CN110256516B - Preparation method of polyinosinic cells - Google Patents
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Abstract
The invention discloses a preparation method of polyinosinic cells, and belongs to the technical field of biological medicines. Firstly, inosine diphosphate is used for preparing polyinosinic acid, and cytidine diphosphate is used for preparing polycytidylic acid; and then the prepared polyinosinic acid reacts with polycytidylic acid to obtain a high-purity polyinosinic acid product. The polyinosinic acid and polycytidylic acid with high purity are prepared by the method, so that the prepared polyinosinic acid is high in purity, and the treatment difficulty in the preparation process is reduced.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a preparation method of polyinosinic cells.
Background
Polyinosinic acid cytidylic acid (polyinosinic cell, PIC for short) is a safe and efficient innate immune activator, has multiple functions of broad-spectrum virus resistance, tumor cell growth inhibition, organism immunity enhancement and the like, and has estimated global market capacity potential of hundreds of billions of dollars per year. Clinically, the medicine can be used for treating viral diseases such as chronic hepatitis B, epidemic hemorrhagic fever, epidemic encephalitis B, viral keratitis and the like, and the auxiliary treatment of tumors such as breast cancer, prostatic cancer and the like is in a stage III clinical experiment stage. The veterinary drug can be clinically used for preventing and treating the virus epidemic diseases such as foot and mouth disease, avian influenza, swine fever and the like, and can also improve the immunity effect of the livestock and poultry vaccine. The polyinosinic-polycytidylic acid is convenient and wide to use, can be used with a plurality of medicines simultaneously, and has synergistic or additive effects. Has no species specificity, can be widely used on animals such as poultry, pigs, cattle and the like, and also has the advantages of wide antiviral spectrum, small toxicity and wide safety range.
In the field, most of the original polyinosinic cells are produced by chemically synthesizing or purchasing semi-finished products of PI (polyinosinic acid) and PC (polycytidylic acid), and then PI and PC are physically mixed, so that the defects of high cost, incapability of large-scale production, incomplete production flow, incorrect molecular structure, incapability of ensuring the quality of final products and the like exist.
Disclosure of Invention
The invention aims to provide a preparation method of polyinosinic acid, which is characterized in that polyinosinic acid is prepared by utilizing inosine diphosphate, and polycytidylic acid is prepared by utilizing cytidine diphosphate; and then the prepared polyinosinic acid reacts with polycytidylic acid to obtain a high-purity polyinosinic acid product.
The technical scheme of the invention is as follows:
a preparation method of polyinosinic cells comprises the following steps:
(1) heating the fine filtrate of polyinosinic acid and polycytidylic acid and the stabilizing agent to 37 ℃;
(2) then mixing the heated fine filtrate of the polyinosinic acid and the polycytidylic acid with a stabilizer, wherein the weight ratio of the polyinosinic acid to the polycytidylic acid to the stabilizer is 1:1:1, and stirring for 30min at the constant temperature of 37 ℃;
(3) filtering the solution after stirring, and carrying out alcohol precipitation treatment on the filtrate by adopting ethanol, wherein the alcohol precipitation time is 0.5-1.5 h;
(4) centrifuging the precipitated material, soaking the centrifuged solid product in ethanol for dehydration, vacuum drying to obtain polyinosinic-polycytidylic acid product,
the polyinosinic acid fine filter liquid is prepared by adding MgCl into inosine diphosphate (IDP for short)2Inosine diphosphate and MgCl2The molar ratio of (A) to (B) is 10:1, and the pH of the mixed solution is adjusted to 7.0; heating for polymerization, fully stirring at 38 ℃, adding curing enzyme, and continuously stirring for 2 hours; collecting reaction stock solution after polymerization reaction, filtering and washing to obtain polyinosinic acid fine filter solution;
the fine filtering solution of polycytidylic acid is prepared by adding MgCl into cytidine diphosphate (CDP for short)2Cytidine diphosphate and MgCl2The molar ratio of (A) to (B) is 10:1, and the pH of the mixed solution is adjusted to 7.0; heating for polymerization, fully stirring at 38 ℃, adding curing enzyme, and continuously stirring for 2 hours; collecting reaction stock solution after polymerization reaction, filtering and washing to obtain polycytidylic acid fine filter solution.
Preferably, the stabilizer is one or more of kanamycin sulfate, polylysine and calcium gluconate.
Preferably, the immobilized enzyme is a polynucleotide phosphorylase, and the addition amount is 0.5-1.5 wt%.
Preferably, the preparation method of the inosine diphosphate solution includes the following steps:
1) deaminating ATP to prepare inosine triphosphate solution, slowly adding 25-35 wt% of sodium nitrite into ATP, reacting for 6 hours, adjusting the pH to 1.2-2.0, and continuing to react for more than 40 hours until no dense smoke is generated, thus obtaining the inosine triphosphate solution;
2) decoloring the creatinine triphosphate solution, namely adjusting the pH value of the inosine triphosphate solution prepared in the step 1) to be neutral, adding activated carbon for decoloring, and filtering to obtain the decolored creatinine triphosphate solution;
3) preparing a degrading enzyme solution, namely preparing a yeast solution with the concentration of 50%, adjusting the pH of the yeast solution to be 6, standing the yeast solution for 2 days at the temperature of 30 ℃, and filtering the yeast solution to obtain the degrading enzyme solution;
4) performing degradation reaction, namely mixing the decolorized creatinine triphosphoric acid solution obtained in the step 2) with the degradation enzyme solution prepared in the step 3), wherein the weight ratio of creatinine triphosphoric acid to degradation enzyme in the mixed solution is 10-15:1, then adding purified water, adjusting the concentration of the creatinine triphosphoric acid in the solution to be 2-3%, and adjusting the pH value of the solution to be neutral; adding glucose accounting for 20 percent of the weight of the creatinine triphosphate, and fully stirring; heating to 36-37 ℃ for enzymolysis, when the weight of creatinine triphosphate in the solution is less than 1% of the total cytidine, the reaction is terminated, and immediately adjusting the pH value of the solution to 2 by hydrochloric acid;
5) cooling the fermentation liquor after the reaction to 25 ℃, adding 2% of perlite powder, fully stirring, cooling the fermentation liquor to below 20 ℃, and filtering, carrying out column chromatography treatment, decoloring and ultrafiltering to obtain the creatinine diphosphoric acid solution.
Preferably, the preparation method of the cytidine diphosphate solution comprises the following steps:
1) phosphorylation, putting 200-500 parts by weight of yeast into a phosphorylation reaction tank, stirring, heating in a water bath to 30-31 ℃, stopping heating, putting 2-10 parts by weight of magnesium chloride, 20-50 parts by weight of potassium dihydrogen phosphate and 20-50 parts by weight of white sugar, controlling the temperature to 31-33 ℃, reacting for 10-20 minutes, then adding 30 parts by weight of cytidine triphosphate, reacting for 2-5 hours, when the content of cytidine triphosphate is more than 85%, immediately adjusting the pH value to 2 to stop the reaction, and cooling with chilled water;
2) performing chromatographic purification, namely performing plate-and-frame filtration, chromatography, adsorption, washing, elution and sodium decoloration filtration on the phosphorylation product cooled in the step 1);
3) degrading, namely adding nucleotide degrading enzyme into the solution subjected to chromatographic purification in the step 2) at 36-37 ℃ for degradation, adjusting the pH to 2 to stop the reaction when the cytidine triphosphate accounts for less than 3% of the total cytidine, and cooling the reaction solution to below 20 ℃;
4) and 3) sequentially carrying out plate-frame filtration, adsorption, washing, elution, decolorization and ultrafiltration nanofiltration on the solution cooled in the step 3) to obtain the cytidine diphosphate solution.
In the preparation process of the cytidine diphosphate solution, the weight ratio of the addition amount of the nucleotide degrading enzyme to the phosphorylated product in the solution after purification in the step 2) is 0.5-2: 1.
Compared with the prior art, the beneficial effects of this application are as follows: the invention prepares polyinosinic acid PI through inosine diphosphate IDP, and polycytidylic acid PC through cytidine diphosphate CDP; and then reacting the prepared polyinosinic acid PI with the polycytidylic acid PC to obtain a high-purity polyinosinic acid PIC product. In the process of preparing PI and PC, impurities such as endotoxin, residual DNA, residual host mycoprotein and the like in PI and PC products are effectively removed by adjusting process means, the production difficulty is reduced in the process of polymerizing PI and PC, the removal of impurities is facilitated, and the prepared PI and PC are used for enabling the PIC prepared by the method to be a double-stranded ribonucleotide polyinosinic cell product with a stable structure, so that the purity of the product and the content of effective components are greatly improved.
Detailed description of the preferred embodiments
The present invention will be further described with reference to the following examples.
Example 1
A preparation method of polyinosinic cells comprises the following steps:
1. preparation of Cytidine diphosphate CDP solution
1) Phosphorylation, adding 300 parts by weight of yeast into a phosphorylation reaction tank, stirring, heating in a water bath to 31 ℃, stopping heating, adding 5 parts by weight of magnesium chloride, 30 parts by weight of potassium dihydrogen phosphate and 20 parts by weight of white sugar, controlling the temperature to 31 ℃ for reaction for 20 minutes, then adding 30 parts by weight of cytidylic acid, reacting for 3 hours, when the content of the cytidine triphosphate is more than 85%, immediately adjusting the pH value to 2 to stop the reaction, and simultaneously cooling with chilled water;
2) performing chromatographic purification, namely performing plate-and-frame filtration, chromatography, adsorption, washing, elution and sodium decoloration filtration on the phosphorylation product cooled in the step 1);
3) degrading, namely adding nucleotide degrading enzyme into the solution after the chromatographic purification in the step 2) at 37 ℃ for degradation, wherein the weight ratio of the addition amount of the nucleotide degrading enzyme to the phosphorylation product in the solution after the purification in the step 2) is 1:1, completing the reaction when the cytidine triphosphate accounts for less than 3 percent of the total cytidine, adjusting the pH value to 2 to terminate the reaction, and cooling the reaction solution to below 20 ℃;
4) and 3) sequentially carrying out plate-frame filtration, adsorption, washing, elution, decolorization and ultrafiltration nanofiltration on the solution cooled in the step 3) to obtain the cytidine diphosphate solution.
2. Preparation of creatinine diphosphate IDP solution
1) Slowly adding 30 wt% of sodium nitrite into ATP, reacting for 6 hours, adjusting the pH value to 1.2, and continuing to react for more than 40 hours until no dense smoke is generated, and terminating the reaction to obtain inosine triphosphate solution;
2) decoloring the creatinine triphosphate solution, namely adjusting the pH value of the inosine triphosphate solution prepared in the step 1) to be neutral, adding activated carbon for decoloring, and filtering to obtain the decolored creatinine triphosphate solution;
3) preparing a degrading enzyme solution, namely preparing a yeast solution with the concentration of 50%, adjusting the pH of the yeast solution to be 6, standing the yeast solution for 2 days at the temperature of 30 ℃, and filtering the yeast solution to obtain the degrading enzyme solution;
4) performing degradation reaction, namely mixing the decolorized creatinine triphosphoric acid solution obtained in the step 2) with the degradation enzyme solution prepared in the step 3), wherein the weight ratio of creatinine triphosphoric acid to degradation enzyme in the mixed solution is 13:1, then adding purified water, adjusting the concentration of the creatinine triphosphoric acid in the solution to be 2%, and adjusting the pH value of the solution to be neutral; adding glucose accounting for 20 percent of the weight of the creatinine triphosphate, and fully stirring; heating to 36-37 ℃ for enzymolysis, when the weight of creatinine triphosphate in the solution is less than 1% of the total cytidine, the reaction is terminated, and immediately adjusting the pH value of the solution to 2 by hydrochloric acid;
5) cooling the fermentation liquor after the reaction to 25 ℃, adding 2% of perlite powder, fully stirring, cooling the fermentation liquor to below 20 ℃, and filtering, carrying out column chromatography treatment, decoloring and ultrafiltering to obtain the creatinine diphosphoric acid solution.
3. Preparation of polycytidylic acid fine filter liquid by using cytidine diphosphate
Adding MgCl into the cytidine diphosphate solution prepared in the step 12Cytidine diphosphate and MgCl2The molar ratio of (A) to (B) is 10:1, and the pH of the mixed solution is adjusted to 7.0;
heating for polymerization, fully stirring at 38 ℃, adding solidified enzyme, wherein the solidified enzyme is polyglycoside phosphorylase, the adding amount of the solidified enzyme is 1.0 wt%, and continuously stirring for 2 hours after adding;
collecting reaction stock solution after polymerization reaction, filtering and cleaning to obtain polycytidylic acid fine filter solution;
4. preparation of polyinosinic acid fine filter liquid by using inosine diphosphate
Adding MgCl into inosine diphosphate solution prepared in step 22Inosine diphosphate and MgCl2The molar ratio of (A) to (B) is 10:1, and the pH of the mixed solution is adjusted to 7.0;
heating for polymerization, fully stirring at 38 ℃, adding solidified enzyme, wherein the solidified enzyme is polynucleotide phosphorylase, the adding amount of the solidified enzyme is 0.5 wt%, and continuously stirring for 2 hours after adding;
and collecting the reaction stock solution after the polymerization reaction, filtering and washing to obtain the polyinosinic acid fine filter solution.
5. Preparation of polyinosinic acid from polycytidylic acid refined filtrate and polyinosinic acid refined filtrate
Heating the fine filtrate of the polycytidylic acid prepared in the step 3, the fine filtrate of the polyinosinic acid prepared in the step 4 and a kanamycin sulfate stabilizer to 37 ℃;
then mixing the heated fine filtrate of the polyinosinic acid and the polycytidylic acid with a stabilizer, wherein the weight ratio of the polyinosinic acid to the polycytidylic acid to the stabilizer is 1:1:1, and stirring for 30min at the constant temperature of 37 ℃;
filtering the solution after stirring, and carrying out alcohol precipitation treatment on the filtrate by adopting ethanol, wherein the alcohol precipitation time is 1.0 h;
and (3) carrying out centrifugal separation on the material subjected to alcohol precipitation, soaking and dehydrating the centrifuged solid product by adopting ethanol, and carrying out vacuum drying to obtain a polyinosinic product.
Example 2
A preparation method of polyinosinic cells comprises the following steps:
1. preparation of Cytidine diphosphate CDP solution
1) Phosphorylation, putting 200 parts by weight of yeast into a phosphorylation reaction tank, stirring, heating in a water bath to 30 ℃, stopping heating, putting 2 parts by weight of magnesium chloride, 20 parts by weight of potassium dihydrogen phosphate and 20 parts by weight of white sugar, controlling the temperature to 31 ℃ for reaction for 10 minutes, then adding 30 parts by weight of cytidylic acid, reacting for 2 hours, when the content of the cytidine triphosphate is more than 85%, immediately adjusting the pH value to 2 to stop the reaction, and simultaneously cooling with chilled water;
2) performing chromatographic purification, namely performing plate-and-frame filtration, chromatography, adsorption, washing, elution and sodium decoloration filtration on the phosphorylation product cooled in the step 1);
3) degrading, namely adding nucleotide degrading enzyme into the solution after the chromatographic purification in the step 2) at 36 ℃ for degradation, wherein the weight ratio of the addition amount of the nucleotide degrading enzyme to the phosphorylation product in the solution after the purification in the step 2) is 1:1, completing the reaction when the cytidine triphosphate accounts for less than 3 percent of the total cytidine, adjusting the pH value to 2 to terminate the reaction, and cooling the reaction solution to below 20 ℃;
4) sequentially carrying out plate-frame filtration, adsorption, washing, elution, decolorization and ultrafiltration nanofiltration on the solution cooled in the step 3) to obtain a cytidine diphosphate solution;
2. preparation of inosine diphosphate IDP solution
Slowly adding 25 wt% of sodium nitrite into ATP, reacting for 6 hours, adjusting the pH value to 1.2, and continuing to react for more than 40 hours until no dense smoke is generated, and terminating the reaction to obtain inosine triphosphate solution;
2) decoloring the creatinine triphosphate solution, namely adjusting the pH value of the inosine triphosphate solution prepared in the step 1) to be neutral, adding activated carbon for decoloring, and filtering to obtain the decolored creatinine triphosphate solution;
3) preparing a degrading enzyme solution, namely preparing a yeast solution with the concentration of 50%, adjusting the pH of the yeast solution to be 6, standing the yeast solution for 2 days at the temperature of 30 ℃, and filtering the yeast solution to obtain the degrading enzyme solution;
4) performing degradation reaction, namely mixing the decolorized creatinine triphosphoric acid solution obtained in the step 2) with the degradation enzyme solution prepared in the step 3), wherein the weight ratio of creatinine triphosphoric acid to degradation enzyme in the mixed solution is 10:1, then adding purified water, adjusting the concentration of the creatinine triphosphoric acid in the solution to be 2%, and adjusting the pH value of the solution to be neutral; adding glucose accounting for 20 percent of the weight of the creatinine triphosphate, and fully stirring; heating to 36-37 ℃ for enzymolysis, when the weight of creatinine triphosphate in the solution is less than 1% of the total cytidine, the reaction is terminated, and immediately adjusting the pH value of the solution to 2 by hydrochloric acid;
5) cooling the fermentation liquor after the reaction to 25 ℃, adding 2% of perlite powder, fully stirring, cooling the fermentation liquor to below 20 ℃, and filtering, carrying out column chromatography treatment, decoloring and ultrafiltering to obtain the creatinine diphosphoric acid solution.
3. Preparation of polycytidylic acid fine filter liquid by using cytidine diphosphate
Adding MgCl into the cytidine diphosphate solution prepared in the step 12Cytidine diphosphate and MgCl2The molar ratio of (A) to (B) is 10:1, and the pH of the mixed solution is adjusted to 7.0;
heating for polymerization, fully stirring at 38 ℃, adding solidified enzyme, wherein the solidified enzyme is polyglycoside phosphorylase, the adding amount of the solidified enzyme is 1.5 wt%, and continuously stirring for 2 hours after adding;
collecting reaction stock solution after polymerization reaction, filtering and cleaning to obtain polycytidylic acid fine filter solution;
4. preparation of polyinosinic acid fine filter liquid by using inosine diphosphate
Adding MgCl into inosine diphosphate solution prepared in step 22Inosine diphosphate and MgCl2The molar ratio of (A) to (B) is 10:1, and the pH of the mixed solution is adjusted to 7.0;
heating for polymerization, fully stirring at 38 ℃, adding solidified enzyme, wherein the solidified enzyme is polyglycoside phosphorylase, the adding amount of the solidified enzyme is 1.0 wt%, and continuously stirring for 2 hours after adding;
collecting reaction stock solution after polymerization reaction, filtering and washing to obtain polyinosinic acid fine filter solution
5. Preparation of polyinosinic acid from polycytidylic acid refined filtrate and polyinosinic acid refined filtrate
Heating the fine filtrate of the polycytidylic acid prepared in the step 3, the fine filtrate of the polyinosinic acid prepared in the step 4 and the polylysine stabilizer to 37 ℃;
then mixing the heated fine filtrate of the polyinosinic acid and the polycytidylic acid with a stabilizer, wherein the weight ratio of the polyinosinic acid to the polycytidylic acid to the stabilizer is 1:1:1, and stirring for 30min at the constant temperature of 37 ℃;
filtering the solution after stirring, and carrying out alcohol precipitation treatment on the filtrate by adopting ethanol, wherein the alcohol precipitation time is 1.5 h;
and (3) carrying out centrifugal separation on the material subjected to alcohol precipitation, soaking and dehydrating the centrifuged solid product by adopting ethanol, and carrying out vacuum drying to obtain a polyinosinic product.
Example 3
A preparation method of polyinosinic cells comprises the following steps:
1. preparation of Cytidine diphosphate CDP solution
1) Phosphorylation, putting 500 parts by weight of yeast into a phosphorylation reaction tank, stirring, heating in a water bath to 31 ℃, stopping heating, adding 10 parts by weight of magnesium chloride, 50 parts by weight of potassium dihydrogen phosphate and 50 parts by weight of white sugar, controlling the temperature to be 33 ℃ for reaction for 20 minutes, then adding 30 parts by weight of cytidylic acid, reacting for 5 hours, finishing the reaction when the content of the cytidine triphosphate is more than 85%, immediately adjusting the pH value to 2 to stop the reaction, and simultaneously cooling with chilled water;
2) performing chromatographic purification, namely performing plate-and-frame filtration, chromatography, adsorption, washing, elution and sodium decoloration filtration on the phosphorylation product cooled in the step 1);
3) degrading, namely adding nucleotide degrading enzyme into the solution after the chromatographic purification in the step 2) at 36 ℃ for degradation, wherein the weight ratio of the addition amount of the nucleotide degrading enzyme to the phosphorylation product in the solution after the purification in the step 2) is 0.5:1, when the cytidine triphosphate accounts for less than 3 percent of the total cytidine, the reaction is finished, adjusting the pH value to 2 to terminate the reaction, and simultaneously cooling the reaction solution to below 20 ℃;
4) sequentially carrying out plate-frame filtration, adsorption, washing, elution, decolorization and ultrafiltration nanofiltration on the solution cooled in the step 3) to obtain a cytidine diphosphate solution;
2. preparation of inosine diphosphate IDP solution
1) Slowly adding 35 wt% of sodium nitrite into ATP, reacting for 6 hours, adjusting the pH value to 1.8, and continuing to react for more than 40 hours until no dense smoke is generated, and stopping the reaction to obtain inosine triphosphate solution;
2) decoloring the creatinine triphosphate solution, namely adjusting the pH value of the inosine triphosphate solution prepared in the step 1) to be neutral, adding activated carbon for decoloring, and filtering to obtain the decolored creatinine triphosphate solution;
3) preparing a degrading enzyme solution, namely preparing a yeast solution with the concentration of 50%, adjusting the pH of the yeast solution to be 6, standing the yeast solution for 2 days at the temperature of 30 ℃, and filtering the yeast solution to obtain the degrading enzyme solution;
4) performing degradation reaction, namely mixing the decolorized creatinine triphosphate solution obtained in the step 2) with the degradation enzyme solution prepared in the step 3), wherein the weight ratio of creatinine triphosphate to degradation enzyme in the mixed solution is 15:1, adding purified water, adjusting the concentration of creatinine triphosphate in the solution to 3%, and adjusting the pH value of the solution to be neutral; adding glucose accounting for 20 percent of the weight of the creatinine triphosphate, and fully stirring; heating to 36-37 ℃ for enzymolysis, when the weight of creatinine triphosphate in the solution is less than 1% of the total cytidine, the reaction is terminated, and immediately adjusting the pH value of the solution to 2 by hydrochloric acid;
5) cooling the fermentation liquor after the reaction to 25 ℃, adding 2% of perlite powder, fully stirring, cooling the fermentation liquor to below 20 ℃, and filtering, carrying out column chromatography treatment, decoloring and ultrafiltering to obtain the creatinine diphosphoric acid solution. .
3. Preparation of polycytidylic acid fine filter liquid by using cytidine diphosphate
Adding MgCl into the cytidine diphosphate solution prepared in the step 12Cytidine diphosphate and MgCl2The molar ratio of (A) to (B) is 10:1, and the pH of the mixed solution is adjusted to 7.0;
heating for polymerization, fully stirring at 38 ℃, adding solidified enzyme, wherein the solidified enzyme is polynucleotide phosphorylase, the adding amount of the solidified enzyme is 0.8 wt%, and continuously stirring for 2 hours after adding;
collecting reaction stock solution after polymerization reaction, filtering and cleaning to obtain polycytidylic acid fine filter solution;
4. preparation of polyinosinic acid fine filter liquid by using inosine diphosphate
Adding MgCl into inosine diphosphate solution prepared in step 22Inosine diphosphate and MgCl2The molar ratio of (A) to (B) is 10:1, and the pH of the mixed solution is adjusted to 7.0;
heating for polymerization, fully stirring at 38 ℃, adding solidified enzyme, wherein the solidified enzyme is polyglycoside phosphorylase, the adding amount of the solidified enzyme is 1.2 wt%, and continuously stirring for 2 hours after adding;
collecting reaction stock solution after polymerization reaction, filtering and washing to obtain polyinosinic acid fine filter solution
5. Preparation of polyinosinic acid from polycytidylic acid refined filtrate and polyinosinic acid refined filtrate
Heating the fine filtrate of the polycytidylic acid prepared in the step 3, the fine filtrate of the polyinosinic acid prepared in the step 4 and a calcium gluconate stabilizer to 37 ℃;
then mixing the heated fine filtrate of the polyinosinic acid and the polycytidylic acid with a stabilizer, wherein the weight ratio of the polyinosinic acid to the polycytidylic acid to the stabilizer is 1:1:1, and stirring for 30min at the constant temperature of 37 ℃;
filtering the solution after stirring, and carrying out alcohol precipitation treatment on the filtrate by adopting ethanol, wherein the alcohol precipitation time is 0.8 h;
and (3) carrying out centrifugal separation on the material subjected to alcohol precipitation, soaking and dehydrating the centrifuged solid product by adopting ethanol, and carrying out vacuum drying to obtain a polyinosinic product.
When XRD detection is carried out on the polyinosinic product prepared in the embodiments 1-3, the three products have the strongest absorption peak at 265.5nm and the smallest absorption peak at 229.5nm, and market requirements are met.
Meanwhile, the polyinosinic cell product prepared by the invention is placed for 6-12 months, the hyperchromic effect is not obviously changed, and the polyinosinic cell product has stronger stability.
The principles and embodiments of the present invention have been described herein using specific examples, which are provided only to help understand the method and the core concept of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, the specific embodiments and the application range may be changed. In view of the above, the present disclosure should not be construed as limiting the invention.
Claims (4)
1. A preparation method of polyinosinic cells is characterized by comprising the following steps:
(1) heating the fine filtrate of polyinosinic acid and polycytidylic acid and the stabilizing agent to 37 ℃;
(2) then mixing the heated fine filtrate of the polyinosinic acid and the polycytidylic acid with a stabilizer, wherein the weight ratio of the polyinosinic acid to the polycytidylic acid to the stabilizer is 1:1:1, and stirring for 30min at the constant temperature of 37 ℃;
(3) filtering the solution after stirring, and carrying out alcohol precipitation treatment on the filtrate by adopting ethanol, wherein the alcohol precipitation time is 0.5-1.5 h;
(4) centrifuging the precipitated material, soaking the centrifuged solid product in ethanol for dehydration, vacuum drying to obtain polyinosinic-polycytidylic acid product,
the polyinosinic acid fine filter liquid is prepared by adding MgCl into inosine diphosphate solution2Inosine diphosphate and MgCl2The molar ratio of (A) to (B) is 10:1, and the pH of the mixed solution is adjusted to 7.0; heating for polymerization, fully stirring at 38 ℃, adding curing enzyme, and continuously stirring for 2 hours; collecting reaction stock solution after polymerization reaction, filtering and washing to obtain polyinosinic acid fine filter solution;
the preparation method of the inosine diphosphate solution comprises the following steps:
1) deaminating ATP to obtain inosine triphosphate solution;
2) decoloring creatinine triphosphate solution;
3) preparing degrading enzyme solution, preparing 50% yeast solution, adjusting pH to 6, standing at 30 deg.C for 2 days,
filtering to obtain a degrading enzyme solution;
4) performing degradation reaction, namely mixing the decolorized creatinine triphosphoric acid solution obtained in the step 2) with the degradation enzyme solution prepared in the step 3), wherein the weight ratio of creatinine triphosphoric acid to degradation enzyme in the mixed solution is 10-15:1, then adding purified water, adjusting the concentration of the creatinine triphosphoric acid in the solution to be 2-3%, and adjusting the pH value of the solution to be neutral; adding glucose accounting for 20 percent of the weight of the creatinine triphosphate, and fully stirring; heating to 36-37 ℃ for enzymolysis, when the weight of creatinine triphosphate in the solution is less than 1% of the total cytidine, the reaction is terminated, and immediately adjusting the pH value of the solution to 2 by hydrochloric acid;
5) cooling the fermentation liquor after the reaction to 25 ℃, adding 2% of perlite powder, fully stirring, cooling the fermentation liquor to below 20 ℃, and filtering, carrying out column chromatography treatment, decoloring and ultrafiltering to obtain a creatinine diphosphoric acid solution;
the fine filtration of polycytidylic acid is to add MgCl into cytidine diphosphate solution2Cytidine diphosphate and MgCl2The molar ratio of (A) to (B) is 10:1, and the pH of the mixed solution is adjusted to 7.0; heating for polymerization, fully stirring at 38 ℃, adding curing enzyme, and continuously stirring for 2 hours; collecting reaction stock solution after polymerization reaction, filtering and cleaning to obtain polycytidylic acid fine filter solution,
the preparation method of the cytidine diphosphate solution comprises the following steps:
1) phosphorylation, putting 200-500 parts by weight of yeast into a phosphorylation reaction tank, stirring, heating in a water bath to 30-31 ℃, stopping heating, putting 2-10 parts by weight of magnesium chloride, 20-50 parts by weight of potassium dihydrogen phosphate and 20-50 parts by weight of white sugar, controlling the temperature to 31-33 ℃, reacting for 10-20 minutes, then adding 30 parts by weight of cytidine triphosphate, reacting for 2-5 hours, when the content of cytidine triphosphate is more than 85%, immediately adjusting the pH value to 2 to stop the reaction, and cooling with chilled water;
2) performing chromatographic purification, namely performing plate-and-frame filtration, chromatography, adsorption, washing, elution, decolorization and nanofiltration on the cooled phosphorylation product obtained in the step 1);
3) degrading, namely adding nucleotide degrading enzyme into the solution subjected to chromatographic purification in the step 2) at 36-37 ℃ for degradation, adjusting the pH to 2 to stop the reaction when the cytidine triphosphate accounts for less than 3% of the total cytidine, and cooling the reaction solution to below 20 ℃;
4) and 3) sequentially carrying out plate-frame filtration, adsorption, washing, elution, decolorization and ultrafiltration nanofiltration on the solution cooled in the step 3) to obtain the cytidine diphosphate solution.
2. The method for preparing polyinosinic-polycytidylic acid according to claim 1, wherein the stabilizer is one or more of kanamycin sulfate, polylysine and calcium gluconate.
3. The method for preparing polyinosinic-polycytidylic acid according to claim 1, wherein the immobilized enzyme is a polyglycoside phosphorylase added in an amount of 0.5-1.5 wt%.
4. The method for preparing polyinosinic-polycytidylic acid according to claim 1, wherein the weight ratio of the nucleotide degrading enzyme to the phosphorylated product in the solution after purification in step 2) is 0.5-2: 1.
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| CN103599071A (en) * | 2013-11-08 | 2014-02-26 | 杭州美亚药业有限公司 | Preparation method of polyinosinic-polycytidylic acid dry powder |
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