CN110885847A - Method for efficiently expressing hypersensitive protein by using T4 phage display technology - Google Patents
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- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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Abstract
The invention relates toA method for efficiently expressing hypersensitive proteins using T4 phage display technology, said method comprising the steps of: (1) sequentially connecting the encoding gene of the hypersensitive protein with TEV protease and T4 phage Soc protein gene fragments to prepare a fusion recombinant protein encoding gene, wherein the encoding region of the hypersensitive protein is positioned at the upstream of the TEV-Soc fusion protein gene and has consistent expression frames; (2) transferring the coding gene of the fusion recombinant protein into a pUC18 plasmid; (3) transferring the plasmid into an escherichia coli host; (4) use of Soc protein-deleted Soc‑Infecting said E.coli host with a T4 bacteriophage, culturing the host to express a T4 bacteriophage displaying said fusion recombinant protein; (5) isolating the T4 phage displaying the fusion recombinant protein; (6) separating the fusion recombinant protein from the T4 bacteriophage, and obtaining the hypersensitive protein after TEV protease cleavage.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for efficiently expressing a hypersensitive protein by using a T4 phage display technology.
Background
Chemical fertilizers and pesticides are the main products for improving crop yield and preventing and treating plant diseases and insect pests, but simultaneously have problems in the aspects of environment, energy, cost and the like, become important for the economic and social development of countries in the world at present, and seriously restrict the sustainable development of agriculture. Biological products (including biological fertilizers and biological pesticides) prepared by using proteins from microorganisms or fungi are the main measures for the high-quality, high-efficiency and pollution-free production of modern crops. In recent years, a great deal of scientific research has been carried out at home and abroad in this regard. The early microbial protein is mainly Insecticidal Crystal Protein (ICP) from bacillus thuringiensis, the novel microbial protein is mainly protein elicitor substances, and the representative novel microbial proteins reported in the current research mainly comprise allergenic protein (Harpin), cryptic protein (Cryptogenin) and Activator protein (Activator), which do not directly kill pests and pathogens, do not directly provide nutrients, but have wide application prospects in the aspects of promoting plant growth and development and absorption of nutrients, enhancing broad-spectrum disease resistance of plants (including resistance to various bacteria, fungi and viruses), enhancing insect repellency of plants, enhancing stress resistance of plants (tolerance of plants to adverse environments such as drought, severe cold and saline alkali), prolonging storage time of crop products, and shaping effects on potted flowers and seedlings. Therefore, they can be formulated into biologicals, and applied to the above aspects.
The novel microbial protein or polypeptide is produced in large scale by fermenting and recombining bioengineering bacteria such as escherichia coli, and the next step after fermentation is to dissolve bacterial cells in bacterial suspension to release the microbial protein or polypeptide. Methods for cell lysis include non-chemical methods such as high pressure or ultrasonic treatment, which require high equipment, consume high energy and are difficult to apply on a large scale. Alternatively, most manufacturers use a cell suspension in contact with lysozyme, followed by digestion at 40-42 ℃, centrifugation to remove cell debris and denatured protein, and protein purification processes to achieve the desired purity. These processes are complicated, expensive, and have many steps and lengthy processes, which reduce the yield of microbial proteins and polypeptides and reduce their biological activity and stability.
Therefore, there is a need for a simple, cost-effective method for the production of such novel microbial proteins or polypeptides. .
Disclosure of Invention
In order to solve the above problems, the present invention provides a simple and rapid method for preparing a hypersensitive protein, and provides the following technical solutions.
The present invention firstly relates to a method for expressing a protein of interest using T4 phage display, said method comprising the steps of:
(1) sequentially connecting the coding gene of the target protein with TEV protease and T4 phage Soc protein gene fragments to prepare a fusion recombinant protein coding gene, wherein the coding region of the target protein is positioned at the upstream of the TEV-Soc fusion protein gene, and the expression frames are consistent;
(2) transferring the coding gene of the fusion recombinant protein into a pUC18 plasmid;
(3) transferring the plasmid into an escherichia coli host;
(4) use of Soc protein-deleted Soc-Infecting said E.coli host with a T4 bacteriophage, culturing the host to express a T4 bacteriophage displaying said fusion recombinant protein;
(5) isolating the T4 phage displaying the fusion recombinant protein;
(6) separating the fusion recombinant protein from the T4 phage, and obtaining the target protein after TEV protease digestion.
The target protein in the step (1) is a hypersensitive protein, preferably harpinEa protein which is derived from a GeneBank with the serial number of M92994.3 of a strain Erwinia amylovora;
introducing a Soc promoter sequence into the upstream of a coding DNA sequence of the fusion recombinant protein in the step (1), and introducing a Soc terminator sequence into the downstream;
the method for transferring the plasmid into the escherichia coli host in the step (3) is a chemical conversion method;
infecting the cultured escherichia coli cells with a phage concentration of MOI (multiplicity of infection) ═ 1;
the method for isolating the T4 phage displaying the fusion recombinant protein in step (5) is as follows,
1) precooling the culture solution, centrifuging the culture solution at a low temperature for 30min by using 43000g of centrifugal force, and removing the supernatant;
2) adding an appropriate amount of PI-Mg buffer (3.7g/L Na)2HPO4、4g/L NaCl、3g/L KH2PO4、1mM MgSO4) Resuspending the pellet thoroughly, adding DNase I to a final concentration of 10. mu.g/ml and several drops of chloroform, and culturing at 37 ℃ and 200rpm for 30 min;
3) removing cell debris by centrifugation at 4000-6000g, wherein the obtained supernatant is the suspension of the T4 phage particles displaying the recombinant protein;
the method for separating the fusion recombinant protein in the step (6) is as follows: centrifuging the suspension of T4 phage particles displaying recombinant protein (suspension is PI-Mg buffer) at low temperature for 30min by using 43000g of centrifugal force, removing supernatant, and suspending the precipitate with TEV enzyme digestion buffer (50mM Tris-HCl pH8.0,0.5mM EDTA,1mM DTT);
the method for obtaining the target protein by TEV protein enzyme digestion in the step (6) comprises the following steps: incubating for 6h at 30 ℃ with 1000U of enzyme for enzyme digestion treatment, and centrifuging for 30min at low temperature by using 43000g of centrifugal force, wherein the supernatant is the target protein solution.
Drawings
FIG. 1 is a schematic diagram of a plasmid pVB-harpinEa-TEV-Soc map, showing the circular plasmid DNA molecule in a circle, with the direction of gene expression shown by arrows at the ends of the fragment; the structural regions of the plasmid include: harpinEa-TEV-Soc recombinant protein (harpinEa-TEV-Soc fusion), gene promoter (Soc promoter), terminator (Soc terminator) and Ribosome Binding Site (RBS); a TEV protease coding region in the middle of the harpinEa-TEV-Soc recombinant protein gene; ampicillin resistance gene (AmpR) and replication origin (ori).
FIG. 2 electrophoresis chart of recombinant protein displayed on the phage surface of T4 and isolated hypersensitive protein,
FIG. 2A: lane 1 is a protein molecular weight standard in Kilodaltons (KD); lane 2 shows a suspension of T4 phage particles displaying harpinEa-TEV-Soc recombinant protein; lane 3: a2-fold dilution of the T4 phage particle suspension displaying harpinEa-TEV-Soc recombinant protein (about 54kD) was performed.
FIG. 2B: lane 1 is a protein molecular weight standard in Kilodaltons (KD); lanes 2-5 show 2-fold gradient dilutions of harpinEa hypersensitive protein solution (44kD) from stock to 8-fold dilution after digestion of T4 phage particle suspension displaying harpinEa-TEV-Soc recombinant protein (about 54kD) with TEV protease; lanes 6-9 show 2-fold gradient dilutions of calf serum protein (BSA) from 2mg/ml to 0.125 mg/ml.
FIG. 3 shows the "red-looking" strawberry crop plant traits under different treatments, the left image being a control group, namely a real map of the strawberry crop plant traits in the clear water treatment area for dilution; the right panel is a physical picture of the agronomic characteristics of the strawberry plants in the test group, i.e. in the treated area where harpinEa protein was purified in the manner described above (applied by spraying at a final concentration of 50. mu.g/ml). The average plant height and the open disc degree of a treatment area of harpinEa protein (sprayed according to a final concentration of 50 mu g/ml) are respectively 8.5cm and 20.7cm, are respectively increased by 10.23 percent and 9.20 percent compared with a control, the leaf width and the leaf length are respectively 4.9cm and 6.4cm, are respectively increased by 11.4 percent and 10.3 percent compared with the control, and the average fresh weight of fruit setting and fruit setting is 733 and 1559g, is respectively increased by 9.5 percent and 12.2 percent compared with the control.
Detailed Description
Example 1 sequence acquisition of hypersensitivity protein (harpinEa)
The hrpN sequence of the gene encoding harpinEa protein derived from strain Erwinia amylovora has been published (GeneBank accession No.: M92994.3). The hypersensitive protein (harpinEa) gene can be obtained by the known technologies in the industries of gene synthesis, PCR amplification, chromosome DNA extraction, library establishment and the like. The present invention adopts gene synthesis process to synthesize amino acid sequence of hypersensitivity protein (hrapieea) (Suzhou Jinwei Zhi Biotech. of China).
Example 2 construction of pVB-harpinEa-TEV-Soc plasmid
The backbone of the plasmid was derived from the pUC18 plasmid. Ampicillin-resistant gene and plasmid replication region were amplified by PCR using the pUC18 plasmid as a template and KOD high fidelity DNA polymerase. The PCR forward primer was 18 bases in the upstream sequence of pUC18 ampicillin resistance gene, and the reverse primer was the 18 bases reverse complement of the downstream sequence of pUC18 plasmid replication. BglII restriction sites were added to the 5' ends of both the forward and reverse primers. Meanwhile, the T4 phage DNA is used as a template, and a specific primer is used for obtaining the Soc protein gene fragment through PCR amplification, wherein the 5' end of the forward primer is introduced with the gene sequence of TEV protease. And fusing the synthesized harpinEa protein gene segment and the TEV-Soc protein gene segment into a DNA molecule by using bypass PCR, wherein the coding region of the harpinEa protein is positioned at the upstream of the TEV-Soc fusion protein gene, and the expression frames are consistent. The upstream of the DNA sequence of the harpinEa-TEV-Soc fusion recombinant protein is introduced with Soc promoter sequence by PCR primer, the downstream is introduced with Soc terminator sequence, and BglII restriction sites are added at the 5' ends of the forward and reverse primers.
After the plasmid backbone DNA fragment obtained by the method and the gene fragment of the harpinEa-TEV-Soc fusion recombinant protein are treated by BglII restriction endonuclease, the plasmid backbone DNA fragment and the gene fragment of the harpinEa-TEV-Soc fusion recombinant protein are connected into circular DNA by T4 ligase and are transformed into escherichia coli DH5 α competent cells by a chemical transformation method, plasmids are extracted by cell culture and an alkaline lysis method, a T4 phage synchronous display plasmid pVB-harpinEa-TEV-Soc is obtained, a plasmid map is shown in figure 1, and the correctness of the plasmid sequence is confirmed by sequencing (Suzhou Jinzhi Biotech company).
Example 3 in vivo expression and phage display of fusion recombinant proteins
The T4 phage synchronous display expression plasmid pVB-harpinEa-TEV-Soc is transformed into competent cells of Escherichia coli strain P301 by a chemical transformation method, and is cultured in a shaker at 37 ℃ and a rotating speed of 200rpm under the pressure of ampicillin until the bacterial density reaches 3 x 108One per ml. Soc deleted with Soc protein-The T4 phage (assigned by professor Rao, university of Nature, university of Nature, USA) infected cultured E.coli cells with MOI (multiplicity of infection) 1 and continued to be cultured in a shaker at 37 ℃ for 30min at 200 rpm. In this process, harpinEa-TEV-Soc recombinant protein is expressed under the control of the T4 phage particle assembly pathway and displayed on the capsid.
After completion of the demonstration, the culture broth was immediately dispensed into pre-cooled centrifuge tubes and centrifuged at low temperature for 30min using 43000g of centrifugal force. Centrifuging, discarding the supernatant, and adding appropriate amount of the supernatantPI-Mg buffer (3.7g/L Na)2HPO4、4g/L NaCl、3g/L KH2PO4、1mMMgSO4) The pellet was resuspended thoroughly and DNase I was added to a final concentration of 10. mu.g/ml and several drops of chloroform, incubated at 37 ℃ for 30min at 200rpm and centrifuged again at 4300g to remove cell debris, and the resulting supernatant was the recombinant protein-displayed suspension of T4 phage particles, see FIG. 2A. After determination of the phage titer, the phage was stored at 4 ℃ and the titer test results are shown in Table 1.
TABLE 1 results of phage titer determination in triplicate experiments
Note: phage titer was tested for three experimental phage display experiments and triplicate replicates were performed each time. The results show that the plaque number is between 50 and 150pfu, and the experimental data show that the infection of the phage does not influence the growth of the host.
Example 4 isolation of Harpinea protein
The suspension of T4 phage particles displaying recombinant protein (suspension is PI-Mg buffer) was centrifuged at low temperature for 30min using 43000g of centrifugal force, the supernatant was removed, and then resuspended in an appropriate amount of TEV protease 1 × reaction buffer (50mM Tri-HClpH8.0,0.5mM EDTA,1mM DDT). harpinEa protein was isolated from the above T4 phage particles displaying harpinEa-TEV-Soc recombinant protein using TEV protease. The harpinEa protein has thermal stability, 1000U enzyme amount is selected for incubation for 6h at 30 ℃ after small-amount protein enzyme digestion efficiency test is carried out according to TEV protease specification, and then 43000g centrifugal force is used for low-temperature centrifugation for 30min, and the supernatant is harpinEa protein solution, which is shown in figure 2. The harpinEa protein solution was supplemented to 50% glycerol and stored at-20 ℃ for a long period.
Example 5 application and Effect of harpinEa protein on strawberry crops
Selecting a strawberry crop of which the variety is 'red-color' in a certain farmer greenhouse in an agricultural demonstration garden of Changfeng county, Anhui, and transplanting and planting the strawberry crop in 2017, 9 and 1 days, wherein the ridge height is 15cm, the ridge distance is 80cm, the ridge surface width is 45cm, 2 rows are arranged on each ridge, the row distance is 18cm, the plant spacing is 12cm, and the planting density is 10000 plants/mu. The soil basal fertility levels are shown in table 2.
TABLE 2 basic soil fertility level of "Red-skin" strawberry crops
Cell area 15m according to random block experimental design2. The harpinEa protein purified by the method (sprayed according to the final concentration of 50 mu g/ml) is designed to be used as an experimental group, and the diluted clear water is used as a control group to be respectively used for 2 groups of parallel groups. Spraying once when 8-11 leaves of strawberries emerge and 10% buds appear at the base, and spraying for 2 times after 15 days from the first spraying. The vegetative characters (plant height, disc opening degree, length, width and petiole of orthotopic leaves) of the strawberries are investigated 50 days after the first spraying is started, and the number of grey mould disease fruits, the number of fruit holes and the fresh weight of the fruit holes of the strawberries are investigated 55 days, 60 days, 65 days and 70 days after the first spraying is started. The experiment was repeated 3 times. Data were counted and analyzed using SPSS 17.0. See table 3 and fig. 3 for results.
TABLE 3 phytology of "Red-look" strawberry crops under different treatments
The influence of the hypersensitivity protein on the physiological properties of fruits of the strawberry crops with the red color is analyzed through indoor experimental research, the sugar degree of the strawberry fruits sprayed with the HarpinEa hypersensitivity protein is improved by 21.6 percent, and the increasing proportion of the content of the vitamin C is 18.95 percent, as shown in table 4.
TABLE 4 fruit physiological indices of strawberry crop for "Red color" under different treatments (content per 100 g)
Test results show that the greenhouse strawberries can be sprayed with the hypersensitivity protein for 2 times in the bud period, so that the quality of the strawberries can be improved. Promoting plant growth, improving disease resistance, and preventing gray mold of strawberry by over 59.38%. Meanwhile, the HarpinEa hypersensitive protein is nontoxic and pollution-free, is simple and easy to spray, is easy to accept by farmers and is convenient to popularize.
Finally, it should be noted that the above examples are only used to help those skilled in the art understand the essence of the present invention, and should not be used to limit the protection scope of the present invention.
Claims (10)
1. A method of using a T4 phage display to express a hypersensitive protein, said method comprising the steps of:
(1) sequentially connecting the encoding gene of the hypersensitive protein with TEV protease and T4 phage Soc protein gene fragments to prepare a fusion recombinant protein encoding gene, wherein the encoding region of the hypersensitive protein is positioned at the upstream of the TEV-Soc fusion protein gene and has consistent expression frames;
(2) transferring the coding gene of the fusion recombinant protein into a pUC18 plasmid;
(3) transferring the plasmid into an escherichia coli host;
(4) use of Soc protein-deleted Soc-Infecting said E.coli host with a T4 bacteriophage, culturing the host and expressing the T4 bacteriophage displaying said fusion recombinant protein;
(5) isolating the T4 phage displaying the fusion recombinant protein;
(6) separating the fusion recombinant protein from the T4 bacteriophage, and obtaining the hypersensitive protein after TEV protease cleavage.
2. The method according to claim 1, wherein the hypersensitive protein is harpinEa protein from the strain erwinia amylovora in GeneBank accession number M92994.3.
3. The method according to claim 1 or 2, wherein a Soc promoter sequence is introduced upstream and a Soc terminator sequence is introduced downstream of the DNA sequence encoding the fusion recombinant protein of step (1).
4. The method according to claim 1 or 2, wherein the method for transforming the plasmid into the E.coli host in step (3) is a chemical transformation method.
5. The method according to claim 1 or 2, wherein the infection in step (4) is an infection of cultured E.coli cells with a phage concentration of MOI (multiplicity of infection) ═ 1.
6. The method according to claim 1 or 2, wherein the method for isolating the T4 phage displaying the fusion recombinant protein in step (5) is as follows:
1) after precooling the culture solution, centrifuging the culture solution at a low temperature for 20 to 40min by using a centrifugal force of 40000g to 50000g, and removing the supernatant;
2) fully resuspending the precipitate with an appropriate amount of buffer solution, adding DNase I and a plurality of drops of chloroform, and continuously culturing at 37 ℃ for 0.5-1 hour;
3) cell debris was removed by centrifugation at 4000-6000g and the resulting supernatant was the suspension of T4 phage particles displaying recombinant protein.
7. The method according to claim 6, wherein the steps 1) and 2) are specifically:
1) precooling the culture solution, centrifuging the culture solution at a low temperature for 30min by using 43000g of centrifugal force, and removing the supernatant;
2) adding a proper amount of PI-Mg (3.7g/L Na)2HPO4、4g/L NaCl、3g/L KH2PO4、1mM MgSO4) The buffer was thoroughly resuspended and the pellet was incubated at 37 ℃ for 30min at 200rpm with the addition of DNase I to a final concentration of 10. mu.g/ml and several drops of chloroform.
8. The method according to claim 1 or 2, wherein the method for isolating the fusion recombinant protein of step (6) is: centrifuging the T4 phage particle suspension displaying the recombinant protein for 30min at 40000 g-50000 g at low temperature, removing the supernatant, and suspending the precipitate with TEV enzyme digestion buffer (50mM Tris-HCl pH8.0,0.5mM EDTA,1mM DTT);
the method for obtaining the hypersensitive protein by TEV protein enzyme digestion in the step (6) comprises the following steps: incubating for 6h at 30 ℃ with 1000U of enzyme for enzyme digestion treatment, and centrifuging for 30min at the low temperature of 40000-50000 g to obtain supernatant which is the hypersensitive protein solution.
9. Use of a method according to any one of claims 1 to 8 in the preparation of a plant growth promoting formulation and/or a plant insect resistant formulation.
10. Use according to claim 9, wherein the plant is a rosaceous plant.
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| US20060029615A1 (en) * | 2004-08-04 | 2006-02-09 | Ren Zhao-Jun | Novel recombinant T4 phage particle and uses thereof |
| CN103074359A (en) * | 2013-01-29 | 2013-05-01 | 连云港脂立方生物医药研究所有限公司 | Method for showing foreign protein macromolecule on surface of T4 bacteriophage by using intracellular synchronous expression method |
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