CN113322222A - Production process of genetic engineering bacteria for expressing feline omega interferon - Google Patents
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Abstract
The invention discloses a production process of a genetic engineering bacterium for expressing feline omega interferon, which comprises the following steps: the construction of the cat interferon engineering bacteria and the induction expression production process of the recombinant escherichia coli sequentially comprise fermentation, tank fermentation, centrifugation and crushing, inclusion body washing, denaturation, purification, renaturation and filtration sterilization. The process is simple and effective, and the cells of the negative control hole of the prepared genetically engineered bacteria do not produce anyThe obtained interferon has no toxic or side effect on cells; and a warp 104Dilute cat omega interferon inhibits cytopathic effects by 100%, while 105The diluted interferon had 3 wells with cytopathic effect, 106Cytopathic effect of 5 wells of the double-diluted interferon was observed, and the specific activity of cat omega interferon against VSV was found to be 1.42X 107U/mg。
Description
Technical Field
The invention relates to the technical field of pet drugs, in particular to a production process of a genetic engineering bacterium for expressing feline omega interferon.
Background
With the rapid development of the economy and the continuous improvement of the living standard of people in China, the pet industry in China is also developed rapidly. At present, the number of pet dogs and cats in China is about 1 hundred million, the number of the pet dogs and cats still increases sharply, the consumption around the pets also increases sharply, and the pet dogs and cats show a huge market prospect. However, it is also clear that more and more complex canine and feline diseases, particularly various viral diseases, seriously compromise their health and also threaten human health. The 560 th bulletin of the ministry of agriculture abolishes the use of antiviral western medicines including amantadine, rimantadine, acyclovir, moroxydine (moroxydine), ribavirin and the like, so that extensive veterinarians and culturists urgently need a residue-free and safe antiviral medicine, and interferon serving as a safe biological product fills the gap of the market. The biological activity of interferon and its quick-acting and multifunctional characteristics make it have important significance in preventing viral infectious diseases, inhibiting tumor growth, regulating immunity, etc.
The project aims to construct efficient expression engineering bacteria to induce and express the feline interferon, and prepare an effective feline interferon biological agent, and has important guiding significance for preventing and treating feline diseases, especially various viral diseases.
In general, interferon genes of related cells in an animal body are in a static state, can be expressed only under the induction of factors such as viruses and the like, and have extremely small expression amount, so that direct extraction and purification are difficult. Therefore, when applying interferon to prevent and treat viral diseases, research and development of a way for efficiently producing interferon must be considered. The development and development of recombinant interferon by utilizing genetic engineering technology is an efficient and economic way, and the production of interferon with the advantages of low price, broad-spectrum antiviral activity, no toxicity, no residue and the like is an effective way for promoting the clinical popularization and application of interferon in veterinarians.
At present, the soluble expression quantity of the cat omega interferon engineering bacteria is lower than that of an inclusion body, and the cat omega interferon engineering bacteria are easily influenced by environmental factors, so that the protein coagulation phenomenon is caused, and the expanded production difficulty is increased; the invention aims to provide a production and preparation process of a cat omega interferon inclusion body.
Disclosure of Invention
The invention aims to provide a production process of a genetically engineered bacterium for expressing feline omega interferon aiming at the defects and shortcomings of the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that: the production process of the gene engineering bacteria for expressing the feline omega interferon has the innovation points that the production process comprises the following steps: the method comprises the following steps of constructing cat interferon engineering bacteria and a recombinant escherichia coli induced expression production process, wherein the recombinant escherichia coli induced expression production process sequentially comprises fermentation, tank fermentation, centrifugation and crushing, inclusion body washing, denaturation, purification, renaturation and filtration sterilization, and the specific steps are as follows;
s1, construction of cat interferon engineering bacteria: logging in GenBank, searching a gene sequence (the sequence number is NM-001102440.1) for coding mature protein of cat omega interferon by using SignalP 3.0Server software to predict that the gene may contain a signal peptide sequence which is the first 23 amino acids of the amino acid sequence, using targetP 1.1Server software to detect the function of the sequence, finding that the sequence is necessary for a signal peptide for assisting protein secretion but not a protein structure, thus removing the signal peptide sequence, and leaving a gene sequence for coding mature cat omega interferon, wherein the nucleotide length of the gene sequence is 543; comparing the encoding codon of the cat omega interferon with the preferential codon of escherichia coli, modifying the known gene sequence encoding the cat omega interferon according to the degeneracy of the codon on the basis of not changing the composition and arrangement of amino acids, replacing the modified gene sequence with the preferential codon of escherichia coli, then adding NdeI and XhoI enzyme cutting sites to the 5 'and 3' ends of the replaced gene sequence respectively for complete gene synthesis, and connecting the ends into a prokaryotic expression plasmid pET28a to obtain a recombinant prokaryotic expression plasmid pET28 a-FeIFN-omega; transferring the recombinant plasmid into escherichia coli BL21 to construct and screen out the feline interferon engineering bacteria;
s2, recombinant escherichia coli induced expression production process:
2.1, fermentation: inoculating 1mL of glycerin tube with recombinant plasmid into 200mL of LB culture solution (tryptone 10%, yeast extract powder 5%, sodium chloride 10%) containing kanamycin, culturing at 37 ℃ for 12-18 hours at 200r/min, and preparing first-grade seeds; inoculating the first-stage seed solution to 600mL LB culture solution containing kanamycin in an inoculation amount of 4%, inoculating two bottles together, and culturing at 37 ℃ for 4h at a speed of 200r/min to prepare second-stage seeds;
2.2, fermentation in a tank: after a culture medium and a defoaming agent are filled in a fermentation tank, a secondary seed solution is inoculated according to 5% of the volume of the culture medium after sterilization, aerated culture is carried out at 37 ℃, IPTG with the final concentration of 0.3mM is added for induction expression when the culture is carried out until the OD600 value is 4.0-6.0, then culture is continued for 5-6h, the pH is controlled to be about 6.5, the dissolved oxygen is controlled to be above 30%, and when the solution rises rapidly, feeding is carried out;
2.3, centrifuging and crushing: after the culture is finished, centrifugally collecting thalli, weighing wet and heavy thalli, re-suspending the thalli by using a solution I (pH value is 8.0, 10mmol/L Tris & Cl and 1.0mmol/L EDTA) according to a ratio of 1:20(w/v), crushing the thalli for 3-5 times at 800-1000 bar by using a high-pressure homogenizer, and centrifuging the crushed thalli for 30min at 4 ℃ and 8000r/min to obtain a precipitate, namely an inclusion body;
2.4, washing of inclusion bodies: washing the inclusion bodies with solution II (pH 8.0, 10mmol/L Tris & Cl, 1.0mmol/L EDTA, 1% TritonX-100) and solution III (pH 8.0, 10mmol/L Tris & Cl, 1.0mmol/L EDTA, 2.0mol/L urea) respectively, centrifuging at 4 ℃ for 20 minutes at 8000r/min, and collecting the precipitate, namely the preliminarily purified interferon inclusion bodies:
2.5, denaturation: adding a denaturing solution according to a ratio of 1:40(w/v) to fully dissolve the inclusion body, stirring for 48 hours at 2-8 ℃ to completely dissolve the inclusion body, centrifuging for 10 minutes at 8000r/min at 4 ℃ to collect supernatant, and filtering by 0.22 mu m to obtain the denatured solution of the inclusion body;
2.6, purification: the operation was carried out according to the instructions of the protein purification apparatus using chemical SepharoseTMFast Flow affinity chromatography packing is pre-packed into a column, washed by PBS until the base line is stable, and then balanced to the base line stability by a binding buffer solution (pH value is 8.0, 500mmol/L imidazole, 50mmol/L Tris & Cl, 100mmol/L NaCl and 8.0mol/L urea); after balancing, 10mL of denatured solution is taken for sample loading, and then the mixed protein which is not combined with the chromatographic column is washed away by the combined buffer solution; finally using elution buffer (pH 8.0, 500mmol/L imidazole)50mmol/L Tris & Cl, 100mmol/L NaCl and 8.0mol/L urea) to collect target protein;
2.7, renaturation: measuring the protein content of the collected target protein according to the instructions of a Bradford protein quantitative kit; slowly adding a proper amount of denatured protein to the renaturation solution in batches to ensure that the final concentration of the protein in the renaturation solution is 0.1mg/ml, stirring at the temperature of 2-8 ℃, carrying out renaturation for 24-48 hours, and removing small molecular substances by dialysis after renaturation;
2.8, filtration sterilization: filtering and sterilizing by 0.22 mu m to obtain the interferon semi-finished product.
Further, the denaturing solution in the step of denaturing: pH 8.0, 10mmol/L Tris & Cl, 6.0-8.0mol/L guanidine hydrochloride.
Further, the denaturant: pH 8.0, 10mmol/L Tris. Cl, 7.0mol/L guanidine hydrochloride.
Further, the flow rate at the time of loading and elution in the purification of said step was controlled at 0.5 mL/min.
Further, the renaturation liquid in the renaturation of the step: pH 8.0, 10mmol/L Tris, 400-600mmol/L L-Arg, 500mmol/L urea, 1.0mmol/L EDTA, 2mmol/L cystine, 0.4mmol/L cysteine, 10-15% glycerol, 0.1-0.5% PEG 6000.
Further, the renaturation liquid: pH 8.0, 10mmol/L Tris, 400mmol/L L-Arg, 500mmol/L urea, 1.0mmol/L EDTA, 2mmol/L cystine, 0.4mmol/L cysteine, 10% glycerol, 0.1% PEG 6000.
The invention has the beneficial effects that:
the process is simple and effective, the cells of the negative control hole of the prepared genetic engineering bacteria do not produce any pathological changes, and the obtained interferon has no toxic action on the cells; and a warp 104Dilute cat omega interferon inhibits cytopathic effects by 100%, while 105The diluted interferon had 3 wells with cytopathic effect, 106Cytopathic effect of 5 wells of the double-diluted interferon was observed, and the specific activity of cat omega interferon against VSV was found to be 1.42X 107U/mg。
Drawings
FIG. 1 is an SDS-PAGE protein electrophoresis of cell disruption precipitates;
FIG. 2 is a Western Blot image.
In the figure, M: a protein Marker; 1: IPTG induced expression; 2: expression without IPTG induction.
Detailed Description
The invention will be further described with reference to the accompanying drawings.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and the detailed description. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
A production process of a gene engineering bacterium for expressing feline omega interferon comprises the following steps:
1. construction of cat interferon engineering bacteria:
logging in GenBank, searching a gene sequence (the sequence number is NM-001102440.1) for coding mature protein of cat omega interferon by using SignalP 3.0Server software to predict that the gene may contain a signal peptide sequence which is the first 23 amino acids of the amino acid sequence, using targetP 1.1Server software to detect the function of the sequence, finding that the sequence is necessary for a signal peptide for assisting protein secretion but not a protein structure, thus removing the signal peptide sequence, and leaving a gene sequence for coding mature cat omega interferon, wherein the nucleotide length of the gene sequence is 543; comparing the encoding codon of the cat omega interferon with the preference codon of the escherichia coli, modifying the known gene sequence of the cat omega interferon according to the degeneracy of the codon on the basis of not changing the composition and arrangement of amino acids, and replacing the gene sequence with the preference codon of the escherichia coli, aiming at improving the expression level of the gene in the escherichia coli, wherein the optimized gene sequence is as follows:
ATGTGTGCGCTGCCGGGTAGCCACGCGCAGGTTAGCCGTGATAACCTGGTTCTGCTGGGCCAGATGCGTCGTCTGAGCCCGTTCCTGTGCCTGCGTGCGCGTAAAGATTTCCGTTTCCCGCGTGAAATGCTGGAAGGTGGACAGCTGCGTGAAGCGCAGGCGGCGGCGGCGGTTCTGCGTGAACTGCTGCAGCAGACCTTCAACCTGCTGCACACCGAACGTTCTAGCGCAGCGTGGAGCCCGGCGCCGCTGCACGGCCTGCGTAGCGGCCTGCACCGTCAGCTGGAAGCGCTGGATGCGTGCCTGCTGCAGGCGACTGGCGAAGGTGAACGTGCTACCGGCGAAGGTGAACGTGCGCCGGGCATGCACGGTCCGGTTCTGGCGATCAAACGTTACTTCCAGGATATCCGTGTTTACCTGGAAGATGAAGGCTACTCTGATTGCGCGTGGGAAATCGTTCGTCTGGAAATCATGCGTGCGTTAAGCAGCTCCGCCACCCTGCAAGATTCCTTAGCCATCAAAGACGGCGATCTGGGTAGCAGCTAA
then NdeI and XhoI restriction enzyme sites are respectively added at the 5 'end and the 3' end of the replaced gene sequence, the gene sequence is sent to Shanghai bio-engineering company for whole gene synthesis, and the gene sequence is connected into a prokaryotic expression plasmid pET28a to obtain a recombinant prokaryotic expression plasmid pET28 a-FeIFN-omega; transferring the recombinant plasmid into escherichia coli BL21 to construct and screen the feline interferon engineering bacteria.
2. The recombinant escherichia coli induced expression production process comprises the following steps:
2.1 fermentation
Inoculating 1mL of glycerin tube with recombinant plasmid into 200mL of LB culture solution (tryptone 10%, yeast extract powder 5%, sodium chloride 10%) containing kanamycin, culturing at 37 ℃ for 12-18 hours at 200r/min, and preparing first-grade seeds; the primary seed solution was inoculated into 600mL LB medium containing kanamycin at an inoculum size of 4%, two bottles were inoculated together, and cultured at 37 ℃ for 4 hours at 200r/min to prepare secondary seeds.
2.2 fermentation in tank
After a culture medium and a defoaming agent are filled in a fermentation tank, a secondary seed solution is inoculated according to 5% of the volume of the culture medium after sterilization, aerated culture is carried out at 37 ℃, IPTG with the final concentration of 0.3mM is added for induction expression when the culture is carried out until the OD600 value is 4.0-6.0, then culture is continued for 5-6h, the pH is controlled to be about 6.5, the dissolved oxygen is controlled to be above 30%, and when the solution rises rapidly, feeding is carried out.
2.3 centrifugation and disruption
After the culture is finished, the thalli are collected centrifugally, wet and heavy thalli are weighed, the thalli are resuspended by using solution I (pH value is 8.0, 10mmol/L Tris & Cl and 1.0mmol/L EDTA) according to the proportion of 1:20(w/v), a high-pressure homogenizer is used for crushing 3-5 times at 800-1000 bar, the crushed thalli are centrifuged for 30min at 8000r/min at 4 ℃, and the obtained precipitate is the inclusion body.
2.4 Inclusion body washes
The inclusion bodies are respectively washed once by solution II (pH value is 8.0, 10mmol/L Tris & Cl, 1.0mmol/L EDTA, 1 percent TritonX-100) and solution III (pH value is 8.0, 10mmol/L Tris & Cl, 1.0mmol/L EDTA, 2.0mol/L urea), and the inclusion bodies are centrifuged at 4 ℃ at 8000r/min for 20 minutes to collect precipitates, thus obtaining the preliminarily purified interferon inclusion bodies.
2.5 denaturation
Adding a denaturing solution (pH value of 8.0, 10mmol/L Tris & Cl and 7.0mol/L guanidine hydrochloride) according to a ratio of 1:40(w/v) to fully dissolve the inclusion body, stirring for 48 hours at 2-8 ℃ to completely dissolve the inclusion body, centrifuging for 10 minutes at 8000r/min at 4 ℃ to collect supernatant, and filtering by 0.22 mu m to obtain the denatured solution of the inclusion body.
2.6 purification
The operation was carried out according to the instructions of the protein purification apparatus using the chemical Sepharose of GETMFast Flow affinity chromatography packing is pre-packed into a column, washed by PBS until the base line is stable, and then balanced to the base line stability by a binding buffer solution (pH value is 8.0, 500mmol/L imidazole, 50mmol/L Tris & Cl, 100mmol/L NaCl and 8.0mol/L urea); after balancing, 10mL of denatured solution is taken for sample loading, and then the mixed protein which is not combined with the chromatographic column is washed away by the combined buffer solution; finally eluting with elution buffer solution (pH value 8.0, 500mmol/L imidazole, 50mmol/L Tris & Cl, 100mmol/L NaCl, 8.0mol/L urea) to collect target protein; wherein the flow rate during loading and elution is controlled at 0.5 mL/min.
2.7 renaturation
The protein content of the collected target protein is measured according to the instructions of the Bradford protein quantification kit. Adding a proper amount of denatured protein to renaturation solution (pH value 8.0, 10mmol/L Tris, 400mmol/L L-Arg, 500mmol/L urea, 1.0mmol/L EDTA, 2mmol/L cystine, 0.4mmol/L cysteine, 10% glycerol, 0.1% PEG6000) slowly in batches to ensure that the final concentration of the protein in the renaturation solution is 0.1mg/ml, stirring at the temperature of 2-8 ℃ while adding, carrying out renaturation for 24-48 hours, and removing small molecular substances by dialysis after renaturation.
2.8 Filter Sterilization
Filtering and sterilizing by adopting a 0.22 mu m filter to obtain the interferon semi-finished product.
Furthermore, in the test process, the denatured liquid, the binding liquid in the purification process and the eluent are respectively collected, and after 10-fold dilution, SDS-PAGE and Western Blot detection are carried out to investigate the protein expression effect.
The experimental results are as follows: the results of detecting feline interferon expression by SDS-PAGE and Western Blot are shown in FIGS. 1 and 2.
Further, feline omega interferon activity assays
The recombinant cat omega interferon sample prepared after renaturation is subjected to antiviral activity detection, a micro cytopathy inhibition method is adopted for determination, CRFK is inoculated into a 96-well plate, culture solution is discarded after the CRFK grows into a single layer, 10-time serial dilution of recombinant cat omega interferon is added, 6 times of each dilution are repeated, supernatant is discarded after the culture is carried out for 18-24 hours, 10TCID50 Vesicular Stomatitis Virus (VSV) is used for counteracting the toxicity, meanwhile, a negative control (only 10-time dilution of interferon is added, no virus is added), a positive control (only virus is added, no interferon is added), a blank control (no interferon is added, no virus is added), counteracting and the result is determined after the cytopathy appears in the positive control hole at 75 percent.
The experimental results are as follows: (1) the cells in the negative control wells did not produce any lesions, indicating that the interferon obtained in this example itself had no toxic effect on the cells; (2) warp 104Dilute cat omega interferon inhibits cytopathic effects by 100%, while 105The diluted interferon had 3 wells with cytopathic effect, 106Cytopathic effects occurred in 5 wells of the double-diluted interferon, and the results indicated that the specific activity of feline omega interferon against VSV was 1.42X 107U/mg。
The above description is only for the purpose of illustrating the technical solutions of the present invention and not for the purpose of limiting the same, and other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (6)
1. A production process of a genetic engineering bacterium for expressing feline omega interferon is characterized by comprising the following steps: the method comprises the following steps of constructing cat interferon engineering bacteria and a recombinant escherichia coli induced expression production process, wherein the recombinant escherichia coli induced expression production process sequentially comprises fermentation, tank fermentation, centrifugation and crushing, inclusion body washing, denaturation, purification, renaturation and filtration sterilization, and the specific steps are as follows;
s1, construction of cat interferon engineering bacteria: logging in GenBank, searching a gene sequence (the sequence number is NM-001102440.1) for coding mature protein of cat omega interferon by using SignalP 3.0Server software to predict that the gene may contain a signal peptide sequence which is the first 23 amino acids of the amino acid sequence, using targetP 1.1Server software to detect the function of the sequence, finding that the sequence is necessary for helping the signal peptide secreted by the protein but not for the protein structure, thus removing the signal peptide sequence, and leaving the gene sequence for coding mature cat omega interferon, wherein the length of the nucleotide is 543 bp; comparing the encoding codon of the cat omega interferon with the preferential codon of escherichia coli, modifying the known gene sequence encoding the cat omega interferon according to the degeneracy of the codon on the basis of not changing the composition and arrangement of amino acids, replacing the modified gene sequence with the preferential codon of escherichia coli, then adding NdeI and XhoI enzyme cutting sites to the 5 'and 3' ends of the replaced gene sequence respectively for complete gene synthesis, and connecting the ends into a prokaryotic expression plasmid pET28a to obtain a recombinant prokaryotic expression plasmid pET28 a-FeIFN-omega; transferring the recombinant plasmid into escherichia coli BL21 to construct and screen out the feline interferon engineering bacteria;
s2, recombinant escherichia coli induced expression production process:
2.1, fermentation: inoculating 1mL of glycerin tube with recombinant plasmid into 200mL of LB culture solution (tryptone 10%, yeast extract powder 5%, sodium chloride 10%) containing 50. mu.g/mL kanamycin, culturing at 37 ℃ for 12-18 hours at 200r/min, and preparing first-grade seeds; inoculating the first-stage seed solution to 600mL LB culture solution containing kanamycin in an inoculation amount of 4%, inoculating two bottles together, and culturing at 37 ℃ for 4h at a speed of 200r/min to prepare second-stage seeds;
2.2, fermentation in a tank: filling culture medium and defoaming agent into fermentation tank, sterilizing, inoculating secondary seed solution at 5% of culture medium volume, culturing at 37 deg.C under aeration, and culturing to OD600When the value is 4.0-6.0, adding IPTG with final concentration of 0.3mM for induction expression, then continuing culturing for 5-6h, controlling pH at about 6.5 and dissolved oxygen at above 30%, and feeding when the solution rises rapidly;
2.3, centrifuging and crushing: after the culture is finished, centrifugally collecting thalli, weighing wet and heavy thalli, re-suspending the thalli by using a solution I (pH value is 8.0, 10mmol/L Tris & Cl and 1.0mmol/L EDTA) according to a ratio of 1:20(w/v), crushing the thalli for 3-5 times at 800-1000 bar by using a high-pressure homogenizer, and centrifuging the crushed thalli for 30min at 4 ℃ and 8000r/min to obtain a precipitate, namely an inclusion body;
2.4, washing of inclusion bodies: washing the inclusion bodies with solution II (pH 8.0, 10mmol/L Tris & Cl, 1.0mmol/L EDTA, 1% TritonX-100) and solution III (pH 8.0, 10mmol/L Tris & Cl, 1.0mmol/L EDTA, 2.0mol/L urea) respectively, centrifuging at 4 ℃ for 20 minutes at 8000r/min, and collecting the precipitate, namely the preliminarily purified interferon inclusion bodies:
2.5, denaturation: adding a denaturing solution according to a ratio of 1:40(w/v) to fully dissolve the inclusion body, stirring for 48 hours at 2-8 ℃ to completely dissolve the inclusion body, centrifuging for 10 minutes at 8000r/min at 4 ℃ to collect supernatant, and filtering by 0.22 mu m to obtain the denatured solution of the inclusion body;
2.6, purification: the operation was carried out according to the instructions of the protein purification apparatus using chemical SepharoseTMA Fast Flow affinity chromatography filler prepacked column is washed by PBS until the base line is stable, and then is balanced to the base line stability by a binding buffer solution (pH value is 8.0, 500mmol/L imidazole, 50mmol/L Tris & Cl, 100mmol/L NaCl and 8.0mol/L urea); after balancing, 10mL of denatured solution is taken for sample loading, and then the mixed protein which is not combined with the chromatographic column is washed away by the combined buffer solution; finally eluting with elution buffer solution (pH value 8.0, 500mmol/L imidazole, 50mmol/L Tris & Cl, 100mmol/L NaCl, 8.0mol/L urea) to collect target protein;
2.7, renaturation: measuring the protein content of the collected target protein according to the instructions of a Bradford protein quantitative kit; slowly adding a proper amount of denatured protein to the renaturation solution in batches to ensure that the final concentration of the protein in the renaturation solution is 0.1mg/ml, stirring at the temperature of 2-8 ℃, carrying out renaturation for 24-48 hours, and removing small molecular substances by dialysis after renaturation;
2.8, filtration sterilization: filtering and sterilizing by adopting a 0.22 mu m filter to obtain the interferon semi-finished product.
2. The process for producing genetically engineered bacteria expressing feline omega interferon according to claim 1, wherein the denaturing solution in the step of denaturing: pH 8.0, 10mmol/L Tris & Cl, 6.0-8.0mol/L guanidine hydrochloride.
3. The production process of the genetically engineered bacterium for expressing feline omega interferon according to claim 2, wherein the denaturant solution comprises: pH 8.0, 10mmol/L Tris. Cl, 7.0mol/L guanidine hydrochloride.
4. The production process of the genetically engineered bacterium for expressing feline omega interferon according to claim 1, wherein the genetically engineered bacterium comprises: the flow rate of the sample loading and elution in the purification of the step is controlled at 0.5 mL/min.
5. The production process of the genetically engineered bacterium expressing feline omega interferon according to claim 1, wherein the renaturation solution in the renaturation step is: pH 8.0, 10mmol/L Tris, 400-600mmol/L L-Arg, 500mmol/L urea, 1.0mmol/L EDTA, 2mmol/L cystine, 0.4mmol/L cysteine, 10-15% glycerol, 0.1-0.5% PEG 6000.
6. The production process of the genetically engineered bacterium expressing feline omega interferon according to claim 5, wherein the renaturation solution: pH 8.0, 10mmol/L Tris, 400mmol/L L-Arg, 500mmol/L urea, 1.0mmol/L EDTA, 2mmol/L cystine, 0.4mmol/L cysteine, 10% glycerol, 0.1% PEG 6000.
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| CN113430220A (en) * | 2021-08-17 | 2021-09-24 | 江苏恒丰强生物技术有限公司 | Synthesis method, construction method and application of genetic engineering bacteria for expressing soluble feline omega interferon |
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