CN102206679A - Shuttle vector of vaccinia virus and its application - Google Patents
Shuttle vector of vaccinia virus and its application Download PDFInfo
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Abstract
The invention relates to a shuttle vector of vaccinia virus used for constructing recombinant vaccinia virus. The shuttle vector comprises homologous recombinant arms TKL and TKR designed according to vaccinia virus thymidine kinase gene TK and its flanking sequence, three or above individual foreign gene expression cassettes are existed between TKL and TKR. Recombinant vaccinia virus prepared in the invention is capable of effectively expressing at least three different target genes in an early stage or a late stage of the whole process, virus is attenuated by knockouting TK as well as the security of a clinical application is raised. The shuttle vector of vaccinia virus and its application provided in the invention solve the problems that the security of the recombinant vaccinia virus is low, the expressive efficiency of the target genes are incoherent and the insert exogenous gene fragment is less, thereby solid foundations of preparation of novel gene engineering active viral vector vaccine as well as the research and development of the biological and technical medicines are established.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of vaccinia virus shuttle vectors and application thereof.
Background technology
Exterminating smallpox is greatest achievement on the physianthropy history, and poxvirus has played conclusive effect in this process.And vaccinia virus is a kind of virus the most complicated on the present known structure as a member of Poxviridae orthopoxvirus, and its vaccine as the prevention smallpox used more than 200 year, also was the deposit vaccine that the our times various countries are used for preventing smallpox.Vaccinia virus Tiantan strain (vaccinia virus Tian Tan, VTT) be Chinese distinctive vaccinia virus strain, Ceng Zuowei prevention smallpox popular vaccine strain used in a large amount of crowd of China midium or long term, it has weak, the safe in utilization characteristics of relative virus force, is the desirable strain that development is applicable to the genetically engineered live vector vaccine that China uses.
Along with the development of genetically engineered and Protocols in Molecular Biology, on the basis that vaccinia virus is constantly studied and resolved, this virus deepens continuously in the application of medical science and field of biology.Nineteen eighty-two particularly, Paoletti and Moss study group first the successful Application vaccinia virus as carrier in Mammals since the expression alien gene, this carrier is widely used in the preparation research of expression of exogenous gene regulation and control, novel polypeptide, genetic engineering subunit vaccine and reorganization live vector vaccine.1987, express the vaccinia virus recombinant of rabies virus G gene and at first get permission in wildlife, to use, and successfully controlled Europe and north America region wildlife rabies; Nineteen ninety is expressed the vaccinia virus recombinant of F gene of NDV strain at U.S. Register; Expressing the vaccinia virus recombinant of newcastle disease virus HN and F gene in 1994 gets permission to produce in batches in the U.S. as first genetically engineered live vector vaccine.Along with the successful Application of vaccinia virus recombinant in the veterinary drug field, people begin to turn one's attention to its medical value.The U.S. in 1996 have started the I clinical trial phase of the vaccinia virus recombinant Aldesleukin that is used for the treatment of malignant melanoma, and have finished the II clinical trial phase of this project in calendar year 2001; The vaccinia virus recombinant ITV that was used to prevent AIDS in 2003 has finished the I/II clinical trial phase.At present, the research that it is biotechnological formulation with the vaccinia virus recombinant that the whole world still has over one hundred item is in the medical clinical test stage, relates to multiple prevention and treatment of diseases fields such as cancer, acquired immune deficiency syndrome (AIDS), hepatitis, malaria and tuberculosis.
With vaccinia virus (vaccinia virus, VV) as carrier for expression of eukaryon, with other mammalian virus expression vector such as retrovirus, the human herpes simplex vicus compares with adenovirus etc., has self special advantages, mainly show: 1. bale capacity is big, many dispensable genes that duplicate are arranged in the vaccinia virus gene group, account for 15% of whole open reading frame, their disappearance does not influence virus normal replicative cycle in cell, so it can insert the foreign gene that total amount reaches 25kb, have very big bale capacity, and heredity that the foreign gene that inserts can be stable and expression; 2. immunogenicity height, foreign protein can be expressed by loyal in host cell, and can carry out correct translation post-treatment and modification, and expressed proteins has natural conformation and biologic activity fully, can induce body to produce long lasting cellular immunization and humoral immunization; 3. safe, duplicate in the strict endochylema, not with the genome conformity of host cell, non-carcinogenesis, this heredity of having eliminated after vaccinia virus recombinant is used threatens; 4. host range is wide, and vaccinia virus can infect most of Mammalss and the bird that comprises people, mouse, pig, dog, monkey, rabbit, chicken etc., also can breed in corresponding clone; 5. be easy to make up polyvalent vaccine, the nonessential regions different in vaccinia virus can insert different foreign genes simultaneously, expression between these genes is separate, interference-free each other, therefore, vaccinia virus is particularly suitable for making up polyvalent vaccine, improves the protection efficient of vaccine with the combination that realizes different effective constituents; 6. easy and simple to handle, do not need helper virus or packing cell; 7. the security of conduct prevention or therapeutic biotechnological formulation and validity are by a large amount of animal and human's body evidences.
Given this, making up stable, efficient, safe vaccinia virus expression vector has great importance.Because the very huge people of vaccinia virus gene group, and its DNA do not have infectivity, thereby can not be by the genome of direct gene-splicing method with external source goal gene importing vaccinia virus.The shuttle vectors that goal gene is carried in utilization carries out the general and main method that homologous recombination becomes the acquisition recombinant vaccinia with vaccinia virus in the cell that infects vaccinia virus.Therefore, the structure of vaccinia virus shuttle vectors is the basis and the key point of vaccinia virus recombinant preparation.
Summary of the invention
The purpose of this invention is to provide a kind of can be simultaneously and efficiently express the vaccinia virus recombinant shuttle vectors of at least three kinds of foreign genes.
Another object of the present invention provides the application of above-mentioned vaccinia virus shuttle vectors in genetically engineered live vector vaccine, polypeptide and subunit vaccine preparation and bio-pharmaceuticals.
In order to realize the object of the invention, a kind of vaccinia virus shuttle vectors of the present invention, it contains the homologous recombination arm TKL and the TKR of with good grounds vaccinia virus thymidine kinase gene TK and flanking sequence design thereof, in the multiple clone site that has between TKL and the TKR more than 3 or 3, the two ends of each multiple clone site are connected with PE/L promotor and T5nT terminator respectively, and described shuttle vectors also contains intestinal bacteria replication origin pUC ori and resistance screening mark.
Aforesaid shuttle vectors, its carrier that sets out is pBluescipt Sfi.
Aforesaid shuttle vectors, described resistance screening is labeled as ampicillin resistance gene.
Aforesaid shuttle vectors, its nucleotide sequence is shown in SEQ ID No.1.
The preparation method of above-mentioned vaccinia virus shuttle vectors comprises step:
1) thymidine kinase gene TK and the flanking sequence thereof according to vaccinia virus Tiantan strain VTT (available from CDC virus Control Study institute) designs and synthesizes homologous recombination arm TKL and TKR;
2) TKL and the TKR after will synthesizing is connected among the carrier pBluescipt Sfi, will connect product called after pSTK plasmid;
3) manually design and synthesize the potent combined promoter sequence of vaccinia virus PE/L, 3 or 3 above MCS multiple clone site and termination signal sequence, form 3 or 3 above expression cassettes, be connected in the pSTK plasmid, promptly get the vaccinia virus shuttle vectors, called after pSTKE.
The present invention also provides the application of above-mentioned vaccinia virus shuttle vectors in genetically engineered live vector vaccine, polypeptide and subunit vaccine preparation and bio-pharmaceuticals.
The present invention further provides the genetically engineered live vector vaccine, polypeptide and the subunit vaccine that utilize above-mentioned vaccinia virus shuttle vectors to prepare.
The element of vaccinia virus shuttle vectors relates to many aspects such as reorganization arm, promotor, termination signal, recombination form, reporter gene, intestinal bacteria replicon, and each selection of components is all closely bound up with recombination efficiency and expression efficiency.Vaccinia virus shuttle vectors pSTKE provided by the invention, duplicate nonessential region TK gene as the reorganization arm with the vaccinia virus strictness, not only can keep the replication of parent's strain and the immune efficacy of vaccinia virus recombinant to greatest extent, and realized attenuation to vaccinia virus having improved security; Choose vaccinia virus early, late period powerful combined promoter, not only guarantee goal gene whole process, efficiently express, also keep it and start actively in early days, realized company's inertia of destination gene expression; Contain more than three or three independently exogenous gene expression box, can insert and express at least three goal gene simultaneously, interference-free each other and influence has avoided using the trouble of different carriers, more helps the structure and different expression of exogenous gene of polyvalent vaccine; All add potent combined promoter of poxvirus and transcription termination signal T5nT (TTTTTNT) in all expression cassettes, can guarantee the efficient transcript and expression of goal gene; Adopt the homologous recombination mode to make up vaccinia virus recombinant, avoid the huge genomic inconvenience of direct control; Respectively three expression cassettes are verified that by green fluorescence protein gene (EGFP) result shows that three expression cassettes all can efficiently expressing exogenous gene, shows that shuttle vectors successfully constructs.
The invention has the advantages that:
(1) vaccinia virus shuttle vectors provided by the invention as reorganization arm and selection markers, had both reached the purpose of screening vaccinia virus recombinant with thymidine kinase gene TK, had knocked out the TK gene again, had realized attenuation, had improved the clinical application security;
(2) vaccinia virus shuttle vectors provided by the invention contains three or three above exogenous gene expression boxes, can simultaneously and efficiently express at least three kinds of foreign genes, can realize the needs of multi-gene expression;
(3) vaccinia virus shuttle vectors provided by the invention adopt have early, late period expression activity powerful combined promoter, solved that the vaccinia virus recombinant destination gene expression is weak, incoherent problem of destination gene expression cycle.
Description of drawings
Fig. 1 is the embodiment of the invention 1 expression cassette mode configuration nucleotide sequence figure, wherein contains three independently expression cassettes, and each expression cassette is formed by compound strong promoter sequence (PE/L), multiple clone site (MCS) and strong transcription termination sequence (T5NT).
Fig. 2 is the embodiment of the invention 2 vaccinia virus shuttle vectors pSTKE synoptic diagram.
Fig. 3 A～Fig. 3 D is the embodiment of the invention 3 recombinant poxvirus are expressed EGFP in the BHK-21 cell fluoroscopic examination result, and wherein, A～D is respectively pSTKE-EGFP1, pSTKE-EGFP2, pSTKE-EGFP3 and negative control.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Genetically engineered such as the screening of being connected of the recovery of the extraction of the preparation of competent escherichia coli cell and conversion, plasmid and digestion with restriction enzyme, dna fragmentation, linear DNA fragment, recombinant plasmid and evaluation, PCR and molecular biology experiment are translated " molecular cloning experiment guide " second edition related Sections with reference to Jin Dongyan, Li Mengfeng etc. and are carried out in following examples.
The reagent and the material that use in following examples are the commercial goods.
The structure of embodiment 1 vaccinia virus shuttle vectors pSTKE
1.1 the design of vaccinia virus shuttle vectors reorganization arm TKL and TKR is with synthetic
According to the vaccinia virus complete genome sequence of announcing among the GenBank, to the thymidine kinase gene TK of vaccinia virus Tiantan strain VTT (available from CDC virus Control Study institute) and flanking sequence thereof analyze with homology relatively after, according to the length of homologous sequence and the relation of recombination efficiency, according to the reorganization right arm is 2 times of left arm, select two sections nucleotide sequences, and interpolation proper restriction site, send JaRa biotechnology company limited (Shanghai) to synthesize, the nucleotide sequence of vaccinia virus vector pSTKE TKL and TKR is respectively shown in SEQ ID No.2 and 3.
1.2pSTK the structure of plasmid
With BamH I and Xho I double digestion composition sequence, and be connected among the cloning vector pBluesciptSfi (available from NEB company), obtain containing the pSTK plasmid of arm of recombinating.
1.3 the design of three expression cassettes is with synthetic
Select vaccinia virus early, late period efficient promoter and transcribe strong terminator sequence, and screen suitable multiple clone site, design three independently exogenous gene expression boxes (Fig. 1), send JaRa biotechnology company limited (Shanghai) to synthesize.
1.4 the structure of vaccinia virus shuttle vectors pSTKE
With enzyme EcoR V and enzyme Spe I the synthetic expression cassette is connected to the corresponding restriction enzyme site of pSTK, makes up vaccinia virus shuttle vectors pSTKE (Fig. 2).PSTKE carrier total length 4740bp mainly is made of following 7 parts: 1. make up the required reorganization arm of vaccinia virus recombinant, i.e. TKL and TKR; 2. the required primer binding sequence of sequencing, i.e. M13 forward and reverse primer binding sequence; 3. insert the required multiple clone site of foreign gene, i.e. MCS1, MCS2 and MCS3; 4. express goal gene required PE/L morning, late period powerful combined promoter, i.e. PE/L promotor; 5. transcription termination sequence T5nT, i.e. terminator; 6. ampicillin resistance gene, i.e. Amp; 7. shuttle plasmid duplicates necessary intestinal bacteria replication origin, i.e. pUC ori.
Embodiment 2 contains the structure of the serial vaccinia virus recombinant shuttle plasmid of EGFP gene
In order to verify the validity of constructed shuttle vectors, in three expression cassettes, connected enhanced green fluorescence protein gene (EGFP) respectively, step is as follows:
EcoR I+Not I double digestion pVL-EGFP and pSTKE reclaim the band of 750bp and 4885bp size respectively, and connect, and EGFP are inserted into corresponding restriction enzyme site among the MCS1 of pSTKE, called after pSTKE-EGFP1.
Kpn I+Sa1I double digestion pMD18T-EGFP and pSTKE reclaim the band of 750bp and 4885bp size respectively, and connect, and EGFP are inserted into corresponding restriction enzyme site among the MCS2 of pSTKE, called after pSTKE-EGFP2.
Hind III+Pst I double digestion pVL-EGFP and pSTKE reclaim the band of 750bp and 4885bp size respectively, and connect, and EGFP are inserted into corresponding restriction enzyme site among the MCS3 of pSTKE, called after pSTKE-EGFP3.
The screening and the evaluation of embodiment 3 vaccinia virus recombinants
3.1 the preparation of recombinant plasmid
Utilize preparation pSTKE-EGFP1, the pSTKE-EGFP2 of " no intracellular toxin plasmid prepares test kit (centrifugal column type) in a large number " (Beijing hundred Tyke Bioisystech Co., Ltd) and three kinds of recombinant plasmids of pSTKE-EGFP3, and the concentration of employing spectrophotometry nucleic acid, and OD260/280 is between 1.8-2.0, and-20 ℃ of preservations are standby.
3.2 cell cultures and had digestive transfer culture
The cell that the present invention uses is baby hamster kidney cell (BHK-21), is a kind of adherent inoblast.With the MEM complete culture solution that contains 5%FCS, in 37 ℃, 5%CO
2Cultivate in the incubator.After treating that the BHK-21 cell grows up to individual layer in culturing bottle, under aseptic condition, the MEM complete culture solution in the culturing bottle is inclined to, adding a spot of 0.25% pancreatin solution washes twice, adding a small amount of pancreatin then leaves standstill and digests that cell all splits away off to the wall, the MEM complete culture solution that adds the 5%FCS of certain volume in each culturing bottle, behind the piping and druming mixing, be sub-packed in the culture hole of six orifice plates in (every hole 2ml) or the culturing bottle (the enchylema volume is decided on the culturing bottle size), put 37 ℃, 5%CO
2Cultivate in the incubator.
3.3 cell counting
Obtained cell suspension 0.5ml suitably splashes in the blood cell counting plate after the dilution, by the four big lattice inner cell sums at four angles of white blood cell count(WBC) method.Only count fine karyon and the complete cell of cytoplasm during counting, cell in heaps calculates by a cell.Total cellular score in the 4 big grids is converted into cell count in every ml cells suspension by following formula.
3.4 vaccinia virus titer determination
Preparation individual layer BHK-21 cell is with 3 * 10
4/ ml cell is covered in 96 porocyte culture plates, and every hole 100 μ l are cultured to cell and grow up to the fusion individual layer.Discard nutrient solution, wash cell twice, VTT virus liquid is kept liquid by 10 with the MEM that does not contain serum with the MEM nutrient solution that does not contain serum
3～10
12Do 10 times of dilutions of going forward one by one, every then hole connects 100 μ l viral dilution liquid successively, and each gradient is established three repetitions, establishes blank simultaneously, 37 ℃, 5%CO
2Incubator absorption 2h.Add 100 μ l then and contain the MEM complete culture solution of 5%FCS, at 37 ℃, 5%CO
2Incubator continues to cultivate 96h.Each dilution plaque of direct viewing forms situation under inverted microscope, be calculated as follows out plaque forming unit contained in every milliliter of viral liquid (Plaque formingunits, PFU).
3.5 transfection and homologous recombination
Inoculation BHK21 cell (1 * 10 in 6 * 30mm culture plate
5～3 * 10
5Individual/as ml), when cell grows to 80% fusion, to infect the vaccinia virus Tiantan strain (VTT) of 0.1MOI (Multiplicity of infection), 37 ℃, 5%CO
2Infect 1-2h (jiggle culture plate once per half an hour), use recombinant plasmid transfected cell then.Get 100 μ lOpti-MEM and add recombinant plasmid dna 10 μ g mixings; In addition in 100 μ l Opti-MEM, add 8 μ l liposomes, mixing gently, room temperature leaves standstill 5min.Then the former is slowly dripped in last liquid, mixing gently, room temperature effect 20min (15-30min) back adds in the cell hole, and 37 ℃, 5%CO2 are cultivated 6h (5-8h), change the freshly prepared MEM that contains 5%FCS, continuation cultivation 48-72h.Behind 48h or the 72h, whether observation has green fluorescence to occur under fluorescent microscope.
3.6 the evaluation of vaccinia virus recombinant
The virus that has fluorescence more than the results is frozen appearance repeatedly, and lysing cell discharges virus, centrifugal removal cell of 3000-6000r/min or cell debris.Get supernatant and adopt " UNIQ-10 pillar viral genome extraction agent box " (worker's biotechnology Services Co., Ltd is given birth in Shanghai) to extract the vaccinia virus recombinant genome, method is referring to this test kit specification sheets.
As template, utilize the primer of EGFP gene with the genome of said extracted:
Upstream primer: 5 '-ATCGATCGATGGTGAGCAAGGGCGAGGAG-3 '
Downstream primer: 5 '-GCGTCGACTTACTTGTACAGCTCGTCCATG.-3 '
Whether amplification EGFP gene is incorporated in the genome of vaccinia virus to identify the EGFP gene.Reaction system is as follows with reaction conditions:
The PCR reaction system:
The PCR reaction conditions:
94 ℃, 5min; 94 ℃, 1min, 68 ℃, 50s, 72 ℃, 50s, 30 circulations; 72 ℃, 10min; 4 ℃, preserve.
3.7 detected result
Fluoroscopic examination is the result show, vaccinia virus shuttle vectors pSTKE can efficiently expressing exogenous gene (Fig. 3 A～Fig. 3 C presents bright green fluorescence), and (Fig. 3 D) appears in the no fluorescence of negative control (vaccinia virus of untransfected recombinant plasmid); Genome PCR detected result shows that foreign gene has been integrated in the vaccinia virus gene group.
Above detected result shows that the present invention has prepared a kind of vaccinia virus recombinant shuttle vectors of efficiently expressing exogenous gene, for vaccinia virus recombinant becomes application serial, on a large scale to lay the foundation at vaccine with bio-pharmaceutical research and development field.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (7)
1. vaccinia virus shuttle vectors, it is characterized in that, it contains the homologous recombination arm TKL and the TKR of with good grounds vaccinia virus thymidine kinase gene TK and flanking sequence design thereof, in the multiple clone site that has between TKL and the TKR more than 3 or 3, the two ends of each multiple clone site are connected with PE/L promotor and T5nT terminator respectively, and described shuttle vectors also contains intestinal bacteria replication origin pUC ori and resistance screening mark.
2. shuttle vectors according to claim 1 is characterized in that, its carrier that sets out is pBluescipt Sfi.
3. shuttle vectors according to claim 1 and 2, described resistance screening is labeled as ampicillin resistance gene.
4. shuttle vectors according to claim 3 is characterized in that, its nucleotide sequence is shown in SEQ ID No.1.
5. the preparation method of each described shuttle vectors of claim 1-4 is characterized in that, comprises step:
1) designs and synthesizes homologous recombination arm TKL and TKR according to vaccinia virus thymidine kinase gene TK and flanking sequence thereof;
2) TKL and the TKR after will synthesizing is connected among the carrier pBluescipt Sfi, will connect product called after pSTK plasmid;
3) manually design and synthesize vaccinia virus PE/L promoter sequence, 3 or 3 above MCS multiple clone site and termination signal sequence, form 3 or 3 above expression cassettes, be connected in the pSTK plasmid, promptly.
6. each described vaccinia virus shuttle vectors application in bio-pharmaceuticals of claim 1-4.
7. the genetically engineered live vector vaccine, polypeptide and the subunit vaccine that utilize each described vaccinia virus shuttle vectors of claim 1-4 to prepare.
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| CN105861558A (en) * | 2016-06-24 | 2016-08-17 | 西安医学院 | Vaccinia virus shuttle vector and preparation method and application thereof |
| CN106795527A (en) * | 2014-04-01 | 2017-05-31 | 玛丽女王伦敦大学 | Oncolytic virus |
| CN107604004A (en) * | 2017-09-22 | 2018-01-19 | 西安医学院 | Tracer target practice plasmid for vaccinia virus Tiantan strain TK genes and preparation method thereof |
| CN108676814A (en) * | 2018-04-20 | 2018-10-19 | 西安医学院 | A kind of fluorescent marker shuttle vector of Tiantan strain vaccinia virus and preparation method thereof |
| CN110157721A (en) * | 2019-01-07 | 2019-08-23 | 西安医学院 | A kind of tracer target practice plasmid of vaccinia virus Tiantan strain and preparation method thereof |
| CN111206022A (en) * | 2020-02-20 | 2020-05-29 | 中国人民解放军军事科学院军事医学研究院 | Recombinant virus for expressing Lassa fever virus empty capsid and preparation method thereof |
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| CN101775410A (en) * | 2009-01-09 | 2010-07-14 | 中国人民解放军军事医学科学院军事兽医研究所 | Fowlpox virus vector shuttle plasmid and application thereof |
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| CN1654665A (en) * | 2005-01-11 | 2005-08-17 | 武汉大学 | Attenuated vaccinia virus Tiantan strain vector and its preparation and application |
| CN1654666A (en) * | 2005-01-11 | 2005-08-17 | 武汉大学 | Attenuated vaccinia virus Tiantan strain vector and its preparation and application |
| CN101775410A (en) * | 2009-01-09 | 2010-07-14 | 中国人民解放军军事医学科学院军事兽医研究所 | Fowlpox virus vector shuttle plasmid and application thereof |
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| CN106795527A (en) * | 2014-04-01 | 2017-05-31 | 玛丽女王伦敦大学 | Oncolytic virus |
| CN106795527B (en) * | 2014-04-01 | 2020-10-30 | 玛丽女王伦敦大学 | Oncolytic virus |
| CN105861558A (en) * | 2016-06-24 | 2016-08-17 | 西安医学院 | Vaccinia virus shuttle vector and preparation method and application thereof |
| CN107604004A (en) * | 2017-09-22 | 2018-01-19 | 西安医学院 | Tracer target practice plasmid for vaccinia virus Tiantan strain TK genes and preparation method thereof |
| CN108676814A (en) * | 2018-04-20 | 2018-10-19 | 西安医学院 | A kind of fluorescent marker shuttle vector of Tiantan strain vaccinia virus and preparation method thereof |
| CN110157721A (en) * | 2019-01-07 | 2019-08-23 | 西安医学院 | A kind of tracer target practice plasmid of vaccinia virus Tiantan strain and preparation method thereof |
| CN111206022A (en) * | 2020-02-20 | 2020-05-29 | 中国人民解放军军事科学院军事医学研究院 | Recombinant virus for expressing Lassa fever virus empty capsid and preparation method thereof |
| CN113481241A (en) * | 2021-06-18 | 2021-10-08 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector with H3L gene knocked out |
| CN113481240A (en) * | 2021-06-18 | 2021-10-08 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector with L1R gene knocked out |
| CN114058645A (en) * | 2021-06-22 | 2022-02-18 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo |
| CN114058643A (en) * | 2021-06-22 | 2022-02-18 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo |
| CN114058644A (en) * | 2021-06-22 | 2022-02-18 | 苏州工业园区唯可达生物科技有限公司 | Recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibody existing in vivo |
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