CN105582532A - Preparing method and application of soluble Ebola virus envelope protein vaccine - Google Patents
Preparing method and application of soluble Ebola virus envelope protein vaccine Download PDFInfo
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- CN105582532A CN105582532A CN201510539663.5A CN201510539663A CN105582532A CN 105582532 A CN105582532 A CN 105582532A CN 201510539663 A CN201510539663 A CN 201510539663A CN 105582532 A CN105582532 A CN 105582532A
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Abstract
The invention relates to a preparing method and application of soluble Ebola virus envelope protein vaccine. The disclosed preparing method of the soluble Ebola virus envelope protein vaccine includes the concrete steps that two thrombin cleavage sites (GSGLVPRGSGLVPRGS) are inserted behind the 629<th> amino acid of Ebola virus envelope protein, the recombinant protein is expressed in a cell 293, or a cell CHO or a cell huh7, then the Ebola virus envelope protein on the outer side of a cell membrane is cut with thrombin, supernatant is collected, and antibody affinity is purified. The purified protein can be used for preparing the Ebola virus envelope protein vaccine with pure protein characteristics.
Description
Technical field
The invention belongs to viral vaccine technology of preparing and medical biotechnology research field. The present invention relates to sequence composition and the use in prevention from suffering from the diseases thereof of the Ebola virus envelope protein (GP albumen) of restructuring.
Background technology
Ebola virus (Ebolavirus) is the Causative virus that causes the mankind and primate generation Ebola hemorrhagic fever, the most fatal hemorrhagic fever viruse in the Ta Shi world today, the infected's symptom is nausea,vomiting,diarrhea, colour of skin change, Muscular stiffness, body internal haemorrhage, heating etc., its fatal rate reaches 50～70%, and the cause of death is mainly apoplexy, miocardial infarction, hypovolemic shock or multiple organ failure. Since 1976 are found first, Ebola hemorrhagic fever is multiple countries and regions outbreak of epidemic in Africa repeatedly, causes serious public health event. Within 2013, rise, Ebola virus cases of infection appear at the west African states such as Guinea, Liberia, Sierra Leone in succession, and propagate with surprising rapidity. By on December 15th, 2014, World Health Organization declaration Ebola virus was caused 6841 people's death in the world, and infected case sum reaches 18464 people, becomes maximum since the dawn of human civilization Ebola's epidemic situation. Although Ebola belongs to regional locality epidemic disease, each state all by it as No.1 defence object, once import this state into, can have influence on this state and other national economic and trade contact and tourism. Ebola propagates anywhere on a large scale, all can lead to a humanitarian catastrophe, even if only have minority case, also can cause economic turmoil in this serious area that relies on trade.
Vaccine is the best strategy of prevention Ebola virus. European Union dropped into and exceeded 200,000,000 Euros of promotion Ebola vaccine researches in January, 2015. Ebola virus has a uniqueness and unique envelope protein (envelopeglycoprotein, GP), and this GP albumen has immunogenicity, can produce in antibody and Ebola virus after immune mouse. The various research of Ebola vaccine of research and development are all for GP albumen at present.
At present, the research of Ebola vaccine that enters clinical testing mainly contains two kinds: a kind of is Ebola's vaccine " rVSV Δ G-EBOVGP " of attenuation vesicular stomatitis virus carrier, Ebola's vaccine " cAd3-EBOV " of another kind of replication defect type 3 type adenovirus vectors. These two kinds of vaccines are viral vaccine alive, after injection human body, carry out the immune system of human activin by the expressed GP albumen of vaccine, thereby in producing and the antibody of Ebola virus.
But the vaccine taking virus as carrier, after injection human body, can produce one and cross the malaise symptoms such as heat pyrexia and myalgia. Hospital of University of Geneva calls time and tests Merck KGaA company (Merck) and jointly Ebola's vaccine of research and development of Niu Lin company of the U.S. (NewLink) on December 11st, 2014, comes from 4 vaccinated volunteers and has occurred arthralgia symptom.
Can prepare vaccine with simple GP albumen? this is in the certain difficulty of current technical existence. This is because GP albumen was translated into before this 676 complete amino acid large proteins in born of the same parents, is cut into two albumen: GP1 and GP2 subsequently by furin enzyme in born of the same parents after 501 amino acids. GP2 albumen is membrane-spanning protein, be anchored on cell membrane, and GP1 albumen is by disulfide bond and the GP2 albumen formation complex that is linked. Meanwhile, three GP1/GP2 protein complexes mutually combine and form trimer, have formed the thorn lug structure on Ebola virus coating. Therefore, as can be seen from above, GP albumen is non-secretory albumen, complex structure, and need depend on cell membrane could correct assembling.
Summary of the invention
The present invention inserts two fibrin ferment cleavage site Leu-Val-Pro-Arg-Gly-Ser(LVPRGS after Ebola virus envelope protein the 629th amino acids), the sequence of inserting after the 629th amino acids is specially GSGLVPRGSGLVPRGS, the complete amino acid number of Ebola virus envelope protein after restructuring is 692 (SEQIDNO:1), and the concrete sequence of complete recombinant protein is:
MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSST60
NQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSE120
CLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGV180
VAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLT240
YVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRK300
IRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREA360
AVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDT420
PSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLG480
LITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAE540
GIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGT600
CHILGPDCCIEPHDWTKNITDKIDQIIHDGSGLVPRGSGLVPRGSFVDKTLPDQGDNDNW660
WTGWRQWIPAGIGVTGVIIAVIALFCICKFVF692
This restructuring Ebola virus envelope protein enters host cell (can be 293 cells, huh7 cell or Chinese hamster ovary celI) by gene transfection, and the recombinant protein after expression is still the outside that is positioned at cell membrane. Subsequently, in cell conditioned medium, add fibrin ferment, with the Ebola virus envelope protein on fibrin ferment incising cell film outside, collect supernatant, the outer section of this film of antibody affinity purifying albumen.
Above-mentioned fibrin ferment cleavage site also can change to enterokinase cleavage site or Xa factor cleavage site. Like this, the cleavage site inserting after Ebola virus envelope protein the 629th amino acids is enterokinase cleavage site or Xa factor cleavage site.
Solubility Ebola virus envelope protein after purifying, its purposes is to prepare the research of Ebola vaccine of pure protein character.
Brief description of the drawings
Fig. 1. insert the restructuring Ebola virus envelope protein structural representation after two fibrin ferment cleavage sites.
Fig. 2. the expression of the Ebola virus envelope protein after restructuring on huh7 cell membrane, what primary antibodie was used is the anti-GP protein antibodies of polyclone mouse, how anti-the goat-anti mouse of the FITC mark of two anti-uses is.
The expression of Ebola virus envelope protein after the core-restructuring of Fig. 3 .DAPI staining cell on huh7 cell membrane.
Fig. 4. the solubility Ebola virus envelope protein after purifying, through 8% non-sex change gel electrophoresis transferring film, uses the anti-GP protein antibodies of polyclone mouse to carry out developing on film.
Claims (4)
1. the preparation method of a solubility Ebola virus envelope protein vaccine, it is characterized by after Ebola virus envelope protein the 629th amino acids and insert two fibrin ferment cleavage sites (GSGLVPRGSGLVPRGS), the amino acid number of the Ebola virus envelope protein after restructuring is 692 (SEQIDNO:1).
2. recombinant protein according to claim 1, the host cell that this restructuring Ebola virus envelope protein is expressed can be 293 cells, huh7 cell or Chinese hamster ovary celI, Ebola virus envelope protein on fibrin ferment incising cell film outside for cell after expression, collect supernatant, the outer section of this film of antibody affinity purifying albumen.
3. the cleavage site inserting after Ebola virus envelope protein the 629th amino acids in claim 1 can be also enterokinase cleavage site or Xa factor cleavage site.
4. the solubility Ebola virus envelope protein after purifying, its purposes is to prepare the research of Ebola vaccine of pure protein character.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106589078A (en) * | 2017-01-06 | 2017-04-26 | 德诺杰亿（北京）生物科技有限公司 | Purifying method and application of ebola-virus antigenic protein |
| CN113416714A (en) * | 2021-06-24 | 2021-09-21 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Recombinant virus for expressing Ebola GP protein and preparation method and application thereof |
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| WO2006113452A2 (en) * | 2005-04-13 | 2006-10-26 | University Of Medicine And Dentistry Of New Jersey | Glycoprotein hormone analogs |
| CA2870293A1 (en) * | 2012-04-12 | 2013-10-17 | The Trustees Of The University Of Pennsylvania | Filovirus consensus antigens, nucleic acid constructs and vaccines made therefrom, and methods of using same |
| CN103864904A (en) * | 2014-03-04 | 2014-06-18 | 中国人民解放军军事医学科学院生物工程研究所 | Antigen fragment and truncation based on ebola virus envelope protein as well as application |
| US20150098956A1 (en) * | 2013-10-03 | 2015-04-09 | Oregon Health & Science University | RECOMBINANT POLYPEPTIDES COMPRISING MHC CLASS II a1 DOMAINS |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006113452A2 (en) * | 2005-04-13 | 2006-10-26 | University Of Medicine And Dentistry Of New Jersey | Glycoprotein hormone analogs |
| CA2870293A1 (en) * | 2012-04-12 | 2013-10-17 | The Trustees Of The University Of Pennsylvania | Filovirus consensus antigens, nucleic acid constructs and vaccines made therefrom, and methods of using same |
| US20150098956A1 (en) * | 2013-10-03 | 2015-04-09 | Oregon Health & Science University | RECOMBINANT POLYPEPTIDES COMPRISING MHC CLASS II a1 DOMAINS |
| CN103864904A (en) * | 2014-03-04 | 2014-06-18 | 中国人民解放军军事医学科学院生物工程研究所 | Antigen fragment and truncation based on ebola virus envelope protein as well as application |
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| BEREND JAN BOSCH等: "The Coronavirus Spike Protein Is a Class I Virus Fusion Protein:Structural and Functional Characterization of the Fusion Core Complex", 《JOURNAL OF VIROLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106589078A (en) * | 2017-01-06 | 2017-04-26 | 德诺杰亿（北京）生物科技有限公司 | Purifying method and application of ebola-virus antigenic protein |
| CN113416714A (en) * | 2021-06-24 | 2021-09-21 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Recombinant virus for expressing Ebola GP protein and preparation method and application thereof |
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