CN110194793B - Polypeptide for inhibiting actin recombination and application - Google Patents
Polypeptide for inhibiting actin recombination and application Download PDFInfo
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- CN110194793B CN110194793B CN201810159838.3A CN201810159838A CN110194793B CN 110194793 B CN110194793 B CN 110194793B CN 201810159838 A CN201810159838 A CN 201810159838A CN 110194793 B CN110194793 B CN 110194793B
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses a polypeptide for inhibiting actin recombination and application thereof, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO.1, and the nucleotide sequence of a gene for coding ABS1p is shown as SEQ ID NO. 2. ABS1p is based on ABS of ABD sequence in non-muscle alpha-actin and can be used for matching with primary CD4+F-actin binds in T cells. Experiments prove that ABS1p inhibits the recombination of T cell F-actin and the infection of HIV-1 to T cells, and is expected to be developed into an inhibitor with the function of treating AIDS, or can be combined with other inhibitors to achieve the ideal treatment effect.
Description
Technical Field
The invention belongs to the technical field of biomedical engineering, and particularly relates to a polypeptide for inhibiting actin recombination and application thereof, wherein the polypeptide provided by the invention inhibits HIV-1 infection by inhibiting actin recombination.
Background
Human Immunodeficiency Virus (HIV) is a lentivirus that infects cells of the Human Immune system, belonging to the family of retroviridae, the genus lentivirus, and can cause the body to produce the "acquired Immunodeficiency Syndrome" (AIDS), i.e. AIDS. Causing damage to the immune system of the organism and causing great threat to human health.
At present, anti-AIDS drugs are mainly divided into: reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and entry inhibitors. Unlike the first three classes of drugs, the entry inhibitor targets the early stages of viral infection. Entry inhibitors are further classified into adsorption inhibitors, co-receptor inhibitors and fusion inhibitors according to three steps of drug targeting HIV into cells.
The cytoskeleton is a dynamic structure and comprises three components, namely a microwire, a middle wire and a microtubule. The main functions include maintaining the morphology of the cell, mediating the movement of the cell, and the transportation of macromolecules and organelles. Microfilaments are composed of actin (actin), actin-binding protein (actin), and myosin (myosin). The Actin monomers form a string of Actin chains one after another, and the spiral fiber formed by winding and assembling two strings of Actin chains is called Fibrous Actin (F-Actin). Actin F-actin is the basis for cell motility and migration, and is involved in many processes of host cell immune response. During infection of the target cell by the virus, actin of the host cell is the first line of defense for the virus to enter the host cell. The actin scaffold, an integral part of the intracellular molecular network, is also the target of pathogens. The study of cytoskeleton is of little interest, since actin can interact non-specifically with many proteins and the mechanism of actin influence on HIV-1 infection is not well defined. Furthermore, the basic composition of the targeted cells can cause severe cytotoxicity, and thus antiviral therapy against actin is often questioned.
Recent studies have shown that F-actin is highly variable in mammalian cell cortex with half-lives of 20-25s, regulated by cofilin, alpha-actin, and the like. Alpha-actinin is a conserved protein for bundling Actin, has two Actin binding sites which are far away from each other, belongs to a highly conserved Actin binding protein family, namely a spectrin superfamily, and is an important Actin cross-linking protein in cytoskeleton. Alpha-actin can stabilize cell adhesion and regulate cell shape and movement under the synergistic action of intracellular integrin, cadherin, signal molecules in a signal transduction pathway and the like.
According to the analysis of the applicant, the alpha-Actin consists of 3 structural domains, the N terminal is an Actin-binding domain (ABD), the ABD contains 3 Actin-binding sites (ABS), the ABD is distributed on the surface of the ABD and respectively comprises ABS1, ABS2 and A BS 3. Wherein ABS1 is distributed at the boundary of CH1 and CH 2; the ABS2 is distributed on the surface divided into CH1 and is partially positioned at the junction of C H1 and CH 2; the ABS3 is located on the CH2 surface. Because ABS is the binding site of actin and alpha-actin and is positioned on the surface of ABD structural domain of alpha-actin protein, no steric hindrance exists when ABS is combined with actin. Therefore, screening for inhibitors of actin and α -actin binding can inhibit endogenous α -actin binding by competitively binding to actin by introducing exogenous ABS peptide fragments. The design of the polypeptide inhibitor can be optimized on the basis of 3 amino acid sequences of ABS1, ABS2 and ABS3, and a candidate inhibitor with stronger affinity between the inhibitor and actin than between alpha-actin and actin is obtained by screening, so that competitive binding and inhibition effects are achieved.
At present, no polypeptide drug which can inhibit F-actin recombination by competitively inhibiting the combination of alpha-actin and actin exists at home and abroad. Therefore, the invention firstly proposes the concept of the competitive inhibitor. The polypeptide inhibitor is designed by taking ABS as a basic agent, other amino acids are not added, unnecessary immunogenicity can be avoided, and the polypeptide inhibitor is more suitable for clinical application. The inhibitor is derived from ABS of alpha-actin, has low immunogenicity, is soluble and is beneficial to pharmacy. Biochemical experiments and real-time tracing prove that the ABS1p inhibitor screened on the basis of ABS can influence the recombination of F-actin and alpha-actin in T cells, and has obvious influence on HIV-1 infection. In addition, the polypeptide inhibitor designed by the invention indicates the direction for inhibiting F-actin recombination.
Disclosure of Invention
The invention provides a polypeptide ABS1p for inhibiting actin recombination and HIV-1 infection, and the amino acid sequence is shown in SEQ ID NO. 1.
Another object of the present invention is to provide a nucleotide sequence encoding the polypeptide ABS1p shown in SEQ ID NO.1, preferably the nucleotide sequence shown in SEQ ID NO.2, as long as the nucleotide sequence encoding the polypeptide is protected by the present invention.
The final purpose of the invention is to provide application of the polypeptide ABS1p, which comprises that the polypeptide ABS1p of the invention is prepared into a medicine for inhibiting actin recombination, or is prepared into a medicine for inhibiting HIV-1 infection, or the polypeptide A BS1p of the invention is combined with other effective components to be prepared into a medicine for inhibiting actin recombination and HIV-1 infection.
In order to achieve the purpose, the invention adopts the following technical measures:
the polypeptide ABS1p for inhibiting actin recombination and HIV-1 infection has the amino acid sequence shown in SEQ ID NO.1, and is obtained through commercial synthesis.
The nucleotide sequence of the polypeptide ABS1p shown in SEQ ID NO.1 is also protected by the invention, and preferably, the nucleotide sequence is shown in SEQ ID NO. 2.
The application of the polypeptide ABS1p comprises that the polypeptide ABS1p is prepared into a medicine for inhibiting actin recombination, or is prepared into a medicine for inhibiting HIV-1 infection, or the polypeptide ABS1p is combined with other effective components to be prepared into a medicine for inhibiting actin recombination and HIV-1 infection.
Compared with the prior art, the invention has the following advantages and effects:
A. compared with other actin related inhibitors, the polypeptide provided by the invention has smaller molecular weight (1819Da) and stronger specific binding capacity.
B. The invention is based on ABS on alpha-actin, and is expected to be developed into an entry inhibitor for inhibiting HIV-1 by introducing exogenous ABS peptide fragments to competitively combine with actin, and the ABS1p is derived from alpha-actin, so that the immunogenicity is relatively weak.
C. The polypeptide ABS1p only contains 15 amino acid residues, has short length, low cost and short period, and is suitable for rapid large-scale production
And (4) production.
Drawings
FIG. 1A shows that different concentrations of ABS1p are responsible for HIV-1 entry into CD4+Results of T cell influence.
FIG. 1B is a graph showing the effect of various concentrations of ABS1p on HIV-1 entry into CEM-ss cells.
FIG. 2A is a Kymograph analysis of HIV-1 infection with CD4+Graphs relating F-actin and a-actin to each other in T cells.
FIG. 2B is a Kymograph analysis of ABS1p for HIV-1 infection CD4+Graphs showing the relationship of F-actin and alpha-actin to each other under the influence of T cells.
FIG. 3A is a graph showing the relationship between F-actin and α -actin in Kymograph assay of HIV-1 infected CEM-ss cells.
FIG. 3B is a graph showing the relationship between F-actin and α -actin in Kymograp assay ABS1p under the influence of HIV-1 infecting CEM-ss cells.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way. The technical solutions described in the embodiments of the present invention are all conventional solutions in the art, unless otherwise specified. The amino acid sequence of the polypeptide ABS1p used in the embodiment of the invention is shown in SEQ ID NO.1, and the polypeptide ABS1p is obtained through commercial synthesis.
Example 1:
ABS1p for HIV-1 entry into CD4+Inhibition of T cells or CEM-ss cells
Study of ABS1p on HIV-1 entry into CD4+In inhibiting T cells or CEM-ss cells, the cell concentration is first adjusted to 1X 106cells/mL, each group of cells volume is 2 mL; adding polypeptide ABS1p into the cell system, and incubating in a 37 ℃ incubator for 1 h; after pretreatment, 200ng of p24HIV-1 was added to the cells, which were placed at 37 ℃ in 5% CO2Infecting for 3 hours under the condition; after the infection is completed, the bacteria are infected,the cells were rinsed 3 times with PBS and fresh medium was added to bring the cell concentration to 1X 106cells/mL, continued at 37 ℃ with 5% CO2Culturing under the condition for 48 h.
When the infection effect of the virus on the cells is measured, the content of p24 in the cells after the virus infection is detected by adopting Intracellular p24 stating. In the experiment, cells after virus infection are collected firstly, the cells are rinsed 3 times by PBS, and then the intracellular p24staining is carried out according to the reaction of Anti-HIV-1p24antibody and the intracellular p24 antigen antibody. The experimental steps are as follows: (1) fixing: centrifuging the cleaned cells at 200 Xg for 5min, and collecting cell precipitates; (2) adding 4% paraformaldehyde into the collected cells, slightly blowing, mixing, and fixing the cells at room temperature for 30 min; (3) permeability: centrifuging the fixed cells at 200 Xg for 5min, collecting cell precipitate, adding 0.5% Triton PBS, and allowing to permeate at room temperature for 10 min; (4) washing: washing the cells 2 times with 4% BSA in PBS, and collecting the cell pellet; (5) primary antibody binding: the wash was discarded, leaving about 100. mu.L of PBS containing 4% BSA, the cells were resuspended, and 0.5. mu.L of Anti-HIV-1p24antibody (Abcam, ab9044) was added and incubated overnight at 4 ℃; (6) cells were washed 3 times with 4% BSA in PBS; (7) and (3) binding of a secondary antibody: the wash solution was discarded, and Anti-mouse IgG Fab2Alexa Fluor (R)555Molecular Probes (Cell Signaling Technology, 4409S) diluted at 1:1000 were added to the cells and incubated at room temperature for 30 min; (8) washing: washing the cells with 4% BSA in PBS 2 times, and then adding Hank solution to wash the cells 2 times; (9) the wash was discarded, the cells were resuspended in 500. mu.L of 1% paraformaldehyde and analyzed by FACS (BD biosciences).
The results are shown in FIGS. 1A-1B, at CD4+In T cells, ABS1p at concentrations of 10. mu.g/mL and 1. mu.g/mL had significant effects on HIV-1 infection (FIG. 1A). In T cell line CEM-ss cells, ABS1p at a concentration of 10. mu.g/mL had a significant effect on HIV-1 infection (FIG. 1B).
Example 2:
ABS1p affects the recombination of F-actin and alpha-actin in T cells:
to in CD4+Study on dynamic relationship between F-actin and alpha-actin in T cells and T cell line CEM-ss cells, plasmid pECFP-C1-life and pirFP-alpha-actin were co-transfected into CD4+T is thinCells or CEM-ss cells, can realize the fluorescent labeling of living cells F-actin and alpha-actin. The specific implementation process comprises the following steps: (1) to room temperature of 100. mu.L
Solution resuspended CD4+Adding 2 mu g of pECFP-C1-Life and 2 mu g of pirFP-alpha-actin into the T cells or the CEM-ss cells, and gently mixing; (2) adding the cell plasmid mixture into the small groove of the electric rotating cup, and covering the electric rotating cup cap; (3) setting a LONZA nuclear transfer instrument program: selecting a V-024 program, inserting the electric rotor containing cells and plasmids into a slot of a nuclear rotation instrument, and operating the selected program; (4) at the end of the procedure, the cuvette was immediately removed, 500. mu.L of RPMI-1640 medium was added to the cuvette, the cell suspension was gently aspirated, and the cell suspension was placed in a pre-equilibrated dish of 10% FBS in RPMI 1640, at 37 ℃ and 5% CO2Culturing for 6h under the condition to express the gene; (5) after 6h, 10% FBS in RPMI 1640 was replaced at 37 ℃ with 5% CO2Performing condition culture; (6) collecting cells after 24h, centrifuging at 200 Xg for 10min, adjusting cell volume to 100 μ L, adding 100 μ L HIV-1-QDs virus, mixing, placing in a laser confocal glass bottom small dish, sealing with paraffin, and incubating at 4 deg.C for 30 min. When the influence of ABS1p on virus infection is verified, 10 mug/mL polypeptide ABS1p is added into a cell system, the cell system is placed in an incubator at 37 ℃ for incubation for 1h, then the cells are centrifuged at 200 Xg for 10min, the cell volume is adjusted to 100 mug, 100 mug of pretreated HIV-1-QDs virus is added, the mixture is mixed evenly, a glass bottom small dish is sealed by paraffin, and the incubation is carried out for 30min at 4 ℃. (7) And (4) observing under a transposed laser confocal microscope, and statistically analyzing image data by adopting a volume software.
The results are shown in FIGS. 2A-2B and 3A-3B, and the change of the fluorescence signal of the virus-adsorbed cell region is observed in real time by adding HIV-1-QDs into the labeled cell system, and the research shows that: in the viral entry area, CD4+F-actin and alpha-actin present asynchronous tendency in T cells (FIG. 2A) and T cell line CEM-ss cells (FIG. 3A), and alpha-actin precedes F-actin aggregation; and the virus-adsorbing region, CD4, on cells treated with the polypeptide ABS1p+F-actin and alpha-actin are in synchrony in T cells (FIG. 2B) and T cell line CEM-ss cells (FIG. 3B). TheThe result shows that the polypeptide ABS1p has good competitive inhibition activity, and the polypeptide ABS1p as a F-actin recombinant inhibitor with potential deserves further research and development, and indicates a new research direction for inhibiting HIV-1 infection.
Sequence listing
<110> Wuhan Virus institute of Chinese academy of sciences
<120> polypeptide for inhibiting actin recombination and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Arg Lys Thr Phe Thr Ala Trp Cys Asn Ser His Leu Arg Lys Ala
1 5 10 15
<210> 2
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agaaagacat tcacggcatg gtgtaactcc cacctccgga aggcg 45
Claims (5)
1. A polypeptide for inhibiting actin recombination has an amino acid sequence shown as SEQ ID NO. 1.
2. A nucleotide sequence encoding the polypeptide of claim 1.
3. The nucleotide sequence of claim 2, which is represented by SEQ ID No. 2.
4. Use of the polypeptide of claim 1 or the nucleotide sequence of claim 2 for the preparation of a recombinant drug for inhibiting actin.
5. Use of the polypeptide of claim 1 or the nucleotide sequence of claim 2 for the manufacture of a medicament for inhibiting HIV-1 infection.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342668A (en) * | 2000-09-12 | 2002-04-03 | 上海博德基因开发有限公司 | Polypeptide-F actin binding factor 32.78 and polynucleotide for coding it |
| CN1425687A (en) * | 2001-12-19 | 2003-06-25 | 复旦大学 | Polypeptide-protein-70.95 containing actin binding structural domain and polynucleotide for encoding such polypeptide |
| CN106397548A (en) * | 2015-07-29 | 2017-02-15 | 复旦大学 | Polypeptide for inhibiting HIV infection, and pharmaceutical uses thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2651317A1 (en) * | 2006-05-05 | 2007-11-15 | George Mason Intellectual Properties, Inc. | Compositions and methods for detecting and treating hiv infection |
| US11028138B2 (en) * | 2016-07-02 | 2021-06-08 | Virongy L.L.C. | Compositions and methods for using actin-based peptides to modulate cellular bioactivity and cellular susceptibility to intracellular pathogens |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342668A (en) * | 2000-09-12 | 2002-04-03 | 上海博德基因开发有限公司 | Polypeptide-F actin binding factor 32.78 and polynucleotide for coding it |
| CN1425687A (en) * | 2001-12-19 | 2003-06-25 | 复旦大学 | Polypeptide-protein-70.95 containing actin binding structural domain and polynucleotide for encoding such polypeptide |
| CN106397548A (en) * | 2015-07-29 | 2017-02-15 | 复旦大学 | Polypeptide for inhibiting HIV infection, and pharmaceutical uses thereof |
Non-Patent Citations (5)
| Title |
|---|
| Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection;Mónica Gordón-Alonso等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20130927;第288卷(第39期);28382-28397 * |
| B. Sjçblom等.a-Actinin structure and regulation.《Cellular and Molecular Life Sciences》.2008,第65卷2688-. * |
| HIV-1与CD4+T细胞F-actin相互作用的实时动态成像研究;任敏;《万方数据》;20151012;1-85 * |
| HIV感染者趋化因子变化及其对杀伤细胞功能和HIV感染的作用及机制研究;王卓;《中国博士学位论文全文数据库（电子期刊） 医药卫生科技辑》;20190115(第12期);全文 * |
| Real-time imaging of individual virion-triggered cortical actin dynamics for human immunodeficiency virus entry into resting CD4 T cells;Wen Yin等;《Nanoscale》;20200107;第12卷(第1期);115-129 * |
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