CN107753941A - Research of Ebola vaccine based on chimpanzee adenoviral vector - Google Patents
Research of Ebola vaccine based on chimpanzee adenoviral vector Download PDFInfo
- Publication number
- CN107753941A CN107753941A CN201610696322.3A CN201610696322A CN107753941A CN 107753941 A CN107753941 A CN 107753941A CN 201610696322 A CN201610696322 A CN 201610696322A CN 107753941 A CN107753941 A CN 107753941A
- Authority
- CN
- China
- Prior art keywords
- ebola
- research
- vaccine
- ebovgp
- carrier
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10041—Use of virus, viral particle or viral elements as a vector
- C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/14011—Filoviridae
- C12N2760/14111—Ebolavirus, e.g. Zaire ebolavirus
- C12N2760/14134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The present invention relates to the research of Ebola vaccine based on chimpanzee adenoviral vector.The present invention constructs new research of Ebola vaccine carrier based on replication defect type chimpanzee adenoviral vector first.Described vaccine carrier can be applied to prepare can high efficient expression and the viral vaccine with good immunogenicity, described research of Ebola vaccine prepares that simple, cost is low, security is good, without adding adjuvant.
Description
Technical field
The invention belongs to field of immunology, more particularly it relates to the Ebola based on chimpanzee adenoviral vector
Viral vaccine.
Background technology
Ebola disease viral disease (Ebola virus disease, EVD) is also referred to as Ebola hemorrhagic fever (Ebola
Hemorrhagic fever, EHF) it is the high fatal infectious disease complex caused by Ebola virus (Ebola virus, EBOV).
1976, Ebola hemorrhagic fever was first in the Yambuku areas of cd (preceding Zaire) and the Nzara of the Sudan
Area is broken out simultaneously, and hereafter, Ebola virus causes multiple prevalence in the world.2014-2016 in the multiple countries in West Africa,
Including Guinea, Libya, Sierra Leone and Nigeria etc., there is largest since the dawn of human civilization, infection and death toll
A most Ebola hemorrhagic fever great outbursts.According to the World Health Organization (WHO) statistics, by the end of March 27 in 2016
Day, this time outburst, which shares 28,646 infection, to be included making a definite diagnosis and suspected case, wherein totally 11,323 death, case fatality rate are up to
39.5%.It can be seen that rich draw the prevalence of virus or outburst worldwide to cause serious public health problem.
Ebola virus belong to filamentous virus section (Filoviridae), Ebola virus category (Ebolavirus) without section
Section, there are coating, the RNA virus of sub-thread minus strand.Ebola virus category shares the virus of 5 kinds of hypotypes, including Ben Dibujiao types
(Bundibugyoebolavirus, BDBV), Zaire's type (Zaire ebolavirus, EBOV), the Sudan type (Sudan
Ebolavirus, SUDV), Reston type (Reston ebolavirus, RESTV), Ta Yisen crop types (Forest
Ebolavirus, TAFV).The infectivity of wherein Zaire's type Ebola virus is most strong, and the number of outburst is most, dead number
Also at most, this time West Africa outburst is also to be caused by this type virus.
The full-length genome of Ebola virus is about 19kb, and its structure is 3 '-UTR-NP-VP35-VP40-GP-VP30-
VP24-L-5 '-UTR., include the non-translational region (UTR) at both ends, also encode 7 kinds of albumen：Nucleoprotein (NP), polymerase auxiliary because
Sub (VP35, VP40), glycoprotein (GP), activating transcription factor (VP30, VP24), RNA polymerase (L).As Ebola virus
Unique structural proteins on envelope membrane surface, GP spinous process can be with the acceptor combination mediate retroviral of host cell absorption and thin with host
The fusion of after birth.GP glycoprotein is the main immunogens of inducing specific neutralizing antibody, and causing a disease for Ebola virus
Property has most important influence, therefore the exploitation of nearly all Ebola's vaccine is all to utilize GP glycoprotein；And different shaped angstrom is rich
The GP albumen of virus is drawn, there is very high conservative in DNA sequence dna and amino acid sequence.
So far commercialized Ebola's vaccine is there is no, related vaccine research enters including for clinical stage：Science disease
Poisonous carrier vaccine, replication-defective virus carrier bacterin, DNA vaccination and recombinant protein etc..With Merck Sharp＆Dohme
The rVSV-ZEBOV conducts of Corp, NewLink Genetics and Public Health Agency of Canada joint developments
The duplicating virus carrier bacterin of representative comes into third stage, and single immunization has good immunogenicity, but peace be present
Full sex chromosome mosaicism；Lacked by the duplication that the ChAd3.EBOZ/ChAd3.EBO of Glaxo Smith Kline and NIH cooperative research and development is representative
Swaged adenovirus carrier vaccine has been enter into the clinical II phases, the problem of in the absence of in terms of security, but needs secondary immunity to increase
Its immunogenicity；Vaccine based on replication-defective adenoviral vector also has Ad26-ZEBOV, Ad5-EBOV etc. also to have been enter into face
Bed research；With VRC-EBODNA023-00-VP, VRC-EBODNA012-00-VP and VRC-MARDNA025-00-VP etc. for representative
DNA recombinant vaccines enter the clinical I phases, but need electroporation technology and secondary immunity when it is immune.
Ebola virus belongs to some dangerous very high viruses, and this area is necessary to carry out in-depth study to it, made
It is standby go out the research of Ebola vaccine with good result.
The content of the invention
It is an object of the invention to provide the research of Ebola vaccine based on chimpanzee adenoviral vector.
In the first aspect of the present invention, there is provided a kind of research of Ebola vaccine, the research of Ebola vaccine is by such as lower section
Method prepares：Research of Ebola vaccine carrier is packed, processing obtains the research of Ebola vaccine with immunogenicity；
It is preferred that by virus transfection virus production cell, packed in the cell so as to viral；Wherein, described research of Ebola vaccine
Carrier is replication defect type chimpanzee adenoviral vector, and wherein includes the expression cassette of Ebola virus glycoproteins (EBOVgp)
(Expression element).
In a preference, described replication defect type chimpanzee adenoviral vector includes the chimpanzee adenovirus of transformation
AdC68 genome sequences, wherein E1 lack；It is preferred that E1 most coded sequence is replaced by catenation sequence；Described connection
Restriction enzyme site I-Ceu I and PI-Sce I are set in sequence.
In another preference, described replication defect type chimpanzee adenoviral vector includes the chimpanzee adenovirus of transformation
AdC7 genome sequences, wherein E1 and E3 lack；It is preferred that part E1 code areas are deleted with whole E3 code areas, E1 is deleted
Area increases I-Ceu I and PI-Sce I restriction enzyme sites.
In another preference, described Ebola virus glycoproteins (EBOVgp) have SEQ ID NO:Core shown in 1
Nucleotide sequence.
In another preference, the expression cassette of described Ebola virus glycoproteins (EBOVgp) is inserted into described duplication
In restriction enzyme site I-Ceu I and the PI-Sce I of deficiency chimpanzee adenoviral vector.
In another preference, described research of Ebola vaccine carrier has SEQ ID NO:2 or SEQ ID NO:3 institutes
The nucleotide sequence shown.
In another aspect of this invention, there is provided a kind of research of Ebola vaccine carrier, it is replication defect type chimpanzee adenovirus
Viral vector, and wherein contain the expression cassette (Expression element) of Ebola virus glycoproteins (EBOVgp).
In a preference, described Ebola virus glycoproteins (EBOVgp) have SEQ ID NO:Core shown in 1
Nucleotide sequence.
In another preference, described research of Ebola vaccine carrier has SEQ ID NO:2 or SEQ ID NO:3 institutes
The nucleotide sequence shown.
In another aspect of this invention, there is provided a kind of medicine box, contain described research of Ebola vaccine in described medicine box；
It is preferred that contain described research of Ebola vaccine in described medicine box simultaneously.
In another aspect of this invention, there is provided a kind of kit for being used to prepare vaccine, the kit include：Described
Research of Ebola vaccine carrier.
In a preference, the kit also includes：Virus production cell.
In another preference, described virus production cell can be achieved on the cell of virus packaging；It is preferably comprised：
HEK293 cells, 293T cells.
The other side of the present invention is apparent to those skilled in the art due to this disclosure
's.
Brief description of the drawings
Fig. 1, restructuring chimpanzee adenovirus plasmid map.
A, chimpanzee adenovirus DNA pAdC68-EBOVgp is recombinated；
B, chimpanzee adenovirus DNA pAdC7-EBOVgp is recombinated.
Fig. 2, restructuring chimpanzee adenovirus plasmid construction and digestion are identified.
A, electrophoretograms of the 1 DNA pUC57-EBOVgp after Xba I and Kpn I double digestions.
B, 2 be electrophoretogram of the pShuttle-EBOVgp DNAs after I-Ceu I, PI-Sce I and Nsi I digestions.
C, 3,4,5 be that recombinant adenovirus plasmid DNA pAdC68-EBOVgp use Bgl II, Xho I, Mfe I digestions respectively
Electrophoretogram afterwards, 6,7,8 be that recombinant adenovirus plasmid DNA pAdC7-EBOVgp use Bgl II, Xho I, Mfe I digestions respectively
Electrophoretogram afterwards.
Fig. 3, Western Blot detection restructuring chimpanzee adenovirus expression EBOVgp.Wherein, detection is 6D8 with antibody.
Fig. 4, mouse immune experimental period table.
1) mouse is exempted from the beginning of the 0th week, using intramuscular injection, the 2nd Tuesday exempted from mouse, equally using intramuscular injection；
2) respectively at the 0th week, the 2nd week (two weeks after just exempting from), the 4th week (two exempt from two weeks afterwards) and the 6th week (two exempt from rear surrounding)
Blood is taken through eyelid；
3) mouse is divided into 6 immune treatment groups：1st group, just exempt from injection restructuring chimpanzee adenovirus AdC68-EBOVgp,
Two exempt to inject PBS；2nd group, just exempt to inject chimpanzee adenovirus control AdC68-empty, two exempt to inject PBS；3rd group, just exempt from
Injection restructuring chimpanzee adenovirus AdC7-EBOVgp, two exempt to inject PBS；4th group, just exempt to inject chimpanzee adenovirus control
AdC7-empty, two exempt to inject PBS；5th group, just exempt from injection restructuring chimpanzee adenovirus AdC7-EBOVgp, two exempt from injection restructuring
Chimpanzee adenovirus AdC68-EBOVgp；6th group, blank control group, just exempt from, two exempt to inject PBS.
Fig. 5, mice serum ELISA testing results.
A. the mice serum of different disposal group is taken to detect the expression water of anti-Ebola GP protein specific antibodies after just exempting from 2 weeks
It is flat；
B.4 the mice serum of different disposal group is taken to detect anti-Ebola GP protein specific antibodies after all (two exempt from 2 weeks)
Expression；
C.6 the mice serum of different disposal group is taken to detect anti-Ebola GP protein specific antibodies after all (two exempt from 4 weeks)
Expression；
D. take anti-angstrom of the mice serum detection of different time points rich after restructuring chimpanzee adenovirus AdC68-EBOVgp is immunized
Draw the expression of GP protein specific antibodies；
E. the mice serum of different time points is taken to detect anti-Ebola after restructuring chimpanzee adenovirus AdC7-EBOVgp is immunized
The expression of GP protein specific antibodies；
F. just exempt to recombinate chimpanzee adenovirus AdC7-EBOVgp, booster immunization is restructuring chimpanzee adenovirus after 2 weeks
AdC68-EBOVgp, detection different time points mice serum moderate resistance Ebola GP protein specific antibody are horizontal.
Embodiment
The present inventor passes through in-depth study, is constructed first based on replication defect type chimpanzee adenovirus plasmid (carrier)
New research of Ebola vaccine carrier.Described vaccine carrier, which can be applied to preparation, high efficient expression and to have good immunogenicity
Viral vaccine.
The present invention utilizes the new research of Ebola vaccine carrier of replication defect type chimpanzee adenovirus plasmid construction.
As a kind of preferred embodiment, described replication defect type chimpanzee adenoviral vector includes the chimpanzee adenovirus of transformation
Malicious AdC68 genome sequences, wherein E1 missings；It is preferred that E1 most coded sequence is replaced by catenation sequence；Described company
Connect and restriction enzyme site I-Ceu I and PI-Sce I are set in sequence.For adenovirus AdC68 genomes, using restriction enzyme site I-Ceu
Insertion points of the I and PI-Sce I as foreign gene, so as to which the other positions in adenovirus expression carrier will not be caused to cause
Shearing.It is preferred that the replication defect type chimpanzee adenoviral vector of the chimpanzee adenovirus AdC68 genome sequences including transformation
Preparation method be：Chimpanzee adenovirus AdC68 genomes are divided into 4 fragments, are sequentially loaded into skeleton carrier, also,
Most coded sequence of E1 in AdC68 genomes is replaced with catenation sequence；4 described fragments are respectively：Chimpanzee adenovirus
Malicious AdC68 genome 1-6025 positions；Chimpanzee adenovirus AdC68 genome 6026-17279 positions；Chimpanzee adenovirus
AdC68 genome 17280-34196 positions；With chimpanzee adenovirus AdC68 genome 34197-36519 positions.
As a kind of preferred embodiment, described replication defect type chimpanzee adenoviral vector includes the chimpanzee adenovirus of transformation
Malicious AdC7 genome sequences, wherein E1 and E3 missings；It is preferred that part E1 code areas are deleted with whole E3 code areas, E1 is deleted
Except area increases I-Ceu I and PI-Sce I restriction enzyme sites.Described deletion part E1 code areas refer to delete wild type AdC7 bases
Because of the sequence of 458-3026 positions in group.It is preferred that the full sequence in E3 areas is deleted, namely delete wild type AdC7 genomes
In 27094-31799 positions sequence.It is preferred that prepare the duplication for the chimpanzee adenovirus AdC7 genome sequences for including transformation
The method of deficiency chimpanzee adenoviral vector includes：(1) by from pNEB193 origin sequences, from AdC7 glands
Virus genomic LITR fragments, the Nde I-Age I fragments from AdC7 adenoviral gene groups are fused into by fusion DNA vaccine
Fragment OLN, with Spe I and Age I endonuclease bamhis OLN；, will with Nde I, Age I and Spe I digestion AdC7 adenoviral gene groups
Fragment Age (the 4028)-SpeI (10610) cut is connected to form plasmid pOIN with the fragment OLN of digestion；(2) use
Hind III and Avr II digestion AdC7 genomes, purpose fragment Hind III (the 7153)-Avr II (23363) cut are inserted
Enter into plasmid pOIN Hind III and Avr II sites, the plasmid of acquisition is named as pOINH；(3) from AdC7 adenovirus bases
Because amplification is located at the fragment between 31800-36535 in AdC7 adenoviral genes in group, Avr II and Rsr II are introduced at 5 ' ends
Restriction enzyme site, Pac I and Asis I sites are introduced at 3 ' ends, the fragment of amplification is inserted into pOINH Avr II and Asis I
In site, the plasmid of acquisition is named as pOINHR；(4) amplification is located at AdC7 adenoviral genes from AdC7 adenoviral gene groups
Fragment between middle 23165-27093, Rsr II sites are introduced at 3 ' ends, the fragment of amplification is inserted into pOINHR Avr II
With in Rsr II sites, obtaining the recombinant adenoviral expressing vector of replication defect type.
Due to chimpanzee adenovirus in crowd it is unpopular, therefore the vaccine of the present invention will not prestore anti-human blood in by human body
The influence of clear type adenovirus neutralizing antibody, its immune effect will be better than the epidemic disease that common human serotype's adenovirus (such as Ad5) is carrier
Seedling；Secondly, AdC68 and AdC7 immunogenicity are similar with Ad5, and better than Ad26, therefore, new Ebola's vaccine of the invention is not
Only there is originality, and will have preferable immunogenicity and immune protective effect.In addition, adenovirus carrier vaccine prepares letter
List, cost is low, security is good, without adding adjuvant, can use the immunization routes such as intramuscular immunisation, oral immunity or nasal cavity immunity,
By with good market competitiveness.
Present invention also offers the coded sequence of the Ebola virus glycoproteins of codon optimization (EBOVgp) antigen, such as
SEQ ID NO:Shown in 1, it can realize efficient expression.In terms of codon optimization, the present inventor follows codon in people
On the basis of predominant expression principle in cell, personalized, unique optimization has also been carried out.By considering and specificity choosing
Select, make to reach optimum state with the important parameter before optimization after codon optimization.
In the present invention, described " expression cassette for including Ebola virus glycoproteins " refers to include expression Ebola virus
The gene expression system of all necessary elements needed for glycoprotein, usual it include elements below：Promoter, coding ebola disease
The gene order of malicious glycoprotein, terminator；Additionally alternative is including signal coding sequence etc..These elements are operability
Connected.
Present invention also offers a kind of method for preparing research of Ebola vaccine, methods described includes：By Ebola virus
Vaccine carrier is packed, and processing obtains the research of Ebola vaccine with immunogenicity.
After obtaining the research of Ebola vaccine carrier, by transfected virus produce cell, carry out virus breeding.Transfection
After a period of time afterwards, virus can be harvested.As the preferred embodiment of the present invention, the virus of harvest can repeated infection virus production
Cell, lasting passage.The measure of virus titer can be carried out according to this area conventional method.
Present invention also offers a kind of kit for being used to prepare vaccine, the kit includes described Ebola virus
Vaccine carrier.
Described kit may also include virus production cell, such as HEK293 cells.In addition, may be used also in described kit
Operation instructions including illustrating vaccine preparation method.
In a particular embodiment of the present invention, the present inventor constructs the two of expression Zaire type Ebola virus glycoproteins
Kind restructuring chimpanzee adenovirus, i.e. AdC68-EBOVgp and AdC7-EBOVgp, and pass through digestion recombinant adenovirus plasmid DNA, survey
Determine recombined adhenovirus titre, identify expressing, specific antibody in mouse, measure immunized mice serum being immunized for purpose glycoprotein
The experiment such as reaction, two kinds of candidate vaccines of checking are AdC68-EBOVgp and AdC7-EBOVgp immunogenicity.As a result prove, this
Inventor successfully build and identify new Ebola's candidate vaccine AdC68-EBOVgp based on chimpanzee adenoviral vector and
AdC7-EBOVgp.Then, mouse is immunized in AdC68-EBOVgp, AdC7-EBOVgp respectively, mice serum after being immunized 2 weeks, 4 weeks
In can detect high titre anti-Ebola virus specific antibody；In addition the present inventor is small with AdC7-EBOVgp initial immunities
Mouse, AdC68-EBOVgp booster immunization mouse are used after 2 weeks again, just exempt from 2 weeks, can be detected after 2 weeks, 4 weeks after booster immunization it is anti-
The specific antibody of Ebola virus.
Clinical applicable Ebola's vaccine is not yet successfully obtained in the market.The chimpanzee adenovirus used in the present invention
Poison there is high immunogenicity, can not only inducing specific humoral immune reaction, and energy inducing specific cell immune response, this
Outside, it will not be influenceed by the anti-human serum type adenovirus neutralizing antibody that prestored in human body, therefore be a kind of preferable vaccine carrier.With
New Ebola's vaccine of this research and development will induce preferable immunoprotection to react.Two kinds of different chimpanzee adenoviral vectors of the invention
Ebola's vaccine, can be used as just exempt from-booster immunization uses.The inventors discovered that just exempted from using AdC7-EBOVgp, be spaced one
After the section time (2 weeks or so), by the use of AdC68-EBOVgp as booster immunization, it can so induce more preferable immune response and be immunized
Protecting effect.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as J. Pehanorm Brookers etc. are write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or
According to the condition proposed by manufacturer.
Materials and methods
1st, experiment material
Various restriction enzymes, T4DNA ligases, DNA ladder are obtained from New England Biolabs.
The small extraction reagent kit of plasmid is obtained from TIANGEN Biotech.Glue reclaim kit is obtained from xygen Biosciences.
Extraction reagent kit is obtained from MACHEREY-NAGEL in plasmid.
Replication defect type chimpanzee adenovirus empty plasmid pAdC68-empty is (referring in 201310362921.8 patents
The replication-defective adenoviral vector pAdC68 (pAdC68-E1-deleted) of E1 missings) and pAdC7-empty (pAdC7 (ginsengs
The pAdC7- Δ E1 Δ E3 seen in 201310364528.2 patents).
Human embryonic kidney cell HEK 293 is obtained from Shanghai Sheng Ke institutes of Chinese Academy of Sciences cell bank.
Transfection reagent Lipofectamine2000 is obtained from Invitrogen.
The 6D8 antibody and Ebola GP albumen of anti-Ebola GP albumen are referring to Hart, M.K., J.A.Wilson, and
A.L.Schmaljohn.Oct.7,2003 2003.Monoclonal antibodies to Ebola glycoprotein.US
patent US 6,630,144B1。
Cesium chloride, desalting column, the anti-human and anti-mouse IgG of HRP marks are obtained from Sigma-Aldrich.
BALB/c female mices are obtained from Shanghai Si Laike animal experimental centers.
2nd, chimpanzee adenovirus plasmid construction and digestion identification are recombinated
The Ebola Zaire strain virus sequence separated in patient body from Ebola according to 2014 announced on NCBI
(GI:674810552) gene (GenBank of EBOVgp albumen, will wherein be encoded:KJ660347.2, SEQ ID NO:4) carry out
Codon optimization, and Xba I restriction enzyme sites (tctaga) and Kozak sequences (gccacc) are added in 5 ' ends, 3 ' ends add Kpn I
Restriction enzyme site (ggtacc).
The inventors discovered that the plasmid that EBOVgp original genes recombinate such as is transferred into HEK293 cells, its codon turns over
Translate that efficiency is very low, so as to cause the expression quantity for recombinating chimpanzee adenovirus to reduce.Compare by substantial amounts of research, the present inventor's pin
To EBOVgp, expression has carried out codon optimization and has devised the optimisation strategy of uniqueness in people (human), as follows：
(1) by PolyT (TTTTTT) domains in original series and antiviral module (Antiviral Motifs) etc.
CIS action sites (cis-acting sites) are optimized for not having because these structures can influence ribosomal joint efficiency and
MRNA stability.
(2) codon adaptation indexI (Codon Adaptation Index, CAI) is risen into 0.93, CAI for 1.0 to recognize
Be set to it is fabulous, CAI be more than 0.8 assert preferably.And it have adjusted frequency (the Frequency of Optimal of optimization codon
Codons) it is distributed.
(3) average G/C content is optimized for 57.55%, so as to extend mRNA half-life period.
(4) adjustment (Remove Repeat Sequences) of repetitive sequence：Original maximum is directly repeated into (Max
Direct Repeat) it is adjusted to size 13, distance 552, frequency 2.
(5) the Kozak sequences (GCCACC) and efficient translation terminator codon (TGA) of efficient translation starting are added.
The EBOVgp fragments through codon optimization are cloned into pShuttle (Clonetech) carrier using digestion, then
The whole Expression element comprising CMV promoter is cloned into pAdC68 and pAdC7 respectively using I-Ceu I and PI-Sce I digestions
In carrier, recombinant adenovirus plasmid DNA, i.e. pAdC68-EBOVgp and pAdC7-EBOVgp are obtained.
Recombinant adenovirus plasmid pAdC68-EBOVgp and pAdC7-EBOVgp are carried out with Bgl II, Xho I and Mfe I
Digestion is identified.
3rd, packaging and the amplification of chimpanzee adenovirus are recombinated
Chimpanzee adenovirus plasmid pAdC68-EBOVgp, pAdC7-EBOVgp and sky are recombinated first by Pac I single endonuclease digestions
Charge material grain pAdC68-empty, pAdC7-empty, the plasmid transfection reagent Lipofectamine 2000 of linearisation is transfected
Density is 80% HEK293 cells.Before transfection, antibiotic-free culture medium is changed into, DNA/ liposome complexes are added, after transfection
5h changes normal culture medium into.
Observe cytopathy daily under the microscope, collected when obvious lesion occurs in cell and 60% plaque occurs thin
Born of the same parents, multigelation 3 times between room temperature and -80 DEG C of ultra low temperature freezers, 3500rpm/min centrifugation 5min, collect supernatant, infection
HEK293 cells, infection harvest virus by the above process after 24 hours, final to harvest what about 1.0L infected through 3-4 amplification
HEK293 cells, through multigelation 3 times, acquisition contains virulent supernatant.
4th, chimpanzee adenovirus purifying and titre detection are recombinated
Above-mentioned restructuring chimpanzee adenovirus supernatant is taken, with cesium chloride density gradient centrifugation, and is obtained after purification using desalting column
Obtain and largely recombinate chimpanzee adenovirus AdC68-EBOVgp, AdC7-EBOVgp, AdC68-empty and AdC7-empty.
Restructuring chimpanzee adenovirus is measured in A260 light absorption value, the light absorption value that will be measured with Nanodrop spectrophotometers
It is multiplied by 1.1 × 1012The titre (vp/ml) of restructuring chimpanzee adenovirus is obtained, restructuring chimpanzee adenovirus is added into glycerine point
Dress is frozen in -80 DEG C of ultra low temperature freezers.
5th, Western Blot detect EBOVgp expression
Take different titers restructuring chimpanzee adenovirus AdC68-EBOVgp, AdC7-EBOVgp, AdC68-empty and
AdC7-empty infects HEK293 cells respectively, after infecting 24 hours, is split on ice with the protein lysate containing protease inhibitors
Solve cell, centrifuging and taking supernatant.Take appropriate albumen supernatant to be mixed with 5 × sample-loading buffer, SDS-PAGE electricity is carried out after boiling cooling
Swimming, after electrophoresis terminates, the albumen on gel is transferred on pvdf membrane.It is small with 5% skim milk closing 2 after the completion of transferring film
When, with TBST by the antibody that the anti-Ebola GP albumen of 6D8 is added after Membrane cleaning 3 times or β-Actin 4 DEG C of overnight incubations of antibody.
Film was cleaned after 3 times with TBST in 2nd day and add the anti-human igg secondary antibody room temperatures of HRP marks and act on 1 hour, with TBST by Membrane cleaning 3
Appropriate ECL chemiluminescences agent is added after secondary, western developing results are obtained with LAS4000 scanning analysis instrument.
6th, mouse immune is tested
6-8 week old BALB/c female mices are divided into 6 groups：
Exempt from the beginning of 1st group, the 0th week, intramuscular injection restructuring chimpanzee adenovirus AdC68-EBOVgp (1 × 1010Vp/, left and right
Leg respectively injects 50ul), the 2nd Tuesday exempted from the PBS of intramuscular injection same volume；
Exempt from the beginning of 2nd group, the 0th week, intramuscular injection chimpanzee adenovirus control AdC68-empty (1 × 1010Vp/, left and right
Leg respectively injects 50ul), the 2nd Tuesday exempted from the PBS of intramuscular injection same volume；
Exempt from the beginning of 3rd group, the 0th week, intramuscular injection restructuring chimpanzee adenovirus AdC7-EBOVgp (1 × 1010Vp/, left and right
Leg respectively injects 50ul), the 2nd Tuesday exempted from the PBS of intramuscular injection same volume；
Exempt from the beginning of 4th group, the 0th week, intramuscular injection chimpanzee adenovirus control AdC7-empty (1 × 1010Vp/, left and right leg
Each injection 50ul), the 2nd Tuesday exempted from the PBS of intramuscular injection same volume；
Exempt from the beginning of 5th group, the 0th week, intramuscular injection restructuring chimpanzee adenovirus AdC7-EBOVgp (1 × 1010Vp/, left and right
Leg respectively injects 50ul), the 2nd Tuesday exempted from, intramuscular injection restructuring chimpanzee adenovirus AdC68-EBOVgp (1 × 1010Vp/, left and right
Leg respectively injects 50ul)；
6th group, blank control group, exempt from the beginning of the 0th week, the 2nd Tuesday exempted from the PBS of equal intramuscular injection same volume.
Respectively at the 0th week, the 2nd week (just exempting from two weeks afterwards), the 4th week (two exempt from two weeks afterwards), (two exempt from rear surrounding) was through eye within the 6th week
Eyelid collects mice serum.
7th, mice serum ELISA is detected
Specific antibody caused by the mouse after immune restructuring chimpanzee adenovirus is detected using ELISA.Use ebola disease
Malicious GP albumen (100ng/ holes) is coated with elisa plate, 4 DEG C of overnight incubations.2nd day, with 10% skim milk after PBST board-washings 3 times
(200ul/ holes) room temperature is closed 1 hour, and different dilution factors (1 are added after PBST board-washings 3 times:10,1:100,1:1000,1:5000,
1:10000) mice serum 100ul, it is incubated at room temperature 2 hours, after PBST board-washings 5 times, 100ul is added per hole through 1:5000 dilutions
The sheep anti-mouse igg of HRP marks, is incubated at room temperature 1 hour, adds 50ul TMB nitrite ions, room temperature lucifuge after PBST board-washings 7 times per hole
Effect 10 minutes, 50ul1M phosphoric acid terminating reactions are added per hole.With Thermo multi-function microplate readers determination sample OD450 reading
Number.
The structure and disease of embodiment 1, restructuring chimpanzee adenovirus DNA pAdC68-EBOVgp and pAdC7-EBOVgp
Malicious titre detection
As shown in figure 1, EBOVgp whole Expression element is contained into CMV promoter and ploy A tails, AdC68 is cloned into respectively
With AdC7 carriers, to obtain restructuring chimpanzee adenovirus DNA, i.e. pAdC68-EBOVgp (Figure 1A) and pAdC7-EBOVgp (scheme
1B)。
PAdC68-EBOVgp full length nucleotide sequence such as SEQ ID NO:2；PAdC7-EBOVgp full length nucleotide sequence
Row such as SEQ ID NO:3.
EBOVgp fragments (fragment that Fig. 2A is obtained) are cloned into pShuttle carriers by the present inventor first, then utilize I-
Whole Expression element (fragment that Fig. 2 B are obtained) is cloned into AdC68 and AdC7 carriers by Ceu I and PI-Sce I respectively, and is used more
Kind digestion identification (Fig. 2 C), shows that the present inventor successfully builds recombinant adenovirus plasmid DNA pAdC68-EBOVgp and pAdC7-
EBOVgp.DNA sequence dna such as SEQ ID NO through codon optimization and the EBOVgp synthesized:Shown in 1.
The amplification of chimpanzee adenovirus poisons, purifying and detection are recombinated, the titre of two kinds of recombined adhenovirus is respectively AdC68-
EBOVgp, 6.82 × 1012vp/ml；AdC7-EBOVgp, 2.5 × 1012vp/ml。
Embodiment 2, recombinate chimpanzee adenovirus EBOVgp albumen detection of expression
The present inventor is with 108, 109, 1010Vp restructuring chimpanzee adenovirus AdC68-EBOVgp and AdC7-EBOVgp, with
And 109, 1010HEK293 cells are infected in the unloaded controls of vp AdC68-empty and AdC7-empty respectively, after infecting 24 hours,
With the cell lysis on ice of the protein lysate containing protease inhibitors, centrifuging and taking albumen supernatant, then detected using Western
The expression of EBOVgp albumen.As shown in figure 3, the unloaded control infection of the AdC68-empty and AdC7-empty of different titers
HEK293 cell detections are expressed less than EBOVgp；And infect 109, 1010Vp restructuring chimpanzee adenovirus AdC68-EBOVgp and
AdC7-EBOVgp HEK293 cells can detect the EBOVgp albumen substantially expressed.EBOVgp sequences after codon optimization
Arranging can efficiently express for recombinating chimpanzee adenoviral vector.
The above results illustrate that the present inventor successfully obtains the restructuring chimpanzee adenovirus of expression Ebola virus glycoproteins
AdC68-EBOVgp and AdC7-EBOVgp.
The detection of specific antibody of anti-Ebola GP albumen after embodiment 3, mouse immune restructuring chimpanzee adenovirus
The mouse of different disposal group is immunized according to the mouse immune timetable shown in Fig. 4 by the present inventor.As a result such as Fig. 5 institutes
Show, AdC68-EBOVgp groups (just exempting from AdC68-EBOVgp, two exempt from PBS) and AdC7-EBOVgp groups (just exempt from AdC7- after just exempting from 2 weeks
EBOVgp, two exempt from PBS) mice serum in can detect anti-Ebola GP protein specific antibodies (Fig. 5 A) compared with high titre；4
All (two exempt from 2 weeks) AdC68-EBOVgp groups, AdC7-EBOVgp groups and AdC7-EBOVgp+AdC68-EBOVgp groups (are just exempted from afterwards
AdC7-EBOVgp, two exempt from AdC68-EBOVgp) mice serum in can equally detect anti-Ebola GP albumen compared with high titre
Specific antibody (Fig. 5 B)；6 weeks (two exempt from 4 weeks) AdC68-EBOVgp groups afterwards, AdC7-EBOVgp groups and AdC7-EBOVgp+
The anti-Ebola GP protein specific antibodies (figure compared with high titre can be still detected in the mice serum of AdC68-EBOVgp groups
5C)；And corresponding control group(just exempting from, two exempt to use PBS), AdC68-empty groups (just exempt from AdC68-empty, two
Exempt from PBS), AdC7-empty groups (just exempt from AdC7-empty, two exempt from PBS) group can not inducing specific antibody generation (Fig. 5 A,
5B、5C)。
The above results illustrate that recombined adhenovirus chimpanzee adenovirus AdC68-EBOVgp, AdC7-EBOVgp have preferable
Immunogenicity, can the anti-Ebola virus antibody of inducing specific.
In Fig. 5 D, 5E, 5F, the present inventor compares AdC68-EBOVgp groups, AdC7-EBOVgp groups and AdC7-
EBOVgp+AdC68-EBOVgp groups were just exempted from 2 weeks, 4 weeks (two exempt from 2 weeks) is small after 6 weeks (two exempt from 4 weeks) at different time points
Specific antibody level in mouse serum, it can be found that more high titre is special after being detected compared to 2 weeks in mice serum after 4 weeks, 6 weeks
Heterogenetic antibody.This explanation restructuring chimpanzee adenovirus AdC68-EBOVgp, AdC7-EBOVgp, which can be used, just exempts from-booster immunization
Strategy to obtain stronger immune effect.
Discuss
Commercialization Ebola's vaccine is there is no at present, also without other effectively preventing measures.Reply may break out again
Ebola virus epidemic situation, accelerating the new Ebola's vaccine of research and development turns into the task of top priority.The present inventor utilizes the black orangutan of replication defect type
Orangutan adenovirus vector successfully builds and identifies new Ebola's candidate vaccine AdC68-EBOVgp and AdC7-EBOVgp.Then,
The present inventor using single or just exempt from-strategy of booster immunization is immunized mouse, in the immune rear different time point mice serums of detection
The specific antibody of anti-Ebola GP albumen, as a result show that AdC68-EBOVgp and AdC7-EBOVgp single immunizations can just induce
Good specific immunity, just exempt from-booster immunization after can induce stronger specific immune response.Therefore, the present invention not only carries
For two kinds of new Ebola's candidate vaccines, and different immunization strategies is provided, established for succeeding in developing for Ebola's vaccine
Basis.
All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (12)
1. a kind of research of Ebola vaccine, it is characterised in that the research of Ebola vaccine prepares by the following method：General angstrom
Rich to draw Viral vaccine vectors to be packed, processing obtains the research of Ebola vaccine with immunogenicity；Wherein, described Ai Bo
It is replication defect type chimpanzee adenoviral vector to draw Viral vaccine vectors, and wherein includes the expression of Ebola virus glycoproteins
Box.
2. research of Ebola vaccine as claimed in claim 1, it is characterised in that described replication defect type chimpanzee adenovirus
Carrier includes the chimpanzee adenovirus AdC68 genome sequences of transformation, wherein E1 missings；It is preferred that E1 most code sequence
Row are replaced by catenation sequence；Restriction enzyme site I-Ceu I and PI-Sce I are set in described catenation sequence.
3. research of Ebola vaccine as claimed in claim 1, it is characterised in that described replication defect type chimpanzee adenovirus
Carrier includes the chimpanzee adenovirus AdC7 genome sequences of transformation, wherein E1 and E3 missings；It is preferred that part E1 code areas with
Whole E3 code areas are deleted, and E1 deletes area and increases I-Ceu I and PI-Sce I restriction enzyme sites.
4. research of Ebola vaccine as claimed in claim 1, it is characterised in that described Ebola virus glycoproteins have
SEQ ID NO:Nucleotide sequence shown in 1.
5. research of Ebola vaccine as claimed in claim 2 or claim 3, it is characterised in that described Ebola virus glycoproteins
Expression cassette is inserted into restriction enzyme site I-Ceu I and the PI-Sce I of described replication defect type chimpanzee adenoviral vector.
6. research of Ebola vaccine as claimed in claim 1, it is characterised in that described research of Ebola vaccine carrier has
SEQ ID NO:2 or SEQ ID NO:Nucleotide sequence shown in 3.
A kind of 7. research of Ebola vaccine carrier, it is characterised in that it is replication defect type chimpanzee adenoviral vector, and wherein
Expression cassette containing Ebola virus glycoproteins.
8. research of Ebola vaccine carrier as claimed in claim 7, it is characterised in that described Ebola virus glycoproteins tool
There are SEQ ID NO:Nucleotide sequence shown in 1.
9. research of Ebola vaccine carrier as claimed in claim 7, it is characterised in that described research of Ebola vaccine carrier
With SEQ ID NO:2 or SEQ ID NO:Nucleotide sequence shown in 3.
10. a kind of medicine box, it is characterised in that contain any described Ebola virus epidemic diseases of claim 1-6 in described medicine box
Seedling；It is preferred that in described medicine box simultaneously containing claim 2 and 3 described in research of Ebola vaccine.
11. a kind of kit for being used to prepare vaccine, it is characterised in that the kit includes：Any one of claim 7-9 institutes
The research of Ebola vaccine carrier stated.
12. kit as claimed in claim 11, it is characterised in that the kit also includes：Virus production cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610696322.3A CN107753941A (en) | 2016-08-19 | 2016-08-19 | Research of Ebola vaccine based on chimpanzee adenoviral vector |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610696322.3A CN107753941A (en) | 2016-08-19 | 2016-08-19 | Research of Ebola vaccine based on chimpanzee adenoviral vector |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107753941A true CN107753941A (en) | 2018-03-06 |
Family
ID=61262907
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610696322.3A Pending CN107753941A (en) | 2016-08-19 | 2016-08-19 | Research of Ebola vaccine based on chimpanzee adenoviral vector |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107753941A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112410375A (en) * | 2019-08-22 | 2021-02-26 | 苏州相奕生物技术有限公司 | Adc68XY adenovirus vector, virus packaged by same and application |
| CN112760341A (en) * | 2020-07-13 | 2021-05-07 | 中国科学院微生物研究所 | Recombinant vector and application of chimpanzee adenovirus packaged with recombinant vector in preparation of 2019 novel coronavirus vaccine |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101014613A (en) * | 2004-01-23 | 2007-08-08 | P·安杰莱蒂分子生物学研究所 | Chimpanzee adenovirus vaccine carriers |
| US20130101618A1 (en) * | 2010-04-16 | 2013-04-25 | Okairos Ag | Chimpanzee adenoviral vector-based filovirus vaccines |
| CN103923943A (en) * | 2013-08-19 | 2014-07-16 | 中国科学院上海巴斯德研究所 | Expression vector based on adenovirus AdC7 and its construction method |
| CN103937835A (en) * | 2013-08-19 | 2014-07-23 | 中国科学院上海巴斯德研究所 | Expression vector based on adenovirus AdC68 and construction method thereof |
| CN105483140A (en) * | 2016-01-31 | 2016-04-13 | 中国人民解放军军事医学科学院生物工程研究所 | Ebola virus disease vaccine taking human replication deficient adenovirus as vector |
-
2016
- 2016-08-19 CN CN201610696322.3A patent/CN107753941A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101014613A (en) * | 2004-01-23 | 2007-08-08 | P·安杰莱蒂分子生物学研究所 | Chimpanzee adenovirus vaccine carriers |
| US20130101618A1 (en) * | 2010-04-16 | 2013-04-25 | Okairos Ag | Chimpanzee adenoviral vector-based filovirus vaccines |
| CN103923943A (en) * | 2013-08-19 | 2014-07-16 | 中国科学院上海巴斯德研究所 | Expression vector based on adenovirus AdC7 and its construction method |
| CN103937835A (en) * | 2013-08-19 | 2014-07-23 | 中国科学院上海巴斯德研究所 | Expression vector based on adenovirus AdC68 and construction method thereof |
| CN105483140A (en) * | 2016-01-31 | 2016-04-13 | 中国人民解放军军事医学科学院生物工程研究所 | Ebola virus disease vaccine taking human replication deficient adenovirus as vector |
Non-Patent Citations (7)
| Title |
|---|
| BAIZE,S.等: "Zaire ebolavirus isolate H.sapiens-wt/GIN/2014/Makona-Gueckedou-C07, complete genome", 《GENBANK》 * |
| DAPHNE A STANLEY等: "Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge", 《NAT MED》 * |
| ELAHEH SABAGHIAN等: "In Silico Design of a Multimeric Polytope as a Highly munogenic DNA Vaccine Against Human Cytomegalovirus", 《JOURNAL OF APPLIED BIOTECHNOLOGY REPORTS》 * |
| KOBINGER, GP等: "Chimpanzee adenovirus vaccine protects against Zaire Ebola virus", 《VIROLOGY》 * |
| OLGADE SANTISMRES等: "Safety and immunogenicity of a chimpanzee adenovirus-vectored Ebola vaccine in healthy adults: a randomised, double-blind, placebo-controlled, dose-finding, phase 1/2a study", 《LANCET INFECT DIS》 * |
| XI YANG等: "Chimpanzee adenoviral vector prime-boost regimen elicits potent immune responses against Ebola virus in mice and rhesus macaques", 《EMERG MICROBES INFECT》 * |
| 杨茜: "基于黑猩猩腺病毒载体的新型埃博拉疫苗研究", 《万方医学网》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112410375A (en) * | 2019-08-22 | 2021-02-26 | 苏州相奕生物技术有限公司 | Adc68XY adenovirus vector, virus packaged by same and application |
| CN112760341A (en) * | 2020-07-13 | 2021-05-07 | 中国科学院微生物研究所 | Recombinant vector and application of chimpanzee adenovirus packaged with recombinant vector in preparation of 2019 novel coronavirus vaccine |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021254327A1 (en) | Envelope replacement-type viral vector vaccine and construction method therefor | |
| Mayr et al. | Development of replication-defective adenovirus serotype 5 containing the capsid and 3C protease coding regions of foot-and-mouth disease virus as a vaccine candidate | |
| Sun et al. | Molecular characterization of infectious clones of the minute virus of canines reveals unique features of bocaviruses | |
| Havenga et al. | Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER. C6 cells | |
| Gall et al. | Construction and characterization of hexon-chimeric adenoviruses: specification of adenovirus serotype | |
| Santos et al. | A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants | |
| CN111218459A (en) | Recombinant novel coronavirus vaccine taking human replication-defective adenovirus as vector | |
| Sakai et al. | Accommodation of foreign genes into the Sendai virus genome: sizes of inserted genes and viral replication | |
| Sanz-Parra et al. | Evidence of partial protection against foot-and-mouth disease in cattle immunized with a recombinant adenovirus vector expressing the precursor polypeptide (P1) of foot-and-mouth disease virus capsid proteins. | |
| CN109536461B (en) | O-type foot-and-mouth disease virus mutant strain and preparation method and application thereof | |
| CN104962580B (en) | A kind of recombinant plant Rhabdovirus vector and its construction method | |
| CN103923943B (en) | A kind of expression vector and its construction method based on adenovirus AdC7 | |
| CN110951699B (en) | Recombinant rabies virus for expressing canine distemper virus structural protein and application thereof | |
| CN107190013A (en) | A kind of zika virus disease vaccine using people Ad5 replication-defective adenovirals as carrier | |
| Dus Santos et al. | Transgenic plants for the production of veterinary vaccines | |
| CN108504686A (en) | A kind of expression African swine fever virus EP153R gene recombinant adenovirus vectors, construction method and recombined adhenovirus preparation method | |
| Belsham | Influence of the Leader protein coding region of foot-and-mouth disease virus on virus replication | |
| CN106317208B (en) | Target protein of P structural domain of monkey-derived GII.17 type norovirus and variant thereof | |
| WO2023050484A1 (en) | Expression vector, recombinant adeno-associated virus, and use thereof in preparation of 2019 novel coronavirus vaccine | |
| CN103468743B (en) | A kind of Rabies Vaccine and preparation method thereof | |
| CN107753941A (en) | Research of Ebola vaccine based on chimpanzee adenoviral vector | |
| CN113308493A (en) | Novel coronavirus Ad26 adenovirus vector vaccine and preparation method and application thereof | |
| CN108514635A (en) | Recombinate trivalent adenovirus vaccine and its construction method | |
| Kim et al. | Full-length ORF2 sequence-based genetic and phylogenetic characterization of Korean feline caliciviruses | |
| CN106636001B (en) | Construction method and application of recombinant enterovirus phenotype mixing system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180306 |
|
| WD01 | Invention patent application deemed withdrawn after publication |