CN100393872C - Reproduction of defect type Tiantan strain vaccinia virus - Google Patents
Reproduction of defect type Tiantan strain vaccinia virus Download PDFInfo
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Abstract
The present invention uses a gene recombination technique to remove 26 genes associated with the host range and toxicity from a Tiantan strain vaccinia virus genome, the total length of the genes is 21, the genes have 243 nucleotides, and a replication-deficient Tiantan strain vaccinia virus named NTV. The virus is well propagated in a chick-embryo cell, is capable of generating a high-titer virus and is incapable of effectively propagating in a human cell; the virus does not generate or only generate a progeny virus with very low titer but reserves the capability of DNA replication, RNA transcription and protein expression which are similar to those of the original Tiantan strain vaccinia virus. The virus has the advantages of obvious reduction of toxicity, high extraneous gene expression and good immune effect. The virus can be widely used for the research of extraneous gene expression and gene engineering vaccines.
Description
Technical field
The present invention relates generally to genetic engineering technique, particularly is the recombinant vaccine of carrier with the vaccinia virus.
Background technology
Vaccinia virus host is general to be enclosed extensively, breeding titre height, dispensable gene is many, foreign gene capacity big (can reach 25Kb in theory) duplicates non-carcinogenesis at endochylema, immunizing power is lasting, the long term human applicating history is arranged, and this unique biological and molecular biology proterties have caused that virologist and molecular biologist are developed to the very big interest of engineering carrier with it.Since nineteen eighty-two, existing 100 various exogenous genes obtain to express in vaccinia virus, the vaccinia virus recombinant of wherein expressing virus antigens such as hepatitis A, hepatitis B, measles, human papilloma, HIV, EBV has carried out human immunity experiment in a small amount, and vaccinia virus has become one of virus vector with fastest developing speed in recent ten years [1-4].Yet, strong local acne reaction and a small amount of severe complication sent out that vaccinia virus exists, as: carrying out property is sent out acne and vaccination encephalitis etc., and seriously having hindered with this virus is the widespread use of the recombiant vaccine of carrier.Therefore, reduce the toxic side effect of vaccinia virus, keep even strengthen that its immune effect has become the virologist and molecular biologist is paid close attention to and the major issue of research.
Early stage in research, reduce the vaccinia virus toxic side effect and mainly carry out from following two aspects.The one, some virulence associated gene of deletion vaccinia virus.Have data to show: some vaccinia virus dispensable genes are relevant with this viral virulence.Deletion vaccinia virus host range gene C 7L, K1L can reduce its fecundity [5-7] in different hosts (comprising the people) cell to some extent.Vaccinia virus immunosuppression genes involved is by its coding class lymphokine acceptor (TNFR, IL-1R, IFN γ R), interferon-resistant albumen (F3L, K3L), anticomplementary activity albumen VCP and serpin (SPI) etc., thereby disturb, weaken or block specificity or the nonspecific reaction of host directly or indirectly, strengthen its virulence [8] vaccinia virus.In addition, thymidine kinase gene J2R, ribonucleotide reductase gene I4L, F4L etc. strengthen virulence [8] by different links.The deletion said gene also can reduce the toxic side effect of vaccinia virus to some extent.The 2nd, on purpose introduce some and have the immunoreactive lymphokine of particular adjustments.Known, the generation of long-term (or throughout one's life) protective immunity depends on effective immunization with keeping, and to many mankind and Animal diseases, the immune response type that induces is depended in the acquisition of protectiveness.And the cytokine control [9] that immune response type or trend (cell or humoral immunization) are produced by the Th cell subtype mainly.Some studies show that: the vaccinia virus recombinant of the express cell factor such as IL-2, IL-5, IL-6, IL-10, IFN-γ, TNF etc. can reduce the virulence of vaccinia virus or optionally strengthen the immunity anti-[10-14] of exogenous antigen.
Yet above-mentioned research all is can carry out under the prerequisite of human body cell breeding keeping vaccinia virus, because could obtain effectively to exempt from by the classical inoculation method this fecundity of only withing a hook at the end.This has just produced the problem of two aspects: the one, for obtaining effective immunity, just must keep viral proliferation ability preferably, and this just is difficult to solve the serious complication of smallpox vaccination problem that the due to illness a large amount of breedings of poison cause; The 2nd, after the viral proliferation ability obviously reduced, toxicity and complication had reduced, but can not reach effective immunity.Therefore, in the framework of classical inoculation method, (promptly breed on one's body the experimenter and bring out effective immunity), be difficult under the prerequisite of guaranteeing effectively immunity, solve severe complication or toxicity problem that the vaccinia virus immunity causes by virus.
In order to address this problem, Taylor etc. 1988 [15] have proposed the notion of " non-replicating poxvirus vector ".They utilize the bird pox virus (FPV) of only breeding in the bird cell as carrier, have made up the recombinant Borrel virus of expressing the rabies virus envelope glycoprotein.This recombinant virus is not all bred in people and other non-avian cell, and is therefore as safe as a house, and can better express exogenous antigen, and causes that non-fowl kind is the effective immune response of animal, but a little less than the immune response.They found again in 1991, canary pox virus (CPV) with and attenuated vaccine strain ALVAC more effective than FPV, and carried out the short run human trial, obtained better effects [16,17].This carrier is fool proof, but a little less than the overall immune reaction, viral large usage quantity is brought certain difficulty to practical application.1992, Tartaglia etc. were used in this strategy in the vaccinia virus vector.They select as vaccine once in human body used vaccinia virus Copenhagen (Copenhagen) strain as setting out strain, remove 18 genes of this virus by gene recombination technology, successfully construct non-replicating Copenhagen (Copenhagen) strain vaccinia virus recombinant NYVAC[18 that loses fecundity at human archeocyte].Comprise 2 host range gene K1L and C7L and 16 virulence associated gene J2R, I4L, A56R, A26L, B13R/B14R, C1-6L, N1-2L and M1-2L in 18 genes removing.The some of them gene relates to duplicating of viral DNA, as basic pattern gene I4L of ribonucleotide reductase Dare etc.Basic proterties and the FPV of NYVAC are close with ALVAC, also are that virus vector is fool proof, but a little less than the immune response, viral large usage quantity is brought certain difficulty to practical application.Particularly be not suitable for those and need bring out the vaccine research that very strong immune response just can reach immunoprotection.
In sum, non-replicating poxvirus vector progress is brought and is watched from a height or a distance for fundamentally solving its toxic side effect.But existing this viroid carrier all has a common feature: the replication of virus vector DNA in human archeocyte is subjected to serious sound, just is blocked [16,18] at early stage its dna replication dna of virus infection.This be on the one hand its as the foolproof reason of vaccine for man carrier, also be a little less than its immune response on the other hand, viral large usage quantity, and in chick-embryo cell breeding titre also not high major reason.The solution of the problems referred to above might cause the generation of non-replicating recombinant poxvirus carrier of new generation safely and efficiently.
Summary of the invention
Goal of the invention has problems in order to solve in the above-mentioned prior art, order of the present invention is: utilize the stronger Chinese Tiantan strain vaccinia virus of immunogenicity to be the strain that sets out, be structured in the chick-embryo cell and can well breed, and it is irreproducible or breed very poor in human archeocyte, but kept the reproduction of defect type Tiantan strain vaccinia virus safely and efficiently of good dna replication dna, rna transcription and protein translation function, be used for the research of exogenous gene expression and recombinant vaccine.
Implementation principle the present invention can realize by following method and principle: 1. use the stronger Chinese Tiantan strain vaccinia virus of immunogenicity to be the strain that sets out; 2. by gene recombination technology, the gene that removal is relevant with host range and virulence when these genes of disappearance, avoids damaging the gene relevant with viral dna replication, rna transcription and protein translation as far as possible; 3. virus is binned in its chick-embryo cell that can breed and carries out, in chick-embryo cell and human archeocyte, screen simultaneously then, obtaining eugonic in chick-embryo cell, but irreproducible or breed very poor reproduction of defect type Tiantan strain vaccinia virus in human archeocyte; 4. again these viral genome characteristics, fecundity, virulence variation, gene expression dose and immune effect etc. are examined and determine, determined biology and molecular biological characteristics that it is basic.
Major advantage major advantage of the present invention is: owing to kept good dna replication dna, rna transcription and protein translation function, reproduction of defect type Tiantan strain vaccinia virus not only possesses the characteristics of general non-replicating poxvirus, but also possesses the unexistent exogenous gene expression height of general non-replicating poxvirus, good immune effect, advantage that viral consumption is few.
Summary of the invention is to the effect that of the present invention: use Chinese Tiantan strain vaccinia virus, with reference to Copenhagen strain vaccinia virus sequence [19], pass through gene recombination technology, to the K fragment, 26 genes relevant have been removed from the C of its genome HindIII restriction endonuclease mark with host range and virulence, totally 21,243 nucleotide sequences have successfully constructed the reproduction of defect type Tiantan strain vaccinia virus recombinant, called after NTV.21 of removal, 243 Nucleotide are positioned at Tiantan strain vaccinia virus genomic nucleic acid sequence the 5th, 497 to 26,741,26 genes removing are respectively: 19 of C1L to C19L genes in the C fragment, 2 of N1L to N2L genes in the N fragment, 2 of M1L to M2L genes in the M fragment, 3 of K1L to K3L genes in the K fragment.This virus has kept good fecundity in chick-embryo cell, can produce infectious titer, in human archeocyte, can not effectively breed, do not produce or only produce the progeny virus of extremely low titre, but the dna replication dna close, rna transcription and protein translation ability have been kept with former Tiantan strain vaccinia virus, virus virulence obviously descends, exogenous gene expression height, good immune effect.Reproduction of defect type Tiantan strain vaccinia virus recombinant NTV is a kind of new virus carrier that can be used for exogenous gene expression and recombinant vaccine research.
Description of drawings
Accompanying drawing 1 explanation
This figure is that recombinant plasmid pNeoCK and pNeoCKLac make up and structural representation. Bp is nucleotide base pair among the figure, and kb is Thousand nucleotide bases pair, J6R and P11 and P25 are the vaccinia virus promoter, BamHI, SacI, KpnI, SmaI, EcoRI, SalI, XbaI, XhoI, BglII, PstI and SstI are restriction enzyme, and Neo is neomycin gene, and Amp is amino Parasiticin resistant gene, Lac are galactosidase gene, and Klenow is dna polymerase gene.
The structure of plasmid pNeoCK and pNeoCKLac:
Through the correct C ' that is used for the Tiantan strain vaccinia virus homologous recombination of pcr amplification, Cloning and sequencing (530bp) and K ' (370bp) Fragment connects and is cloned in the pBR-Neo plasmid in direction of poxvirus genome group by it, obtains plasmid pNeoCK. Will Lac gene with the vaccinia virus promoter in the pGFM-34XRLac plasmid cuts out rear clone to C and the K of pNeoCK plasmid through restriction endonuclease Obtain plasmid pNeoCKLac among the cloning site BamHI between the fragment.
The explanation of plasmid structure:
Plasmid pBR-Neo: this plasmid be classical plasmid pBR322 after restriction endonuclease EcoRI and BglII hydrolysis, will contain replication orgin and aminobenzylpenicillin antibiosis (Amp
r) carrier segments (2.3kb) and vaccinia virus J6R promoter fragment (0.1kb) be connected reorganization with neomycin gene Neo (1kb) and form, the plasmid size is 3,400bp (or 3.4kb).
Plasmid pGEM-34XRLac: this plasmid is to insert vaccinia virus P11 and P25 bidirectional promoter fragment (0.2kb) and Lac gene (4.85kb) in the multiple clone site of classical pGEM plasmid (2.95kb) to make up and form, and the plasmid size is 8,000bp (or 8.0kb).
Plasmid pNeoCK: this plasmid is to insert by the vaccinia virus gene prescription to the homologous recombination sequence C that connects together at pBR-Neo plasmid multiple clone site SmaI and SalI ' (0.530bp) and K ' (0.370bp) make up and form.Except that C ' and K ' composition (what be designated as C in the plasmid is C ', and what be designated as K is K '), other composition of this plasmid is all identical with pBR-Neo, and the plasmid size is 4,300bp (or 4.3kb).
Plasmid pNeoCKLac: this plasmid is to be with Lac gene (4.85kb) structure of vaccinia virus promotor P11 and P25 (0.2kb) to form at the C of pNeoCK plasmid and the intersegmental multiple clone site BamHI insertion of K sheet.Except the Lac gene of band vaccinia virus promotor, other composition is all identical with pNeoCK, and the plasmid size is 9,350bp (or 9.4kb).
Accompanying drawing 2 explanations
This figure is the comparison of reproduction of defect type Tiantan strain vaccinia virus recombinant VHFIL2 Δ CKSS1 and rf the Temple of Heaven strain vaccinia virus recombinant VHFIL2SS1 foreign gene SS1 expression level.Column type figure represents the height of hepatitis B surface antigen SS1 expression level among the figure, solid post figure represents replication-defective virus VHFIL2 Δ CKSS1, open tubular column figure represents duplicating virus VHFIL2SS1, the viral number (pfu/cell) of each cell infection of numeral on the column diagram.(CEF is former generation chick-embryo cell to the different cell of the letter and number representative of X-coordinate below, and 143 is people's myeloma cell 143TK
-, 2BS is the human embryo lung (HEL) primary cell, 293 is the human embryo kidney (HEK) passage cell, SMMC7721 is a human liver cancer cell), the ordinate zou numeral detects the count per minute value (cpm) that hepatitis B surface antigen SS1 expresses for radioimmunity.As seen, use the experiment of different cells and different virus infective dose to show among the figure, between the replication defect type of identical infective dose and the rf vaccinia virus recombinant, the expression level of exogenous antigen SS1 does not have obvious difference.
Accompanying drawing 3 explanations
This figure is the genome structure synoptic diagram of reproduction of defect type Tiantan strain vaccinia virus NTV.The genome of reproduction of defect type Tiantan strain vaccinia virus NTV is represented with thick horizontal line in the circle, the segmental Name ﹠ Location that capitalization English letter on the genome produces after by the hydrolysis of Hind III restriction endonuclease for this genome, the artificial for simplicity herein weak point of drawing in the interval that oblique stroke " // " expression A between A and B and the intersegmental reality of B sheet are very big, dotted line in the genome is the position of gene in genome of disappearance, and bp is that nucleotide base is right.Removing the gene total length is 21,243 Nucleotide, be positioned at Tiantan strain vaccinia virus genome the 5th, 497 to the 26th, 741 Nucleotide contain 26 genes altogether, are respectively: segmental 19 genes of Tiantan strain vaccinia virus HindIII restriction endonuclease C (TC19-1L), segmental 2 genes of N (TN1-2L), segmental 3 genes of segmental 2 genes of M (TM1-2L) and K (TK1-3L).
Embodiment
The present invention is further elaborated below in conjunction with chart.
One. select Tiantan strain vaccinia virus as the original strain of transforming usefulness.
It is original strain that the present invention uses Tiantan strain vaccinia virus, and this strain is provided by Chinese medicine and biological products assay institute, lot number 7601.
Tiantan strain vaccinia virus is once to be extensive use of 50 years in China, is used for the vaccine strain that vaccination is a preventive against smallpox.Have good immune effect in human body, but vaccination back skin reaction is bigger, the millionth probability of having an appointment produces vaccination encephalitis, whole body is sent out severe complications such as acne and vaccina gangrenosa.Surpass several hundred million person-times because this strain has used at human body, its immune effect and side effect are all more clearly.The whole genome sequence data of this strain has deposited in the international gene pool, is numbered Gene Bank, No.AF095689.
Because the biological character and the molecular biology characteristics of Tiantan strain vaccinia virus are clearer, it is again the vaccine strain of the distinctive prevention smallpox of China, use modern molecular biology technique that it is transformed, the vaccine strain of new generation of good immune effect, few side effects might be obtained, and engineering carrier safely and efficiently may be become.
Two. structure can be removed the recombinant plasmid pNeoCK and the pNeoCKLac of Tiantan strain vaccinia virus virulence associated gene by homologous recombination
(1) the definite important gene relevant that will remove with the Tiantan strain vaccinia virus virulence
Over nearly 20 years, vaccinia virus biology and Progress on Molecular Biology are very fast, and many genes relevant with virus virulence are clear and definite, as genes [8] such as virus replication, host range, immunosuppression.According to these progress, U.S. scientist Tartaglia etc. use vaccinia virus Copenhagen strain (Copenhagen Strain, Gene Bank, No.M35027) be original strain, by removing its virulence relevant 18 genes (J2R, I4L, A56R, A26L, B13R/B14R, C1-7L, N1-2L, M1-2L and K1L), obtained that a strain can be bred but in the irreproducible non-replicating vaccinia virus of human body in chick-embryo cell.Though this strain safety, immune effect is very weak.Analyzing reason may be: related to the gene relevant with viral dna replication in the gene of disappearance, as basic pattern gene I4L of ribonucleotide reductase Dare etc., cause the strain of new generation in people's cell, not have duplicating of DNA, cause the exogenous gene expression level low, the virus consumption is big, and immune effect is poor.
Can be when transforming vaccinia virus, make it lose the ability of breeding generation infectious virus in people's cell on the one hand, keep its efficient exogenous gene expression and good immune effect on the other hand? data according to vaccinia virus biology and molecular biology research, we have taked the transformation thinking different with existing document, promptly only remove in the vaccinia virus and human host's scope, the gene that immunosuppression and other virulence are relevant and do not go to transform and viral dna replication, rna transcription and the synthetic relevant gene of albumen, with obtain a kind of can good virus of proliferation on chick-embryo cell, but on human archeocyte, only keep good dna replication dna, rna transcription and albumen synthesis capability, but can not pack the New Replication defective type vaccinia virus that produces progeny virus, this virus will possess characteristics safely and efficiently.On basis to vaccinia virus biology and molecular biological analysis research, the present invention determines between the C and K fragment of Tiantan strain vaccinia virus genome Hind III restriction endonuclease mark, remove 21,243 Nucleotide, the Nucleotide of removing is positioned at genomic nucleic acid sequence the 5th, 497 to 26,741, contain 26 genes altogether, be respectively: 19 of C1L to C19L genes in the C fragment, 2 of N1L to N2L genes in the N fragment, 2 of M1L to M2L genes in the M fragment, 3 of K1L to K3L genes in the K fragment.
(2) the definite homologous recombination sequence that can remove the important virulence gene of Tiantan strain vaccinia virus
According to 26 genes will removing, determine that 26 gene left side homologous recombination sequences are 522 Nucleotide, be positioned at 5,497 Nucleotide of the 4th, 976 Nucleotide to the of Tiantan strain vaccinia virus genome, called after C ' fragment, right side homologous recombination sequence is 367 Nucleotide, is positioned at the 26th, 741 Nucleotide to the 27 of Tiantan strain vaccinia virus genome, 107 Nucleotide, called after K ' fragment.According to homologous recombination C ' and K ' fragment nucleotide sequence, segmental primer 1 of pcr amplification C ' and primer 2 have been determined, the amplification segmental primer 3 of K ' and 4.The nucleotide sequence of C ' and K ' fragment nucleotide sequence and primer 1,2,3,4 and the position on C ' and K ' fragment thereof are as follows:
1.C ' sequence location of fragment homologous recombination sequence and primer 1 and 2
(1) C ' fragment homology primer sequence and the comparison of initial and terminating nucleotide position in the Temple of Heaven strain (TC20L, TC19L) and Copenhagen strain (C23L, C12L) vaccinia virus gene group thereof:
Primer 15 '-----G
GGATGATTYTGAAGACT-------3 '
4976bp 5’-----A
GGATGATTGCTGAAGACT------3’
4993bp?TC20L(4274bp-5008bp)
4976bp 5’-----A
GGATGATTGCTGAAGACT------3’4993bp?C23L(4274bp-5008bp)
Primer 23 '-----
GGACACCACAGAAACCTAGG-----5 '
5484bp 5’-----
CCTGTGTTGTCTTTAATCAT-----3’
5497bp?TC19L(5444bp-6505bp)
12712bp?5’-----
CCTGTGGTGTCTTTAATCAT-----3’12725bp?C12L(12672bp-13733bp)
(2) sequence of C ' fragment homologous recombination and primer 1 and primer 2 position therein:
Primer 1 g
4976
ggcaggcacatgeatgccaggacgatatattgtttcatgattgctat?tgattgagta
5051?ctgttcttta?tgattctact?tccttaccgt?gcaataaatt?agaatatatt?ttctactttt?acgagaaatt
5121?aattattgta?tttatgggtg?aaaaacttac?tataaaaagc?gggtgggttt?ggaattagtg?atcagtttat
5191?gtatatcgca?actaccgggc?atatggctat?cgacatcgag?aacattaccc?acatgataag?agattgtatc
5261?agtttcgtag?tcttgagtat?tggtattact?atatagtata?tagatgtcgc?ccactagagt?tactgtctcc
5331?gaatgcggca?tgataagtat?cattctttgc?tttcgttaac?tgtttagttt?atactattac?tatttgtaat
5401?atttagacat?agataaacgt?gataaaagtc?tatttgttta?tatttattgc?ggatagcagt?atttccctat
5471?aaaaagtata?cgt
Annotate: cat is the initiator codon of TC20L in the sequence, and tta is the terminator codon of TC19L
2.K ' sequence location of fragment homologous recombination sequence and primer 3 and 4
(1) K ' fragment homology primer sequence and the comparison of initial and terminating nucleotide position in the Temple of Heaven strain (TK3L, TK4L) and Copenhagen strain (K2L, K3L) vaccinia virus gene group thereof:
Primer 35 '-----GGATCCTA
GACACTGAACAC------3 '
26741bp 5’-----GATAGGCG
GACGCTGAACAC------3’
26752bp?TK3L(25672bp-26781bp)
30265bp 5’-----GATAGGTA
GACACTGAACAC------3’30284bp?K2L?(29204bp-30313bp)
Primer 43 '-----
TCGTAGAACAATTGCAGCTG------5 '
27093bp 5’-----
AGCATCTTGTTAACGGGCTC------3’
27107bp?TK4L(26831bp-27097bp)
30625bp 5’-----
AGCATCTTGTTAACGGGCTC------3’30638bp?K3L?(30363bp-30629bp)
(2) sequence of K ' fragment homologous recombination and primer 3 and primer 4 position therein:
Primer 3ggatccta
26741
gctaacga
26761?tagtatcaat?aacgcaatca?tgattttatg?gtattaataa?ttaaccttat?ttttatgttc?ggtataaaaa
26831?ttattgatgt?ctacacatcc?ttttgtaatt?gacatctata?tatccttttg?tataatcaac?tctaatcact
26901?ttaactttta?cagttttccc?taccagttta?tccctatatt?caacatatct?atccatatgc?atcttaacac
26971?tctctgccaa?gatagcttca?gagtgaggat?agtcaaaaag?ataaatgtat?agagcataat?ccttctcgta
27041?tactctgccc?tttattacat?cacccgcatt?gggcaacgaa?taacaaaatg?ca
gggc?t
c
Annotate: ca t and cat are respectively the initiator codon of TK3L and TK4L, and tta is the terminator codon of TK4L
(3) can remove the recombinant plasmid pNeoCK of the important virulence gene of Tiantan strain vaccinia virus and the structure of pNeoCKLac
Use aforementioned primer 1 and 2, and primer 3 and 4, be template with the Tiantan strain vaccinia virus virus genom DNA, pcr amplification obtains corresponding C ' and K ' dna fragmentation.These two fragments are inserted among the plasmid pBR-Neo, and the plasmid called after pNeoCK of acquisition sees Fig. 1 for details.This plasmid can be used for and the Tiantan strain vaccinia virus homologous recombination, removes 26 intersegmental genes of this virus C and K sheet, and 21,243 Nucleotide of length overall (seeing this explanation two () for details) obtain the desired reproduction of defect type Tiantan strain vaccinia virus NTV of the present invention.
More smooth reliable for what viral retrofit work was carried out, on pNeoCK plasmid basis, galactosidase gene (being called for short Lac) is inserted in the pNeoCK plasmid, obtain to contain the recombinant plasmid of Lac gene, called after pNeoCKLac sees Fig. 1 for details.When transforming vaccinia virus Tiantan strain, can at first use this plasmid and viral homologous recombination, removing virus 21, in the time of 243 nucleotide fragments, inserted the Lac gene in this disappearance district, acquisition can be expressed the reproduction of defect type Tiantan strain vaccinia virus NTVLac of Lac gene, and this virus plaque is blue under X-gal dyeing, easily distinguishes mutually with original strain plaque colourless or title white.And then carry out the homologous recombination second time with pNeoCK and NTVLac virus, and remove the Lac gene in the NTVLac virus, obtain only to lack the reproduction of defect type Tiantan strain vaccinia virus NTV of 21,243 nucleotide fragments.This virus plaque is colourless (also claiming hickie) under X-gal dyeing, easily differentiate mutually with blue plaque.
Three. the structure of reproduction of defect type Tiantan strain vaccinia virus NTV
Described in this explanation two (three), at first use the reorganization of pNeoCKLac plasmid and Tiantan strain vaccinia virus, acquisition has lacked 21,243 nucleotide fragments can be expressed the reproduction of defect type Tiantan strain vaccinia virus NTVLac of Lac gene again, and then use pNeoCK plasmid and virus N TVLac to recombinate, remove the Lac gene, final acquisition has only lacked the reproduction of defect type Tiantan strain vaccinia virus NTV of C and intersegmental 21,243 nucleotide fragments of K sheet.Specific implementation process such as following:
(1) makes up vaccinia virus strain and the cell that NTV uses
1. Tiantan strain vaccinia virus: this strain is once to be widely used in the vaccine strain that vaccination is a preventive against smallpox in China, identifies that by Chinese medicine and biological products institute provides lot number 7601.
2. cell: former generation chick-embryo cell (CEF), human embryonic lung cell (2BS) is available from Beijing Biological Product Inst..143TK
-Cell (human myeloma cell) is so kind as to give by the Shanghai biochemical research.
(2) structure of reproduction of defect type Tiantan strain vaccinia virus NTVLac and screening
As reorganization virus, pNeoCKLac is the reorganization plasmid, adopts G418-Neo resistance and indigo plant/hickie double-tagging to screen with Tiantan strain vaccinia virus.The virus liquid of recombinating is cultivated 1-2 generation in containing the growth media of G418, do not containing keeping continuation cultivation 1-3 generation in the liquid of G418 then, again with the nutrient agar medium shop spot that contains X-gal, the isolated locus coeruleus of picking at random, in 3 generations of continuous single spot purifying, obtain that plaque be the reproduction of defect type Tiantan strain vaccinia virus NTVLac of blueness under X-gal dyeing.
(3) structure of reproduction of defect type Tiantan strain vaccinia virus NTV and screening
Use and obtain NTVLac viral identical structure and triage techniques, use viral as reorganization with NTVLac, pNeoCK is the reorganization plasmid, adopt G418-Neo resistance and the dual screening index of indigo plant/hickie, the isolated white plaque of picking at random, continuous purification is after 3 generations, and acquisition plaque under X-gal dyeing is the reproduction of defect type Tiantan strain vaccinia virus NTV of white.
Four. the evaluation of reproduction of defect type Tiantan strain vaccinia virus NTV
(1) genome of NTV calibrating
1. conventional PCR identifies with order-checking.Lacked 21,243 Nucleotide because the genomic C of NTV and K sheet are intersegmental, thus when using primer 1 and 4 (seeing this explanation two (two) for details) to carry out the PCR evaluation, can amplify the characteristic fragment of about 900 Nucleotide, and original Tiantan strain vaccinia virus (abbreviation TK
+) containing 21,243 nucleotide sequences between C and K, conventional PCR can't amplify this big fragment.Therefore, use primer 1 of the present invention and 4 pairs of NTV genomes to carry out conventional pcr amplification, produce the nucleotide fragments of about 900 base pairs, for identifying the key index of NTV disappearance C and intersegmental 21,243 Nucleotide of K sheet.These 900 Nucleotide are checked order the nucleotide sequence 5 ' that is joined together to form again behind 21,243 Nucleotide of the intersegmental disappearance of C and K sheet in the sequence
CCTGTGGTGT CTTTGGATCCGGGTACCGAGCTCGAATTGGATCCTA
GACACTGAACAC3 ', be binned in together characteristic sequence for identifying behind NTV disappearance C and intersegmental 21,243 Nucleotide of K sheet again by design.The restriction enzyme site that primer 2 that the present invention that the nucleotide sequence of two underscores is complementary after being respectively and reconnecting with C and K fragment in this sequence uses and 3 sequence, the sequence of no underscore add during for design.
2. restriction enzyme is identified.Use restriction enzyme HindIII hydrolyzing N TV genomic dna, as seen formerly contain the C fragment of about 18,000 Nucleotide and the K fragment of about 4,500 Nucleotide all disappears, reappeared a new segment that reconnects about 9,000 Nucleotide that together form by remaining C and K fragment.This fragment also can be used as identifies the important indicator that connects together again by design again behind NTV disappearance C and intersegmental 21,243 Nucleotide of K sheet.
(2) growth characteristics of NTV calibrating
1.NTV the comparison of continuous passage time breeding titre on different cells: use the Tiantan strain vaccinia virus of 5,000 plaque forming units (pfu) and NTV to infect a big square vase (about 1 * 10 respectively
7Cell) CEF, 143TK
-Behind (human archeocyte) and the 2BS cell (human archeocyte), continuous passage 3 times, in the titre of each generation virus of titration on the CEF cell on different cells.As shown in table 1, NTV eugonic and effectively going down to posterity on the CEF cell, irreproducible and go down to posterity on the 2BS cell, at 143TK
-Can only produce the virus of low titre in the cell.
Table 1.NTV and Tiantan strain vaccinia virus (TK
+) on different cells during continuous passage the breeding titre relatively
2.NTV the comparison of viral plaque form on different cells: respectively with the Tiantan strain vaccinia virus of 5000pfu and CEF, the 143TK of a big square vase of NTV infection
-With the 2BS cell, observe viral plaque form.NTV is at 143TK
-With all can not form the typical viral plaque the same on these two kinds of human archeocytes of 2BS with former Tiantan strain vaccinia virus.
(3) virulence of NTV calibrating
1. virulence in the rabbit: as shown in table 2, NTV compares with Tiantan strain vaccinia virus, and the red and swollen and skin of rabbit decreases habituation, reaction peak period shortening 5-6 days, and virulence obviously reduces.
Table 2.NTV and Tiantan strain vaccinia virus (TK
+) in the comparison of rabbit endogenous toxic material power
Be the relation of clear and definite viral proliferation titre and dermatosis, carried out the interior injection site of rabbit viral proliferation and dynamic observed.The result is as shown in table 3, and the NTV virus titer of injection site first day is the highest, by day descending, can not detect virus on the 6th day, and Tiantan strain vaccinia virus reaches every injection point 4.2 * 10 at the 6th day subsequently
6Virus.This conforms to red and swollen variation tendency in the two rabbit, illustrates that it is the major reason that its virulence descends that NTV does not breed in rabbit.
Table 3.NTV and Tiantan strain vaccinia virus (TK
+) in the rabbit inoculation position the viral proliferation titre relatively
2. virulence in the suckling mouse brain: in the NTV and Tiantan strain vaccinia virus inoculation suckling mouse brain with the difference amount, judge virus virulence by the lowest dose level (LD50) that causes 50% suckling mouse death.The result shows that the LD50 of Tiantan strain vaccinia virus is 15 viruses, and the LD50 of NTV is greater than 1.5 * 10
6Virus, promptly the virulence of NTV in suckling mouse brain is than Tiantan strain vaccinia virus at least 5 logarithms that descended.
(4) calibrating of foreign gene expression level and immune effect in NTV
With Measles virus H (hemagglutinin) and F (blood lysin) gene, and hepatitis B virus SS1 (hepatitis B virus surface antigen that contains PreS1) gene is recombinated in NTV or the former Tiantan strain vaccinia virus simultaneously, made up the replication defect type reorganization Tiantan strain vaccinia virus VHFIL2 Δ CKSS1 and the rf reorganization Tiantan strain vaccinia virus VHFIL2SS1 that can express Measles virus H gene, F gene and hepatitis B virus SS1 gene simultaneously, external source gene expression dose and immune effect in these two kinds of recombinant viruses have been examined and determine.The result is as follows:
1. the calibrating of foreign gene expression level in NTV
As shown in Figure 2, in the replicative cycle, no matter infect dissimilar cells with low titre recombinant virus (average 0.1 virus of each cell) or high titre recombinant virus (average 10 viruses of each cell), hepatitis B virus SS1 expression of gene level does not all have significant difference between replication defect type (VHFIL2 Δ CKSS1) and rf (VHFIL2SS1) the Temple of Heaven strain vaccinia virus recombinant.
2.NTV the immune effect of middle expressed exogenous gene calibrating
As shown in table 4, same dose virus (every rabbit 10
7Virus) under the intramuscular injection rabbit epidemic situation condition, replication defect type (VHFIL2 Δ CKSS1) and rf (VHFIL2SS1) the Temple of Heaven strain vaccinia virus recombinant bring out between the vaccinia virus antibody of carrier self and at all not having significant difference between the Measles virus H antibody (looking into hemagglutination inhibition antibody HI) of external source and the F antibody (molten inhibition antibody HLI has a blood test).
The comparison of antibody response after table 4. recombinant virus VHFIL2 Δ CKSS1 and the VHFIL2SS1 intramuscular injection
Annotate: A. rf the Temple of Heaven strain vaccinia virus recombinant RVJVHFIL2SS1
B. reproduction of defect type Tiantan strain vaccinia virus recombinant RVJVHFIL2 Δ CKSS1
Five. reproduction of defect type Tiantan strain vaccinia virus NTV genome structure and principal feature brief summary
Use Chinese Tiantan strain vaccinia virus, with reference to Copenhagen strain vaccinia virus sequence [19], pass through gene recombination technology, to the K fragment, 26 genes relevant have been removed from the C of its genome HindIII restriction endonuclease mark with host range and virulence, totally 21,243 nucleotide sequences have successfully constructed the reproduction of defect type Tiantan strain vaccinia virus recombinant, called after NTV.21 of removal, 243 Nucleotide are positioned at Tiantan strain vaccinia virus genomic nucleic acid sequence the 5th, 497 to 26,741,26 genes removing are respectively: 19 of C1L to C19L genes in the C fragment, 2 of N1L to N2L genes in the N fragment, 2 of M1L to M2L genes in the M fragment, 3 of K1L to K3L genes in the K fragment.Fig. 3 is seen in the title and the position of its genome structure and missing gene.This virus has kept good fecundity in chick-embryo cell, can produce infectious titer, in human archeocyte, can not effectively breed, do not produce or only produce the progeny virus of extremely low titre, but the dna replication dna close, rna transcription and protein translation function have been kept with former Tiantan strain vaccinia virus, virus virulence obviously descends, exogenous gene expression height, good immune effect.Reproduction of defect type Tiantan strain vaccinia virus recombinant NTV is a kind of new virus carrier that can be used for exogenous gene expression and recombinant vaccine research.
Reference:
1.Panicali?D?and?Paoletti?E.1982.Construction?of?poxviruses?as?cloning?vectors：Insertion?of?the?thymidinekinase?Gene?from?herpes?simple?virus?into?the?DNA?of?infectious?vaccinia?virus.Proc?Natl?Acad?Sci?USA.79：4927-4931
2.Perkus?M?E，Piccini?A，Lipinskas?B?R，et?al.1985.Recombinant?Vaccinia?virus：Immunizationagainst?multiple?pathogens.Science.229：981-983
3.Konishi?E?and?Mason?P?W.1993.Proper?maturation?of?the?Japanese?encephalitis?virus?envelopeglycoprotein?requires?cosyntheses?with?the?premembrane?protein.J?Virol.67：1672-1675
4. Lou Yuan plum, Liu Qifu, Zhu Dezhong etc.1995, the antigen presentation of encephalitis vaccinia virus recombinant and immune protective thereof.The virus journal, 11 (4): 317-321
5.Perkus?M?E，Limbach?k?and?Paoletti?E.1989，Cloning?and?expression?offoreign?genes?in?vaecinia?virus?using?ahost?range?selection?system.J?Virol.64：3829-3836
6.Perkus?M?E，Goebel?S?J，Davis?S?W，et?al.1990，Vaccinia?virus?host?range?genes.Virology.179：276-286
7.Sutter?G，Ramsey-Ewing?A，Rosales?R，et?al.1994，Stable?expression?of?the?vaccinia?virus?K1L?gene?in?rabbitcells?complements?the?host?range?defects?of?a?vaccinia?virus?mutant.J?virol.68(7)：4109-4116
8. Lou Yuan plum, Ruan Li, Zhu Jiming.1996, the progress of vaccinia virus virulence associated gene.The virus journal, 12:88-96
9.Mosmann?T?R?and?Coffman?R?L.1989，TH1?and?TH2?cells：different?patterns?of?Lymphokine?secretion?leadto?different?functional?properties.Annu?Rev?Lmmunol.7：145-173
10.Flexner?C，Murphy?B?R，London?W?T，1990，Attenuation?and?immunogenicity?imprimates?of?vaccinia?virusrecombinants?expressing?human?interleukin?2.Vaccine.8：17-24
11.Kohonen-Corish?M?R?J，Long?N?J?C，Woodhams?C?E，et?al.1990，Immuno-deficient?mice?recover?frominfection?with?vaccinia?virus?expressing?interferon?gamma.Eur?J?Immunol.20：157-163
12. Ruan Li, Yan Xiujun, Zhu Jiming etc.1993, express hepatitis B vaccinia virus recombinant (RVJ123KIL-2) a small amount of anthroposcopy result of IL-2, the Ins of Virology.Medicine is evaluated declaration material.
13.Kotwal?G?J，Hugin?A?W，and?Moss?B.1989，Mapping?and?insertional?mutagenesis?of?a?vaccinia?virus?geneencoding?a?13800-Da?protein.Virology.171：579-587.
14.Buller?R?M?L?and?Palumbo?G?J.1991，Poxvirus?pathogenesis.Microbiol?Rev.55：80-122.
15.Taylor?J，Weinberg?R，Languet?B，et?al.1988，Recombinant?fowlpox?virus?inducing?protective?immunity?innon-ayian?species.Vaccine.6：497-503
16.Tarlaglia?J，Cox?W?I，Taylor?J，et?al.1992，Live?vectors?as?vaccines：highly?attenuated?poxvirus?vectors.AIDSResearch?and?Human?Retrovirus.8(8)：1445-1447
17.Taylor?J，Trimarchi?C，Weinbery?R，et?al.1991，Efficacy?studies?on?a?canarypox-rabies?recombinant?virus.Vaccine.9：190-193
18.Tartaglia?J，Perkus?M?E，Taylor?J，et?al.1992，NYNAC：A?highly?attenuated?strain?of?vaccinia?virus.Virology188：217-232
19.Goebel?S?J，Johnson?G?P，Perkus?M?E，et?al.1990.The?complete?DNA?sequence?of?Vaccinia?virus.Virology.179：247-266.
Appendix: related nucleotide sequences list machine readable form copy content in the specification sheets
＜110〉China Sickness Prevention Control Center Virus Disease Prevention Control Institute
＜120〉reproduction of defect type Tiantan strain vaccinia virus
<160>6
<210>1
<211>522
<212>DNA
＜213〉vaccinia virus Tiantan strain (vaccinia virus Tian Tan strain) C ' fragment homologous recombination sequence
<400>1
ggatgattgc?tgaagactgg?caggcacatg?catgccagga?cgatatattg?tttcatgatt 60
gctattgatt?gagtactgtt?ctttatgatt?ctacttcctt?accgtgcaat?aaattagaat?120
atattttcta?cttttacgag?aaattaatta?ttgtatttat?gggtgaaaaa?cttactataa?180
aaagcgggtg?ggtttggaat?tagtgatcag?tttatgtata?tcgcaactac?cgggcatatg?240
gctatcgaca?tcgagaacat?tacccacatg?ataagagatt?gtatcagttt?cgtagtcttg?300
agtattggta?ttactatata?gtatatagat?gtcgcccact?agagttactg?tctccgaatg?360
cggcatgata?agtatcattc?tttgctttcg?ttaactgttt?agtttatact?attactattt?420
gtaatattta?gacatagata?aacgtgataa?aagtctattt?gtttatattt?attgcggata?480
gcagtatttc?cctataaaaa?gtatacgtcc?tgtgttgtct?tt 522
<210>2
<211>367
<212>DNA
＜213〉vaccinia virus Tiantan strain (vaccinia virus Tian Tan strain) K ' fragment homologous recombination sequence
<400>2
gacgctgaac?acgctaacga?tagtatcaat?aacgcaatca?tgattttatg?gtattaataa 60
ttaaccttat?ttttatgttc?ggtataaaaa?ttattgatgt?ctacacatcc?ttttgtaatt?120
gacatctata?tatccttttg?tataatcaac?tctaatcact?ttaactttta?cagttttccc?180
taccagttta?tccctatatt?caacatatct?atccatatgc?atcttaacac?tctctgccaa?240
gatagcttca?gagtgaggat?agtcaaaaag?ataaatgtat?agagcataat?ccttctcgta?300
tactctgccc?tttattacat?cacccgcatt?gggcaacgaa?taacaaaatg?caagcatctt?360
gttaacg 367
<210>3
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design according to the segmental nucleotide sequence of vaccinia virus C ', as polymerase chain reaction (PCR) the amplification segmental upstream primer 1 of vaccinia virus Tiantan strain C ' (primer 1), 18 Nucleotide and C ' fragment 5 ' terminal sequence coupling in the primer sequence, unmatched 1 Nucleotide is as restriction endonuclease SmaI site.
<400>3
gggatgattg?ctgaagact
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design according to the segmental nucleotide sequence of vaccinia virus C ', as polymerase chain reaction (PCR) the amplification segmental downstream primer 2 of vaccinia virus Tiantan strain C ' (primer 2), 14 Nucleotide and C ' fragment 3 ' terminal sequence coupling in the primer sequence, unmatched 6 Nucleotide are as restriction endonuclease BamHI site.
<400>4
ggatccaaag?acaccacagg
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
Appendix: related nucleotide sequences list machine readable form copy content in the specification sheets
＜223〉design according to the segmental nucleotide sequence of vaccinia virus K ', as polymerase chain reaction (PCR) the amplification segmental upstream primer 3 of vaccinia virus Tiantan strain K ' (primer 3), 12 Nucleotide and vaccinia virus Copenhagen strain K ' fragment 5 ' terminal sequence coupling in the primer, wherein 11 with vaccinia virus Tiantan strain K ' fragment 5 ' terminal sequence coupling, unmatched in addition 8 Nucleotide are as restriction endonuclease BamHI site and auxiliary sequencel.
<400>5
ggatcctaga?cactgaacac
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉design according to the segmental nucleotide sequence of vaccinia virus K ', as polymerase chain reaction (PCR) the amplification segmental downstream primer 4 of vaccinia virus Tiantan strain K ' (primer 4), 15 Nucleotide and K ' fragment 3 ' terminal sequence coupling wherein, unmatched 5 Nucleotide are as restriction endonuclease SalI site.
<400>6
gtcgacgtta?acaagatgct
Claims (1)
- One kind in human archeocyte, can not effectively breed but in chick-embryo cell eugonic reproduction of defect type Tiantan strain vaccinia virus NTV, it is characterized in that: the original strain of NTV is a Tiantan strain vaccinia virus, compare the NTV genome with original strain and removed Hind III restriction endonuclease C to the intersegmental genome the 5th that is positioned at of K sheet, 497 to the 26th, between 741 21,243 nucleotide sequences, contain TC1L to TC19L in the nucleotide sequence of removing, TN1L to TN2L, TM1L to TM2L and TK1L to TK3L be 26 genes altogether, NTV is eugonic in chick-embryo cell, can produce infectious titer, in human archeocyte, can not effectively breed, do not produce or only produce the progeny virus of extremely low titre, but kept the dna replication dna close with original strain, rna transcription and albumen synthetic ability, the exogenous gene expression height, good immune effect, virulence obviously descends.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5077213A (en) * | 1988-11-10 | 1991-12-31 | Shanghai Institute Of Biochemistry, Chinese Academy Of Sciences | Recombinant vaccinia virus |
| CN1560248A (en) * | 2004-03-10 | 2005-01-05 | 中国疾病预防控制中心性病艾滋病预防 | Tiantan remocined vaccine virus of IFN-alpha receptor gene (B8R) deletion and application thereof |
-
2006
- 2006-03-09 CN CNB2006100568000A patent/CN100393872C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5077213A (en) * | 1988-11-10 | 1991-12-31 | Shanghai Institute Of Biochemistry, Chinese Academy Of Sciences | Recombinant vaccinia virus |
| CN1560248A (en) * | 2004-03-10 | 2005-01-05 | 中国疾病预防控制中心性病艾滋病预防 | Tiantan remocined vaccine virus of IFN-alpha receptor gene (B8R) deletion and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| 痘苗病毒天坛株非必需区TA25R…TA36L的两步重组表达载体的构建. 邹艳等.病毒学报，第14卷第2期. 1998 |
| 痘苗病毒天坛株非必需区TA25R…TA36L的两步重组表达载体的构建. 邹艳等.病毒学报，第14卷第2期. 1998 * |
| 缺失IFN-γ受体基因的天坛株减毒重组痘苗病毒载体的构建. 黄薇等.中华实验和临床病毒学杂志，第18卷第1期. 2004 |
| 缺失IFN-γ受体基因的天坛株减毒重组痘苗病毒载体的构建. 黄薇等.中华实验和临床病毒学杂志，第18卷第1期. 2004 * |
| 表达乙型肝炎病毒表面抗原的非复制型重组痘苗病毒疫苗株RVJ123ΔCK的构建及其生物学性状鉴定. 陆柔剑等.病毒学报，第19卷第1期. 2003 |
| 表达乙型肝炎病毒表面抗原的非复制型重组痘苗病毒疫苗株RVJ123ΔCK的构建及其生物学性状鉴定. 陆柔剑等.病毒学报，第19卷第1期. 2003 * |
| 非复制重组痘苗病毒天坛株C-K缺失区表达载体的构建及重组病毒生物学性状的研究. 郭斐等.病毒学报，第17卷第1期. 2001 |
| 非复制重组痘苗病毒天坛株C-K缺失区表达载体的构建及重组病毒生物学性状的研究. 郭斐等.病毒学报，第17卷第1期. 2001 * |
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