CN103966263A - Recombinant human adenovirus 3, and preparation method and application thereof - Google Patents
Recombinant human adenovirus 3, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a novel enterovirus 71-recombinant human adenovirus 3 vaccine candidate strain with human adenovirus 3 (HAdv3) as a carrier, and a preparation method thereof. Two EV71 neutralizing epitopes are embedded to the hexon of the human adenovirus 3, and the VP1 protein cassette of EV71 is inserted to the genome E3 region of the human adenovirus 3. The vaccine candidate strain can induce a strong anti-EV71 infection and anti-HAdv3 infection immunization reaction, and can be used for making bivalent vaccines for preventing the EV71 infection and the HAdv3 infection.
Description
Technical field
The invention belongs to recombinant viral genome field of engineering technology, be specifically related to a kind of newtype enteroviru 71 type-restructuring human 3-type adenovirus vaccine Candidate Strains that human 3-type adenovirus (HAdv3) is carrier and preparation method thereof of take.
Background technology
Enterovirns type 71 (EV71) is to cause hand foot mouth disease (hand-foot-mouth disease, HFMD) one of main pathogens, and often cause as serious central nervous system complications such as aseptic meningitis, encephalitis, poliomyelitis sample paralysis and neurogenic pulmonary edemas, and cause death.Within 1969, outburst EV71 in the reported first whole world infects, and in recent years, the report of EV71 eruption and prevalence increases, and popularity degree is obvious ascendant trend, has become the important pathogen body that the Asian-Pacific area comprises domestic threat public health.EV71 propagates through approach such as the air spittle, and tool is highly pathogenic, and relative death rate is higher, there is no at present effective medicine and method, and vaccine inoculation prevents the optimal path of EV71 virus infection beyond doubt.
EV71 belongs to Picornaviridae enterovirus genus, nonencapsulated single strand plus RNA virus, and its capsid is comprised of 4 kinds of protein: VP1, VP2, VP3 and VP4.Wherein VP1, VP2, VP3 be on virus particle surface, and VP4 is inner in virus particle.The research of relevant EV71 subunit vaccine shows; VP1 is comprised of 297 amino acid; the albumen that has maximum and maximum surfaces to expose in capsid protein; comprised the main neutrality epitope of EV71 virus; can stimulate body to produce the protectiveness neutralizing antibody of high titre, be the most important albumen of preparation EV71 subunit vaccine.The EV71 vaccine that also there is no at present clinical application.Epiposition vaccine is to use the vaccine of preparing based on epitope, is the direction of developing at present infectious diseases and malignant tumour vaccine.Epitope is the sequence that determines antigen-specific in antigen molecule, claims again antigenic determinant, comprises t cell epitope and B cell epitope, is respectively the fundamental unit of T cell antigen receptor and B cell antigen receptor and antibody specific combination.Compare traditional vaccine, epiposition vaccine has been removed unwanted even harmful antigen and epi-position and has been made immunne response accurately for specific epi-position.The epi-position that has had in recent years a lot of uniquenesses is out identified and be applied to the vaccine research of transmissible disease, cancer and autoimmune disorder.Yet a little less than independent epitope peptide immunogenicity, can not induce immune response and immunological memory, thereby development in recent years a lot of methods, as novel adjuvant, lipopeptid coupling, application Universal T-cell epitopes, multiple antigenic peptide, nanometer are delivery system and virus vector etc., promoted the development of epiposition vaccine.Have been found that at present a plurality of neutrality epitopes of EV71 virus, as the 163-177 amino acids (PESRESLAWQTATNPC, SP55) of VP1 albumen and 208-222 amino acids (YPTFGEHKQEKDLEYC, SP70).
Adenovirus carrier can effectively be induced body fluid and cellullar immunologic response, has been widely used in the gene therapy and vaccine research of cancer and infectious diseases.Adenovirus molecular weight is large, itself is a kind of fabulous immunological adjuvant, can effectively stimulate body to produce specific cellular immunization and humoral immunization; And adenovirus carrier has and easily carries out genetic manipulation, the easy feature of production and high yield, high security.Traditional adenovirus carrier vaccine is as transgene carrier, utilizes host cell expression exogenous antigen, stimulates body to produce body fluid or the cellullar immunologic response of antigen-specific.Due to the pre-existing immunity of ubiquity in crowd for human adenovirus type 5 carrier, and the strong immunogenicity of adenovirus carrier, cause the scavenging(action) of immunity system to carrier, suppressed the expression of transgenosis antigen, traditional adenovirus carrier is very restricted as the application of vaccine carrier.In recent years, adopt the recombinant adenovirus vaccine of " antigen displaying " strategy preparation developed, do not affecting under the prerequisite of viral infectivity and stability, thereby embed exogenous antigen in adenovirus carrier capsid, obtaining the recombinant adenovirus particle with new antigenic characteristic.After object antigen peptide insertion capsid, as a part for capsid, repeatedly immune body is induced effective immunne response, and does not need to carry out transgene expression, has overcome the shortcoming that traditional adenovirus transgene carrier is subject to the anti-carrier immunity of body restriction.
Adenovirus particles is an icosahedral symmetrical structure, and housing is by 12 penton pedestal and scleroproeins that are positioned at drift angle, and six adjacent bodies (hexon) of 240 non-drift angle particles and structure accessory protein polypeptide form.Six adjacent bodies are tripolymer structures, are immunogens the abundantest in adenovirus particles, insert a Surface Display of Foreign Epitopes in hexon, and each adenovirion just can be shown 720 epitopes, and epitope can be given full expression to.The aminoacid sequence of the hexon of different type adenovirus has the homology up to 78-95%, its difference mainly concentrates on 7 hypervariable regions (HVRs), these hypervariable regions are exposed to capsid surface, do not relate to the interaction between six adjacent body monomers, do not interact with other adenovirus protein yet.Existing a plurality of research based on 5 type adenovirus shows that a plurality of hypervariable regions of six adjacent bodies have enough snappinesies, can embed the exogenous peptide of different lengths and does not affect the copying of virus, stability and function; And the adenovirus immunity that likely can be in vivo prestores with external escape of the chimeric adenovirus carrier that enters some specific HVR district of epi-position, stronger immunogenicity is provided.
Therefore, capsid is shown EV71 neutralizing epitope, the major antigen VP1 albumen of transgene expression EV71 simultaneously, and the enterovirns type 71-restructuring human 3-type adenovirus vaccine Candidate Strain that retains human 3-type adenovirus major antigen is expected to for prevent EV71 to infect and HAdv3 infection simultaneously.
Summary of the invention
The invention provides a kind of restructuring human 3-type adenovirus and its preparation method and application.
The invention provides a kind of restructuring human 3-type adenovirus, described restructuring human 3-type adenoviral gene ZuE3 has inserted in district the VP1 protein expression frame of EV71, in the adjacent body of described human 3-type adenovirus six, two kinds of EV71 neutralizing epitope SP70 and SP55 have been embedded, the aminoacid sequence of described EV71 neutralizing epitope SP70 is YPTFGEHKQEKDLEYC, and the aminoacid sequence of described neutralizing epitope SP55 is PESRESLAWQTATNPC.
The replacement district of described EV71 neutralizing epitope SP70 is the HVR1 district of HAdv3 six adjacent bodies, and the replacement district of described EV71 neutralizing epitope SP55 is the HVR2 district of HAdv3 six adjacent bodies.
The replacement amino acid of described EV71 neutralizing epitope SP70 is the NRDNAV in HVR1 district, and the replacement amino acid of described EV71 neutralizing epitope SP55 is the TTEGEE in HVR2 district.
The present invention also provides the bivalent vaccine candidate of a kind of human 3-type adenovirus-EV71, and described bivalent vaccine candidate contains above-mentioned restructuring human 3-type adenovirus.
The present invention also provides a kind of carrier, and described carrier comprises above-mentioned restructuring human 3-type adenovirus.
The present invention also provides a kind of method of preparing above-mentioned restructuring human 3-type adenovirus, comprise the steps: to have inserted in restructuring human 3-type adenoviral gene ZuE3 district the VP1 protein expression frame of EV71, two kinds of EV71 neutralizing epitopes on the adjacent body of human 3-type adenovirus six, have been embedded simultaneously, the amino acid of replacing is respectively the NRDNAV in HVR1 district, the TTEGEE in HVR2 district.
Recombinant virus provided by the present invention, allows the indivedual amino acid whose disappearances of EV71 neutralizing epitope, replacement or interpolation, but need to keep the immunogenicity of neutralizing epitope; Allow the indivedual amino acid whose disappearances of replaced HVR1 district and HVR2 region sequence, replacement or interpolation, but need to keep the stability of adenovirus, guarantee the immunocompetence of foreign epitope simultaneously; The EV71 epi-position embedding in the present invention is not limited to SP70 and SP55, can be also other protectiveness t cell epitope or B cell epitope, but need to keep the stability of adenovirus and the immunogenicity of assurance foreign epitope.
In the present invention, insert in the exogenous gene expression frame in HAdv3 genome E3 district, comprise CMV promotor and SV40po1yA, the major antigen VP1 protein gene sequence of EV71 is the full gene of VP1 of EV71-C4 hypotype; Meanwhile, allow to use other eukaryotic expression cassette and VP1 total length or the portion gene of other EV71 hypotype.
In the present invention; in the major antigen VP1 of transgene expression EV71 albumen; two kinds of main neutralizing epitopes simultaneously showing EV71 on six adjacent body capsids; this recombinant viral vector can be escaped the adenovirus immunity prestoring; produce the protective immune response that strong anti-EV71 infects, multiple injection energy booster immunization.Meanwhile, recombinant virus of the present invention has retained the major antigen activity of HAdv3, and this recombinant virus can be simultaneously for preventing the infection of HAdv3.
Accompanying drawing explanation
Fig. 1 infects EV71 suckling mouse survivorship curve figure.
Embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described, in the following example, NM specific experiment method, carries out according to normal experiment method conventionally.
HAdv3 described in this specification sheets embodiment is HAdv3-gz01 virus strain, No. Genbank, full genome: DQ099432.EV71 described in this specification sheets embodiment is EV71-08-02 virus strain, No. GenBank, whole genome sequence: FJ360545.EV71VP1 gene amplification in this specification sheets embodiment is from EV71-08-02 virus strain, and aminoacid sequence is without sudden change.
Embodiment 1: the surperficial exposure zone of bioinformatic analysis prediction human 3-type adenovirus six adjacent bodies
Use BLAST-P program in Protein Data Bank (PDB), to search for the formwork structure of the adjacent body homology of HAd3 six modeling, and use Modeller9.9 to carry out homology modeling, build the 3D model of HAd3 hexon.Image file experiment PYMOL processes.Selection is exposed to the hypervariable region sequence on capsid surface and carries out next step experiment.
By 3D model is analyzed, can in potential on the adjacent body of human 3-type adenovirus six and epitope position, analyze and be wherein exposed to that capsid surface most probable is substituted and the sequence that do not affect six adjacent body structure stability.HVR1, HVR2, HVR4, HVR5, HVR7 are potential Zhong He epitope districts, wherein partial amino-acid is likely substituted and does not affect six adjacent body structure stability, two hypervariable region sequences are as follows, and wherein the amino acid of underscore mark is in the present invention, to be the sequence that SP70 and SP55 replace.
HVR1：IVTT
NRDNAVTTTTNT
HVR2：KEGLQIGKDIT
TTEGEEKPIYADK
Embodiment 2: construction of recombinant virus carrier
Disappearance shuttle vectors in E3 district builds take HAdv3-gz01 virus strain genome (Genbank accession no.DQ099432) and distinguishes on pcr amplification genome 26 for template, 782 to 27, 736, on the E3L fragment (KpnI+ClaI) of long 973bp and genome 30, 900 to 31, 901, the E3R fragment (SpeI+NotI) of long 1020bp, take pEGFP C2 carrier as template pcr amplification CMV-eGFP-SV40 expression cassette (ClaI+SpeI), enzyme is cut and is connected on pBluescript II SK (+) carrier successively, acquisition comprises the carrier pSKE3LCMV-eGFP-SV40E3R (disappearance E3 district 3203bp sequence) of left and right, E3 district two arm sequences.
Human 3-type adenovirus skeleton plasmid builds: use primer pair AdLU1 (5 '-GAATTCGCGATCGCTATCTATATAATATACCTTATAGATGG-3 ', introduce EcoRI and AsisI restriction enzyme site) and AdLD1 (5 '-CTGCTGTGGATAAGCTTGAG-3 ', comprise HindIII restriction enzyme site) the Segment A 3L of amplification HAdv3-gz01 virus strain genome left end 1391bp, EcoRI+HindIII double digestion is connected to pBR322 carrier and obtains pBRAL carrier; With primer pair AdRU (5 '-AACAAAGCTTACACTATGCATAGTCATAGTATC-3 ', comprise HindIII restriction enzyme site) and AdRD (5 '-AGTCGACGCGATCGCTATCTATATAATATACCTTATAGATG-3 ', introduce Sal I and AsiSI restriction enzyme site) the Segment A 3L of amplification HAdv3-gz01 virus strain genome right-hand member 2025bp, EcoRI+HindIII double digestion is connected to pBRAL carrier and obtains pBRALR carrier.HindIII linearizing pBRALR carrier, obtains human 3-type adenovirus skeleton plasmid pBRAdv3 with the full genome of HAdv3-gz01 virus strain homologous recombination in bacterium BJ5183 of extracting purifying.
The human 3-type adenovirus plasmid pBRAd △ E3GFP that E3 district inserts eGFP expression cassette builds: EcoRV+NotI double digestion shuttle vectors pSKE3LCMV-eGFP-SV40E3R, in E.coli BJ5183, carry out homologous recombination with the human 3-type adenovirus skeleton plasmid pBRAdv3 of RsrlI linearization for enzyme restriction again, PCR identifies, enzyme is cut evaluation and order-checking evaluation obtains recombinant plasmid pBRAd △ E3GFP.
VP1 shuttle plasmid builds: carry out rite-directed mutagenesis pcr amplification EV71-08-02 strain VP1 protein gene and (the EcoRV restriction enzyme site sudden change silence on VP1 gene is convenient to subclone, primer the Vp1-ageI:5 '-gctaccggtcgccaccatgggagatagggtggcagatgtaattg-3 ' using, Vp1-pstI:5 '-aaactgcagTcAGTGATGGTGATGGTGGTGaagagtagtgatcgccgtgcggct-3 ', VP1U:5 '-gcgagtgcttatCaatggttttatgacgggtatcc-3 ', VP1R:5 '-ctcctgtttgtgttctccgaatgtgggataccc-3 '), after AgeI+PstI double digestion, be connected with shuttle vectors pSKE3LCMV-eGFP-SV40E3R, EV71VP1 protein gene replaces eGFP gene order and obtains shuttle plasmid pSKE3LCMV-VP1-SV40E3R.
Overlapping PCR (overlapping PCR) method builds the restructuring human 3-type adenovirus carrier that six adjacent bodies are modified: take pBRAd △ E3GFP as template respectively on pcr amplification pBRAd △ E3GFP carrier 16764-21140 be about the L1 fragment of 4.34kb and the L2 fragment that 21125-24569 is about 3.44kb, with EcoRI+BamHI and BamHI+SalI double digestion, be connected to carrier pBR322 respectively upper, obtain carrier pBRHSS.The six adjacent body genes of modifying with the amplification of overlapping PCR (overlapping PCR) method, first the DNA sequence dna that the HAdv3 genome of take obtains SP70 (5 '-tatcccacattcggagaacacaaacaggagaaagatcttgaatat-3 ') as overlapping PCR method for template is replaced the adjacent body fragment of sudden change six of the HVR1 corresponding sequence of the adjacent body gene of HAdv3 six, is connected in T carrier and order-checking is identified; Take these six adjacent body genes that suddenly change obtains two sudden change six adjacent body fragments that SP55 (5 '-ccagattccagggaatcccttgcatggcaaactgccaccaacccc-3 ') replaces the HVR2 corresponding sequence of the adjacent body gene of HAdv3 six as overlapping PCR method for template again, after ClaI+BamHI double digestion, be connected on pBRH3S carrier, obtain shuttle vectors pBRSP70R1SP55R2H3S.With EcoRI+SalI double digestion shuttle vectors, in E.coli BJ5183, carry out homologous recombination with the skeleton plasmid pBRAd △ E3GFP of AvRII+PacI double digestion, obtain recombinant plasmid pAdSP70R1SP55R2 △ E3GFP.The primer using is PCR Screening and Identification special primer in Table 1, Sp70r and Sp55r.
The primer that the adjacent body gene of HAdv3 six that the overlapping PCR method amplification of table 1 foreign epitope embeds is used
Recombinant viral vector builds: the pAdSP70R1SP55R2 △ E3GFP plasmid that the six adjacent bodies of take are modified is as skeleton plasmid and use RsrII linearization for enzyme restriction, EcoRV+NotI double digestion shuttle plasmid pSKE3LCMV-VP1-SV40E3R, the two carries out homologous recombination in E.coli BJ5183, and PCR identifies, enzyme is cut and identified and order-checking evaluation acquisition recombinant plasmid pAdSP70R1SP55R2 △ E3VP1.
Transfection AD293 cell after the plasmid AsisI linearization for enzyme restriction that above-mentioned restructuring is obtained, rescue is packaged to be recombinant viral vector AdSP70R1SP55R2 △ E3VP1, a large amount of cultivation and CsCl density gradient centrifugation purification of Recombinant virus particle.Purified Ad3dE3VP1 virus particle reaches 1 * 10
12vPs/ml, measures the about 1.2-1.4 of 0D260/0D280 ratio, and intracellular toxin detects < 10EU/m1.Recombinant virus AdSP70R1SP55R2 △ E3VP1 continuous passage in HEp-2 cell is extracted viral genome and is carried out that enzyme is cut evaluation, order-checking is identified after 10 generations, show that recombinant virus genomes keeps stable, there is no that gene lacks, Reorganization.
Embodiment 3: recombinant viral vector Study On Immunogenicity
Prepare antiserum(antisera), carry out serum neutralization test, the immunogenicity of checking recombinant viral vector.Recombinant virus AdSP70R1SP55R2 △ E3VP1 intramuscular injection immune mouse by purifying, obtains polyclonal serum, then carries out microneutralization test, with detect antibody that recombinant virus produces whether have external in and the ability of EV71 virus and HAdv3 virus.
The experimental result of the mice serum neutralization reaction gathering for 21 days after single immunization is in Table 2, and single immunization can induce the anti-EV71 virus of high titre and the neutralizing effect antibody of anti-HAdv3 virus; The experimental result of the mice serum neutralization reaction gathering for the 14th day after immunity 3 times is in Table 3, and table 3 is tired for immune serum neutralization repeatedly, and after experimental result data shows repeatedly immunity, anti-EV71 immunne response viral and anti-HAdv3 virus is all strengthened.
The neutralization of table 2 single immunization serum is tired
Many immune serum neutralizations of table 3 are tired
Embodiment 4: suckling mouse Protection
Owing to being greater than the mouse infection EV71 in one week age, after virus, there is no obvious symptom, in order further to verify that recombinant virus injection induces the immunne response of Protective effect, have the potentiality as vaccine candidate strain application, carry out suckling mouse Protection.The concrete steps of this experiment are as follows:
1, mouse respectively muscle immunity recombinant virus AdSP70R1SP55R2 △ E3VP1 and contrast HAdv3 virus, contrast PBS of immune programme for children routinely, the deactivation EV71 virus of abdominal injection adjuvant emulsion, gathered mice serum, antibody purification after 21 days.
2, the mode of abdominal injection is given the EV71 virus that 1 age in days suckling mouse infects lethal dose, and viral level is 1000TCID
50.
3, infect latter 4 hours abdominal injection immune mouse antibody, set up simultaneously do not attack the contrast of malicious suckling mouse and not injection of antibodies suckling mouse attack poison contrast.The survival condition of every day entry mouse.
Experimental result is shown in Fig. 1, and Fig. 1 infects EV71 suckling mouse survivorship curve figure.As seen from the figure, the mouse antibodies of the relative PBS immunity of mouse antibodies of recombinant virus AdSP70R1SP55R2 △ E3VP1 immunity or not injection of antibodies suckling mouse is had to significant provide protection (P < 0.05).This experimental result shows that recombinant virus induces the immunne response of anti-EV71.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention is explained in detail with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not depart from essence and the scope of technical solution of the present invention.
Claims (6)
- One kind restructuring human 3-type adenovirus, it is characterized in that, the genome E3 district of described restructuring human 3-type adenovirus has inserted the VP1 protein expression frame of EV71, in six adjacent bodies of described restructuring human 3-type adenovirus, two kinds of EV71 neutralizing epitope SP70 and SP55 have been embedded, the aminoacid sequence of described EV71 neutralizing epitope SP70 is YPTFGEHKQEKDLEYC, and the aminoacid sequence of described neutralizing epitope SP55 is PESRESLAWQTATNPC.
- 2. the human 3-type adenovirus of recombinating according to claim 1, is characterized in that, the replacement district of described EV71 neutralizing epitope SP70 is the HVR1 district of HAdv3 six adjacent bodies, and the replacement district of described EV71 neutralizing epitope SP55 is the HVR2 district of HAdv3 six adjacent bodies.
- 3. the human 3-type adenovirus of recombinating according to claim 2, is characterized in that, the replacement aminoacid sequence of described EV71 neutralizing epitope SP70 is the NRDNAV in HVR1 district, and the replacement aminoacid sequence of described EV71 neutralizing epitope SP55 is the TTEGEE in HVR2 district.
- 4. a bivalent vaccine candidate of human 3-type adenovirus-EV71, is characterized in that, described bivalent vaccine candidate contains the described restructuring human 3-type adenovirus of one of claim 1-3.
- 5. a carrier, is characterized in that, described carrier comprises the described restructuring human 3-type adenovirus of one of claim 1-3.
- 6. a method of preparing the described restructuring human 3-type adenovirus of one of claim 1-3, is characterized in that, comprises the steps:The VP1 protein expression frame that has inserted EV71 in restructuring human 3-type adenoviral gene ZuE3 district has embedded two kinds of EV71 neutralizing epitope SP70 and SP55 simultaneously on the adjacent body of human 3-type adenovirus six; Wherein,The aminoacid sequence of described EV71 neutralizing epitope SP70 is YPTFGEHKQEKDLEYC, and the aminoacid sequence of described neutralizing epitope SP55 is PESRESLAWQTATNPC;The replacement amino acid of described EV71 neutralizing epitope SP70 is the NRDNAV in HVR1 district, and the replacement aminoacid sequence of described EV71 neutralizing epitope SP55 is the TTEGEE in HVR2 district.
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| CN105219740A (en) * | 2015-09-29 | 2016-01-06 | 东莞市第八人民医院 | A kind of restructuring human 3-type adenovirus and preparation method and application |
| CN105238767A (en) * | 2015-10-15 | 2016-01-13 | 东莞市第八人民医院 | Application and preparation method of candidate strain of human type-3 adenovirus expressed human type-55 adenovirus neutralization epitope vaccine |
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| CN106754760A (en) * | 2017-01-20 | 2017-05-31 | 华南农业大学 | A kind of preparation method and application of the restructuring human 3-type adenovirus of chimeric IBDV neutralizing epitopes |
| CN110283795A (en) * | 2019-05-30 | 2019-09-27 | 广州市妇女儿童医疗中心 | The recombined adhenovirus of EV71 virus and CVA16 virus Neutralization and crystallization is shown simultaneously |
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| CN111108192A (en) * | 2017-07-05 | 2020-05-05 | Nouscom股份公司 | Non-human simian adenovirus nucleic acid sequences and amino acid sequences, vectors containing same, and uses thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219740A (en) * | 2015-09-29 | 2016-01-06 | 东莞市第八人民医院 | A kind of restructuring human 3-type adenovirus and preparation method and application |
| CN105238767A (en) * | 2015-10-15 | 2016-01-13 | 东莞市第八人民医院 | Application and preparation method of candidate strain of human type-3 adenovirus expressed human type-55 adenovirus neutralization epitope vaccine |
| CN105238766A (en) * | 2015-10-15 | 2016-01-13 | 东莞市第八人民医院 | Application and preparation method of candidate strain of human type-3 adenovirus expressed human type-14 adenovirus neutralization epitope vaccine |
| CN105238766B (en) * | 2015-10-15 | 2018-10-19 | 东莞市第八人民医院 | Human 3-type adenovirus shows the preparation method and application of 14 type adenovirus Neutralization and crystallization vaccine candidate strain of people |
| CN105238767B (en) * | 2015-10-15 | 2018-10-19 | 东莞市第八人民医院 | Human 3-type adenovirus shows the preparation method and application of 55 type adenovirus Neutralization and crystallization vaccine candidate strain of people |
| CN106220738A (en) * | 2016-07-31 | 2016-12-14 | 中国医学科学院医学生物学研究所 | The structure of EV71 Neutralization and crystallization and norovirus P-structure territory chimeric vector and expression |
| CN106754760A (en) * | 2017-01-20 | 2017-05-31 | 华南农业大学 | A kind of preparation method and application of the restructuring human 3-type adenovirus of chimeric IBDV neutralizing epitopes |
| CN111108192A (en) * | 2017-07-05 | 2020-05-05 | Nouscom股份公司 | Non-human simian adenovirus nucleic acid sequences and amino acid sequences, vectors containing same, and uses thereof |
| CN111108192B (en) * | 2017-07-05 | 2023-12-15 | Nouscom股份公司 | Non-human simian adenovirus nucleic acid sequences and amino acid sequences, vectors containing same and uses thereof |
| CN110283795A (en) * | 2019-05-30 | 2019-09-27 | 广州市妇女儿童医疗中心 | The recombined adhenovirus of EV71 virus and CVA16 virus Neutralization and crystallization is shown simultaneously |
| CN110699380A (en) * | 2019-09-04 | 2020-01-17 | 广州医科大学附属第一医院 | EV71 virus nucleic acid plasmid and construction method and application thereof |
| CN110699380B (en) * | 2019-09-04 | 2023-12-15 | 广州医科大学附属第一医院 | EV71 virus nucleic acid plasmid, construction method and application thereof |
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