase chain reaction (PCR).Result: use the variola virus special primer that positive band is arranged on the U160K sequence template, and on negative control, do not have (seeing accompanying drawing 5b).Originally experiment showed, the U160K sequence can as the sign gene carry out the variola virus specific detection.
The invention provides the nucleotide sequence U160K of one section variola virus genome specific, long 920 bases of this sequence, extensively be present in the variola virus genome, different in coventional type with its distribution mode in the milk-pox virus, but nucleotide homology is greater than 90%, not having obvious homology with the virus beyond the variola virus, is the distinctive genetic material sign of variola virus, can be used for the research and development of variola virus specific detection technology.
Reference
1.Sergei?N.Shchelkunov,Robert?F.Massung,Joseph?J.Esposito.Comparison?of?the?genome?DNAsequences?of?Bangladesh-1975?and?India-1967?variola?viruses.Virus?Research?36(1995)107-118
2.Shchelkunov?SN,Totmenin?AV,Loparev?VN,Safronov?PF,Gutorov?VV,Chizhikov?VE,KnightJC,Parsons?JM,Massung?RF,Esposito?JJ.Alastrim?smallpox?variola?minor?virus?genome?DNAsequences.Virology.2000Jan?20;266(2):361-86.
3.Massung?RF,Liu?LI,Qi?J,Knight?JC,Yuran?TE,Kerlavage?AR,Parsons?JM,Venter?JC,EspositoJJ.Analysis?of?the?complete?genome?of?smallpox?variola?major?virus?strain?Bangladesh-1975.Virology.1994?Jun;201(2):215-40.
4.EMBL?Nucleotide?Sequence?Database?Release?71(established?in?28-MAY-2002,worked?inSeptember-2002).
5.Espy?MJ,Cockerill?III?FR,Meyer?RF,Bowen?MD,Poland?GA,Hadfield?TL,Smith?TF.Detection?of?smallpox?virus?DNA?by?LightCycler?PCR.J?Clin?Microbiol.2002?Jun;40(6):1985-8.
6.Shchelkunov?SN,Resenchuk?SM,Totmenin?AV,Blinov?VM,Marennikova?SS,Sandakhchiev?LS.Comparison?of?the?genetic?maps?of?variola?and?vaccinia?viruses.FEBS?Lett.1993;327(3):321-4.
7.Shchelkunov?SN,Blinov?VM,Resenchuk?SM,Totmenin?AV,Olenina?LV,Chirikova?GB,Sandakhchiev?LS.Analysis?of?the?nucleotide?sequence?of?53?kbp?from?the?right?terminus?of?thegenome?of?variola?major?virus?strain?India-1967.Virus?Res.1994Dec;34(3):207-36.
8.Lapa?S,Mikheev?M,Shchelkunov?S,Mikhailovich?V,Sobolev?A,Blinov?V,Babkin?I,Guskov?A,Sokunova?E,Zasedatelev?A,Sandakhchiev?L,Mirzabekov?A.Species-level?identification?oforthopoxviruses?with?an?oligonucleotide?microchip.J?Clin?Microbiol.2002?Mar;40(3):753-7.
9.Derrick?Baxby.Human?poxvirus?infection?after?the?eradication?of?smallpox.Epidem.Inf.(1988),100,321-334.
10.Shchelkunov?SN,Totmenin?AV,Babkin?IV,Safronov?PF,Ryazankina?OI,Petrov?NA,GutorovVV,Uvarova?EA,Mikheev?MV,Sisler?JR,Esposito?JJ,Jahrling?PB,Moss?B,Sandakhchiev?LS.Human?monkeypox?and?smallpox?viruses:genomic?comparison.FEBS?Lett.2001?Nov?30;509(1):66-70.
Table 1. uses other Virus Name and the genome sequence source of Pairwise BLAST software and variola virus genome alignment
Virus Name Recognition sequence in the GenBank database number
The camel poxvirus  GI18640237
The strain of vaccinia virus Copenhagen  GI335317
Vaccinia virus Tiantan strain  GI6969640
Vaccinia virus  GI20178370
Monkey pox virus  GI17529780
Molluscum contagiosum virus's 1 type  GI1491943
Mouse pox virus  GI22123748
Fowlpox virus  GI9634679
Sheep pine skin virus  GI22595533
Rabbit fibroma virus  GI9633809
Pig pox virus  GI18640086
Inferior bar monkey sample virus  GI12084983
Herpes simplex virus type 1  GI1944536
Herpes simplex virus type 2  GI6572414
Varicella virus  GI9625875
Measles virus  GI9181912
Rubella virus  GI16923736

Claims (2)

1. the nucleotide sequence U160K of one section variola virus genome specific, it is characterized in that: U160K derives from coventional type variola virus India strain India-1967, be positioned at this strain genome 5 '-end and play 161600 Nucleotide of the 160681st Nucleotide to the, totally 920 Nucleotide; U160K contains two encoding histone frames, lays respectively at 5 ' of U160K-end and plays 500 Nucleotide of the 207th Nucleotide to the and 713 Nucleotide of the 516th Nucleotide to the.
2. the application of the nucleotide sequence U160K of variola virus genome specific according to claim 1 in variola virus detects.
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Title
Genbank Ac: L22579. 1995;Genbank Ac: X69198. 1993;Genbank Ac: Y16780. 1998;天花及其他正痘病毒基因组结构研究. 王艳等.国外医学、流行病学传染病学分册,第29卷第5期. 2002 *

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