CN113430220A - Synthesis method, construction method and application of genetic engineering bacteria for expressing soluble feline omega interferon - Google Patents
Synthesis method, construction method and application of genetic engineering bacteria for expressing soluble feline omega interferon Download PDFInfo
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Abstract
The invention discloses a synthetic method, a construction method and application of a genetic engineering bacterium for expressing soluble cat omega interferon, wherein a gene sequence for coding mature protein of cat omega interferon is searched, software is utilized to predict that the gene may contain a signal peptide sequence which is the first 23 amino acids of the amino acid sequence of the gene, the function of the sequence is detected by the software, the function of the sequence is found to be necessary for helping the signal peptide secreted by protein rather than the protein structure, so that the signal peptide sequence is removed, the gene sequence for coding mature cat omega interferon is left, and the nucleotide length is 543 bp; the codon for encoding the cat omega interferon is compared with the preferential codon of the escherichia coli, and the known gene sequence for encoding the cat omega interferon is modified and replaced by the preferential codon of the escherichia coli on the basis of not changing the composition and arrangement of amino acids according to the degeneracy of the codon. The cells of the negative control hole of the gene engineering bacteria prepared by the invention do not produce any pathological changes, so the interferon itself has no toxic action on the cells.
Description
Technical Field
The invention relates to the technical field of pet medicines, in particular to a synthetic method, a construction method and application of a genetic engineering bacterium for expressing soluble 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 publication of Ministry of agriculture abolishes the use of antiviral drugs including amantadine, rimantadine, acyclovir, moroxydine (moroxydine), ribavirin, etc., so that extensive veterinarians and culturists urgently need a residue-free and safe antiviral drug, and interferon as a safe biological product fills the gap of the market. The biological activity and the quick-acting and multifunctional characteristics of the interferon make the interferon have important significance in the aspects of inhibiting virus proliferation, inhibiting tumor growth, regulating immunity and the like.
The project aims to construct the high-efficiency expression recombinant feline interferon engineering bacteria and prepare the effective feline interferon biological agent, and has important guiding significance for preventing and treating feline diseases, particularly 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.
Disclosure of Invention
The invention aims to provide a synthetic method, a construction method and application of a genetic engineering bacterium for expressing soluble 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 method for synthesizing the gene engineering bacteria for expressing the soluble cat omega interferon has the innovation points that the method comprises the following specific steps:
s1, logging in GenBank, and searching a gene sequence for encoding the mature protein of the feline omega interferon;
s2, comparing the codon for cat omega interferon with the preferred codon of colibacillus, and according to the degeneracy of the codon. Replacing the cat omega interferon codon with a preferred codon of escherichia coli;
s3, adding NdeI and XhoI restriction enzyme sites at the 5 'end and the 3' end of the replaced gene sequence respectively, carrying out whole-gene synthesis, connecting into a prokaryotic expression plasmid pET28a to obtain a recombinant prokaryotic expression plasmid pET28 a-FeIFN-omega, transferring into Rosetta-gami2(DE3) competent cells by a heat shock transformation method, identifying to obtain a positive recombinant strain, and optimizing through fermentation conditions to obtain the genetic engineering bacteria for high expression of the soluble protein.
The construction method of the genetic engineering bacteria for expressing the soluble cat omega interferon is innovative and comprises the following specific steps:
s1, taking out competent cells of a Rosetta-gami2(DE3) strain from-80 ℃, quickly inserting the competent cells into ice, melting the strain after 5 minutes, adding a recombinant vector pet28 a-FeIFN-omega 10 mu L, stirring the EP tube bottom by hands, gently mixing the mixture evenly and avoiding sucking the mixture by a gun, and standing the mixture in the ice for 25 minutes;
s2, thermally shocking the water bath at 42 ℃ for 90 seconds, quickly putting the water bath back on ice, standing the water bath for 2 minutes, and reducing the conversion efficiency by shaking;
s3, adding 1mL of antibiotic-free sterile medium (LB) into the centrifuge tube, uniformly mixing, and recovering at 37 ℃ and 200rpm for 60 minutes;
s4, centrifuging at 5000rpm for 1 minute to collect bacteria, leaving about 100 mu L of supernatant, slightly blowing and beating the heavy suspension bacteria, and coating the heavy suspension bacteria on an LB culture medium containing 50 mu g/mL of kanamycin (the LB culture medium is tryptone 1%, yeast powder 0.5% and sodium chloride 1%);
s5, placing the flat plate upside down in an incubator at 37 ℃ for overnight culture;
s6, randomly selecting strains on 10 LB plates, respectively inoculating the strains into 1mL of LB culture solution containing kanamycin, culturing at 37 ℃ and 200rpm for 8h, and then carrying out PCR identification.
The application of the genetic engineering bacteria for expressing the soluble feline omega interferon has the innovation point that the genetic engineering bacteria specifically for inducing expression comprise the following steps:
s1, adding 0.5mL of bacterial liquid with recombinant plasmids into 100mL of LB culture solution (containing kanamycin with the final concentration of 50 mug/mL), culturing and activating at 37 ℃ at 150r/min overnight; inoculating the bacterial liquid with the inoculation amount of 8% into 200mL LB culture liquid (containing kanamycin with the final concentration of 50 mug/mL), culturing at 22 ℃ at 200r/min, adding 200ul IPTG with the concentration of 100mM into every 100mL of bacterial liquid to induce expression when the OD600 value of the bacterial liquid is about 0.4, and inducing the expression of the target protein at 22 ℃ at 200r/min overnight (16 h);
s2, treating the sample by an ultrasonic crushing method: taking 100mL of bacterial liquid, carrying out centrifugation at 8000r/min for 5min, removing supernatant, washing the precipitate with 10mL of 1 XPBS for 2 times, carrying out centrifugation at 8000r/min for 5min, leaving the precipitate, adding 10mL of 1 XPBS, mixing uniformly, carrying out ultrasonic crushing (working for 2s, gap 2s and working for 10min), centrifuging the cell crushing liquid at 10000r/min, and centrifuging for 10 min;
s3, taking the supernatant, centrifuging for 1 time again, filtering the supernatant with 0.45 micron to remove impurities, taking 40 microliter of the supernatant, adding 10 microliter of 5 xSDS loading buffer solution, carrying out boiling water bath for 10min, and detecting;
s4, taking 15 mu L of a sample to be detected to carry out SDS-PAGE electrophoresis detection, and finding out high-expression soluble feline omega interferon protein;
s5, operating according to the instructions of the protein purification instrument, and adopting Chelating SepharoseTMProtein purification is carried out by Fast Flow affinity chromatography pre-packed column, SDS-PAGE electrophoresis is carried out on purified interferon samples, the purification result is detected by Western Blot, and the protein concentration is detected by Bradford method.
The invention has the beneficial effects that:
the process is simple and effective, and the cells of the negative control hole of the prepared genetically engineered bacteria do not produce any pathological changes, so that the interferon has no toxic action on the cells; warp 104Dilute cat omega interferon inhibits cytopathic effects by 100%, while 105The diluted interferon had 2 wells with cytopathic effect, 106The results of the double dilution of interferon were all pathological, and the specific activity of feline omega interferon against VSV was found to be 1.78X 107U/mg。
Drawings
FIG. 1 shows the restriction enzyme digestion of a vector;
FIG. 2 is an SDS-PAGE protein electrophoresis of cell debris;
FIG. 3 is a Western Blot chart;
FIG. 4 is an SDS-PAGE protein electrophoresis of protein purified samples.
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.
Synthesis of feline omega interferon gene
Logging in GeneBank, 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 so as to improve the expression level of the gene in the escherichia coli;
the DNA sequence is shown below:
ATGTGTGCGCTGCCGGGTAGCCACGCGCAGGTTAGCCGTGATAACCTGGTTCTGCTGGGCCAGATGCGTCGTCTGAGCCCGTTCCTGTGCCTGCGTGCGCGTAAAGATTTCCGTTTCCCGCGTGAAATGCTGGAAGGTGGACAGCTGCGTGAAGCGCAGGCGGCGGCGGCGGTTCTGCGTGAACTGCTGCAGCAGACCTTCAACCTGCTGCACACCGAACGTTCTAGCGCAGCGTGGAGCCCGGCGCCGCTGCACGGCCTGCGTAGCGGCCTGCACCGTCAGCTGGAAGCGCTGGATGCGTGCCTGCTGCAGGCGACTGGCGAAGGTGAACGTGCTACCGGCGAAGGTGAACGTGCGCCGGGCATGCACGGTCCGGTTCTGGCGATCAAACGTTACTTCCAGGATATCCGTGTTTACCTGGAAGATGAAGGCTACTCTGATTGCGCGTGGGAAATCGTTCGTCTGGAAATCATGCGTGCGTTAAGCAGCTCCGCCACCCTGCAAGATTCCTTAGCCATCAAAGACGGCGATCTGGGTAGCAGCTAA
then NdeI and XhoI enzyme cutting 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 is connected into a prokaryotic expression plasmid pET28a to obtain a recombinant prokaryotic expression plasmid pET28 a-FeIFN-omega, and the enzyme cutting verification picture is shown in figure 1;
prokaryotic expression of feline omega interferon
(1) Construction of cat omega interferon engineering bacteria
1.1, Rosetta-gami2(DE3) strains competent cells were taken out from-80 ℃, quickly inserted into ice, 5 minutes later, after the pellet was thawed, recombinant vector pet28 a-FeIFN-. omega.10. mu.L was added and gently mixed by hand-tapping the bottom of an EP tube (avoiding pipetting with a gun), and left to stand on ice for 25 minutes.
Heat shock in water bath at 1.2 and 42 deg.c for 90 sec, fast returning to ice and standing for 2 min to avoid shaking.
1.3, adding 1mL of antibiotic-free sterile medium (LB) into the centrifuge tube, mixing uniformly, and recovering at 37 ℃ and 200rpm for 60 minutes.
1.4, 5000rpm centrifugation for 1 minutes to collect bacteria, left 100 u L supernatant gently blow heavy suspended bacteria block and spread to Carna containing LB medium.
1.5, place the plate upside down in a 37 ℃ incubator overnight.
1.6, randomly selecting positive clone strains on 10 LB plates, respectively inoculating the positive clone strains into 1mL of LB culture solution containing cana, culturing at 37 ℃ and 200rpm for 8h, and then carrying out PCR identification.
The PCR reaction system was 25. mu.l: 2 × taq PCR Mix 12.5 μ l, upstream primer 0.5 μ l, downstream primer 0.5 μ l, bacterial liquid 0.5 μ l, ddH2O 11μl。
The PCR reaction conditions were 95 ℃ for 5min, 95 ℃ for 60s, 55 ℃ for 60s, and 72 ℃ for 90s, and the PCR amplification products were detected by 1.5% agarose electrophoresis, resulting in a band of about 700 bp.
(2) Inducible expression
2.1, 0.5mL of the recombinant plasmid-containing bacterial suspension was added to 100mL of LB medium (containing 50. mu.g/mL of kanamycin to the final concentration), and cultured and activated at 37 ℃ at 150 rpm overnight. The bacterial solution was inoculated into 200mL of LB medium (containing 50. mu.g/mL kanamycin) at an inoculum size of 8%, cultured at 22 ℃ at 200r/min, and when the OD600 value of the bacterial solution was about 0.4, 200ul of IPTG at a concentration of 100mM was added to 100mL of the bacterial solution to induce expression, and expression of the target protein was induced overnight at 22 ℃ at 200r/min (16 h). (LB medium: tryptone 1%, Yeast powder 0.5%, sodium chloride 1%)
2.2, treating a sample by an ultrasonic crushing method: taking 100mL of bacterial liquid, carrying out centrifugation at 8000r/min for 5min, removing supernatant, washing the precipitate with 10mL of 1 XPBS for 2 times, carrying out centrifugation at 8000r/min for 5min, leaving the precipitate, adding 10mL of 1 XPBS, mixing uniformly, carrying out ultrasonic crushing (working for 2s, gap 2s and working for 10min), centrifuging the cell crushing liquid at 10000r/min, and centrifuging for 10 min;
2.3, taking the supernatant, centrifuging for 1 time again, filtering the supernatant with 0.45 micron to remove impurities, taking 40 microliter of the supernatant, adding 10 microliter of 5 xSDS loading buffer solution, carrying out boiling water bath for 10min, and detecting;
2.4, taking 15 mu L of sample to be detected to carry out SDS-PAGE electrophoresis detection, and finding out the high-expression soluble feline omega interferon protein. See fig. 2.
2.5 protein purification
According to the proteinInstructions for operation of the purification apparatus Using chemical SepharoseTMProtein purification is carried out by Fast Flow affinity chromatography prepacked column, SDS-PAGE electrophoresis is carried out on purified interferon samples, the purification result is detected by Western Blot, and the protein concentration is detected by Bradford method
The experimental results are as follows: the results of SDS-PAGE and Western Blot of the purified cat interferon are shown in figures 3 and 4, and the purity is qualified.
2.7 detection of feline omega Interferon Activity
Diluting the recombinant cat omega interferon sample into 0.1mg/mL solution, detecting antiviral activity, determining by a micro cytopathy inhibition method, inoculating CRFK into a 96-well plate, removing culture solution after growing into a single layer, adding 10-fold serial diluted recombinant cat omega interferon, repeating 6 times for each dilution degree, culturing for 18-24 hours, removing supernatant, using 10TCID50 Vesicular Stomatitis Virus (VSV) to detoxify, simultaneously setting up a negative control (only 10-fold diluted interferon and no virus), a positive control (only virus and no interferon), a blank control (no interferon and no virus), performing toxicity counteracting culture for 24 hours, observing cytopathy under an inverted microscope, and determining a result when 75% of lesions appear in the positive control hole.
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 2 wells with cytopathic effect, 106The results of the double dilution of interferon were all pathological, and the specific activity of feline omega interferon against VSV was found to be 1.78X 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 (3)
1. A synthetic method of a genetic engineering bacterium for expressing soluble cat omega interferon is characterized by comprising the following specific steps:
s1, logging in GenBank, and searching a gene sequence for encoding the mature protein of the feline omega interferon;
s2, comparing the codon for encoding the cat omega interferon with the preferred codon of the escherichia coli, and replacing the cat omega interferon codon with the preferred codon of the escherichia coli according to the degeneracy of the codon;
s3, adding NdeI and XhoI restriction enzyme sites at the 5 'end and the 3' end of the replaced gene sequence respectively, carrying out whole gene synthesis, connecting the obtained product into a prokaryotic expression plasmid pET28a to obtain a recombinant prokaryotic expression plasmid pET28 a-FeIFN-omega, transferring the recombinant prokaryotic expression plasmid pET 28-FeIFN-omega into Rosetta-gami2(DE3) competent cells by a heat shock transformation method, identifying to obtain a positive strain for expressing recombinant cat omega interferon, and optimizing by fermentation conditions to obtain the genetically engineered bacteria for high expression of soluble protein.
2. The construction method of the genetic engineering bacteria for expressing the soluble feline omega interferon according to claim 1, which is characterized by comprising the following steps:
s1, taking out competent cells of the Rosetta-gami2(DE3) strain from a freezer at the temperature of-80 ℃, quickly inserting the competent cells into ice, melting the strain blocks after 5 minutes, adding 10 mu L of recombinant vector pet28 a-FeIFN-omega, stirring the mixture by hands and gently mixing the mixture at the bottom of an EP tube without sucking the mixture by a gun, and standing the mixture in ice for 25 minutes;
s2, thermally shocking the mixture for 90 seconds by using a water bath at 42 ℃, quickly putting the mixture back on ice and standing the mixture for 2 minutes to avoid shaking;
s3, adding 1mL of antibiotic-free sterile medium (LB) into the centrifuge tube, uniformly mixing, placing the mixture in a 37 ℃ shaking table, and recovering the mixture for 60 minutes at 200 rpm;
s4, centrifuging at 5000rpm for 1 minute to collect bacteria, leaving about 100 mu L of supernatant, slightly blowing and beating the heavy suspension bacteria, and coating the heavy suspension bacteria on an LB culture medium containing 50 mu g/mL of kanamycin (the LB culture medium is tryptone 1%, yeast powder 0.5% and sodium chloride 1%);
s5, placing the flat plate upside down in an incubator at 37 ℃ for overnight culture;
s6, randomly selecting strains on 10 LB plates, respectively inoculating the strains into 1mL of LB culture solution containing kanamycin, culturing at 37 ℃ and 200rpm for 8h, and then carrying out PCR identification.
3. The application of the genetically engineered bacteria expressing soluble feline omega interferon according to claim 1 or 2, in particular to induction of expression, comprising the steps of:
s1, adding 0.5mL of bacterial liquid with recombinant plasmids into 100mL of LB culture solution (containing kanamycin with the final concentration of 50 mu g/mL), placing the mixture in a shaking table at 37 ℃, and culturing and activating overnight at the rotating speed of 150 r/min; inoculating the bacterial liquid with 8% inoculum size into 200mL LB culture solution (containing kanamycin with final concentration of 50 μ g/mL), placing in a shaker at 22 deg.C, culturing at 200r/min, and collecting the bacterial liquid OD600When the value is about 0.4, 200 mul of IPTG with the concentration of 100mM is added into every 100mL of bacterial liquid to induce the expression, and the expression of the target protein is induced overnight under the conditions of 22 ℃ and 200r/min (16 h);
s2, treating the sample by an ultrasonic crushing method: centrifuging 100mL of bacterial solution at 8000r/min for 5min, removing supernatant, washing precipitate with 10mL of 1 XPBS for 2 times at 8000r/min, centrifuging for 5min, leaving precipitate, adding 10mL of 1 XPBS, mixing, performing ultrasonic disruption (working for 2s, gap 2s, total working time 10min), centrifuging cell disruption solution for 10000r/min, and centrifuging for 10 min;
s3, taking the supernatant, centrifuging for 1 time again, filtering the supernatant by using a 0.45 micron filter to remove impurities, taking 40 mu L of the supernatant, adding 10 mu L of 5 xSDS loading buffer solution, carrying out boiling water bath for 10min, and detecting;
s4, taking 15 mu L of a sample to be detected to carry out SDS-PAGE electrophoresis detection, and finding out high-expression soluble feline omega interferon protein;
s5, operating according to the operation instructions of the protein purifier, and adopting chemical
SepharoseTMProtein purification is carried out by Fast Flow affinity chromatography pre-packed column, SDS-PAGE electrophoresis is carried out on purified interferon samples, the purification result is detected by Western Blot, and the protein concentration is detected by Bradford method.
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| CN113322222A (en) * | 2021-07-02 | 2021-08-31 | 江苏恒丰强生物技术有限公司 | Production process of genetic engineering bacteria for expressing feline omega interferon |
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