CN103145802A - Polypeptide infection promoting agent - Google Patents
Polypeptide infection promoting agent Download PDFInfo
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- CN103145802A CN103145802A CN201210336804XA CN201210336804A CN103145802A CN 103145802 A CN103145802 A CN 103145802A CN 201210336804X A CN201210336804X A CN 201210336804XA CN 201210336804 A CN201210336804 A CN 201210336804A CN 103145802 A CN103145802 A CN 103145802A
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Abstract
The invention relates to a polypeptide with HIV infection promoting ability. The HIV infection promoting polypeptide comprises a peptide sequence, containing a terminal C peptide of near extramembrane fragment MPER fragment of an HIV-1 envelope protein GP41. The polypeptide infection promoting agent provided by the invention has significant activity in promoting envelope viral infection, and shows activity superior to that of a common infection promoting agent DEAE at a cellular level. Therefore, the polypeptide infection promoting agent plays an important role in the development of retroviral or lentiviral gene therapy.
Description
Technical field
The present invention relates to a kind of polypeptide that promotes the HIV infection ability that has, peptide sequence is derived from the nearly film outskirt MPER of HIV envelope protein GP41.
Background technology
At present in gene therapy, the means of transferring of goal gene is divided into virus vector and the large class of non-virus carrier two, and is the most commonly used take virus vector such as retrovirus and adenovirus as basic carrier system.The retroviral vector advantage is that the transfection scope is wide, and the foreign gene that changes over to can be integrated into the host cell gene group; Shortcoming is to be integrated into division cells; Can insert exogenous genetic fragment little, be difficult to satisfy the insertion of larger gene; The possibility that produces wild-type virus or auxiliary type virus is arranged, and random integration may produce undesirable action.And the appearance of lentiviral vectors has improved these problems.Lentiviral vectors comes from retrovirus, take HIV-1 virus as the basis, has removed virulent gene, replaces the envelope protein of HIV-1 with the VSV-G envelope protein.Advantage is that host range is wider, and is safer, and persistence is expressed.Can be integrated into division stage and non-division cells, can insert the external source fragment and grow to 5kb.Therefore lentiviral vectors has a very wide range of applications in fields such as RNAi, nucleus Animal Model and gene therapies.But, no matter be retroviral vector or slow virus carrier system, all face a virus packing titre and hang down and the poor problem of efficiency of infection.
Summary of the invention
In order to solve the low problem of efficiency of infection of retroviral vector system and slow virus carrier system in prior art, the invention provides a kind of reagent that promotes virus infection, comprise a peptide sequence, described peptide sequence is the derivative peptide sequence with the nearly film outskirt of the transmembrane protein of enveloped virus.The nearly film outskirt of transmembrane protein (membrane proximal extracellular region, MPER) general enrichment hydrophobic amino acid, can be combined with the lipid bilayer of membrane structure, the performance membrane is upset active (membrane perturbing property).The MPER district of HIV-1 envelope protein Gp41 can be inserted into the outer interface zone of viromembrane, and the high stability of break virus coating is rebuild and merge the formation in hole in order to the film fat of cell and virus.The MPER district of deletion HIV can cause the stagnation of fusion process.Therefore the MPER district has extremely important effect for the performance of HIV virus fusion and infection ability.Based on this, we infer that the derivative synthetic polypeptide of MPER also should be influential to the infection of HIV, and have confirmed that by experiment this type of polypeptide can promote the infectivity with enveloped virus.In the present invention, the derived peptide in the MPER district of preferred HIV-1 transmembrane protein Gp41 is as short infection reagent.
In one embodiment, peptide sequence of the present invention can further increase basic aminoacids on the MPER of Gp41 native sequences basis.Described basic aminoacids comprises arginine, one or more of Methionin and Histidine.The basic aminoacids number that increases can be for one or more.
In one embodiment, the aforementioned polypeptides sequence also can be by amino acid single-point or multipoint mutation transformation, to strengthen its short infection activity.
In one embodiment, can by regulating pH of cushioning fluid and ionic strength, improve the short infection activity of aforementioned polypeptides.
The short reagent that infects of polypeptide class of the present invention has significant short infection activity with enveloped virus, and therefore the development for retrovirus or lentiviral gene therapy has vital role.
Description of drawings
The comparison of the short infection activity of Fig. 1 MPER, NK13 and QW13.In 96 plates, after the polypeptide gradient dilution, ZM214M.PL15 is hatched 1h with the HIV-1 pseudovirus, adds TZM-bl (1 * 10
5) the cell suspension infection.The course of infection serum-free is mended 10% serum after infection 4h.After infecting 48h, detect the luciferase expression amount.Enhancement (n-fold)=sample value/virus control value.Virus final concentration (p24 content) is 3.2ng/mL; The diagram peptide concentration refers to add the final concentration after cell.
The comparison of the short infection activity of Fig. 2 NK16 and NK13.In 96 plates, after the polypeptide gradient dilution, ZM214M.PL15 is hatched 1h with the HIV-1 pseudovirus, adds TZM-bl (1 * 10
5) the cell suspension infection.The course of infection serum-free is mended 10% serum after infection 4h.After infecting 48h, detect the luciferase expression amount.Enhancement (n-fold)=sample value/virus control value.Virus final concentration (p24 content) is 3.2ng/mL; The diagram peptide concentration refers to add the final concentration after cell.
The comparison of short infection activity on different infection times of Fig. 3 NK16 and deae dextran.The short infection ability that NK16 induces is faster stronger than DEAE.The course of infection serum-free is mended 10% serum after infection 4h.The final concentration of NK16 is 60u g/mL, and the final concentration of deae dextran is 16 μ g/mL.Detect luciferase after 48h active.
Fig. 4 Hela cell is by the metainfective GFP fluorescence of FIV/VSV-G flow cytometer showed.FIV/VSV-G infects the Hela cell levels and improves under the NK16 effect.The FIV/VSV-G recombinant virus infects adherent Hela cell under the condition that has or not NK16 to hatch altogether, the course of infection serum-free is washed cell after infection 4h, changes perfect medium.Detect the expression level of GFP after 2 days with flow cytometer.
The short infection activity of Fig. 5 NK16 under the different ionic strength condition.The phosphoric acid buffer ionic strength of NK16 and HIV-1 pseudovirus ZM214M.PL15 preincubate is respectively 0,0.1 and 0.5M (pH 7.68).DMEM refers to use the DMEM of serum-free as dilution buffer liquid.The course of infection serum-free is mended 10% serum after infection 4h.The final concentration of NK16 is 60 μ g/mL.The damping fluid ionic strength is lower, and the short infection ability of NK16 is stronger.
The short infection activity of Fig. 6 NK16 under condition of different pH.The phosphoric acid buffer pH value of NK16 and HIV-1 pseudovirus ZM214M.PL15 preincubate is respectively 6.35,7 and 7.68M (0.1M).The course of infection serum-free is mended 10% serum after infection 4h.The final concentration of NK16 is 60 μ g/mL.The pH value is lower, and the short infection ability of NK16 is stronger.
The comparison of the short infection activity of Fig. 7 NK16 and NK16-D.In 96 plates, after the polypeptide gradient dilution, ZM214M.PL15 is hatched 1h with the HIV-1 pseudovirus, adds TZM-bl (1 * 10
5) the cell suspension infection.The course of infection serum-free is mended 10% serum after infection 4h.After infecting 48h, detect the luciferase expression amount.Enhancement (n-fold)=sample value/virus control value.Virus final concentration (p24 content) is 3.2ng/mL; The diagram peptide concentration refers to add the final concentration after cell.
Embodiment
The nearly film outskirt of the HIV-1gp41 of high conservative (MPER) has vital role to the fusion process of HIV-1 and host cell.Experiment confirm the synthetic peptide of the C end regions NWFDITNWLWYIK of MPER (SEQ ID NO:1) have the absorption that promotes with enveloped virus and host cell, thereby increase the effect of virus infection efficient.
Advantage of the present invention and technique effect hereinafter have been described in conjunction with specific embodiments.Protection scope of the present invention is with being as the criterion that claims limit, and embodiment exemplarily illustrates ideals and principles of the present invention.
The design of polypeptide
Table 1
| Name | Describe | Sequence |
| MPER | MPER district full-length polypeptide | Ac-NEQELLELDKWASLWNWFDITNWLWYIK |
| QW13 | MPER district N holds polypeptide | Ac-QELLELDKWASLW |
| NK13 | MPER district C holds polypeptide | Ac-NWFDITNWLWYIK |
Short infection activity characterizes
The comparison of the short infection activity of Fig. 1 MPER, NK13 and QW13.
The design of polypeptide
Table 2
Short infection activity characterizes
The comparison of the short infection activity of Fig. 2 NK16 and NK13.
Compare with the short agent of activity that infects commonly used
The comparison of short infection activity on different infection times of Fig. 3 NK16 and deae dextran.
Application in other infection of coated virus
Fig. 4 Hela cell is by the metainfective GFP fluorescence of FIV/VSV-G flow cytometer showed.
Embodiment 3
The short infection activity of Fig. 5 NK16 under the different ionic strength condition.
The short infection activity of Fig. 6 NK16 under condition of different pH.
Table 3
| Name | Describe | Sequence |
| NK16 | The derived peptide of NK13 | Ac-NWFDITNWLWYIKKKK |
| NK16-D | The derived peptide of NK16 | Ac-NWFAITNWLWYIKKKK |
The comparison of the short infection activity of Fig. 7 NK16 and NK16-D.
SEQ ID NO：1
NWFDITNWLWYIK
Claims (6)
1. the polypeptide that short infection activity is arranged, comprise a kind of peptide sequence, and described peptide sequence comprises C terminal sequence (SEQ ID NO:1) and the truncated sequence thereof in MPER district of the envelope protein Gp41 of HIV-1.
2. the polypeptide that short infection activity is arranged as claimed in claim 1, further comprise a kind of peptide modified, described peptide modified for increasing one or more basic aminoacidss.Described basic aminoacids comprises Methionin, arginine and Histidine.Described basic aminoacids can number can be one or more.
3. the polypeptide that short infection activity is arranged as described in claim 1-2 any one, further comprise by amino acid single-point or multipoint mutation transformation, to strengthen its short infection activity.
4. the polypeptide that short infection activity is arranged as described in claim 1-3 any one comprises that further the ion condition of appropriate mix and pH value are to improve short infection activity.
5. the polypeptide that short infection activity is arranged as described in claim 1-3 any one, except HIV-1, further comprise being applicable to other tunicary virus.Described virus is naturally occurring virus or recombinant virus.
6. the polypeptide that short infection activity is arranged as described in claim 1-3 any one, comprise its application in research aspect the gene therapy of tissue and live body.
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| CN201210336804.XA CN103145802B (en) | 2012-09-13 | 2012-09-13 | A kind of polypeptide promotees infection reagent |
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| CN201210336804.XA CN103145802B (en) | 2012-09-13 | 2012-09-13 | A kind of polypeptide promotees infection reagent |
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| CN103145802B CN103145802B (en) | 2017-07-21 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294838B (en) * | 2015-09-22 | 2018-07-03 | 徐州市玛泰生物科技有限公司 | A kind of antibacterial peptide and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110124842A1 (en) * | 2003-09-19 | 2011-05-26 | Brunel Florence M | Peptide that binds to a broadly neutralizing anti-HIV antibody-structure of 4E10 Fab fragment complex, uses thereof, compositions therefrom |
| CN102558314A (en) * | 2010-12-08 | 2012-07-11 | 吉林大学 | HIV (human immunodeficiency virus) immunogen and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110124842A1 (en) * | 2003-09-19 | 2011-05-26 | Brunel Florence M | Peptide that binds to a broadly neutralizing anti-HIV antibody-structure of 4E10 Fab fragment complex, uses thereof, compositions therefrom |
| CN102558314A (en) * | 2010-12-08 | 2012-07-11 | 吉林大学 | HIV (human immunodeficiency virus) immunogen and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| JEFFREY R BRENDER ET AL: "Helical conformational of the SEVI precursor peptide PAP248-286,a dramatic enhancer of HIV infectivity,promotes lipid aggregation and fusion", 《BIOPHYSICAL JOURNAL》 * |
| KARAL SALZWEDEL ET AL: "A conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for env-mediated fusion and virus infectivity", 《JOURNAL OF VIROLOGY》 * |
| ZHAI YOUGANG ET AL: "Display of conserved Human Immunodeficiency Virus Type 1 gp41 membrane-proximal external region epitopes on bovine papilloma virus-like particles", 《THE JOURNAL OF IMMUNOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105294838B (en) * | 2015-09-22 | 2018-07-03 | 徐州市玛泰生物科技有限公司 | A kind of antibacterial peptide and its application |
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Inventor after: Dan Yaming Inventor after: Kong Wei Inventor after: Zhang Lishuang Inventor after: Zhang Hao Inventor after: Guan Shanshan Inventor before: Dan Yaming Inventor before: Kong Wei Inventor before: Zhang Lishuang |
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