CN104805061A - Virus adaptive strain capable of infecting Chinese hamster ovary cell and application thereof - Google Patents
Virus adaptive strain capable of infecting Chinese hamster ovary cell and application thereof Download PDFInfo
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Abstract
The present invention relates to a virus adaptive strain capable of infecting Chinese hamster ovary cells and application thereof. The invention discloses a CHO-K adaptive strain of EV71, and the adaptive strain can effectively infect CHO-K cells. The mutant virus strain provided by the invention can be applied to the preparation of a cell model infected by enterovirus 71 for the research of pathogenic mechanism of virus, and antiviral drug evaluation.
Description
Technical field
The invention belongs to field of virology; More specifically, the present invention relates to a kind of energy infection of Chinese hamster ovary cell virus adapted strain and the application in vaccine development thereof.
Background technology
Enterovirus EV 71 type belongs to Picornaviridae enterovirus genus, and the infected is mainly preschool children, mainly causes hand foot mouth disease, can also cause aseptic meningitis, encephalitis simultaneously, the complication that the neural system such as poliomyelitis sample paralysis are relevant.Since reported first in 1974, worldwide cause repeatedly outburst with popular.Within 1997, start popular on a large scale and in rising trend in the Asian-Pacific area, become a kind of serious public health problem.China's Taiwan EV71 great outburst in 1998, there is hand foot mouth disease and erythematous eruption in 129106 examples, 405 examples are serious central nervous system infection, dead 78 examples.
2008, China's Fuyang hand foot mouth disease Epidemic outbreak of disease, wherein 23 examples are dead, subsequently, the report that the area such as Beijing, Shanghai, Zhejiang, Guangdong also has EV71 to infect in succession.Hand foot mouth diseases of reporting 2009 Nian Ji Henan Province Ming Quanxian infect and after having ten routine patient deaths, in succession in Shandong, Beijing, Sichuan, Guangxi, Yunnan, Gansu, multiple provinces and cities such as Guizhou are popular, number of patients and death toll are close on twice more than 2008.Have 1,774 in China in 2010,669 reported cases, cause 905 examples dead and more than 2.1 ten thousand routine severes.The cause of death is mainly the BBE caused by central nervous system infection, pulmonary edema and hemorrhage.
The metainfective medicine of EV71 comprises Interferon, rabbit (IFN-α), intravenous injection of immunoglobulin (IV I G) and a kind of new drug Pleconaril.Although the popular and genetic engineering subunit vaccine having the prevention of EV71 inactivated vaccine active immunity to infect is successfully used to the report of laboratory animal, not still being recognized at present is highly effective vaccine.Therefore, the vaccine developing effective anti-EV71 virus is extremely urgent, to ensureing the life security of children and growing up healthy and sound significant.
The EV71 vaccine research kind of carrying out at present comprises inactivated whole virus vaccines, attenuated live vaccine, virus sample particle vaccines (the VLP vaccine of baculovirus expression), the VP1 subunit vaccine etc. of DNA vaccination and escherichia coli expression.The wherein conformational epitope of attenuated live vaccine not break virus, and the neutralizing antibody producing and there is provide protection can be induced, and there is certain cross-protection ability.But attenuation EV71 also has certain virulence, there is the danger that virulence is recovered, there is certain potential safety hazard.
The VLP that baculovirus expression system is expressed can produce cell and humoral immunization by inducing mouse, has certain protective capability, but also lack the research of VLP cross protection to mouse.The NAT that other VP1 subunit vaccines and DNA vaccination induction body produce is lower, is still difficult to as vaccine development, need studies further.
EV71 inactivated virus vaccine has good immunogenicity, mature production technology, is the fastest vaccine of Recent Progresses In The Development.In March, 2013, the EV71 inactivated vaccine III clinical trial phase of China's independent research completed smoothly, goes on the market thus effectively prevent EV71 type hand foot mouth disease to provide scientific basis for EV71 vaccine.
Utilize in genetic evolution with the distant grey mouse CHO-K cells produce EV71 inactivated virus vaccine of the mankind in theory than mdck cell and CHO-K cell safer.But the EV71 virus vaccines developed also is not carried out in current this area on CHO-K cell.
Summary of the invention
The object of the present invention is to provide a kind of energy infection of Chinese hamster ovary cell virus adapted strain and application thereof.
In a first aspect of the present invention, provide a kind of Enterovirus 71 mutant strain, its genome encoding Structural protein VP1, VP2, VP3, VP4 and NS2 Protein A, 2B, 2C, 3A, 3B, 3C, 3D; Wherein, the 36th amino acids of VP1 is Asn, and the 258th amino acids is Val; 77th amino acids of 2A is Thr; 171st amino acids of 2C is Met, and the 305th amino acids is Ala.
In a preference, the aminoacid sequence of described VP4 is as shown in SEQ ID NO:2; The aminoacid sequence of described VP2 is as shown in SEQ ID NO:3; The aminoacid sequence of described VP3 is as shown in SEQID NO:4; The aminoacid sequence of described VP1 is as shown in SEQ ID NO:5; The aminoacid sequence of described 2A is as shown in SEQ ID NO:6; The aminoacid sequence of described 2B is as shown in SEQ ID NO:7; The aminoacid sequence of described 2C is as shown in SEQ ID NO:8; The aminoacid sequence of described 3A is as shown in SEQ ID NO:9; The aminoacid sequence of described 3B is as shown in SEQ ID NO:10; The aminoacid sequence of described 3C is as shown in SEQ ID NO:11; Or the aminoacid sequence of described 3D is as shown in SEQ ID NO:12.
In another preference, described Enterovirus 71 mutant strain, the aminoacid sequence of the albumen of its genome encoding is as shown in SEQ ID NO:13.
In another preference, the genome of described Enterovirus 71 mutant strain has the nucleotide sequence shown in SEQ ID NO:1.
In another aspect of this invention, provide the purposes of described Enterovirus 71 mutant strain, for setting up Enterovirus 71 virus reverse genetics technology based on Chinese hamster ovary celI; Or transform Enterovirus 71 virus with enhanced virus amplification ability or reduce its infectivity to the cell of people for cloning, further, or change Enterovirus 71 virus to host parent preferendum.
In another aspect of this invention, the purposes of described Enterovirus 71 mutant strain is provided, for the preparation of attenuation EV71 living vaccine or inactivated vaccine; Or
For prevent Enterovirus 71 and other cause the infection of the enterovirus of hand foot mouth disease.
In another aspect of this invention, provide the purposes of described Enterovirus 71 mutant strain, for the preparation of the cell model that Enterovirus 71 infects, or for the protected effect of evaluating vaccine or security, or for the research of attenuation EV71 living vaccine or inactivated vaccine; Preferably, described cell is Chinese hamster ovary celI.
In a preference, the cell model that described Enterovirus 71 infects is used for screening, the drug efficacy study of anti-Enterovirus 71 infection medicine.
In another aspect of this invention, a kind of test kit is provided, wherein comprises container, and be loaded on the described Enterovirus 71 mutant strain in container.
In a preference, the cell model that described test kit infects for the preparation of Enterovirus 71, or for the protected effect of evaluating vaccine or security, or for the research of attenuation EV71 living vaccine or inactivated vaccine.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
There is the CHO-K adapted strain (CAVS) of obvious pathology in the CHO-K that makes that Fig. 1, MAVS573 blind passage CHO-K cell line selection obtains.
Fig. 2, virus titer (TCID50) measure.
Fig. 3, CAVS-5# are for the infection figure of CHO-K cell, and wherein MOCK is normal CHO-K cell.
Fig. 4, CAVS-5#, the growth curve of MAVS, FY573 virus respectively on different clone CHO-K, RD, SK-N-SH.
Fig. 5, MAVS573 is infected CHO-K and RD respectively, then do growth curve, analyze infection conditions.
Fig. 6, viral CAVS-5#, FY573, MAVS are infected RD, CHO-K, SK-N-SH clone, the pathology situation on observation of cell.
Embodiment
The present invention is through studying widely, obtain the CHO-K adapted strain (CAVS-5#) of a strain EV71, can effectively infect CHO-K cell, its infectivity on RD cell is lower than Chinese hamster ovary celI adapted strain MAVS573 and people EV71 strain FY573, there is certain attenuation, this adapted strain is checked order and sequence alignment, has found 5 amino acid mutation sites.Mutated viruses strain of the present invention can be applied to preparing attenuation EV71 living vaccine or inactivated vaccine etc.
Viral protein and encoding gene
EV71 virus mutation strain of the present invention comprises Structural protein VP1, VP2, VP3 and VP4, and Nonstructural Protein comprises 2A, 2B, 2C and 3A, 3B, 3C and 3D; The gene of these albumen of encoding arranges as follows in viral genome: VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D.
Further research finds, in EV71 virus mutation strain, some amino acid mutations in VP1,2A, 2C contribute to the Chinese hamster ovary celI adaptability of this EV71 virus strain, and the infection ability made it for Chinese hamster ovary celI significantly strengthens.Described site is as follows: the 36th amino acids of VP1 is Asn, and the 258th amino acids is Val; 77th amino acids of 2A is Thr; 171st amino acids of 2C is Met, and the 305th amino acids is Ala.
The present invention also comprises the fragment of above-mentioned each mutant virus albumen, derivative and analogue.As used herein, term " fragment ", " derivative " and " analogue " refer to the polypeptide substantially keeping biological function that mutant virus albumen of the present invention is identical or activity.Polypeptide fragment of the present invention, derivative or analogue can be the polypeptide that (i) has one or more conservative or non-conservative amino acid residue (preferred conservative amino acid) and be substituted, and the amino-acid residue of such replacement can may not be and encoded by genetic code, or (ii) has the polypeptide of substituted radical in one or more amino-acid residue, or (iii) additional aminoacid sequence is fused to this peptide sequence and the polypeptide formed (as leader sequence or secretion sequence or be used for the sequence of this polypeptide of purifying or proprotein sequence, or fusion rotein).The known scope of those skilled in the art is belonged to according to these fragments of definition herein, derivative and analogue.But, in described fragment, derivative and analogue, be conservative corresponding to contributing the amino acid in the adaptive site of Chinese hamster ovary celI described in leading portion.
Present invention also offers the polynucleotide sequence of code book invention mutant virus albumen.The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, the sum analogous to general Dedekind sum of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient of non-natural generation.These nucleotide variants comprise and replace varient, Deletion variants and insertion varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of one or more Nucleotide, disappearance or insertion, but can not from the function of polypeptide changing in fact its coding.
The polynucleotide sequence of encode mutant polypeptide of the present invention can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.When sequence is longer, usually needs to carry out twice or repeatedly pcr amplification, and then the fragment that each time amplifies is stitched together by proper order.
In addition, also can synthesize relevant sequence by the method for synthetic, usually, by first synthesizing multiple small segment, and then carry out connect can obtain the very long fragment of sequence.
Virus mutation strain
The present invention, by the separation of EV71 virus strain, cultivates, and the technique means such as cell screening obtain and effectively can infect the virus strain that CHO-K produces adaptive mutation, and the vaccine research and development of the hand foot mouth disease caused for EV71 provide strong support.
First, the present invention uses ICR mouse adaptability strain 573 to be tested by virus infection, and obtaining can the adaptability strain of efficient infection CHO-K.
Secondly, this CHO-K adaptability strain 5# (CHO-Kadapted virus strain5#, CAVS-5#) is further purified by plaque colony screening.
3rd, in order to confirm CAVS-5# gene expression characteristics further, contriver has carried out comparison to its sequencing and analyzing, determines amino acid mutation site, for through engineering approaches vaccine provides modification scheme.
Therefore, the invention provides a kind of EV71 virus mutation strain, greatly strengthen relative to its ability infecting Chinese hamster ovary celI of wild-type starting strain.In described EV71 virus mutation strain, some amino acid mutations in VP1,2A, 2C contribute to the Chinese hamster ovary celI adaptability of this EV71 virus strain, and the infection ability made it for Chinese hamster ovary celI significantly strengthens.
As optimal way of the present invention, the albumen of the genome encoding of described EV71 virus mutation strain has the aminoacid sequence shown in SEQ ID NO:13.More preferably, its genome has the nucleotide sequence shown in SEQ ID NO:1.
Based on genomic information or the amino acid sequence information of virus strain provided by the invention, those skilled in the art can synthesize described gene or albumen completely on this basis, prepare the virus strain that Chinese hamster ovary celI of the present invention adapts to.
The present invention is by the separation of EV71 virus strain, and cultivate, the technique means such as cell screening obtain and can effectively infect CHO-K cell and the virus strain producing adaptive mutation, and the vaccine research and development of the hand foot mouth disease caused for EV71 provide strong support.Can be used for the research of attenuation EV71 living vaccine or inactivated vaccine; For the research of EV71 virusology aspect; For the research of EV71 virus with immunology aspect.
Test kit
The present invention also comprises the test kit for the preparation of EV71 virus infection Chinese hamster ovary celI model, and described test kit comprises: container, and is placed in the described EV71 virus mutation strain of described container.
As optimal way of the present invention, described test kit also comprises other reagent being applied to virus culture and periphery reagent.More preferably, also can comprise the working instructions that using method and lime light are described in described test kit, apply described EV71 virus mutation strain to be conducive to people.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.
I. material
Clone
Chinese hamster ovary line (CHO-K) is available from Beijing institute of Biological Products; Human rhabdomyosarcoma's clone (RD) and human neuroblastomacells (SK-N-SH) obtain from ATCC company of the U.S..
Virus
EV71 mouse adapted strain virus (MAVS573) is that the present inventor passes through to infect the acquisition of young mouse, and (this adapted strain is suitable to breed on Mouse L929 cell; See Chinese patent ZL201110113557.2; People EV71 virus strain (FY573) is that this laboratory obtains from Formations In Fuyang Area hand foot mouth disease Patient Sample A separation in 2008, and sequence is shown in NCBI#HM064456.1.
Reagent
HyClone modified form RPMI-1640 substratum (purchased from
), HyClone DMEM/HIGAGLUCOSE substratum (purchased from
), foetal calf serum (FBS purchased from
), 4% low melting-point agarose.
III. embodiment
Embodiment 1, MAVS573 blind passage CHO-K clone
(1) cell was inoculated in 6 orifice plates in 24 hours in advance, containing 1640 substratum of 1ml2%FBS, 3 × 10
5individual cell per well, hatch in incubator (37 DEG C, CO
2concentration 5%).
(2) discard substratum, PBS washes 2 times, adds 1640 substratum of 300ul without FBS, adds the viral 200ul/ hole of EV71 mouse adapted strain (MAVS573), hatches 1h for 37 DEG C; Discard infection virus liquid, PBS washes 2 times, rejoins 500ul2%FBS1640,37 DEG C, CO
2(5%) cultivate; Infect after 72 hours, collect virus and supernatant liquor, centrifugal after multigelation 3 times, get supernatant liquor frozen; The viral supernatants obtained is infected CHO-K cell again, and after consecutive infection 6 generation, screening makes CHO-K occur the CHO-K adapted strain (CAVS) of obvious pathology, as Fig. 1.
Embodiment 2, mensuration virus titer (TCID50)
(1) CHO-K cell is inoculated into 96 orifice plates, 100ul2%FBS1640 substratum/hole, 7 × 10
3cells/well, 37 DEG C of overnight incubation.
(2) use is without 1640 substratum of FBS by viral gradient dilution, and 100ul/ hole dilution venom, each gradient 9 repeating holes, each gradient has a control wells, hatches 1h for 37 DEG C.
(3) suck venom, PBS washes 1 time, rejoins 2%FBS1640 substratum, 100ul/ hole, observes pathology situation, infects and calculate virus titer (TCID after seven days
50).Result is as Fig. 2.
Embodiment 3, plaque colony screening are tested
(1) do the experiment of plaque colony screening with the CHO-K adapted strain (CAVS) the 6th generation (C-EV-P6) obtained, CHO-K cell is inoculated into 24 orifice plates, in 1ml2%FBS1640 substratum 2 × 10
5/ hole, 37 DEG C of CO
2(5%) overnight incubation;
(2) viral gradient dilution, adds 24 orifice plates, hatches 1h for 37 DEG C, and 4% low melting-point agarose glue mixes with 2%FBS1640 substratum 1:3, adds 24 orifice plates, 1ml/ hole;
(3) observation of cell pathology, picking plaque, is melted into 500ul without FBS1640 substratum; With the plaque clonal virus liquid inductance dye CHO-K cell of picking, observe pathology situation, select pathology and clone significantly.
No. 5 clone picked out, called after " CAVS-5# ", can on CHO-K cell obvious pathology, as Fig. 3.
Embodiment 4, CAVS-5#, MAVS, the FY573 growth curve of virus on different clone CHO-K, RD, SK-N-SH and cell infection capability analysis
Respectively CHO-K, RD, SK-N-SH cell is inoculated in 24 orifice plates (2 × 10
5cells/well), 37 DEG C of CO
2(5%) overnight incubation, MOI=1 cells infected, infects step with aforementioned " embodiment 1 "; Do time curve (time course), collect different time points supernatant and cell, multigelation 3 times, centrifugal, obtain venom ,-80 DEG C frozen.
Detect the virus titer (TCID50) of different time points sample venom.Step is with aforementioned " embodiment 2 ".Result is as Fig. 4.Visible on CHO-K cell, CAVS-5# cell has very strong infection ability, and infection ability is significantly higher than MAVS573, FY573 virus; For other cell, then infection ability is lower.
MAVS573 is infected CHO-K and RD respectively, then on RD, does growth curve, analyze infection conditions.Result is pointed out, and MAVS partly can to enter in CHO-K cytolemma but can not copy on CHO-K, as Fig. 5.
Viral CAVS-5#, FY573, MAVS are infected RD, CHO-K, SK-N-SH clone, the pathology situation on observation of cell.Particularly, by 2 × 10
5individual cell is inoculated in 24 orifice plates, 37 DEG C of overnight incubation, and virus infected cell (MOI=1), observation of cell pathology, takes pictures.Result is as Fig. 6.
Embodiment 5, CHO-K adapt to the amino acid mutation Locus Analysis in Shoots of virus strain
The gene sequencing result of CAVS-5# virus as SEQ ID NO:1, the Structural protein VP1 (SEQ ID NO:5) of its encode viral, VP2 (SEQ ID NO:3), VP3 (SEQ ID NO:4), VP4 (SEQ IDNO:2) and NS2 Protein A (SEQ ID NO:6), 2B (SEQ ID NO:7), 2C (SEQ ID NO:8), 3A (SEQ ID NO:9), 3B (SEQ ID NO:10), 3C (SEQ ID NO:11), 3D (SEQ ID NO:12).Full amino acid sequence after the sequence merging of each albumen is as SEQ ID NO:13.
By CAVS-5# and MAVS sequence alignment, 5 new amino acid mutation sites are found, as table 1.
Table 1
Therefore, CAVS-5# and MAVS two-strain, for the difference infecting CHO-K ability, is caused by the amino acid mutation in above-mentioned site.
Embodiment 6, CHO-K adapt to the cells infected model of virus strain
CHO-K cell is inoculated in 24 orifice plates (2 × 10
5cells/well), 37 DEG C of CO
2(5%) overnight incubation, by CAVS-5# with MOI=1 cells infected, infects step with aforementioned " embodiment 1 ", obtains the cell model infected, can be observed obvious pathology on CHO-K cell.
CAVS-5# virus can be used for exploitation attenuation EV71 living vaccine or inactivated vaccine and improves vaccine producing quantifier elimination.Specific as follows:
The present inventor is viral with the CAVS-5# of acquired high virulence on CHO-K cell, adopt the cell matrix being virus culture with CHO-K cell, adopt Microcarrier Culture Techniques, infect CAVS-5# virus, obtain the virus of high titre, and detect tiring of the neutralizing antibody of virus further, prepare EV71 inactivated vaccine.
Further, CAVS-5# can be infected, goes down to posterity on zooblast or young baby, to obtain the cell of the people not attenuated strain that reduces of the infective virus strain of tool or virulence, for the preparation of attenuated live vaccine.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. an Enterovirus 71 mutant strain, is characterized in that, its genome encoding Structural protein VP1, VP2, VP3, VP4 and NS2 Protein A, 2B, 2C, 3A, 3B, 3C, 3D; Wherein,
36th amino acids of VP1 is Asn, and the 258th amino acids is Val;
77th amino acids of 2A is Thr;
171st amino acids of 2C is Met, and the 305th amino acids is Ala.
2. Enterovirus 71 mutant strain as claimed in claim 1, is characterized in that,
The aminoacid sequence of described VP4 is as shown in SEQ ID NO:2;
The aminoacid sequence of described VP2 is as shown in SEQ ID NO:3;
The aminoacid sequence of described VP3 is as shown in SEQ ID NO:4;
The aminoacid sequence of described VP1 is as shown in SEQ ID NO:5;
The aminoacid sequence of described 2A is as shown in SEQ ID NO:6;
The aminoacid sequence of described 2B is as shown in SEQ ID NO:7;
The aminoacid sequence of described 2C is as shown in SEQ ID NO:8;
The aminoacid sequence of described 3A is as shown in SEQ ID NO:9;
The aminoacid sequence of described 3B is as shown in SEQ ID NO:10;
The aminoacid sequence of described 3C is as shown in SEQ ID NO:11; Or
The aminoacid sequence of described 3D is as shown in SEQ ID NO:12.
3. Enterovirus 71 mutant strain as claimed in claim 1, it is characterized in that, the aminoacid sequence of the albumen of its genome encoding is as shown in SEQ ID NO:13.
4. Enterovirus 71 mutant strain as claimed in claim 1, it is characterized in that, its genome has the nucleotide sequence shown in SEQ ID NO:1.
5. the purposes of the arbitrary described Enterovirus 71 mutant strain of claim 1-4, is characterized in that, for setting up Enterovirus 71 virus reverse genetics technology based on Chinese hamster ovary celI; Or
Transform Enterovirus 71 virus with enhanced virus amplification ability or reduce its infectivity to the cell of people for cloning, further, or change Enterovirus 71 virus to host parent preferendum.
6. the purposes of the arbitrary described Enterovirus 71 mutant strain of claim 1-4, is characterized in that, for the preparation of attenuation EV71 living vaccine or inactivated vaccine; Or
For prevent Enterovirus 71 and other cause the infection of the enterovirus of hand foot mouth disease.
7. the purposes of the arbitrary described Enterovirus 71 mutant strain of claim 1-4, is characterized in that, for the preparation of the cell model that Enterovirus 71 infects, or for evaluating the protected effect of vaccine, or for the research of attenuation EV71 living vaccine or inactivated vaccine; Preferably, described cell is Chinese hamster ovary celI.
8. purposes as claimed in claim 7, is characterized in that, the cell model that described Enterovirus 71 infects is used for screening, the drug efficacy study of anti-Enterovirus 71 infection medicine.
9. a test kit, is characterized in that, wherein comprises container, and is loaded on the arbitrary described Enterovirus 71 mutant strain of claim 1-4 in container.
10. test kit as claimed in claim 9, is characterized in that, the cell model that described test kit infects for the preparation of Enterovirus 71, or for the protected effect of evaluating vaccine or security, or for the research of attenuation EV71 living vaccine or inactivated vaccine.
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| CN108663517A (en) * | 2017-03-31 | 2018-10-16 | 深圳市儿童医院 | It can be used for diagnosing the kit of human enterovirus infection |
| CN112029735A (en) * | 2020-08-31 | 2020-12-04 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease virus non-structural protein 3B dominant epitope deletion marker strain and preparation method and application thereof |
| CN113105529A (en) * | 2020-01-10 | 2021-07-13 | 北京大学 | Nucleic acid sequence, enterovirus containing nucleic acid sequence and application of enterovirus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108663517A (en) * | 2017-03-31 | 2018-10-16 | 深圳市儿童医院 | It can be used for diagnosing the kit of human enterovirus infection |
| CN113105529A (en) * | 2020-01-10 | 2021-07-13 | 北京大学 | Nucleic acid sequence, enterovirus containing nucleic acid sequence and application of enterovirus |
| CN113105529B (en) * | 2020-01-10 | 2022-12-02 | 北京大学 | Nucleic acid sequence, enterovirus containing nucleic acid sequence and application of enterovirus |
| CN112029735A (en) * | 2020-08-31 | 2020-12-04 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease virus non-structural protein 3B dominant epitope deletion marker strain and preparation method and application thereof |
| CN112029735B (en) * | 2020-08-31 | 2022-04-12 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease virus non-structural protein 3B dominant epitope deletion marker strain and preparation method and application thereof |
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