CN113121672A - Soluble prokaryotic expression and purification method of cat interferon gamma and application - Google Patents
Soluble prokaryotic expression and purification method of cat interferon gamma and application Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
The invention provides a soluble prokaryotic expression and purification method of cat interferon gamma and application thereof, belonging to the field of biological genetic engineering. The method comprises the following steps: analyzing a signal peptide sequence and physicochemical properties of FeIFN-gamma protein; constructing a prokaryotic expression plasmid pET28 a-SUMO-FeIFN-gamma; performing induced expression and recombinant protein solubility analysis on BL21 bacteria containing recombinant plasmids; screening the optimal expression condition of SUMO-FeIFN-gamma fusion protein; carrying out mass expression and purification on the SUMO-FeIFN-gamma fusion protein; sixthly, carrying out enzyme digestion and purification on SUMO-FeIFN-gamma fusion protein. The invention clones mature protein gene sequence without signal peptide, constructs prokaryotic expression plasmid, converts BL21 competent cell to express and purify, uses pET28a-SUMO expression vector with SUMO solubilizing-aid label, thereby realizing soluble expression of target protein, having high soluble expression level of foreign protein, having very high biological activity, simple and convenient purification steps, and laying foundation for development of later antiviral drugs.
Description
Technical Field
The invention belongs to the field of biological gene engineering, and particularly relates to a soluble prokaryotic expression and purification method of cat interferon gamma and application thereof.
Background
Interferon (IFN) is a glycoprotein produced by immune cells of the body and secreted by the cells after an inducer acts on living cells, and has various biological activities such as antiviral activity, antitumor activity, immunoregulation activity and the like. Interferons are classified into type I and type II according to their origin, biological properties and activity. Type I interferons have mainly antiviral and antitumor effects, including IFN-alpha, IFN-beta, IFN-omega, IFN-epsilon, IFN-kappa, IFN-tau, IFN-delta and IFN-zeta, and type II interferons, of which only the IFN-gamma subtype, play a key role in host defense against intracellular pathogens. In 1992, Nakamura et al, Tokory, Japan, first isolated a feline interferon gene and classified it as an omega-type interferon, and studies showed that IFN-omega has a significant therapeutic effect on feline co-infected with Feline Leukemia Virus (FLV) and Feline Immunodeficiency Virus (FIV) and parvovirus, and then demonstrated that IFN-omega has an inhibitory effect on FIV replication and is dose-dependent. Because of its broad antiviral ability and significant efficacy, it is of great importance to study antiviral mechanisms of feline IFNs and to practice treatment of viral diseases in cats. IFN-gamma is taken as a novel interferon, plays a strong role in the aspects of antivirus and antitumor, and in 1995, chicken IFN-gamma genes are successfully cloned for the first time, and then IFN-gamma of animals such as recombinant geese, rabbits, pigs, cows and the like is successfully expressed and used for clinical treatment, but research on IFN-gamma of cats is rarely reported.
In recent years, the living standard of people is rapidly improved, the pet raising industry is promoted to develop vigorously, and diseases related to cats, particularly viral diseases, increase year by year. The interferon serving as an important epidemic prevention system member of an organism can resist virus infection, and the interferon expressed in vitro has highly similar biological activity with natural interferon. Therefore, the in vitro expressed cat interferon is expected to enhance the immunity of pet cats and reduce the morbidity and mortality.
Disclosure of Invention
The invention aims to provide a soluble prokaryotic expression and purification method of cat interferon gamma and application thereof, and the cat interferon gamma prepared by the method has good biological activity and can be used for treating cat virus infectious diseases.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a soluble prokaryotic expression and purification method of cat interferon gamma comprises the following steps:
(1) signal peptide sequence and physicochemical property analysis of FeIFN-gamma protein
Respectively predicting and analyzing the signal peptide segment and the physicochemical property of the two proteins by a SignalP-4.0Server and an Expasy online Server;
(2) construction of prokaryotic expression plasmid pET28 a-SUMO-FeIFN-gamma
According to FeIFN-gamma sequences recorded in GenBank, signal peptides are abandoned and then PCR primers are designed, the upstream primer sequence is shown as SEQ ID No.1, the downstream primer sequence is shown as SEQ ID No.2, BamH I and Hind III enzyme cutting sites are respectively introduced through the primers for PCR amplification, then a PCR product is subjected to double enzyme cutting and then a target gene fragment is recovered, meanwhile, pET28a-SUMO vector fragments are subjected to double enzyme cutting and recovered, and are connected through T4DNA ligase and then transferred into DH-5 alpha competent cells, extracted plasmids are subjected to PCR and double enzyme cutting verification and then sequenced, and the extracted plasmids are named as pET28 a-SUMO-FeIFN-gamma after the sequencing is correct;
(3) induced expression of BL21 bacteria containing recombinant plasmid and solubility analysis of recombinant protein
Transferring the recombinant plasmid pET28 a-SUMO-FeIFN-gamma into BL21 competent cells, picking a monoclonal colony, and inoculating the colony containing the monoclonal colonyCulturing overnight in LB culture solution with kanamycin resistance, transferring into new LB culture solution with kanamycin resistance at a ratio of 1:100 the next day, shake culturing for 2h at 37 ℃ and 220r/min in a shaking table until bacterial liquid OD600Taking 1mL of bacterial liquid as a reference, adding IPTG inducer into the rest of bacterial liquid with the final concentration of 1mmol/L, carrying out shake cultivation at 16 ℃ and 220r/min for 10h, centrifuging at 8000rpm for 10min, collecting thalli, discarding thalli sediment after supernatant, adding binding buffer, repeatedly blowing and beating by using a pipette to enable thalli to be resuspended and adding PMSF, wherein the final concentration is 1mmol/L, carrying out ultrasonic crushing on the resuspended bacterial liquid, carrying out operation on the whole process on ice, carrying out ultrasonic treatment for 3s, pausing for 4s and carrying out power of 220W, taking clear and transparent bacterial liquid as a reference, centrifuging the bacterial liquid at 10000rpm for 10min after ultrasonic treatment, collecting supernatant and sediment to prepare samples, and detecting and analyzing the expression level and water solubility of protein by SDS-PAGE electrophoresis;
(4) screening of SUMO-FeIFN-gamma fusion protein optimal expression conditions
In a culture flask, the ratio of 1:100 adding bacterial liquid and LB culture liquid containing kanamycin resistance, shaking and culturing at 37 ℃ and 220r/min in a shaking table, and obtaining OD of bacterial liquid600When the value reaches 0.6-0.8, performing condition screening on each culture bottle according to the IP TG concentration gradient, the induction temperature gradient and the time gradient, and detecting by SDS-PAGE electrophoresis; the IPTG concentration gradient is 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L and 1.0mm ol/L, the induction temperature gradient is 37 ℃, 30 ℃, 22 ℃ and 16 ℃, and the time gradient is 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24 h;
(5) large-scale expression and purification of SUMO-FeIFN-gamma fusion protein
Performing amplification culture after the optimal expression condition is determined, filtering induced and ultrasonically treated supernatant by using a filter membrane with the aperture of 0.45 mu m, purifying, washing a Ni affinity chromatographic column by using distilled water, balancing for many times by using a binding buffer solution, adding the filtrate into the chromatographic column, then placing the chromatographic column at room temperature for binding for 2 hours, and collecting flow-through liquid at the flow rate of 0.5-1 mL/min; washing the liquid of the chromatographic column with a washing buffer solution at a flow rate of 1mL/min to clean the Ni column, and removing impure proteins by using the amount which is 8-10 times of the volume of the column; finally eluting the protein on the chromatographic column by using an elution buffer solution with the volume of 5-10 times of the column volume at the flow rate of 0.5-1 mL/min, placing the collected samples on ice, and finally detecting by using SDS-PAGE electrophoresis;
(6) enzyme digestion and purification of SUMO-FeIFN-gamma fusion protein
The Sumo protease was purified at a ratio of 1: adding the fusion protein solution according to the proportion of 10, carrying out enzyme digestion at the temperature of 4 ℃, collecting enzyme digestion products at different time points of 3h, 6h, 9h and 12h respectively, desalting, concentrating and further purifying the protein after enzyme digestion by using a 30ku interception column and a 10ku interception column, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection, and determining the concentration of the target protein by using a Bradford assay method.
Further, the binding buffer is 50mM Na2HPO4300mM NaCl, 10mM imidazole, pH 8.0.
Furthermore, the optimal conditions for the induction expression of the SUMO-FeIFN-gamma fusion protein are that the concentration of IPTG is 0.8mmol/L, the temperature is 16 ℃, and the time is 9 h.
Further, in the step (6), when a 30ku entrapment column and a 10ku entrapment column are used, firstly, uniformly mixing the enzyme-digested protein and the exchange Buffer, adding 3mL of protein into the 30ku entrapment column, centrifuging for 10min at 4 ℃ and 3000r/min by using a horizontal centrifuge, adding 3mL of exchange Buffer into the entrapment column, concentrating to 200-500 mu L, repeating for multiple times, collecting flow-through liquid for each time, sequentially adding the flow-through liquid into the 10ku entrapment column, adding 3mL of flow-through liquid for each time until all the flow-through liquid is added, and finally trapping the residual volume of 500 mu L to obtain the target protein without the SUMO label; the exchange Buffer is 50mM Na2HPO4300mM NaCl, pH8.0, and ultrafiltered with a 0.22 μm ultrafiltration membrane.
The invention also provides a cat interferon gamma which is prepared by the method.
The invention also provides application of the feline interferon gamma in preparing a medicament for treating feline parvovirus infection diseases.
The beneficial technical effects of the invention are as follows:
(1) the method clones a mature protein gene sequence without signal peptide, constructs a prokaryotic expression plasmid, and converts BL21 competent cells for expression and purification; the escherichia coli expression system has the advantages of high expression level, simplicity and convenience in operation, easiness in culture and control and the like, but in the expression process, the target protein is easy to form an inclusion body, and the functional protein with non-natural conformation needs refolding and renaturation to obtain the original biological activity, so that correct soluble protein can be directly folded needs to be expressed, and the invention uses a pET28a-SUMO expression vector with a SUMO hydrotropy tag, thereby realizing the soluble expression of the target protein, wherein the expression vector is obtained by modifying pET28a to fuse an SUMO tag after an N-end His tag, so that the soluble expression level of the foreign protein can be improved, the correct folding of the foreign protein is promoted, the biological activity is achieved, and the purification step is simpler and more convenient.
(2) The invention also optimizes the SUMO-FeIFN-gamma fusion protein induction expression conditions, when the induction expression conditions are IPTG final concentration of 0.8mmol/L, temperature of 16 ℃ and time of 9h, the expression quantity is highest, and the soluble expression of the fusion protein is improved by methods of reducing the induction temperature, controlling the IPTG concentration, selecting proper carriers and expression host bacteria, optimizing fusion tags and the like.
(3) The invention realizes the soluble expression of the SUMO-FeIFN-gamma fusion protein, uses SUMO protease to perform enzyme digestion for 6h at 4 ℃ after being purified by a nickel column to remove the SUMO label, and obtains FeIFN-gamma without the label after being purified by two times of trapping columns, thereby laying a foundation for the development of later antiviral drugs.
Drawings
FIG. 1 is a diagram showing an analysis of a signal peptide of a FeIFN-. gamma.protein according to an embodiment of the present invention;
FIG. 2 is an electrophoretic analysis chart of the PCR amplification product of the FeIFN-gamma gene according to an embodiment of the present invention, wherein M represents a DNA standard DL5000, and 1 represents a FeIFN-gamma negative control; 2 represents a FeIFN-gamma gene;
FIG. 3 shows the result of double digestion of pET28 a-SUMO-FeIFN-y, where M represents DNA standard DL5000, 1 represents recombinant plasmid pET28 a-SUMO-FeIFN-y, and 2 represents pET28 a-SUMO-FeIFN-y;
FIG. 4 is an SDS-PAGE analysis chart of pET28 a-SUMO-FeIFN-gamma inducible expression product, wherein M represents a protein molecular mass standard, and 1-4 represent a recombinant bacteria pre-induced product, a recombinant bacteria post-induced product, a recombinant bacteria induced product supernatant and a recombinant bacteria induced product precipitate, respectively;
FIG. 5 is an SDS-PAGE electrophoretic analysis of pET28 a-SUMO-FeIFN-. gamma.induced expression products at different IPTG concentrations, wherein M represents a protein molecular mass standard, and 1-9 represent recombinant bacterial products induced by IPTG concentrations of 0mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L, 1.4mmol/L and 1.6mmol/L, respectively;
FIG. 6 is an SDS-PAGE analysis of pET28 a-SUMO-FeIFN-. gamma.induced expression products at different temperatures according to an embodiment of the present invention, wherein M represents a protein molecular mass standard, and 1-9 represent recombinant bacterial products induced at 37 deg.C, 30 deg.C, 22 deg.C, and 16 deg.C, respectively;
FIG. 7 is an SDS-PAGE electrophoretic analysis chart of pET28 a-SUMO-FeIFN-gamma induced expression products at different times according to an embodiment of the present invention, wherein M represents a protein molecular mass standard, and 1-9 represent recombinant bacterial products induced at different time points of 3h, 6h, 9h, 12h, 15h, 18h, 21h, and 24h, respectively;
FIG. 8 shows the result of the purification of SUMO-FeIFN-y fusion protein according to an embodiment of the present invention, wherein M represents the protein molecular mass standard, and 1-3 represent the purified SUMO-FeIFN-y protein;
fig. 9 is a result of enzyme digestion analysis of the SUMO-FeIFN- γ fusion protein according to an embodiment of the present invention, in which M represents a protein molecular mass standard, 1 represents SUMO protease, 2-5 represent enzyme digestion of SUMO-FeIFN- γ fusion protein for 3h, 6h, 9h, and 12h, respectively, and 6 represents before enzyme digestion of SUMO-FeIFN- γ fusion protein;
fig. 10 is a purification result of the SUMO-FeIFN- γ fusion protein after enzyme digestion according to an embodiment of the present invention, in which M represents a protein molecular mass standard, 1-5 represents a 30ku cut-off column retentate of the SUMO-FeIFN- γ fusion protein after concentration, 6h of the SUMO-FeIFN- γ fusion protein after enzyme digestion, a 30ku cut-off column retentate of the SUMO-FeIFN- γ fusion protein, a 30ku cut-off column flow-through liquid of the SUMO-FeIFN- γ fusion protein, and a 10ku cut-off column retentate of the FeIFN- γ protein.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Detailed description of the preferred embodiment 1
1. Materials and reagents
1.1 strains, plasmids and Primary reagents
Competent cells DH5 alpha, BL21 were purchased from Biotech, Inc., of Kyoto Ongzhi, Ltd; t4DNA ligase, IPTG (isopropylthio-. beta. -D-galactoside), and kanamycin were purchased from TaKaRa; restriction enzymes BamHI, HindIII were purchased from New England BioLabs; PMSF (phenylmethylsulfonyl fluoride), SUMO protease from Solebao scientific Co., Ltd, Beijing; Ni-NTA purification column, protein concentration column, and retention column were purchased from Merck.
2. Soluble prokaryotic expression and purification method of cat interferon gamma
2.1 analysis of the Signal peptide sequence and physicochemical Properties of the FeIFN- γ protein
Respectively predicting and analyzing the signal peptide segment and the physicochemical property of the two proteins by a SignalP-4.0Server and an Expasy online Server, wherein,
SignalP-4.0Server site: http:// www.cbs.dtu.dk/services/SignalP/, Expasy website: https:// web. expasy. org/protparam/.
2.2 construction of prokaryotic expression plasmid pET28 a-SUMO-FeIFN-. gamma.
According to FeIFN-gamma (GenBank No. NM-001009873.1) sequences recorded by GenBank, a PCR primer is designed after signal peptides are abandoned, the FeIFN-gamma upstream primer sequence is shown as SEQ ID No.1, the FeIFN-gamma downstream primer sequence is shown as SEQ ID No.2, and BamH I and HindIII enzyme cutting sites are respectively introduced through the primers, and PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 25s, annealing at 56 ℃ for 25s, and extension at 72 ℃ for 35s for 32 cycles; and (2) extending for 10min at 72 ℃, carrying out double enzyme digestion on the PCR amplification product by BamHI and HindIII, recovering a target gene fragment, carrying out double enzyme digestion, recovering a pET28a-SUMO vector fragment, connecting the vector fragment by T4DNA ligase, transferring the vector fragment into a DH-5 alpha competent cell, carrying out PCR and double enzyme digestion verification on the extracted plasmid, then carrying out sequencing, and naming the extracted plasmid as pET28 a-SUMO-FeIFN-gamma after the sequencing is correct.
2.3 inducible expression of BL21 bacterium containing recombinant plasmid and solubility analysis of recombinant protein
Transferring the recombinant plasmid pET28 a-SUMO-FeIFN-gamma into BL21 competent cells, selecting a monoclonal colony, inoculating the colony in LB culture solution containing kanamycin resistance for overnight culture, transferring the colony into new LB culture solution containing kanamycin resistance at a ratio of 1:100 the next day, performing shake culture at 37 ℃ and 220r/min for 2 hours in a shaking table, and waiting for bacterial liquid OD600And (3) taking 1mL of bacterial liquid as a reference, adding IPTG inducer into the residual bacterial liquid with the final concentration of 1mmol/L, carrying out shake culture at 16 ℃ and 220r/min for 10h, centrifuging at 8000rpm for 10min, collecting thalli, discarding thalli sediment after supernatant, adding binding buffer, repeatedly blowing and beating by using a pipette to enable the thalli to be resuspended and adding PMSF with the final concentration of 1mmol/L, carrying out ultrasonic crushing on the resuspended bacterial liquid, carrying out operation on the whole process on ice, carrying out ultrasonic treatment for 3s, pausing for 4s and carrying out power 220W, taking clear and transparent bacterial liquid as a reference, centrifuging the bacterial liquid at 10000rpm for 10min after ultrasonic treatment, collecting supernatant and sediment to prepare a sample, and detecting and analyzing the expression level and water solubility of protein by SDS-PAGE electrophoresis.
2.4 screening of the optimal expression conditions for SUMO-FeIFN- γ fusion proteins
In a culture flask, the ratio of 1:100 adding bacteria solution and K+The LB culture solution is shake-cultured at 37 ℃ and 220r/min in a shaking table, and the OD of the strain solution is obtained600When the value reaches 0.6-0.8, screening each culture bottle according to the conditions of IPTG concentration gradient of 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L and 1.0mmol/L, induction temperature of 37 ℃, 30 ℃, 22 ℃ and 16 ℃ and time gradient of 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h respectively, and detecting the expression level of the fusion protein by SDS-PAGE electrophoresis.
2.5 Mass expression and purification of SUMO-FeIFN-gamma fusion proteins
Performing amplification culture after the optimal expression condition is determined, filtering induced and ultrasonically treated supernatant by using a filter membrane with the aperture of 0.45 mu m, purifying, washing a Ni affinity chromatographic column by using distilled water, balancing for many times by using a binding buffer solution, adding the filtrate into the chromatographic column, then placing the chromatographic column at room temperature for binding for 2 hours, and collecting flow-through liquid at the flow rate of 0.5-1 mL/min; washing the liquid of the chromatographic column with a washing buffer solution at a flow rate of 1mL/min to clean the Ni column, and removing impure proteins by using the amount which is 8-10 times of the volume of the column; and finally eluting the protein on the chromatographic column by using an elution buffer solution with the volume of 5-10 times of the column volume at the flow rate of 0.5-1 mL/min, placing all collected samples on ice, and finally detecting the collected samples by using SDS-PAGE electrophoresis.
2.6 enzyme digestion and purification of SUMO-FeIFN-gamma fusion protein
The Sumo protease was purified at a ratio of 1: adding the fusion protein solution in a proportion of 10, carrying out enzyme digestion at the temperature of 4 ℃, respectively collecting enzyme digestion products of 3h, 6h, 9h and 12h at different time points, and desalting, concentrating and purifying the two enzyme digested proteins by using a 30ku interception column and a 10ku interception column; firstly, uniformly mixing enzyme-digested protein and exchange Buffer, adding 3mL of protein into a 30ku interception column, centrifuging for 10min at 4 ℃ by using a horizontal centrifuge at 3000r/min, adding the 3mL of exchange Buffer into the interception column, concentrating to 200-500 mu L, repeating for multiple times, collecting flow-through liquid for each time, sequentially adding the flow-through liquid into the 10ku interception column, adding 3mL of flow-through liquid for each time until all the flow-through liquid is added, and finally intercepting the residual volume of 500 mu L to obtain target protein without SUMO labels, carrying out SDS-PAGE (sodium dodecyl sulfate electrophoresis) detection, and determining the concentration of the target protein by using a Bradford assay method; the exchange Buffer contains 50mM Na2HPO4300mM NaCl, pH8.0, and ultrafiltered through a 0.22 μm ultrafiltration membrane.
3. Results and analysis
3.1 FeIFN-gamma protein Signal peptide analysis
The signal peptide of FeIFN-gamma protein was analyzed by SignalP-4.0Server on-line Server, see FIG. 1.
The results show that: the signal peptide sequence of the FeIFN-gamma protein is the first 20 amino acids and belongs to secretory proteins, and the 20 th amino acid and the 21 st amino acid are cleavage sites of the signal peptide.
3.2 prediction analysis of the physicochemical Properties of the FeIFN-. gamma.protein
The physicochemical properties of the FeIFN-gamma protein were analyzed using a Protparam on-line server.
The results show that: the FeIFN-gamma protein contains 167 amino acids, has the molecular weight of 19.6ku, has the signal peptide removal rate of 17.3ku, the isoelectric point of 9.1 and the average total hydrophilicity value of-0.483.
3.3 PCR amplification results
The PCR amplification product of the FeIFN-. gamma.gene was detected by 1% agarose gel electrophoresis, see FIG. 2.
The results show that: the size of the obtained specific target band is about 435bp, and the result is consistent with the expectation.
3.4 double restriction enzyme identification of recombinant plasmid pET28 a-SUMO-FeIFN-gamma
After double digestion with BamHI/HindIII for 2h, the DNA was detected by electrophoresis on a 1% agarose gel, see FIG. 3.
The results show that: the recombinant plasmid pET28 a-SUMO-FeIFN-. gamma.appeared as a band at about 5633bp and 435bp, respectively, and the result was in accordance with the expectation.
3.5 induced expression and solubility analysis results of recombinant plasmid pET28 a-SUMO-FeIFN-gamma
Transferring the constructed pET28 a-SUMO-FeIFN-gamma recombinant plasmid into BL21 competent cells for induction expression, carrying out ultrasonic lysis on the thalli, enabling the bacterial liquid to become clear and transparent when the ultrasonic duration is 20min, respectively taking protein supernatant and sediment for sample preparation, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detection, wherein the reference is shown in figure 4.
The results show that: the recombinant bacteria have obvious expression bands after induction, the size of the expression bands is about 34ku, and the bands in the supernatant are obviously more than those in the precipitate, which indicates that the fusion protein is expressed in the escherichia coli in a soluble form.
3.6 optimization results of induced expression conditions for SUMO-FeIFN-gamma fusion protein
In order to optimize the expression condition of the fusion protein, experimental groups with different concentrations of the inducer, induction time and temperature were set, after induction, the cells were collected and prepared, and the expression level of the fusion protein was detected by SDS-PAGE electrophoresis, see FIGS. 5-7.
The results show that: when the final concentration of IPTG is 0.8mmol/L, the fusion protein obtains higher expression quantity; when the IPTG with the optimal concentration is used, the expression level of the fusion protein induced at 16 ℃ is obviously higher than that of the fusion protein induced at other temperatures; similarly, under the induction of 16 ℃, the expression level of the fusion protein reaches the highest level when the expression level is 9h, and then the expression level does not increase obviously along with the increment of time, so that the optimal conditions of the expression level are IPTG0.8mmol/L and the induction of 16 ℃ for 9 h.
3.7 purification results of SUMO-FeIFN-gamma fusion protein
Expressing SUMO-FeIFN-gamma fusion protein in large amount under optimal expression condition, centrifuging, resuspending, performing ice bath ultrasonication, collecting supernatant, purifying protein with nickel column, and detecting with SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) as shown in FIG. 8.
The results show that: the fusion protein eluted showed a clear single band at 34 ku.
3.8 enzyme digestion analysis and purification results of SUMO-FeIFN-gamma fusion protein
The purified fusion protein was cleaved with SUMO protease, see fig. 9-10.
The results show that: after enzyme digestion is carried out for 6h at 4 ℃, the amount of the tag-removed protein does not increase obviously along with the time extension, so the enzyme digestion is carried out for 6h, the purified protein after the enzyme digestion is the FeIFN-gamma protein without the SUMO tag, SDS-PAGE detection shows that the target protein with higher purity and less impurity protein is obtained, and the size of the protein band after the SUMO tag is removed is about 17ku, which is consistent with the expected result. The FeIFN-. gamma.protein content was 0.85mg/mL using the Bradford assay with BSA as the standard protein.
Specific example 2 feline interferon gamma antiviral activity, safety and clinical applications
1. Determination of antiviral Activity
The antiviral activity of the feline interferon gamma obtained by the invention is measured by taking feline parvovirus as a model virus and adopting a trace cytopathic inhibition method.
Cat kidney cells F81 were seeded in 96-well cell plates and incubated at 37 ℃ with 5% CO2Culturing in an incubator until cell monolayer, adding 2-fold diluted cat interferon gamma for 12h, and adding 100TCID per well50The virus solution of (1) is provided with a negative control group and a known cat interferon omega 2 positive control group at the same time, and the cytopathic condition is observed after 24 hours, and the result is shown in table 1.
Table 1 results of recombinant feline interferon gamma antiviral activity
Note: (+) indicates no cytopathy (i.e., antiviral activity), and (-) indicates cytopathy (i.e., no antiviral activity).
The results show that: the cat interferon gamma has good antiviral activity, the effective concentration of the cat parvovirus resistance is 1.20mg/mL and is slightly lower than the known cat omega 2 interferon (0.80mg/mL), and good application potential is shown in the aspect of the cat parvovirus resistance.
2. Safety test
40 cats from 6 months to 1 year old are injected with 1.5mg/kg cat interferon gamma per vein, and the male and female animals are half.
And (5) counting results: the 27 cats slightly sleeps, the symptoms disappear after 8-12 hours, and the body temperature of 31 cats is slightly increased, but the electrolyte index in the body fluid is not abnormal within the normal range.
3. Clinical trial effect
30 cats with artificially infected feline parvovirus are divided into an administration group and a control group, wherein each group comprises 15 cats, 1.5mg/kg of feline interferon gamma is intravenously instilled in the administration group every day, and the control group is injected with physiological saline placebo for three days continuously.
The results show that: the feline interferon gamma can effectively treat feline parvovirus infection, and the illness state of the sick cats is obviously improved, which is specifically shown in that the number of leucocytes is increased to be normal or close to be normal, and the fever symptom disappears.
Claims (6)
1. A soluble prokaryotic expression and purification method of cat interferon gamma is characterized by comprising the following steps:
(1) signal peptide sequence and physicochemical property analysis of FeIFN-gamma protein
Respectively predicting and analyzing the signal peptide segment and the physicochemical property of the two proteins by a SignalP-4.0Server and an Expasy online Server;
(2) construction of prokaryotic expression plasmid pET28 a-SUMO-FeIFN-gamma
According to FeIFN-gamma sequences recorded in GenBank, signal peptides are abandoned and then PCR primers are designed, the upstream primer sequence is shown as SEQ ID No.1, the downstream primer sequence is shown as SEQ ID No.2, BamH I and Hind III enzyme cutting sites are respectively introduced through the primers for PCR amplification, then a PCR product is subjected to double enzyme cutting and then a target gene fragment is recovered, meanwhile, pET28a-SUMO vector fragments are subjected to double enzyme cutting and recovered, and are connected through T4DNA ligase and then transferred into DH-5 alpha competent cells, extracted plasmids are subjected to PCR and double enzyme cutting verification and then sequenced, and the extracted plasmids are named as pET28 a-SUMO-FeIFN-gamma after the sequencing is correct;
(3) induced expression of BL21 bacteria containing recombinant plasmid and solubility analysis of recombinant protein
Transferring the recombinant plasmid pET28 a-SUMO-FeIFN-gamma into BL21 competent cells, selecting a monoclonal colony, inoculating the colony in LB culture solution containing kanamycin resistance for overnight culture, transferring the colony into new LB culture solution containing kanamycin resistance at a ratio of 1:100 the next day, performing shake culture at 37 ℃ and 220r/min for 2 hours in a shaking table, and waiting for bacterial liquid OD600Taking 1mL of bacterial liquid as a reference, adding IPTG inducer into the rest of bacterial liquid with the final concentration of 1mmol/L, carrying out shake culture at 16 ℃ and 220r/min for 10h, centrifuging at 8000rpm for 10min, collecting thalli, removing thalli sediment after supernatant, adding binding buffer, repeatedly blowing and beating the thalli by a pipette, resuspending and adding PMSF, with the final concentration of 1mmol/L, carrying out ultrasonic crushing on the resuspended bacterial liquid, carrying out operation on the whole process on ice, carrying out ultrasonic treatment for 3s, pausing for 4s and carrying out power 220W, taking the clear and transparent bacterial liquid as a reference, centrifuging the bacterial liquid at 10000rpm for 10min after the ultrasonic treatment is finished, collecting supernatant and sediment to prepare a sample, and detecting and analyzing the expression level and the water solubility of protein by SDS-PAGE electrophoresis;
(4) screening of SUMO-FeIFN-gamma fusion protein optimal expression conditions
In a culture flask, the ratio of 1:100 adding bacterial liquid and LB culture liquid containing kanamycin resistance, shaking and culturing at 37 ℃ and 220r/min in a shaking table, and obtaining OD of bacterial liquid600When the value reaches 0.6-0.8, screening each culture bottle according to IPTG concentration gradient, induction temperature gradient and time gradient, and detecting SUMO-FeIFN-gamma fusion by SDS-PAGE electrophoresisThe level of expression of the complex protein;
(5) large-scale expression and purification of SUMO-FeIFN-gamma fusion protein
Performing enlarged culture after the optimal expression condition is determined, filtering induced and ultrasonically treated supernatant by using a filter membrane for purification, washing a Ni affinity chromatographic column by using distilled water, balancing for multiple times by using a binding buffer solution, adding the filtrate into the chromatographic column, then placing the chromatographic column at room temperature for binding for 2 hours, and collecting flow-through liquid at the flow rate of 0.5-1 mL/min; washing the liquid of the chromatographic column with a washing buffer solution at the flow rate of 1mL/min to remove impure proteins by using the amount which is 8-10 times of the volume of the column; finally eluting the protein on the chromatographic column by using an elution buffer solution with the volume of 5-10 times of the column volume at the flow rate of 0.5-1 mL/min, placing the collected samples on ice, and finally detecting by using SDS-PAGE electrophoresis;
(6) enzyme digestion and purification of SUMO-FeIFN-gamma fusion protein
The Sumo protease was purified at a ratio of 1: adding the eluted fusion protein liquid in a proportion of 10, carrying out enzyme digestion at the temperature of 4 ℃, respectively collecting enzyme digestion products in different time periods, desalting, concentrating and further purifying the enzyme digested protein by using a 30ku interception column and a 10ku interception column, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detection, and determining the concentration of the target protein by using a Bradford assay method.
2. The method for the soluble prokaryotic expression and purification of feline interferon gamma according to claim 1 characterized in that: the binding buffer is 50mM Na2HPO4300mM NaCl, 10mM imidazole, pH 8.0.
3. The method for the soluble prokaryotic expression and purification of feline interferon gamma according to claim 1 characterized in that: the SUMO-FeIFN-gamma fusion protein is induced and expressed under the conditions of IPTG concentration of 0.8mmol/L, temperature of 16 ℃ and time of 9 h.
4. The method for the soluble prokaryotic expression and purification of feline interferon gamma according to claim 1 characterized in that: in the step (6), when a 30ku entrapment column and a 10ku entrapment column are used, firstly, the enzyme-digested protein and the exchange Buffer are mixed uniformly, 3mL of protein is added into the 30ku entrapment column, and water is usedCentrifuging for 10min at 4 ℃ by using a flat centrifuge at 3000r/min, adding 3mL of exchange Buffer into an interception column, concentrating to 200-500 mu L, repeating for multiple times, collecting flow-through liquid for each time, sequentially adding the flow-through liquid into the interception column of 10ku, 3mL for each time until all the flow-through liquid is added, and finally intercepting the residual volume of 500 mu L to obtain the target protein without the SUMO label; the exchange Buffer is 50mM Na2HPO4300mM NaCl, pH8.0, and ultrafiltered with a 0.22 μm ultrafiltration membrane.
5. A feline interferon gamma produced by the method of soluble prokaryotic expression and purification of the recombinant feline interferon gamma of any one of claims 1-4.
6. Use of the feline interferon gamma of claim 5 in the preparation of a medicament for treating a feline parvovirus infection disease.
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