A technology of fusion protein and interferon, applied in the field of biological genetic engineering, can solve the problems of unfavorable clinical application, low molecular weight of interferon, high cost of interferon, avoid denaturation and renaturation, shorten expression time, and improve immune response. Effect
Inactive Publication Date: 2017-10-17
WUHU YINGTE FEIER BIOLOGICAL PROD IND RES INST CO LTD
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AI-Extracted Technical Summary
Problems solved by technology
The common long-acting interferon on the market is represented by polyethylene glycol fusion interferon, which only partially solves the problem of short half-life due to the small mol...
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Method used
[0113] A fusion protein composed of sheep interleukin 2 and sheep interferon gamma, the others are the same as in Example 1, except that the competent cells of Escherichia coli BL21 (DE3) are replaced by competent cells of BL21 (DE3) with pGro7 plasmid cell. The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 54.6KD in the supernatant is thicker, indicating that after the introduction of molecular chaperone pGro7, the expression of the target protein in the supernatant is better, and the obtained The amount of fusion protein is higher. Most of the proteins expressed in E. coli exist in inclusion bodies; by introducing molecular chaperones into the expression strains, the co-expressed proteins can be correctly folded to achieve protein soluble expression.
[0158] A fusion protein composed of goat interleukin 2 and goat interfero...
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Abstract
The invention discloses a recombinant sheep interferon gamma and a fusion protein for preparing the peg interferon gamma and a preparation method thereof. The fusion protein is formed by connecting sheep interleukin 2 and sheep interferon gamma through a flexible linker , the fusion protein is mixed with a freeze-drying protective agent and then freeze-dried to obtain the recombinant sheep peginterferon gamma. The recombinant sheep long-acting interferon gamma can significantly increase the half-life of sheep interferon, which is more than 10 times higher than that of ordinary sheep interferon gamma, has broad-spectrum antiviral effect and can improve the immune response of sheep itself.
Application Domain
BacteriaMicroorganism based processes
+8
Technology Topic
Long actingInterleukin II
+7
Image
Examples
- Experimental program(9)
Embodiment 1
[0073] A fusion protein composed of sheep interleukin 2 and sheep interferon gamma, the preparation method of which is as follows:
[0074] 1. Acquisition and amplification primer design of sheep interleukin 2 (IL-2) and sheep interferon gamma (IFN-gamma) target genes:
[0075] Design synthetic primers according to the target gene sequence reported in Genebank, as shown in Table 1. Introduce the EcoRI restriction site and Linker sequence into the upstream primer and downstream primer of sheep interleukin 2, respectively, and introduce the upstream primer and downstream primer of sheep interferon gamma Linker sequence and XhoI restriction site were introduced respectively.
[0076] Table 1PCR amplification primers
[0077]
[0078] RT-PCR to obtain the target gene:
[0079] RNA was extracted from sheep liver tissue, and the target genes of IL-2 and IFN-γ were obtained by reverse transcription. The gene sequences of the two are shown in SEQUENCE LISTING 400<4> and SEQUENCE LISTING 400<5>, respectively;
[0080] RT-PCR reaction system (25μL) see Table 2
[0081] Table 2 RT-PCR reaction system
[0082] RNase Free Water
10 μL
dNTP Mix
10 μL
reverse transcriptase
2.5μL
Upstream and downstream primers
0.5μL each
genomic RNA
1.5μL
[0083] The reaction parameters were: reverse transcription at 50°C for 30 min, pre-denaturation at 95°C for 4 min, and cycle: denaturation at 95°C for 45 s; annealing at 58°C for 45 s, extension at 72°C for 1 kb/min, a total of 35 cycles; final extension at 72°C for 10 min.
[0084] RT-PCR amplification products appear specific bands around 490bp and 520bp through agarose gel electrophoresis, and the results are as follows: figure 1 As shown, it shows that the target gene of sheep interferon IL-2 and the target gene of sheep interferon gamma respectively connected with the flexible linker have been obtained.
[0085] 2. Ligation of the target gene
[0086] Dilute the target gene to 10ug/mL, and use overlapping PCR to connect the target gene. The 25μL reaction system is shown in Table 3:
[0087] Table 3 PCR reaction system
[0088]
[0089] The reaction parameters are: pre-denaturation at 95°C for 4 minutes, entering cycle: denaturation at 94°C for 45 s; annealing at 58°C for 45 s, extension at 72°C at 1 kb/min, a total of 35 cycles; final extension at 72°C for 10 min.
[0090] The PCR amplification product appeared a specific band at about 990bp by agarose gel electrophoresis, and the results were as follows: figure 2 as shown, figure 2The bands of sheep interleukin 2 amplification product and sheep interferon gamma amplification product appeared in , this is because non-specific reaction occurred during the PCR connection process of sheep interleukin 2 gene and sheep interferon gamma gene. The obtained nucleotide sequence of the target gene is shown in SEQUENCE LISTING 400<2>.
[0091] 3. Expression vector construction
[0092] Select the target gene after the ligation and sequence the correct PCR gel recovery product and pET-32a plasmid for double digestion and recovery with EcoRI and XhoI restriction endonucleases, and perform double digestion according to the 20 μL system in Table 4:
[0093] Table 4 Double Enzyme Digestion System
[0094]
[0095]
[0096] Ligate the target gene after ligation with the pET-32a plasmid digested recovery product according to the system in Table 5, and ligate overnight at 4°C:
[0097] table 5
[0098] target DNA
10 μL
Expression vector
3μL
buffer
2μL
Ligase
1μL
RNase Free Water
4μL
[0099] Transform the ligation product into Escherichia coli BL21 (DE3) competent cells, spread the competent cells on LB medium plate containing ampicillin and culture overnight; take the colony grown on the LB plate to identify the target gene by PCR, and the positive cloned bacterial plasmid is passed through EcoRI and XhoI double enzyme digestion identification, positive identification means that the expression vector was successfully constructed, and the engineered bacteria pET-32a/rIL2-IFNγ was obtained; PCR amplification and double enzyme digestion products were subjected to agarose gel electrophoresis, and a single gene appeared at about 990bp strips, the result of which is image 3 shown.
[0100] 4. Expression of Recombinant Proteins
[0101] Pick the engineered bacteria pET-32a/rIL2-IFNγ in the LB medium containing 100 μg/mL ampicillin at 37°C for 1 hour to recover the activity of the engineered bacteria, and scale up the culture in LB medium (containing 100 μg/mL ampicillin) for 4 hours , when the measured OD value reaches 1.0; add IPTG, the final concentration of IPTG is 100 μg/mL, and induce expression at 32°C for 5 hours; collect bacteria and detect them by SDS-PAGE electrophoresis, and the results are as follows: Figure 4 As shown in the figure, it can be seen from the figure that the supernatant and sediment after 5 hours of recombinant bacterial induction were crushed, and a dominant expression band was seen at about 54.6KD, indicating that the fusion protein was successfully expressed in both the sediment and the supernatant.
[0102] Add PBS with a mass volume ratio of 1:1 to resuspend the pellet; freeze-thaw the pellet at room temperature at -20°C for 3 times; ultrasonically lyse the bacterial pellet at 4°C, work for 10s with an interval of 3s, and sonicate for 6min, and repeat the whole process 3 to 4 times; 4 Centrifuge at 12000r/min for 15min, take the supernatant and precipitate, respectively, to obtain the crude fusion protein.
[0103] 5. Fusion protein purification
[0104] 5.1 His affinity chromatography
[0105] After filtering the crude fusion protein with a filter membrane with a pore size of 0.22 μm, the sample was connected to the AKTA explorer 100 protein purification system, and the His affinity chromatography column was equilibrated with Binding Buffer Ⅰ (PBS), and washed with PBS buffer. The unbound protein was eluted with Elution buffer I (50mM tris, 20-500mM imidazole, pH8.0) until the A280nm was stable, and the rIL2-IFNγ protein peak was collected.
[0106] 5.2 DEAE anion exchange chromatography
[0107] Replace the protein collected after His affinity chromatography purification into Binding Buffer II (50mM Tris, pH 6.5), and load the sample through a DEAE anion-exchange chromatography column equilibrated with Binding Buffer II. Then pass through the column with Binding Buffer II until the A280nm value is stable, and then use Elution Buffer II (50mM Tris, 1M NaCl, pH6.5) for linear gradient elution to collect the rIL2-IFNγ protein peak.
[0108] 5.3 Molecular sieve chromatography
[0109] Concentrate the sample collected by ion exchange chromatography and load the sample through the Superdex 200 molecular sieve chromatography column equilibrated with Binding Buffer Ⅲ (50mMNa2HPO4, 0.15M NaCl, PH7.4), elute with Binding Buffer Ⅲ, and collect the rIL2-IFNγ protein peak .
[0110] 5.4 Sample Identification
[0111] Determination of rIL2-IFNγ titer and specific activity, specific activity ≥ 1.0×10 6 IU/mg protein is qualified; aseptically aliquoted and stored at -80°C. A fusion protein composed of sheep interleukin 2 and sheep interferon γ can be obtained, and its amino acid sequence is shown in SEQUENCE LISTING 400<1>.
Embodiment 2
[0113] A fusion protein composed of sheep interleukin 2 and sheep interferon gamma, the others are the same as in Example 1, except that the competent cells of Escherichia coli BL21 (DE3) are replaced by competent cells of BL21 (DE3) with pGro7 plasmid cell. The SDS-PAGE electrophoresis result of the fusion protein is compared with that of Example 1, and the dominant expression band at about 54.6KD in the supernatant is thicker, indicating that after the introduction of molecular chaperone pGro7, the expression of the target protein in the supernatant is better, and the obtained The amount of fusion protein is higher. Most of the proteins expressed in E. coli exist in inclusion bodies; by introducing molecular chaperones into the expression strains, the co-expressed proteins can be correctly folded to achieve protein soluble expression.
[0114] The BL21(DE3) competent cells carrying the pGro7 plasmid were purchased from Shanghai Inshore Science & Technology Co., Ltd./Simbano Biotech, Cat. No. V205.
Embodiment 3
[0116] A fusion protein composed of sheep interleukin 2 and sheep interferon gamma, the preparation method of which is as follows:
[0117] 1. Acquisition and amplification of sheep interleukin 2 (IL-2) and sheep interferon gamma (IFN-gamma) target genes
[0118] IL-2 and IFN-γ in Example 1 are optimized, and IL-2 and IFN-γ target genes are artificially synthesized. After optimization, the nucleotide sequences of the two are as SEQUENCE LISTING 400<6> and SEQUENCE LISTING 400 respectively As shown in <7>.
[0119] 1.1 Codon optimization
[0120] There are 64 genetic codes, but most organisms tend to use a subset of these. Those that are most frequently used are called optimal codons, and those that are not frequently used are called rare or low-usage codons. Virtually every organism commonly used for protein expression or production (including E. coli, yeast, mammalian cells, plant cells, and insect cells) exhibits some degree of difference or bias in codon usage. The expression efficiency of genes containing optimal codons in E. coli, yeast and Drosophila was significantly higher than that of genes containing low utilization codons. Therefore, in the heterologous expression system, the codon bias largely affects the expression of recombinant proteins. Gene synthesis using preferred codons and avoiding rare codons is called codon optimization. Therefore, in this example, the codons of sheep IL-2 and IFN-γ genes were optimized.
[0121] 1.2 Analysis of results after codon optimization
[0122] Generally, when the codon adaptation index (CAI)=1.0, it is considered the optimal high expression state of the gene in the expression system, and the lower the CAI value, the lower the expression level in the host. The optimal distribution range of GC content in genes is 30-70%, any area exceeding this range will affect the efficiency of translation and transcription. The codon adaptation index (CAI) of the original gene of sheep IL-2 and IFN-γ was found to be 0.27, 0.25 in Escherichia coli by software detection, and the GC percentage was 54.0%, 40.9%; After optimization with IFN-γ gene, the codon adaptation index (CAI) of the recombinant gene in Escherichia coli was 1.0, 1.0, and the GC percentage was 53.0%, 44.1%. Gene optimization significantly reduces the usage rate of low codons, avoids the impact of rare codons on protein expression, improves the GC content of genes, and improves transcription and translation efficiency.
[0123] 1.3 Primer design:
[0124] Table 6PCR amplification primers
[0125]
[0126] The optimized genomic DNA of IL-2 and IFN-γ were diluted to 0.05mg/mL respectively. The target gene was obtained by PCR amplification, and the 25 μL reaction system was shown in Table 7:
[0127] Table 7 PCR reaction system
[0128] RNase Free Water
10.5μL
dNTP Mix
10.0 μL
Taq DNA polymerase
2.5μL
Upstream and downstream primers
0.5μL each
Genomic DNA
1.0 μL
[0129] The reaction parameters are: pre-denaturation at 95°C for 4min, entering cycle: denaturation at 95°C for 45s; annealing at 60°C for 45s, extension at 72°C at 1kb/min, a total of 35 cycles; final extension at 72°C for 10min.
[0130] The PCR amplification products of IL-2 and IFN-γ showed specific bands at around 490bp and 520bp respectively after agarose gel electrophoresis, indicating that the optimized sheep IL-2 and sheep IFN- The target gene of γ.
[0131] 2. Ligation of the target gene
[0132] Dilute the target gene to 10ug/mL, and connect the target gene by overlapping PCR. The 25μL reaction system is shown in Table 8:
[0133] Table 8 PCR reaction system
[0134]
[0135] The reaction parameters are: pre-denaturation at 95°C for 4 minutes, entering cycle: denaturation at 94°C for 45 s; annealing at 60°C for 45 s, extension at 72°C at 1 kb/min, a total of 35 cycles; final extension at 72°C for 10 min.
[0136] PCR amplified products were subjected to agarose gel electrophoresis and a specific band appeared at about 990bp, indicating that the target gene after the connection of IL-2 and IFN-γ was successfully obtained, and the nucleotide sequence of the obtained target gene was as follows: SEQUENCE LISTING 400<3 > Shown.
[0137] 3. Expression vector construction
[0138]Select the target gene after the ligation and sequence the correct PCR gel recovery product and pET-32a plasmid to perform double digestion and recovery with NcoI and XhoI restriction endonucleases, and perform double digestion according to the 20 μL system in Table 9:
[0139] Table 9 Double Enzyme Digestion System
[0140] General buffer
2μL
restriction endonuclease (pair)
1μL+1μL
vector or recovered fragment
2μL
RNase Free Water
14μL
[0141] Ligate the target gene after ligation with the pET-32a plasmid digested recovery product according to the system in Table 10, and ligate overnight at 4°C:
[0142] Table 10
[0143] target DNA
10 μL
Expression vector
3μL
buffer
2μL
Ligase
1μL
RNase Free Water
4μL
[0144] Transform the ligation product into Escherichia coli BL21 (DE3) competent cells, spread the competent cells on LB medium plate containing ampicillin and culture overnight; take the colony grown on the LB plate to identify the target gene by PCR, and the positive cloned bacterial plasmid is passed through NcoI and XhoI double enzyme digestion identification, positive identification indicates that the expression vector was successfully constructed, and a single band appeared at about 990bp in the PCR amplification and double enzyme digestion product after agarose gel electrophoresis, indicating that it contained IL-2 and IFN-γ The expression vector of the ligated target gene was constructed successfully, and the engineering bacteria pET-32a/rIL2-IFNγ was obtained.
[0145] 4. Expression of Recombinant Proteins
[0146] Pick the engineered bacteria pET-32a/rIL2-IFNγ in the LB medium containing 100 μg/mL ampicillin at 37°C for 1 hour to recover the activity of the engineered bacteria; scale up the culture in LB medium (containing 100 μg/mL ampicillin) for 4 hours , when the measured OD value reaches 1.0; add IPTG, the final concentration of IPTG is 100μg/mL, and induce expression at 32°C for 5h; collect the bacteria, detect by SDS-PAGE electrophoresis, and the supernatant after the recombinant bacteria are broken for 5h after induction and Predominant expression bands can be seen at about 54.6KD in the precipitate, indicating that the recombinant protein was obtained in both the supernatant and the precipitate.
[0147] Add PBS with a mass volume ratio of 1:1 to resuspend the pellet; freeze-thaw the pellet at room temperature at -20°C for 3 times; ultrasonically lyse the bacterial pellet at 4°C, work for 10s with an interval of 3s, and sonicate for 6min, and repeat the whole process 3 to 4 times; 4 Centrifuge at 12000r/min for 15min, take the supernatant and precipitate, respectively, to obtain the crude fusion protein.
[0148] 5. Fusion protein purification
[0149] 5.1 His affinity chromatography
[0150] After filtering the crude fusion protein with a filter membrane with a pore size of 0.22 μm, the sample was connected to the AKTA explorer 100 protein purification system, and the His affinity chromatography column was equilibrated with Binding Buffer Ⅰ (PBS), and washed with PBS buffer. The unbound protein was eluted with Elutionbuffer I (50mM tris, 20-500mM imidazole, pH8.0) until the A280nm was stable, and the rIL2-IFNγ protein peak was collected.
[0151] 5.2 DEAE anion exchange chromatography
[0152] Replace the protein collected after His affinity chromatography purification into Binding Buffer II (50mM Tris, pH 6.5), and load the sample through a DEAE anion-exchange chromatography column equilibrated with Binding Buffer II. Then pass through the column with Binding Buffer II until the A280nm value is stable, and then use Elution Buffer II (50mM Tris, 1M NaCl, pH6.5) for linear gradient elution to collect the rIL2-IFNγ protein peak.
[0153] 5.3 Molecular sieve chromatography
[0154] Concentrate the sample collected by ion exchange chromatography and load the sample through the Superdex 200 molecular sieve chromatography column equilibrated with Binding Buffer Ⅲ (50mMNa2HPO4, 0.15M NaCl, PH7.4), elute with Binding Buffer Ⅲ, and collect the rIL2-IFNγ protein peak .
[0155] 5.4 Sample Identification
[0156] Determination of rIL2-IFNγ titer and specific activity, specific activity ≥ 1.0×10 6 IU/mg protein is qualified; aseptically aliquoted and stored at -80°C. A fusion protein composed of sheep interleukin 2 and sheep interferon γ can be obtained, and its amino acid sequence is shown in SEQUENCE LISTING 400<1>.
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PUM
| Property | Measurement | Unit |
| Concentration | >= 1.4 | mg/ml |
| tensile | MPa | |
| Particle size | Pa | |
| strength | 10 |
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FAQs
What is recombinant interferon used for? ›
(ree-KOM-bih-nunt in-ter-FEER-on AL-fuh …) A drug used alone or with other drugs to treat adults with AIDS-related Kaposi sarcoma, hairy cell leukemia, certain types of follicular lymphoma or melanoma, genital warts or perianal warts, or chronic hepatitis B or C.
What is recombinant murine ifn γ? ›Recombinant Murine IFN-γ is a 15.6 kDa protein containing 134 amino acid residues. Purity: ≥ 98% by SDS-PAGE gel and HPLC analyses. Biological Activity: Determined by its ability to inhibit the proliferation of murine WEHI-279 cells.
How is recombinant interferon produced? ›Introduction. Interferon-alpha is produced predominantly by B lymphocytes but may be derived from leukocytes, lymphoblasts or by recombinant DNA technology. Recombinant interferon alpha differs from interferon alpha-2 in 20 of 166 amino acids.
Is interferon gamma approved by the FDA? ›ACTIMMUNE® (Interferon gamma-1b) is approved by the FDA to delay time to disease progression in patients with severe malignant osteopetrosis (SMO). ACTIMMUNE® is believed to work by changing how some cells in your body work. This includes the cells that help form your bones.
How does interferon gamma work? ›IFN‐γ augments antigen presentation through both the MHC class I and class II pathways, thereby increasing the immunogenicity of tumor cells and enhancing their detection and killing by immune surveillance and effector mechanisms ( 131, 185, 186, 187).
What is an interferon protein responsible for? ›Interferons are a family of structurally related, species-specific proteins that enhance the immune system released by host cells in response to the presence of pathogens such as viruses, bacteria, parasites, and tumor cells.
What is the role of interferon gamma in viral infection? ›Interferon-gamma (IFNG) is a pleiotropic cytokine that modulates both innate and adaptive immune networks; it is the most potent activator of macrophages and a signature cytokine of activated T lymphocytes.
What is the role of interferon gamma in inflammation? ›IFN-gamma has long been recognized as a signature proinflammatory cytokine that plays a central role in inflammation and autoimmune disease. There is now emerging evidence indicating that IFN-gamma possesses unexpected properties as a master regulator of immune responses and inflammation.
What does IFN-gamma do in inflammation? ›Production of interferon-gamma (IFNgamma) in response to infection is a hallmark of innate and adaptive immunity. In addition to the pivotal role of IFNgamma in host defense, its excessive release has been associated with the pathogenesis of chronic inflammatory and autoimmune diseases.
How are recombinant products made? ›These are manufactured by the expression of equivalent proteins from genetically engineered cell lines.
Where does interferon gamma come from? ›
Interferon-gamma is secreted predominantly by activated lymphocytes such as CD4 T helper type 1 (Th1) cells and CD8 cytotoxic T cells (23–26), γδ T cells (27–33), and natural killer (NK) cells (34, 35) and, to a less extent, by natural killer T cells (NKT), B cells (36–39), and professional antigen-presenting cells ( ...
Where do recombinant proteins come from? ›Most recombinant proteins for therapeutic use are from humans but are expressed in microorganisms such as bacteria, yeast, or animal cells in culture. Human genes are very complex, often containing non-coding DNA sequences known as introns.
Is interferon used anymore? ›As well as occurring naturally in the body, interferon is also used as a treatment for various health disorders, including multiple sclerosis (MS), some types of cancer, and hepatitis C.
Which interferon is used in COVID-19? ›A total of 1,941 patients with risk factors for severe COVID-19 were randomized to receive either a single subcutaneous injection of pegylated interferon lambda 180 mcg or placebo. Eighty-three percent of these patients had received at least 1 dose of COVID-19 vaccine.
Is interferon still used today? ›Interferons are currently used clinically to treat viral infections such as hepatitis C, cancers including non-Hodgkin's lymphoma, and autoimmune diseases such as multiple sclerosis.
How long does interferon gamma take? ›Results can be available within 24 hours.
What is the main action of interferon gamma? ›A primary role for IFN-γ is the activation of macrophages to increase phagocytosis, tumoricidal properties, and intracellular killing of pathogens, particularly bacteria and fungi. IFN-γ induces macrophage production of a variety of inflammatory mediators and reactive oxygen and nitrogen intermediates.
What is the brand name for interferon gamma? ›ACTIMMUNE® (Interferon gamma-1b)
What are side effects of interferon? ›Injection site reactions (pain/swelling/redness), headache, tiredness, diarrhea, upset stomach, loss of appetite, back pain, dizziness, dry mouth, taste changes, nausea, or vomiting may occur. If any of these effects last or get worse, tell your doctor or pharmacist promptly.
Do interferons prevent inflammation? ›Inflammation is an essential physiological process, which enables survival during infection and maintains tissue homeostasis. Interferons (IFNs) and pro- and anti-inflammatory cytokines are crucial for appropriate response to pathogens, damaged cells, or irritants in inflammatory response.
What is difference between protein and interferon? ›
IFNs belong to the large class of proteins known as cytokines, molecules used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens. Interferons are named for their ability to "interfere" with viral replication by protecting cells from virus infections.
Is interferon gamma inflammatory or anti inflammatory? ›Interferon γ (IFN-γ) induces an inflammatory response and apoptotic cell death.
What does interferon do to fight off viruses? ›Interferons act via autocrine and paracrine pathways to induce an antiviral state in infected cells and in neighboring cells containing interferon receptors. Interferons are the frontline defenders against viral infection and their primary function is to locally restrict viral propagation.
How interferon prevents viral infection? ›Interferons provide a first line of defence against virus infections by generating an intracellular environment that restricts virus replication and signals the presence of a viral pathogen to the adaptive arm of the immune response.
What does interferon gamma do in tumor? ›Interferon-γ (IFN-γ) is a pleiotropic cytokine that has long been praised as an important effector molecule of anti-tumor immunity, capable of suppressing tumor growth through various mechanisms.
How does IFNG induce cell death? ›Downstream events of the PI3K/AKT signaling pathway have been explored. IFN-α causes the activation of the ERK1/2 and JNK pathways culminating in apoptosis via the mitochondrial-dependent death pathway and involving the activation of proapoptotic Bcl-2 family members Bax and Bak.
Does interferon suppress the immune system? ›There are at least two types of interferon. Along with their antiviral properties, they have recently been shown to exert a suppressive effect on the humoral and cellular immune response; they affect both B and T lymphocytes.
How do you increase interferon gamma? ›As opposed to endurance training, moderate exercise can enhance IFN-gamma production. This was the case in a clinical trial on 16 healthy men [55]. Sun exposure promotes nitric oxide release, which in turn can increase interferon-gamma levels, as seen in a clinical trial on 24 healthy volunteers [56].
What are the advantages of recombinant proteins? ›The advantages of producing proteins using recombinant DNA technology are: As human gene may be cloned and expressed, it minimizes the risk of immune reaction and the specific activity of the protein is high.
What are the hazards of recombinant DNA technology? ›Safety issues in recombinant DNA technology include: “Gene pollution” of the environment resulting in “superweeds,” antibiotic-resistant microbes. Health effects of foods from GMOs. Allergenicity/adverse immune reactions/effectiveness of pharmaceutical compounds produced using rDNA technology.
Why is a recombinant protein so called? ›
Using recombinant DNA technology, scientists are able to create new DNA sequences that would not naturally exist under normal circumstances and environmental conditions. The resulting recombinant DNA is put into a host cell, where it is expressed into a new protein, which is the so-called recombinant protein.
What immune cells produce interferon gamma? ›IFN-γ is produced by activated NK cells [1], CD4+ Th1 cells [2] and CD8+ cytotoxic T cells [3] and is a critical component of the cell-mediated immune response to intracellular pathogens and tumors (reviewed in [4]).
Where is interferon gamma found? ›IFN-γ is primarily known to be produced by T helper cell type 1 (Th1) lymphocytes, CD8 lymphocytes, B cells, NKT cells, and antigen-presenting cells (monocytes, macrophages and dendritic cells).
Why use recombinant protein instead of native protein? ›Recombinant proteins are invaluable in proteomics research. In comparison to native proteins, recombinant have two primary advantages: 1) more flexibility regarding which and how much recombinant proteins are produced, and 2) purified recombinant proteins are cheaper than purifying native proteins.
What is the difference between native protein and recombinant protein? ›Native antigens are extracted in their natural form from the corresponding source. Recombinant antigens are instead manufactured artificially.
What disease is interferon used to cure? ›Interferon alfa-2b injection is used to treat hepatitis B and C, lymphoma (lymph node cancer), malignant melanoma (skin cancer), genital warts, hairy cell leukemia (blood cell cancer), and Kaposi sarcoma (AIDS-related tumor).
What is interferon most effective against? ›The various forms of interferon are the body's most rapidly produced and important defense against viruses. Interferons can also combat bacterial and parasitic infections, inhibit cell division, and promote or impede the differentiation of cells.
Which interferon destroys tumor? ›MAK cells were shown to exert antitumor activity by killing tumor cells and inhibiting their proliferation. These activities were enhanced by activation with IFN-γ and addition of antitumor antibodies. TNF-α was one of the mediators used by MAK cells to inhibit tumor proliferation.
Is interferon an expensive drug? ›The total cost of the IFN treatment regimen was estimated to range between US$1,120 and US$1,962 and the total cost of the Peg-IFN treatment regimen between US$2,156 and US$5,887 ( Table 3).
What is the new antiviral for COVID Canada? ›PaxlovidTM is intended to reduce the severity of COVID-19 symptoms in people at risk of developing serious complications from this infection. It is estimated to reduce the risk of hospitalization or death from COVID-19. PaxlovidTM treatment must be started within five days of the appearance of COVID-19 symptoms.
Does interferon protect against COVID? ›
A single dose of lambda-interferon reduced hospitalization among COVID-19 outpatients in a late-stage study spearheaded by a Stanford Medicine virologist. A single injection of PEG-lambda interferon proved to be effective against all variants of the coronavirus tested by researchers at Stanford Medicine.
Is interferon like chemo? ›Interferon-alfa2b is different than a chemotherapy drug; it is actually a natural part of your body's immune system. It is known as a cytokine, which are chemicals normally secreted by cells called leukocytes in response to a virus, bacteria, or other foreign intruders.
Does interferon cause depression? ›Therapeutic administration of IFN-α leads to depression in up to 50% of patients, and up to 30% of patients develop IFN-α-induced depression (a major depressive episode according to Diagnostic and Statistical Manual of Mental Disorders, 4th edn diagnostic criteria) within the first 3 months.
Can interferon cause neuropathy? ›Peripheral neuropathy is a rare and uncommon side effect seen in patients treated with IFN-α.
What infection does interferon cure? ›Recombinant IFN-α is now used as the basis for treatment of chronic hepatitis C virus infection and can also be used to treat certain forms of chronic hepatitis B virus infections. IFNs have also been used in other viral infections, although with less success.
Is interferon a type of chemotherapy? ›Interferon-alfa2b is different than a chemotherapy drug; it is actually a natural part of your body's immune system. It is known as a cytokine, which are chemicals normally secreted by cells called leukocytes in response to a virus, bacteria, or other foreign intruders.
What is the use of recombinant cytokines? ›Recombinant cytokines are pharmaceutical analogs of endogenously produced cytokines and colony-stimulating factors and are used therapeutically to increase the activity of the immune system or the production of particular populations of blood cells.
How long does interferon treatment last? ›Why interferons cause long-term side effects. Interferon treatment for hepatitis C would typically last 24–48 weeks (6–12 months). Interferons caused many long-term side effects partly because of this long treatment time. Using the drug for this length of time gave side effects a chance to develop and get worse.
Why is interferon being discontinued? ›Merck announced the permanent discontinuation of Intron A (interfereon alfa-2b). — Intron A is being discontinued due to business reasons and is not due to any safety, efficacy or quality reasons. — Intron A powder for injection is expected to be discontinued on or near March 2022.
What is a serious effect of interferon treatment? ›This drug may cause you to develop serious mental/mood changes that may get worse during treatment or after your last dose. Tell your doctor right away if you have symptoms such as confusion, depression, thoughts of suicide or hurting others, unusual irritability, or aggressive behavior.
Can interferon cause dementia? ›
IFN-a has been associated with reversible, subcortical dementia in other cases where it was used as a treatment, usually at high doses.
Does interferon weaken immune system? ›There are at least two types of interferon. Along with their antiviral properties, they have recently been shown to exert a suppressive effect on the humoral and cellular immune response; they affect both B and T lymphocytes.
Does interferon reduce inflammation? ›Interferon-β1a (IFNβ1a) is the prototypical regulatory cytokine with anti-inflammatory properties largely used in the past two decades to slow down pathological and clinical features of central nervous system (CNS) immune-mediated diseases, such as multiple sclerosis [12,13,14,15,16,17] and its animal model ...
How quickly does interferon work? ›Occurs within 1-2 hours of treatment, may last up to 24 hours (over time the intensity of these symptoms decreases depending on the dose, how it is given, and the schedule of administration). Fatigue , drowsiness. Low blood counts .
Why do we need recombinant proteins? ›Recombinant proteins serve as valuable tools for investigating cellular response to stress, and disease situations. Recombinant proteins and peptides administered to animal models of disease assist researchers in identifying novel potential therapeutic candidates.
Why are recombinant cells useful to humans? ›Recombinant DNA technology has also proven important to the production of vaccines and protein therapies such as human insulin, interferon and human growth hormone. It is also used to produce clotting factors for treating haemophilia and in the development of gene therapy.
What cytokine is used for inflammation? ›Cytokines are signaling proteins that help control inflammation in your body. They allow your immune system to mount a defense if germs or other substances that can make you sick enter your body. Too many cytokines can lead to excess inflammation and conditions like autoimmune diseases.