CN103304671A - Human rotavirus P[8]deltaVP8*-P[6]deltaVP8* recombinant chimeric protein and application thereof - Google Patents
Human rotavirus P[8]deltaVP8*-P[6]deltaVP8* recombinant chimeric protein and application thereof Download PDFInfo
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Abstract
The invention relates to a human rotavirus P[8]deltaVP8*-P[6]deltaVP8* recombinant chimeric protein, wherein the amino acid sequence of the protein is shown in SEQ ID NO.2. Furthermore, the invention provides a coding gene and a preparation method of the protein as well as an application of the protein in preparation of a rotavirus vaccine. Two segments P[8]deltaVP8* and P[6]deltaVP8* are connected in serial to be expressed into the human rotavirus P[8]deltaVP8*-P[6]deltaVP8* recombinant chimeric protein; the protein can simultaneously induce neutralizing antibodies of major P genotypes P [8], P[4] and P[6] of the human rotavirus, overcome the shortcoming of an existing vaccine which can induce the generation of an antibody only capable of resisting one genotype and greatly enhance immune efficiency; and meanwhile, the protein can avoid a potential risk that oral administration of attenuated vaccine rotavirus possibly induces intestinal invagination, and is applicable to preparation of rotavirus vaccine.
Description
Technical field
The invention belongs to molecular biology and genetically engineered field, relate to a kind of human rotavirus P[8] △ VP8*-P[6] △ VP8* restructuring chimeric protein and application thereof.
Background technology
Rotaviral gastroenteritis also claims Diarrhea Caused by Porcine rotavirus, it is the topmost acute infectious intestinal disease of the interior infant of worldwide and multiple young animal diarrhoea, take diarrhoea and dehydration as feature, mortality ratio is high, causes significant damage for human health and animal husbandry development.The annual nearly children below 1.1 hundred million 5 years old in the whole world suffer from rotaviral gastroenteritis, wherein there are 2,500 ten thousand children to go to a doctor, the necessary hospital care of 2,000,000 children, approximately have every year 453000 children to die from Diarrhea Caused by Porcine rotavirus, wherein the death of developing country and less developed country accounts for more than 85% of sum.Take China as example, approximately there are every year 30000 children to die from this disease, rank the first five position of death sum country; The medical expense that is used for the treatment of infant every year just reaches billions of units.After infecting rotavirus (Rotavirus, RV), because dehydration and electrolyte disturbance cause severe diarrhea and death, there is no at present specific medicament, clinical treatment is symptomatic treatment just also, i.e. fluid infusion benefit salt.Therefore develop efficient vaccine and therapeutic strategy is one of top priority of scientific effort, with this reducing sickness rate and mortality ratio, alleviate society and economical load that this disease causes.
Yet current two people's Rotavirus Vaccines:
With
Threaten the most serious place at rotavirus, vaccine immunity is renderd a service barely satisfactory.For example:
Effectiveness in Malawi only has 48%,
Effectiveness in South East Asia also only has about 50%.Also have simultaneously oral
The report of molecular recombination between severe diarrhea and the vaccine strain occurs behind the vaccine; In addition
With
Do not get rid of with the cognation of intussusception.For example monitoring shows after the immunity of Australia
With
Vaccine has improved the sickness rate of intussusception.Therefore the Development of New Generation head that substitutes vaccine and become current vast researcher is wanted hot job.
Rotavirus (Rotavirus, RV) belongs to (Reoviridae) member for arc reovirus virus, and viral genome is comprised of 11 sections dsRNA, encode 6 structural protein (VP1～4, VP6～7) and 6 Nonstructural Proteins (NSP1～6).Ripe virus particle is three layers of capsid structure, and outermost layer forms VP8* and VP5* by spike protein VP4(protease cracking) and glycoprotein VP7 form.VP7 copies directly related in the intestinal epithelial cell of the maturation that is rich in proteolytic enzyme with virus.VP4 albumen not only relates to adhesion, the intrusion of RV, and relevant with physicochemical properties such as RV blood clotting, neutralization activity, virulence and the enhancings of protease cracking postoperative infection.VP7 and VP4 can independently induce virucidin, and the antibody of anti-VP7 and VP4 all can stop the intrusion of virus.Therefore VP7 and VP4 become the preferred object molecule of vaccine development.
The combination of rotavirus gene type is numerous, and the cross immunity between different genotype is very few, and this has brought difficulty to vaccine development.But research finds that a P genotype may cover several G genotype, and namely certain P type can make up from different G types, and same P type can be induced the heterotypic immunity of anti-different G types in theory.And the VP4 albumen that determines the P type contains a plurality of linearizing epitopes, and this albumen need not posttranslational modification, the VP8* fragment that it forms after the pancreatin cracking, comprised VP4 type specificity Main Antigenic, be all linearity and comparatively conservative, this has pointed out us can utilize VP8* albumen to come development of new PRV subunit vaccine.VP8* epi-position collection of illustrative plates according to draftings such as Kapikian AZ, selection has comprised among the VP8* and the zone of epitope, the VP8* gene that makes up brachymemma (followingly represents with △ VP8*, the amino acid that contains the 65-223 position) prokaryotic expression carrier, with the recombinant protein that obtains solubility as immunogen (Construction and characterization of human rotavirus recombinant VP8*subunit parenteral vaccine candidates.Wen X, Cao D, Jones RW, Li J, Szu S, Hoshino Y.Vaccine.2012Sep21; 30 (43): 6121-6).Simultaneously the main P genotype of human rotavirus is P[8], P[4], P[6] three kinds.P[8 is found in our research] △ VP8* can induce anti-P[8] the specificity neutralizing antibody of the multiple rotavirus of genotype, simultaneously can also induce the anti-P[4 of high titre] the special multiple rotavirus cross neutralization antibody of genotype, but still lack at present and a kind ofly can cover P[8], P[4], P[6] three kinds of genotypic effective vaccines, to improve the efficient after the vaccinate.
Summary of the invention
The first purpose of the present invention provides a kind of human rotavirus P[8] △ VP8*-P[6] the △ VP8* chimeric protein of recombinating.
The second purpose of the present invention provides the above-mentioned human rotavirus P[8 of coding] △ VP8*-P[6] gene and the expression vector of △ VP8* restructuring chimeric protein.
The 3rd purpose of the present invention provides above-mentioned human rotavirus P[8] △ VP8*-P[6] preparation method of △ VP8* restructuring chimeric protein.
The 4th purpose of the present invention provides above-mentioned human rotavirus P[8] △ VP8*-P[6] purposes of △ VP8* restructuring chimeric protein.
The present invention is achieved through the following technical solutions:
One, a kind of human rotavirus P[8] △ VP8*-P[6] the △ VP8* chimeric protein of recombinating, its aminoacid sequence is shown in SEQ ID No.2.
Two, the above-mentioned human rotavirus P[8 of coding] △ VP8*-P[6] gene of △ VP8* restructuring chimeric protein, its nucleotide sequence is shown in SEQ ID No.1, and the expression vector that contains said gene.Should be understood that the degeneracy of considering codon and the preferences of different plant species codon, those skilled in the art can use as required and be fit to the codon that specific species are expressed.
Three, above-mentioned human rotavirus P[8] △ VP8*-P[6] preparation method of △ VP8* restructuring chimeric protein, be to be converted into competent escherichia coli cell with the expression vector that contains the said gene fragment, the IPTG abduction delivering obtains recombinant protein, and concrete steps are as follows:
(1) human rotavirus cDNA's obtains;
(2) according to Wa strain (genotype is G1P[8]) (the GenBank searching number is: FJ423116) and 1076 strains (genotype is G3P[6]) GenBank searching number (M88480) sequence, design primer, wherein, amplification P[8] primer of Δ VP8* is as follows:
Upstream primer: 5'-ACT
CATATGTTAGATGGTCCTTATCAGCC-3'(SEQ ID No.3), underlined is the NdeI restriction enzyme site;
Downstream primer: 5'-TA
GGATCCTATCACAGACCATTATTAATATATTCATTAC-3'(SEQ ID No.4), underlined is BamH I restriction enzyme site;
Amplification P[6] primer of Δ VP8* is as follows:
Upstream primer: 5'-AAT
GGA TCCGGC TCA GGC GTA CTC GAT GGT CCT TAT CAA CCA AC-3'(SEQ ID No.5), wherein underlined is BamH I restriction enzyme site;
Downstream primer: 5'-TAG
AGC TCTATC ATA ACC CAG TAT TTA TAT ATT CAT TAC AC-3'(SEQ ID No.6), wherein underlined is Sac I restriction enzyme site;
(3) with purpose fragment P[8] △ VP8* fragment by restriction enzyme NdeI and BamH I directed cloning to the pET28a carrier, obtain carrier pET28a-P[8] △ VP8*, again with P[6] △ VP8* fragment by restriction enzyme BamH I and Sac I directed cloning to pET28a-P[8] in the △ VP8* carrier, obtain carrier pET28a-P[8] △ VP8*-P[6] △ VP8*;
(4) take pET28a-P[8] △ VP8*-P[6] △ VP8* plasmid is template, the primer sequence of amplification is as follows:
Upstream primer:
5'-
CGCGAACAGATTGGAGGTCAGTATATAAAAGCAAATTCTAAATTTATAG-3'（SEQ?ID?No.7），
Downstream primer:
5'-
GTGGCGGCCGCTCTATTATAACCCAGTATTTATATATTCATTACACTTAG-3'（SEQ?ID?No.8），
The underscore sequence is the carrier homology arm,
Amplification is with the PCR fragment of expression vector pETite carrier homology arm, and the PCR fragment of purifying and linearizing expression vector pETite homologous recombination obtain expression plasmid pETite-P[8] △ VP8*-P[6] △ VP8*;
(5) with above-mentioned expression vector pETite-P[8] △ VP8*-P[6] △ VP8* is converted into competent escherichia coli cell HI-control BL21 (DE3), and the IPTG abduction delivering obtains recombinant protein.
Four, above-mentioned human rotavirus P[8] △ VP8*-P[6] application of △ VP8* restructuring chimeric protein in the preparation Rotavirus Vaccine.
Adopt the positively effect of technique scheme: the present invention is with P[8] △ VP8* and P[6] two fragment tandem expression of △ VP8* become P[8] △ VP8*-P[6] the △ VP8* chimeric protein of recombinating, this albumen can be induced the main P genotype of anti-human rotavirus P[8 simultaneously], P[4], P[6] neutralizing antibody, overcome existing vaccine and can only induce the defective that produces anti-a kind of genotypic antibody, greatly improved Immune efficiency; Simultaneously, can overcome the potential risk that oral weak malicious Rotavirus Vaccine brings out intussusception, be applicable to prepare Rotavirus Vaccine.
Description of drawings
Fig. 1 is P[8] Δ VP8* and P[6] Δ VP8*PCR electrophorogram;
Among the figure, 1Marker, 2P[8] Δ VP8* fragment, 3P[6] Δ VP8*
Fig. 2 is pET28a-P[8] Δ VP8*-P[6] Δ VP8* recombinant plasmid double digestion evaluation electrophorogram;
Among the figure, 1Marker, 2pET28a empty plasmid; 3pET28a-P[8] Δ VP8*-P[6] Δ VP8* recombinant plasmid;
Fig. 3 is pETite-P[8] Δ VP8*-P[6] Δ VP8* recombinant plasmid double digestion electrophorogram;
Among the figure, 1Marker, the 2nd, pETite empty plasmid double digestion; 3.pETite-P[8] Δ VP8*-P[6] Δ VP8* recombinant plasmid double digestion;
Fig. 4 is P2-P[8] △ VP8*-P[6] △ VP8* expression of recombinant proteins electrophorogram;
Among the figure, 1Marker, 2P[8] △ VP8*-P[6] △ VP8* albumen;
Fig. 5 is P[8] △ VP8*-P[6] the Western blot of △ VP8* restructuring chimeric protein detects figure;
Among the figure, 1Marker, 2P[8] △ VP8*-P[6] △ VP8* albumen;
Embodiment
Below in conjunction with embodiment and test example technical scheme of the present invention is described further, but should not be construed as limitation of the present invention:
The source of biomaterial among the present invention:
1, human rotavirus Wa and 1076 strains are purchased from ATCC;
2, carrier pET28a is available from Novagen company; Prokaryotic expression carrier pETite is purchased from Lucigen company
3, HI-control BL21 (DE3) competent cell is available from Lucigen company;
4, the primer is designed, designed and entrusts Suzhou gold dimension intelligence bio tech ltd synthetic.
The present embodiment is used for the structure that explanation contains the expression vector of purpose fragment.
Material therefor is the human rotavirus Wa, and genotype is G1P[8]; 1076 strains, genotype are G2P[6].Being classical strains, is to cultivate with former generation African green monkey kidney (AGMK) cell.Used medium is EMEM, and wherein adding pancreatin to final concentration is 0.5 μ g/ml, penicillin 100IU/ml, Streptomycin sulphate 100 μ g/ml, amphotericin B 2.5 μ g/ml.EMEM culture medium prescription such as table 1:
Table 1EMEM culture medium prescription
Pass through TRIzol-LS(Invitrogen) method extraction human rotavirus Wa (G1P[8]) and 1076 strains (G2P[6]) RNA, the RNA that extracts is become cDNA with the RT-PCR reverse transcription.Wherein used primer is according to human rotavirus Wa (the GenBank searching number is respectively: FJ423116(Wa) and M88480(1076)) the gene order of RNA fragment 4 design the gene order of RNA fragment 4 design.
Amplification P[8] primer of Δ VP8* is as follows:
Upstream primer: 5'-ACT
CATATGTTAGATGGTCCTTATCAGCC-3'(SEQ ID No.3), underlined is Nde I restriction enzyme site;
Downstream primer: 5'-TA
GGATCCCAGACCATTATTAATATATTCATTAC-3'(SEQ ID No.4), wherein underlined is BamH I restriction enzyme site;
Amplification P[6] primer of Δ VP8* is as follows:
Upstream primer: 5'-AAT
GGA TCCGGC TCA GGC GTA CTC GAT GGT CCT TAT CAA CCA AC-3'(SEQ ID No.5), wherein underlined is BamH I restriction enzyme site
Downstream primer: 5'-TAG
AGC TCTATC ATA ACC CAG TAT TTA TAT ATT CAT TAC AC-3'(SEQ ID No.6), wherein underlined is Sac I restriction enzyme site
With Superscript III ThermoScript II reverse transcription VP8*cDNA, reaction parameter arranges as follows:
50-200ng geneome RNA and final concentration are that 15% DMSO mixes, 95 ℃ of sex change 3min, then place immediately on ice, reaction system is as follows: the 2pmol gene-specific primer, the total RNA of 10pg-5 μ g, 1 μ L10mMdNTP, deionized water complement to 13 μ L, 65 ℃ of sex change 5min, at least 1min is hatched on the iceberg; Add 4 μ L5 * First-Strand buffer, 1 μ L100mM DTT, 1 μ L RNaseOUT RNase inhibitor, 1 μ L SuperScriptIII (200U/ μ L).50 ℃ of 30-60min, 70 ℃ of 15min, cDNA chain formation.Take cDNA as template, by the specific amplification P[8 of iProof High fidelity PCR system (Bio-Rad)] Δ VP8* and P[6] Δ VP8* fragment.PCR detected result such as Fig. 1.Reclaim test kit by the quick glue of QIA and reclaim every kind of PCR product.With the P[8 that reclaims] Δ VP8*PCR fragment clone through Nde I and BamH I, is cloned into (Novagen) in the pET28a carrier, thus acquisition pET28a-P[8] Δ VP8* recombinant plasmid.Afterwards, pET28a-P[8] Δ VP8* recombinant plasmid and P[6] Δ VP8*PCR fragment is through BamH I and Sac I double digestion, with P[6] Δ VP8* fragment is inserted into pET28a-P[8] in the Δ VP8* recombinant plasmid, made up pET28a-P[8] Δ VP8*-P[6] Δ VP8* recombinant plasmid.The recombinant plasmid double digestion is identified electrophorogram such as Fig. 2.Constructed plasmid is carried out dna sequencing, identify integrity and the exactness of constructed plasmid.Wherein, enzyme is cut the conventional means that can be understood as those skilled in the art of the present technique with the conditional parameter that is connected.
According to business directory (Lucigen) construction expression fusion gene P[8] Δ VP8*-P[6] recombinant plasmid of Δ VP8*, design of primers is as follows:
Upstream primer:
5'-
CGCGAACAGATTGGAGGTCAGTATATAAAAGCAAATTCTAAATTTATAG-3'（SEQ?ID?No.7），
Downstream primer:
5'-
GTGGCGGCCGCTCTATTATAACCCAGTATTTATATATTCATTACACTTAG-3'（SEQ?ID?No.8），
The underscore sequence is the carrier homology arm,
Take pET28a-P[8] Δ VP8*-P[6] Δ VP8* recombinant plasmid is the pcr amplification template, 95 ℃ of denaturation 2min, 95 ℃ of sex change 25s, 55 ℃ of annealing 15s, 72 ℃ are extended 30s, 30 circulations.Last 72 ℃ are extended 10min, amplification is with the PCR fragment P[8 of homology arm] Δ VP8*-P[6] Δ VP8*, mix with linearizing carrier pETite, by the homologous recombination principle external source fragment is cloned into expression vector pETite, obtain recombinant plasmid pETite-P[8] Δ VP8*-P[6] Δ VP8*, double digestion is identified electrophorogram as shown in Figure 3.
The present embodiment is used for the expression of explanation human rotavirus subunit recombinant protein.
Use the heat-shocked method, with recombinant plasmid pETite-P[8] Δ VP8*-P[6] Δ VP8* is transformed in HI-control BL21 (DE3) expression host cell, the picking mono-clonal is inoculated in the LB liquid nutrient medium that contains 50 μ g/ml kantlex and (adds 2% glucose, 1% ethanol) from agar plate.When absorbance reaches 0.5 under the 600nm, add IPTG to its final concentration be 1mM, induce 16-20h for 18 ℃.The SDS-PAGE electrophoresis result as shown in Figure 4.4 ℃ of centrifugal 15min of 10000g collect restructuring E.coli cell, and-80 ℃ of storages are for subsequent use.
Embodiment is used for the purifying of explanation recombinant protein.
Destroy the cell walls of E.Coli cell with the BugBuster Master Mix (Novagen) that contains protease inhibitor cocktail (Roche), collect soluble product ,-80 ℃ of storages are for subsequent use.With ProBond nickel-NTA agar affinity chromatography (Invitrogen), to step every kind of albumen is carried out purifying according to manufacturer.Behind the wash buffer flush away cell protein that contains different concns imidazoles (20-100mM), 250mM imidazoles wash-out recombinant protein.According to business directory, remove P[8 with SUMO expressing protein enzyme (Lucigen)] Δ VP8*-P[6] the SUMO label of Δ VP8*N end.SDS-PAGE analyzes the purity of recombinant protein, then uses centrifugal filter device (Millipore) to remove imidazoles in the solution.According to BCA quantification kit (Thermo) manufacturer explanation, determine recombinant protein concentration behind the purifying with the BCA quantification kit.Recombinant protein behind the purifying-80 ℃ saves backup.According to step that manufacturer gives, with level of endotoxin in the albumen behind every kind of purifying of ToxinsensorTMchromogenic LAL intracellular toxin detection kit (GenScript) detection.The result shows, level of endotoxin is all at 1.8EU/ml, its well below recombinant subunit vaccine maximum allow level of endotoxin (＜20EU/ml).
Embodiment 4
The present embodiment detects albumen with Western blot method.
4-12%NuPAGE glue analysing protein, and protein transduction moved on on the nitrocellulose filter (Whatman).Film and super 4 ℃ of the anti-Wa of cavy (P[8]) the strain serum (1:50) of exempting from are spent the night and hatches altogether.After washing three times, with the anti-cavy IgG(H+L of the goat of film and peroxidase labelling)) (KPL) room temperature hatch altogether 1h.The blue aniline of 3,3 ' diamino (DAB) (Sigma) develops the color.The result as shown in Figure 5.
Embodiment 5
The present embodiment is used for the explanation neutralization test.
Reduce the NAT that every part of serum sample is determined in neutralization (PRN) test by 60% plaque.Virus places on the agar layer of 6 orifice plates after adsorbing 1h with serum, and other processes are all used MA104 cell and 6 orifice plates afterwards.Add AP Adjuvanted vaccines intramuscular injection immune guinea pig with 10 μ g or 20 μ g, as expected, produced high-caliber neutralizing antibody behind the second immunisation, three exempt from rear two all NATs reaches 1:12800.Until research finishes front (three exempted from rear 6 months), Neutralizing antibody response is kept higher level always.
Test example 1
Adopt the 500-550g/ female Hartley cavy of outbreeding system only.The whole process of each experiment, cavy are all raised in the isolation mouse cage of the safe secondary condition of animal organism.Per two weeks are given cavy intramuscularly (IM) immunity once, P[8] Δ VP8*-P[6] the each immune 20 μ g(of Δ VP8* add aluminum phosphate (AP) adjuvant) (ADJU-PHOSTM, Brenntag Biosector) (every dose contains 100 μ g aluminium), immunity is 3 times altogether, each immune front and last immunity afterwards blood sampling in 7 days.P[8] Δ VP8*-P[6] Neutralizing antibody response that produces behind Δ VP8* albumen+AP intramuscular injection immune guinea pig sees Table 2.
Table 2.P[8] Δ VP8*-P[6] Δ VP8* restructuring chimeric protein induces the anti-P[8 of high titre], P[4] and P[6] the neutralizing antibody level of type rotavirus strain
a60% plaque reduces neutralization test serum antibody titer (inverse)
As can be seen from the table, cavy immunity P[8] Δ VP8*-P[6] after Δ VP8* restructuring chimeric protein 3 exempted from, neutralization the most common G1, G3, G4 and G9 type human rotavirus (P[8] homotype) geometric mean titer (GMT) reached 1:1659.9～1:4305; Cross neutralization antibody geometric mean titer for G2, G8, G12 type human rotavirus (P[4] abnormal shape) has also reached 1:1280～1:1522; Reached 1:987～1:1280 for G2, G4 type human rotavirus (P[6] homotype) neutralizing antibody geometric mean titer.The P[10 that anti-human rotavirus is rarer] the special-shaped neutralizing antibody level of strain rotavirus is lower.
This restructuring chimeric protein can be induced the main P genotype of anti-human rotavirus P[8 simultaneously], P[4], P[6] neutralizing antibody, overcome existing vaccine and can only induce the defective that produces anti-a kind of genotypic antibody, greatly improved Immune efficiency.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (5)
1. human rotavirus P[8] △ VP8*-P[6] the △ VP8* chimeric protein of recombinating, its aminoacid sequence is shown in SEQ ID No.2.
2. human rotavirus P[8 claimed in claim 1 encodes] △ VP8*-P[6] gene of △ VP8* restructuring chimeric protein, its nucleotide sequence is shown in SEQ ID No.1.
3. the expression vector that contains gene claimed in claim 2.
4. prepare human rotavirus P[8] △ VP8*-P[6] method of △ VP8* restructuring chimeric protein, comprise with expression vector claimed in claim 3 being converted into competent escherichia coli cell, the IPTG low temperature induction is expressed the acquisition recombinant protein.
5. human rotavirus P[8 claimed in claim 1] △ VP8*-P[6] application of △ VP8* restructuring chimeric protein in the preparation Rotavirus Vaccine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546288A (en) * | 2018-04-13 | 2018-09-18 | 成都迈科康生物科技有限公司 | A kind of human rotavirus VP8 recombinant proteins and people's Rotavirus Vaccine using the VP8 recombinant proteins |
| CN112552397A (en) * | 2020-11-06 | 2021-03-26 | 植链(上海)生物科技有限公司 | Rotavirus VP7 recombinant protein and application thereof |
| CN116355079A (en) * | 2022-08-19 | 2023-06-30 | 上海迈科康生物科技有限公司 | Monoclonal antibody for detecting recombinant human rotavirus VP8 antigen (VP 8P 8) and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101972475A (en) * | 2010-04-12 | 2011-02-16 | 李建平 | Bacterial polysaccharide-protein conjugate vaccine and preparation method thereof |
| CN102643348A (en) * | 2012-04-17 | 2012-08-22 | 中国医学科学院医学生物学研究所 | Recombinant chimeric protein carrying rotavirus antigen epitope and preparation thereof |
-
2013
- 2013-06-06 CN CN2013102220035A patent/CN103304671A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101972475A (en) * | 2010-04-12 | 2011-02-16 | 李建平 | Bacterial polysaccharide-protein conjugate vaccine and preparation method thereof |
| CN102643348A (en) * | 2012-04-17 | 2012-08-22 | 中国医学科学院医学生物学研究所 | Recombinant chimeric protein carrying rotavirus antigen epitope and preparation thereof |
Non-Patent Citations (2)
| Title |
|---|
| XIAOBO WEN ET AL: "Construction and Characterization of Human Rotavirus Recombinant VP8* Subunit Parenteral Vaccine Candidates", 《VACCINE》, vol. 30, no. 43, 21 September 2012 (2012-09-21), pages 6121 - 6126 * |
| 杨艳梅等: "A组轮状病毒多个结构蛋白抗原表位的串联表达及其免疫原性的检测", 《生物化学与生物物理进展》, vol. 38, no. 1, 31 December 2011 (2011-12-31), pages 60 - 66 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546288A (en) * | 2018-04-13 | 2018-09-18 | 成都迈科康生物科技有限公司 | A kind of human rotavirus VP8 recombinant proteins and people's Rotavirus Vaccine using the VP8 recombinant proteins |
| CN112552397A (en) * | 2020-11-06 | 2021-03-26 | 植链(上海)生物科技有限公司 | Rotavirus VP7 recombinant protein and application thereof |
| CN112552397B (en) * | 2020-11-06 | 2024-02-13 | 植链(上海)生物科技有限公司 | Rotavirus VP7 recombinant protein and application thereof |
| CN116355079A (en) * | 2022-08-19 | 2023-06-30 | 上海迈科康生物科技有限公司 | Monoclonal antibody for detecting recombinant human rotavirus VP8 antigen (VP 8P 8) and application thereof |
| CN116355079B (en) * | 2022-08-19 | 2023-12-26 | 上海迈科康生物科技有限公司 | Monoclonal antibody for detecting recombinant human rotavirus VP8 antigen (VP 8P 8) and application thereof |
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