CN109679984B - Application of plant as host in expression of hemoglobin - Google Patents
Application of plant as host in expression of hemoglobin Download PDFInfo
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- CN109679984B CN109679984B CN201710979372.7A CN201710979372A CN109679984B CN 109679984 B CN109679984 B CN 109679984B CN 201710979372 A CN201710979372 A CN 201710979372A CN 109679984 B CN109679984 B CN 109679984B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
Abstract
The invention relates to the field of biotechnology, in particular to application of a plant as a host in expression of hemoglobin. The invention utilizes the recombinant vector and the agrobacterium-mediated vacuum infiltration method to express human hemoglobin (Hb). The expression system can collect the plant exogenous protein after confirming that the agrobacterium is infected for 4 d. The successful expression of recombinant Hb was confirmed by SDS-PAGE and immunoblotting (Western). The CO combined hemoglobin experiment proves that Hb produced by lettuce has biological activity. The present invention provides a method for mass-producing recombinant human Hb having activity at low cost and conveniently.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of a plant as a host in expression of hemoglobin.
Background
Hemoglobin (Hb) is a protein responsible for oxygen transport in higher organisms and is commonly known as Hemoglobin (abbreviated: Hb or Hgb). Hemoglobin is present in almost all vertebrates, and is distributed in certain invertebrate tissues. The hemoglobin in the blood transports oxygen from the respiratory organ to other parts of the body for release, so as to meet the operation requirement of the support function of the body oxidation nutrient substances, and the generated carbon dioxide is brought back to the respiratory organ to be discharged out of the body. In mammals, hemoglobin constitutes 97% of the dry weight of red blood cells and 35% of the total weight. On average, 1.34ml of oxygen can be combined with each gram of hemoglobin, and the oxygen content is 70 times of the dissolved oxygen content of plasma. One mammalian hemoglobin molecule can bind up to four oxygen molecules. Hemoglobin is also involved in the transport of other gases: it can carry part of the carbon dioxide of the body (about 10%). The important regulatory molecule nitric oxide can also be bound to a certain thiol group of the globular protein, releasing oxygen at the same time. Hemoglobin and hemoglobin-like molecules are also distributed in many invertebrates, fungi and plants. In these organisms, hemoglobin may carry oxygen or play a role in the transfer and regulation of, for example, carbon dioxide, nitric oxide, hydrogen sulfide and sulfides. One such variant molecule, known as Leghemoglobin (Leghemoglobin), is used to scavenge oxygen to avoid poisoning the anaerobic system of nitrogen-fixing nodules, such as legumes.
Transfusion therapy is an important means indispensable for clinical treatment. Since the 70's of the 20 th century, safety concerns have gradually come into the eye of people with the large number of clinical applications of blood products. Global blood reserve deficiency, blood product related disease. Moreover, potential pathogens in transfusion plasma, such as hepatitis and HIV, present various risks from Hb, which is currently derived from plasma. In order to eliminate the potential risk of plasma pathogens and viral contamination, the use of genetic engineering techniques to produce recombinant Hb (rhb) may serve as a safer and lower cost Hb for large-scale production. Hemoglobin in a human body is composed of four subunits, namely two alpha subunits and two beta subunits, and the four subunits of hemoglobin can be automatically assembled into a form of alpha 2 beta 2 in an electrolyte solution similar to the human environment. The shortage of blood and the risk of transmitting viruses, make finding new ways to source blood an urgent task. A large amount of cytokine drugs have been developed by utilizing the genetic engineering technology, and the drugs have the advantages of high yield, no virus pollution and the like, so that the production of recombinant human hemoglobin by utilizing the genetic engineering technology is one of the most promising approaches for solving the blood source.
Hemoglobin has been expressed in E.coli. Plants have been studied for nearly thirty years as a system for the expression and production of pharmaceutical proteins. In addition to the advantages of low cost and high yield, plant-based expression systems reduce the risk of transmission of human and animal pathogens to humans from the process of protein production. In addition, plant eukaryotic protein inner membrane expression systems and secretory pathways are similar to those of mammalian cells. The plant expression system can produce large amount of high molecular weight and subunit medicine protein and is superior to prokaryotic expression system, such as colibacillus expression system. Proteins that require post-translational modification, glycosylation, and monoclonal antibodies that require assembly, which cannot be achieved with prokaryotic systems. The use of plant-produced pharmaceutical proteins as biological agents has been commercialized or usedCan be used as vaccine additive for poultry. In 2012, the united states Food and Drug Administration (FDA) approved the protein eleyso for the treatment of the genetic disease type 1 gaucher disease TM (taliglucerase alfa), and this protein is produced from carrot. Over the past decade, the demand for pharmaceutical proteins has increased dramatically, so the number of plant pharmaceutical proteins approved by the FDA for clinical trials has increased.
The plant transient expression system can produce recombinant proteins in large quantities for clinical studies or to cope with paroxysmal diseases. In 2014, the only antibody therapeutic drug used to effectively resist ebola virus outbreaks, ZMappTM, was produced in tobacco leaves by the agrobacterium infiltration method. The efficacy and safety of ZMapp opens the way for the industry to advance the plant pharmaceutical industry. Currently, tobacco transient protein expression is the most common host plant, and various vectors and agroinfiltration methods have been developed for large-scale production in a short time. However, tobacco has a high fiber content and potentially toxic compounds, such as the alkaloid nicotine, significantly increasing the cost of downstream purification processes, greatly impeding the further development of plant foreign protein pharmaceuticals. Compared with a tobacco leaf system, the lettuce contains less phenols and toxic compounds, so that the use of plants, particularly lettuce as a host has important practical significance in expressing human hemoglobin.
Disclosure of Invention
In view of the above, the present invention provides the use of plants as hosts for the expression of hemoglobin. The invention utilizes plants, especially lettuce, as an effective platform for recombinant protein production, eliminates the growth cycle of plants and greatly saves the time for cultivating plants in the early stage. The invention uses the lettuce system to express Hb, and successfully separates active foreign protein under mild conditions, thereby proving that the lettuce expression platform can be used for producing recombinant human hemoglobin.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides the application of a plant as a host in expressing hemoglobin; the plant is selected from lettuce, tobacco, Chinese cabbage, rice, corn, soybean or wheat; the plant organ is selected from the group consisting of seed, leaf, rhizome, or whole plant.
In some embodiments of the invention, the hemoglobin is human Hb.
The invention also provides an expression vector, which comprises the nucleotide sequence of the hemoglobin and a binary plant vector.
In some embodiments of the invention, the hemoglobin in the expression vector is human Hb.
In some embodiments of the present invention, the method for constructing the expression vector comprises the following steps:
step 1: adding Xbal restriction enzyme cutting sites to the 5 'end of the codon optimized human hemoglobin nucleotide sequence and adding Sacl restriction enzyme cutting sites to the 3' end; the hemoglobin is Hb;
step 2: cloning into a vector pUC57 to obtain a cloning vector pUC 57-Hb;
and step 3: obtaining gene fragment Hb from the cloning vector obtained in the step 2 through Xbal/Sacl, cloning to a binary plant vector pCam35S, and obtaining an expression vector p 35S-Hb.
Specifically, the construction method of the expression vector provided by the invention comprises the following steps: the codon for human Hb (Hb- α +2A + Hb- β) was optimized to plant-preferred codons, designed by Integrated DNA Technology (IDT) and synthesized by Kinsry. An Xbal restriction site was added to the 5 '-end of the optimized Hb sequence, a Sacl site was added to the 3' -end, and the resulting product was cloned into pUC57 vector by Kinsley to obtain a cloning vector pUC57-Hb (FIG. 1A). The human hemoglobin gene fragment Hb was isolated from pUC57-Hb by Xbal/Sacl (FIG. 1B) and cloned into binary plant vector pCam35S, yielding transient expression vector p35S-Hb, respectively (FIG. 2). The plant expression constructs were transformed into Agrobacterium tumefaciens GV3101 by electroporation with a multipolator (Eppendorf, Hamburg, Germany), respectively. The resulting strain was spread evenly on selective LB plates containing kanamycin antibiotic (50 mg/L). After incubation in the dark at 28 ℃ for 2 days, a single colony was picked and inoculated into 0.5L YEB (yeast extract broth, 5g/L sucrose, 5g/L tryptone, 6g/L yeast extract, 0.24g/L yeast extract) MgSO 4 pH7.2) and supplemented with antibiotic liquid medium (50mg/L kanamycin). The inoculated culture was incubated in a shaker (220rpm) for 72h at 25-28 ℃. OD600 values were measured by addition of YEB medium and adjusted to 3.5-4.5. The culture broth was then collected and centrifuged (4500 rpm) for 10 min. Resuspending Agrobacterium cells in osmotic Medium (10mM MES, 10mM MgSO) 4 ) The neutral to o.d.600 is 0.5.
In the invention, a Hb alpha subunit cDNA sequence is shown as SEQ ID No. 1; the Hb alpha subunit amino acid sequence is shown as SEQ ID No. 2; hb beta subunit cDNA sequence SEQ ID No. 3; the Hb beta subunit amino acid sequence is shown as SEQ ID No. 4; 2A signal peptide amino acid sequence SEQ ID No. 5; the amino acid sequence of the Hb-alpha +2A + Hb-beta fusion protein sequence is shown as SEQ ID No. 6; the codon-optimized Hb-alpha +2A + Hb-beta DNA sequence is shown in SEQ ID No. 7.
The invention also provides application of the expression vector in expression of hemoglobin. In some embodiments of the invention, the hemoglobin is human Hb.
The invention also provides a method for expressing human hemoglobin by using lettuce as a host, which comprises the steps of transforming the expression vector into agrobacterium, infiltrating the agrobacterium-mediated vacuum into plant tissues, extracting and separating protein to obtain the hemoglobin.
In some embodiments of the invention, the hemoglobin in the method is human Hb.
In some embodiments of the invention, the agrobacterium-mediated vacuum infiltration comprises the steps of:
step 1: vacuumizing for 25-45 s;
step 2: keeping the vacuum (-95kPa) pressure for 30-60 s;
and step 3: releasing the pressure such that the permeate permeates the plant tissue;
repeating the steps for 2-3 times, and carrying out light-proof treatment for 4 d.
In some embodiments of the invention, the agrobacterium is agrobacterium tumefaciens GV 3101.
Specifically, the method for agrobacterium-mediated vacuum infiltration comprises the following steps: the prepared agrobacterium culture suspension was placed in a 2L beaker and placed in a desiccator. Lettuce kept in this laboratory was inverted (core up) and gently swirled into the bacterial suspension and the desiccator was sealed. The Vacuum pump (Welch Vacuum, Niles, IL, USA) was turned on to evacuate and the permeate was visible in the leaf tissue. Keeping the pressure state for 30-60 s. The system is rapidly opened to release pressure and allow the permeate to penetrate into the space within the tissue. The process is repeated for 2-3 times until the clear visible penetrating fluid is obviously diffused in the lettuce tissues. The lettuce tissues were then gently removed from the permeate and rinsed three times in succession with distilled water before being transferred to a plastic film covered container. The treated sample was kept in the dark for 4 d.
After infiltration, most lettuce tissues were submerged during vacuum infiltration, except for the firm intercostal areas, which all showed a light tan area 4 days after vacuum infiltration. To increase the number of Agrobacterium tumefaciens that are immersed in the leaf tissue, 10% of the lettuce leaves were cut off from the head with scissors so that the lettuce leaf tissue was as infiltrated in the permeate as possible and released. This method reduces leaf tissue necrosis compared to longer vacuum exposure times.
In some embodiments of the invention, the extracting and isolating of proteins is in particular:
the lettuce samples permeated by the agrobacterium under vacuum are stirred by a stirrer, and are homogenized for 1-2 min at high speed in an extraction buffer (100mMKPi, pH 7.8; 5 mMEDTA; 10mM beta-mercaptoethanol) stirrer with the volume ratio of 1: 1. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000g for 15min at 4 ℃ to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%) and incubated on ice for 60min with shaking. The separation was again carried out by means of a centrifuge (10,000g) at 4 ℃ for 15 min. The resulting supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended with shaking on ice for 60min, and centrifuged again at 10,000g for 15min at 4 ℃. Then, the supernatant was discarded, and the treatment sample precipitated protein was dissolved in 5mL of a buffer (20mM KPi, pH 7.8; 2mM EDTA; 10 m. beta. -mercaptoethanol) and stored at 4 ℃.
Collecting purified protein extracted from vacuum infiltration lettuce of Agrobacterium, samplingThe sample (5uL) was heat denatured (95 ℃ C.) with loading buffer (Biorad, Hercules, Calif., USA) at 4-12%
Bis-Tris Plus SDS-gel (ThermoFisher Scientific, Waltham, MA, USA) was run and the gel was photographed again after staining with Coomassie blue G250 (Biorad). For Western Blot hybridization of recombinant HB, 10ul of recombinant sample and hHB standard (Biovision) were 10-20%
Bis-Tris Plus polyacrylamide gel separation and electrophoretic transfer to polyvinylidene fluoride (PVDF) membrane, immunoreaction with anti-HB antibody (Abcam), dilution 1: 10000 and goat anti-rabbit IgG labeled with horseradish peroxidase (HRP) (Beyotime), dilution 1:20000, respectively, and visualized using ECL plus (Amersham Biosciences), and the display image was photographed.
Downstream processing of recombinant proteins of plant origin is often difficult and expensive because of the difficulty of lysis of the cellulose cell wall and secondary plant metabolites. The invention uses the stirrer to stir and homogenize, thereby greatly saving the homogenization cost and the process. Recombinant Hb separation by SDS-PAGE we observed bands with estimated molecular weights of approximately 15kDa and 16kDa in the lanes (fig. 3A, lane 1), with no corresponding band evident in the stealth control lane (fig. 3A, lane 2). The protein content of the purified sample was determined to be 1.07mg/g based on the Bradford assay and densitometry controls. In addition, Western blot analysis also detected bands of approximately 15kDa and 16kDa (FIG. 3B, lane 1), and the observed protein molecular weights were consistent with the subunit sizes of human hemoglobin alpha and beta.
Both oxygen and carbon dioxide can form unstable compounds with hemoglobin in the red blood cells in the blood, and such compounds can release oxygen or carbon dioxide. If hemoglobin encounters carbon monoxide, but a more stable compound is produced, which reduces the ability of hemoglobin to deliver oxygen to the tissue, as with oxyhemoglobin, the Fe atom has a valence of 2 and covalently bonds to CO, 4N of the porphyrin, and the histidine residues of the hemoglobin prion to form an octahedral structure. CO was added to 10 grams of oxygen saturated purified recombinant Hb and the standard. Photolytic kinetic analysis demonstrated that oxygen was rapidly released from the purified recombinant Hb as well as the standard upon addition of CO and the release profile was consistent (fig. 4). These results indicate that exogenous Hb, transiently expressed by the lettuce system, is biologically active, and the lettuce system may be a suitable bioreactor for the mass production of biologically active recombinant pharmaceutical proteins.
The growth time of tobacco plants for vacuum agroinfiltration is typically 4 to 6 weeks. The invention utilizes lettuce as an effective platform for recombinant protein production, eliminates the growth cycle of plants and greatly saves the time for cultivating plants in the early stage. The invention uses lettuce system to express human hemoglobin, and successfully separates active foreign protein under mild condition, thus proving that lettuce expression platform can be used for producing human hemoglobin.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1(A) shows a Hb cloning vector (Kingsry construction synthesis);
FIG. 1(B) shows Hb gene fragment cleavage (XbaI/SacI) identification;
FIG. 2 shows a scheme for the construction of plant transient expression vector p 35S-Hb; utilizing restriction endonuclease (XbaI/SacI) to carry out double enzyme digestion, respectively cutting Hb fragments from the cloning vector in the figure 1, connecting the HbaI/SacI fragments into XbaI/SacI sites of pCam35S, and generating a plant transient expression vector p 35S-Hb;
wherein 35S is the CaMV 35S promoter with Tobacco Mosaic Virus (TMV) 5' UTR; NPT II, the expression of the nptII gene encoding for kanamycin resistance; nos 3', terminator;
FIG. 3(A) shows the detection of purified recombinant fibroblast human hemoglobin using polyacrylamide gel electrophoresis (SDS-PAGE); lane 1 purification of recombinant Hb (5. mu.g); lane 2: negative control of non-vacuum osmotic leaf eluent;
FIG. 3(B) shows detection of purified recombinant human hemoglobin by Western blot hybridization; lane 1 purification of recombinant Hb (5. mu.g); lane 2: negative control of non-vacuum osmotic leaf eluent;
FIG. 4 shows a hemoglobin binding CO experiment; the release curve of the purified recombinant Hb combined with CO is consistent with that of the control human hemoglobin combined with CO, and the biological activity of the exogenous Hb instantaneously expressed by the lettuce system is proved.
Detailed Description
The invention discloses the application of plants, especially lettuce, as a host in expressing hemoglobin, and can be realized by appropriately improving process parameters by referring to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The research of the invention shows that the lettuce system can be an effective expression platform and provides a method for quickly and instantaneously expressing recombinant protein. The vacuum agrobacterium infiltration method is simple and rapid, reduces the leaf necrosis and can improve the yield of recombinant protein. Lettuce can increase protein production by withstanding vacuum pressure and allow for more complete penetration of each leaf. Lettuce is easier to grow and cheaper to produce commercially in large quantities than other transiently expressing plants, such as tobacco and the like. And is more cost effective as no special equipment or liquid nitrogen is required. The present invention demonstrates that the method can be used for large-scale production of Hb recombinant proteins in a short time.
The invention provides raw materials and reagents used in the application of lettuce as a host in expressing human hemoglobin, which are all commercially available.
The invention is further illustrated by the following examples:
example 1 construction of plant transient expression vectors
To provide for efficient expression of exogenous proteins in plants, the present invention codon optimizes the human hemoglobin Hb- α +2A + Hb- β fusion protein sequence to plant-preferred codons, designed by Integrated DNA Technology (IDT) and synthesized by Kinsley. An Xbal restriction site was added to the 5 'end of the optimized Hb sequence, a Sacl site was added to the 3' end, and the vector was cloned into pUC57 by Kinsley. The human hemoglobin gene fragment Hb was isolated from pUC57-Hb by Xbal/Sacl and cloned into binary plant vector pCam35S, yielding the transient expression vector p35S-Hb, respectively. The plant expression constructs were transformed into Agrobacterium tumefaciens GV3101 by electroporation with a multipolator (Eppendorf, Hamburg, Germany), respectively. The resulting strain was spread evenly on selective LB plates containing kanamycin antibiotic (50 mg/L). After incubation in the dark at 28 ℃ for 2 days, a single colony was picked and inoculated into 0.5L YEB (yeast extract broth, 5g/L sucrose, 5g/L tryptone, 6g/L yeast extract, 0.24g/L MgSO4, pH7.2) and supplemented with antibiotic liquid medium (50mg/L kanamycin). The inoculated culture was incubated in a shaker (220rpm) for 72h at 25-28 ℃. OD600 values were measured by addition of YEB medium and adjusted to 3.5-4.5. The culture broth was then collected and centrifuged (4500 rpm) for 10 min. The Agrobacterium cells were resuspended in osmotic medium (10mM MES, 10mM MgSO 4 ) The neutral to o.d.600 is 0.5.
Example 2 Agrobacterium-mediated vacuum infiltration
The invention optimizes the method for vacuum infiltration of agrobacterium tumefaciens. The prepared agrobacterium culture suspension was placed in a 2L beaker and placed in a desiccator. Lettuce stored in this laboratory was inverted (core up) and gently swirled into the bacterial suspension and the desiccator was sealed. The Vacuum pump (Welch Vacuum, Niles, IL, USA) was turned on to evacuate and the permeate was visible in the leaf tissue. Keeping the pressure state for 30-60 s. The system is rapidly opened to release pressure and allow the permeate to penetrate into the space within the tissue. The process is repeated for 2-3 times until the clear visible penetrating fluid is obviously diffused in the lettuce tissues. The lettuce tissues were then gently removed from the permeate and rinsed three times in succession with distilled water before being transferred to a plastic film covered container. The treated sample was kept in the dark for 4 d.
After infiltration, most lettuce tissues were submerged during vacuum infiltration, except for the firm intercostal areas, which all showed a light tan area 4 days after vacuum infiltration. To increase the number of Agrobacterium tumefaciens that are immersed in the leaf tissue, 10% of the lettuce leaves were cut off from the head with scissors so that the lettuce leaf tissue was as infiltrated in the permeate as possible and released. This method reduces leaf tissue necrosis compared to longer vacuum exposure times.
Example 3 protein extraction and isolation
The lettuce samples permeated by the agrobacterium under vacuum are stirred by a stirrer, and are homogenized for 1-2 min at high speed in an extraction buffer (100mM KPi, pH 7.8; 5mM EDTA; 10mM beta-mercaptoethanol) stirrer with the volume ratio of 1: 1. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000g for 15min at 4 ℃ to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%) and incubated for 60 minutes on ice with shaking. The separation was again carried out by means of a centrifuge (10,000g) at 4 ℃ for 15 min. The resulting supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended with shaking on ice for 60min, and centrifuged again at 10,000g for 15min at 4 ℃. Then, the supernatant was discarded, and the treatment sample precipitated protein was dissolved in 5mL of a buffer (20mM KPi, pH 7.8; 2mM EDTA; 10 mM. beta. -mercaptoethanol) and stored at 4 ℃.
Example 4SDS-PAGE gel electrophoresis and Western Blot hybridization
The purified protein from vacuum infiltrated lettuce of Agrobacterium was collected and a sample (5uL) was heat denatured (95 ℃) loading buffer (Biorad, Hercules, Calif., USA) at 4-12%
Bis-Tris Plus SDS-gel (ThermoFisher Scientific, Waltham, MA, USA) was run and the gel was photographed again after staining with Coomassie blue G250 (Biorad). For Western Blot hybridization of recombinant Hb, 10ul of recombinant sample and hHb and standard (Biovision) were 10-20%
Bis-Tris Plus polyacrylamide gel separation and electrophoretic transfer to polyvinylidene fluoride (PVDF) membranes, immunoreaction with anti-hHb antibody (Abcam), dilution 1: 10000 and goat anti-rabbit IgG labeled with horseradish peroxidase (HRP) (Beyotime), dilution 1:20000, respectively, and visualized using ECL plus (Amersham Biosciences), and the display image was photographed.
Downstream processing of recombinant proteins of plant origin is often difficult and expensive because of the difficulty of lysis of the cellulose cell wall and secondary plant metabolites. The homogenizer is used for stirring and homogenizing, so that the homogenizing cost and the homogenizing process are greatly saved. Recombinant Hb separation by SDS-PAGE we observed a band with an estimated molecular weight of approximately 15kDa, 16kDa in the lane (fig. 3A, lane 1) and no corresponding band evident in the stealth control lane (fig. 3A, lane 2). The protein content of the purified sample was determined to be 1.07mg/g based on the Bradford assay and densitometry controls. In addition, Western blot analysis also detected a band of approximately 15kDa, 16kDa (FIG. 3B, lane 1), and the observed protein molecular weight was consistent with human hemoglobin alpha, beta subunit size.
Example 5 hemoglobin CO binding assay
Both oxygen and carbon dioxide can form unstable compounds with hemoglobin in the red blood cells in the blood, and such compounds can release oxygen or carbon dioxide. If hemoglobin encounters carbon monoxide, but a more stable compound is produced, thus reducing the ability of the hemoglobin to deliver oxygen to the tissue, as with oxygenated hemoglobin, the Fe atom is 2 valent, covalently bonded to CO, 4N of the porphyrin, and histidine residues of the blood cells in an octahedral structure. CO was added to 10 grams of oxygen saturated purified recombinant Hb and the standard. Photolytic kinetic analysis demonstrated that oxygen was rapidly released from the purified recombinant Hb as well as the standard upon addition of CO and the release profile was consistent (fig. 4). These results indicate that exogenous Hb, transiently expressed by the lettuce system, is biologically active, and the lettuce system may be a suitable bioreactor for the mass production of biologically active recombinant pharmaceutical proteins.
Example 6
Control group: producing human hemoglobin by using tobacco leaves;
experimental groups: the lettuce provided by the invention produces human hemoglobin;
TABLE 1 human hemoglobin
* Shows that P is less than or equal to 0.05 compared with the control group; # shows that P is less than or equal to 0.01 compared with the control group;
as can be seen from Table 1, compared with the tobacco leaf system of the control group, the lettuce instantly expresses human hemoglobin (Hb) obviously (P is less than or equal to 0.05), the production period is shortened, the combined CO multiple is obviously improved (P is less than or equal to 0.05), the expression yield is obviously improved (P is less than or equal to 0.05), the difficulty of protein purification is simplified, and the production cost is greatly reduced (P is less than or equal to 0.01).
The comprehensive test results show that the plant system, especially the lettuce system, is a more economic and efficient expression platform. Can express recombinant protein rapidly and instantaneously, and can produce human hemoglobin in a large scale in a short time.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Beijing Hello bioengineering technology Co., Ltd
Application of <120> plant as host in expression of hemoglobin
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Met Val Leu Ser Pro Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly
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Lys Val Gly Ala His Ala Gly Glu Tyr Gly Ala Glu Ala Leu Glu Arg
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Leu Ser His Gly Ser Ala Gln Val Lys Gly His Gly Lys Lys Val Ala
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Leu Ser Ala Leu Ser Asp Leu His Ala His Lys Leu Arg Val Asp Pro
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Val Asn Phe Lys Leu Leu Ser His Cys Leu Leu Val Thr Leu Ala Ala
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His Leu Pro Ala Glu Phe Thr Pro Ala Val His Ala Ser Leu Asp Lys
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gtggatgaag ttggtggtga ggccctgggc aggctgctgg tggtctaccc ttggacccag 120
aggttctttg agtcctttgg ggatctgtcc actcctgatg ctgttatggg caaccctaag 180
gtgaaggctc atggcaagaa agtgctcggt gcctttagtg atggcctggc tcacctggac 240
aacctcaagg gcacctttgc cacactgagt gagctgcact gtgacaagct gcacgtggat 300
cctgagaact tcaggctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc 360
aaagaattca ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat 420
gccctggccc acaagtatca ctaa 444
<210> 4
<211> 147
<212> PRT
<213> Hb-β
<400> 4
Met Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp
1 5 10 15
Gly Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu
20 25 30
Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp
35 40 45
Leu Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His
50 55 60
Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp
65 70 75 80
Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys
85 90 95
Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val
100 105 110
Cys Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln
115 120 125
Ala Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His
130 135 140
Lys Tyr His
145
<210> 5
<211> 20
<212> PRT
<213> 2A signal
<400> 5
Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 6
<211> 309
<212> PRT
<213> Hb-α+2A+ Hb-β
<400> 6
Met Val Leu Ser Pro Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly
1 5 10 15
Lys Val Gly Ala His Ala Gly Glu Tyr Gly Ala Glu Ala Leu Glu Arg
20 25 30
Met Phe Leu Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp
35 40 45
Leu Ser His Gly Ser Ala Gln Val Lys Gly His Gly Lys Lys Val Ala
50 55 60
Asp Ala Leu Thr Asn Ala Val Ala His Val Asp Asp Met Pro Asn Ala
65 70 75 80
Leu Ser Ala Leu Ser Asp Leu His Ala His Lys Leu Arg Val Asp Pro
85 90 95
Val Asn Phe Lys Leu Leu Ser His Cys Leu Leu Val Thr Leu Ala Ala
100 105 110
His Leu Pro Ala Glu Phe Thr Pro Ala Val His Ala Ser Leu Asp Lys
115 120 125
Phe Leu Ala Ser Val Ser Thr Val Leu Thr Ser Lys Tyr Arg Gln Leu
130 135 140
Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro
145 150 155 160
Gly Pro Met Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala
165 170 175
Leu Trp Gly Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly
180 185 190
Arg Leu Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe
195 200 205
Gly Asp Leu Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys
210 215 220
Ala His Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His
225 230 235 240
Leu Asp Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys
245 250 255
Asp Lys Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val
260 265 270
Leu Val Cys Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro
275 280 285
Val Gln Ala Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu
290 295 300
Ala His Lys Tyr His
305
<210> 7
<211> 930
<212> DNA
<213> The sequence of password suboptimization of Hb-α and 2A and Hb-β
<400> 7
atggttttga gtccagccga taagactaac gtcaaggcag catggggaaa agttggtgca 60
catgctggag agtatggtgc agaggccttg gagcgaatgt ttctctcatt tcctactact 120
aaaacttatt ttccccattt tgatttaagc catggctcag cccaagttaa aggccacggg 180
aagaaggtgg ccgatgccct cactaacgct gtagctcacg ttgacgacat gcccaacgct 240
ttgtcagcac tgagcgattt acatgctcac aagttaaggg tggaccctgt caattttaaa 300
ctcctgagtc attgtttgct cgttactttg gctgcccacc tgcctgctga gtttactcca 360
gcagttcatg cttccttaga caaatttctt gctagtgtct ctacagtgct cactagcaaa 420
tatagacaac tgttaaattt cgacctcctg aaattggctg gtgatgtcga gagtaaccct 480
ggcccaatgg ttcacctcac tcctgaggaa aagtccgcag tcacagcact gtggggcaag 540
gtcaatgtag atgaagttgg aggtgaggcc ttaggaaggt tgctggttgt ttatccctgg 600
actcaaagat ttttcgagtc cttcggcgac ttaagtaccc ctgacgcagt gatggggaac 660
cctaaagtaa aagcccatgg taagaaagtg cttggggctt tttctgacgg attagcacat 720
ttggataacc tgaagggaac atttgcaaca ctgtcagaac ttcattgtga taagctccac 780
gtcgacccag aaaacttcag actcttgggt aatgtattgg tgtgcgttct ggcccatcac 840
ttcggtaaag agtttacccc cccagtacag gctgcctacc agaaagtcgt tgccggagtc 900
gccaacgcct tggctcataa gtaccattaa 930
Claims (1)
1. A method for expressing hemoglobin by using a plant as a host is characterized in that an expression vector is transformed into agrobacterium, and after the vector is permeated into plant tissues in vacuum through mediation of the agrobacterium, protein is extracted and separated to obtain hemoglobin;
the hemoglobin is human Hb;
the agrobacterium-mediated vacuum infiltration comprises the following steps:
step 1: vacuumizing for 25-45 s;
step 2: keeping the vacuum at-95 kPa for 30-60 s;
and step 3: releasing the pressure such that the permeate permeates the plant tissue;
repeating the steps for 2-3 times, and carrying out light-proof treatment for 4 d;
the plant tissue is lettuce;
the expression vector comprises a nucleotide sequence of hemoglobin and a binary plant vector;
the hemoglobin is human Hb;
the construction method comprises the following steps:
step 1: adding an Xbal restriction site at the 5 'end and a Sacl restriction site at the 3' end of the nucleotide sequence of hemoglobin;
step 2: cloning into a vector pUC57 to obtain a cloning vector pUC-Hb;
and step 3: obtaining a gene fragment Hb from the cloning vector obtained in the step 2 through Xbal/Sacl, cloning to a binary plant vector pCam35S, and obtaining an expression vector p 35S-Hb;
the codon-optimized Hb-alpha +2A + Hb-beta DNA sequence of the hemoglobin is shown in SEQ ID No. 7.
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| CN110229822B (en) * | 2019-06-24 | 2023-11-03 | 王跃驹 | Application of plant as host in expression of Albiglutide |
| CN110205337A (en) * | 2019-06-24 | 2019-09-06 | 王跃驹 | Application of the plant as host expresses source of people Telomerase |
| CN111286511B (en) * | 2019-12-20 | 2022-11-25 | 鲁东大学 | Method for producing human epidermal growth factor and ganoderma lucidum immunomodulatory protein and application |
| CN113106098B (en) * | 2021-04-21 | 2022-04-01 | 贵州医科大学 | Recombinant sequence for specifically expressing human beta globin in erythroid cells and application thereof |
| WO2024003668A1 (en) * | 2022-06-29 | 2024-01-04 | Moolec Science Limited | High expression of animal heme protein in plants |
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