阅读说明：本技术 一种干扰素的制备方法 (Preparation method of interferon ) 是由 李冬冬 訾红彦 于 2020-12-25 设计创作，主要内容包括：本发明公开了一种干扰素的制备方法,包括以下步骤：步骤1、分离灰黄层、步骤2、氯化铵处理、步骤3、起动诱生、步骤4、正式诱生、步骤5、收获、步骤6、纯化。该一种干扰素的制备方法一次制备量大,回收率高于60％,经济,简便,易于普及,效价,比活2.2×10°u/mg蛋白,PIFN-A中干扰素含量占回收干扰素的82％,比活也比较高,IIFN的比活较低(5×10’u/mg蛋白)。(The invention discloses a preparation method of interferon, which comprises the following steps: step 1, separating a lime-yellow layer, step 2, treating ammonium chloride, step 3, starting induction, step 4, formal induction, step 5, harvesting, step 6 and purifying. The interferon preparation method has advantages of large preparation amount at one time, high recovery rate higher than 60%, economy, simplicity, easy popularization, high potency, specific activity of 2.2 × 10 u/mg protein, high specific activity of PIFN-A (interferon-alphA) 82%, and low specific activity of IIFN (5 × 10' u/mg protein).)

1. A method for preparing interferon is characterized in that: the method comprises the following steps:

step 1, separating a lime-yellow layer: collecting 100ml blood into a plastic bag containing anticoagulant, centrifuging, separating blood plasma, sucking the lime-yellow layer, and placing the sucked lime-yellow layer in a refrigerator at 4 ℃ for 8-12 hours;

step 2, ammonium chloride treatment: adding 30ml of buffer saline into each gray yellow layer, adding 9 times of cold 0.83% ammonium chloride solution, mixing, standing at 40 deg.C for 10min, centrifuging at 4 deg.C (8,000 rpm) for 20min, discarding hemolysis supernatant, adding appropriate amount of buffer saline, collecting precipitated cells, making into suspension, treating with 9 times of 0.83% ammonium chloride solution once again, dissolving residual erythrocytes, taking precipitated leukocytes, suspending in culture solution, placing in ice bath, sampling for viable cell counting, and diluting with pre-warmed culture solution to contain 107 viable cells per ml;

step 3, starting induction: adding leukocyte interferon into the diluted cell suspension to make the final concentration of the leukocyte interferon 100u/ml, and stirring and culturing in 37 ℃ water bath for 2 h;

step 4, formal induction: adding Sendai virus into the activated leukocytes to make the final concentration of the Sendai virus be 100-;

and 5, harvesting: centrifuging the culture (2, 500r/min) for 30min, sucking the supernatant to obtain crude interferon, sampling for aseptic test and determining titer, wherein each buffy coat can be used for preparing 100 ten thousand units of purified interferon;

and 6, purification: adding potassium thiocyanate into crude human leukocyte interferon to 0.5mol L, adjusting pH to 3-53.5 with 2mol L hydrochloric acid, centrifuging to remove supernatant, adding 1/5 amount of 90-96% cold ethanol into precipitate A, centrifuging to remove precipitate, adjusting pH to 5.5 with hydrochloric acid into supernatant A, centrifuging to remove precipitate, adjusting pH to 5.8, centrifuging to obtain supernatant B and precipitate B, adding 1/50 amount of glycine-hydrochloric acid buffer solution into precipitate B, dissolving, and detecting to obtain interferon.

2. The method of claim 1, wherein the interferon is prepared by: in the step 1, the anticoagulant is one of sodium citrate, heparin and ethylene diamine tetraacetic acid, 13-15ml of 400ml blood is sucked, and the volume of the blood is about 40-50% of the cells in the blood.

3. The method of claim 1, wherein the interferon is prepared by: and 6, adding hydrochloric acid into the supernatant B to reduce the pH value to 3, centrifuging to obtain A precipitate C, adding pH8 with the original volume of 1/5,500 and 0.1mol/L PBS to dissolve the precipitate C, adding NaOH to adjust the pH of 7-7.5, dialyzing the PBS, standing for 12 hours, centrifuging, collecting the supernatant, and detecting to obtain the PIEN-A.

4. The method of claim 1, wherein the interferon is prepared by: and 6, regulating the pH value of the supernatant B to 8, centrifuging to remove the supernatant to obtain a precipitate D, adding 0.1mol/LPBSO.5mol/L potassium thiocyanate of the original volume of 1/50 into the precipitate D for dissolution, reducing the pH to 5.2, centrifuging to obtain a supernatant C and a precipitate E, adding 1/2 of the original volume of the precipitate E, dissolving with 500 of pH8 and 0.1mol L PBS, regulating the pH to 7-7.5, dialyzing the PBS, standing for 12 hours, centrifuging, collecting the supernatant, and detecting to obtain the PIFN-B.

5. The method of claim 1, wherein the interferon is prepared by: the basic component of the culture solution in the step 2 is Eagle's culture medium which contains 4-6% of human plasma protein, no phosphate, 3mg/ml of Tricine and a proper amount of antibiotics.

6. The method of claim 1, wherein the interferon is prepared by: culturing Sendai virus in step 4 in 10-day-old chick embryo for 48-72h, and harvesting allantoic fluid.

7. The method of claim 1, wherein the interferon is prepared by: the titer-determining material in step 5 was assayed after 24h treatment at pH 2.

8. The method of claim 1, wherein the interferon is prepared by: the pH regulator in step 6 is hydrochloric acid.

9. The method of claim 1, wherein the interferon is prepared by: the pH of the glycine-hydrochloric acid buffer in step 6 was 2.

Technical Field

The invention relates to the technical field of interferon production, in particular to a preparation method of interferon.

Background

Interferons are glycoproteins with high species specificity, so that animal interferons are ineffective to humans, and have antiviral, cytostatic, immunomodulating and antitumor effects.

The interferon is mainly used for treating advanced hairy cell leukemia, renal carcinoma, melanoma, Kaposi sarcoma, chronic myelogenous leukemia and low-grade non-Hodgkin lymphoma, and other tumors such as osteosarcoma, breast cancer, multiple myeloma, head and neck cancer and bladder cancer, and is also suitable for acute and chronic viral hepatitis C and chronic active hepatitis B.

The existing preparation method of interferon has small preparation amount at one time, and therefore, a preparation method of interferon is provided.

Disclosure of Invention

The invention aims to provide a preparation method of interferon, which solves the problems.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention provides a preparation method of interferon, which comprises the following steps:

step 1, separating a lime-yellow layer: collecting 100ml blood into a plastic bag containing anticoagulant, centrifuging, separating blood plasma, sucking the lime-yellow layer, and placing the sucked lime-yellow layer in a refrigerator at 4 ℃ for 8-12 hours;

step 2, ammonium chloride treatment: adding 30ml of buffer saline into each gray yellow layer, adding 9 times of cold 0.83% ammonium chloride solution, mixing, standing at 40 deg.C for 10min, centrifuging at 4 deg.C (8,000 rpm) for 20min, discarding hemolysis supernatant, adding appropriate amount of buffer saline, collecting precipitated cells, making into suspension, treating with 9 times of 0.83% ammonium chloride solution once again, dissolving residual erythrocytes, taking precipitated leukocytes, suspending in culture solution, placing in ice bath, sampling for viable cell counting, and diluting with pre-warmed culture solution to contain 107 viable cells per ml;

step 3, starting induction: adding leukocyte interferon into the diluted cell suspension to make the final concentration of the leukocyte interferon 100u/ml, and stirring and culturing in 37 ℃ water bath for 2 h;

step 4, formal induction: adding Sendai virus into the activated leukocytes to make the final concentration of the Sendai virus be 100-;

and 5, harvesting: centrifuging the culture (2, 500r/min) for 30min, sucking the supernatant to obtain crude interferon, sampling for aseptic test and determining titer, wherein each buffy coat can be used for preparing 100 ten thousand units of purified interferon;

and 6, purification: adding potassium thiocyanate into crude human leukocyte interferon to 0.5mol L, adjusting pH to 3-53.5 with 2mol L hydrochloric acid, centrifuging to remove supernatant, adding 1/5 amount of 90-96% cold ethanol into precipitate A, centrifuging to remove precipitate, adjusting pH to 5.5 with hydrochloric acid into supernatant A, centrifuging to remove precipitate, adjusting pH to 5.8, centrifuging to obtain supernatant B and precipitate B, adding 1/50 amount of glycine-hydrochloric acid buffer solution into precipitate B, dissolving, and detecting to obtain interferon.

Preferably, the anticoagulant in step 1 is one of sodium citrate, heparin and ethylene diamine tetraacetic acid, and 13-15ml of 400ml blood is sucked, and is about 40-50% of cells in the blood.

Preferably, hydrochloric acid is added into the supernatant B in the step 6 to reduce the pH value to 3, the mixture is centrifuged to obtain A precipitate C, the precipitate C is added with 1/5,500 of the original volume of pH8 and 0.1mol/L of PBS for dissolution, NaOH is added to adjust the pH value to be 7-7.5, the PBS is dialyzed, the mixture is kept stand for 12 hours and centrifuged, and the supernatant is collected and detected to obtain the PIEN-A.

Preferably, in the step 6, the pH of the supernatant B is adjusted to 8, the supernatant B is centrifuged to be discarded to obtain a precipitate D, 0.1mol/LPBSO.5mol/L potassium thiocyanate in the original volume of 1/50 is added into the precipitate D to be dissolved, the pH is reduced to 5.2, the precipitate D is centrifuged to obtain a supernatant C and a precipitate E, the precipitate E is dissolved by 1/2 in the original volume of 3526, 500 amounts of pH8 and 0.1mol L PBS to be adjusted to pH7-7.5, the mixture is dialyzed against the PBS, the mixture is stood for 12 hours, centrifuged, the supernatant is collected, and the PIFN-B is detected to obtain the PIFN-B.

Preferably, the basic component of the culture solution in step 2 is Eagle's medium, which contains 4-6% of human plasma protein, no phosphate, 3mg/ml Tricine and appropriate amount of antibiotics.

Preferably, the Sendai virus in step 4 is cultured in 10-day-old chick embryos for 48-72h, and allantoic fluid is harvested.

Preferably, the titer-determining material of step 5 is assayed after 24h treatment at pH 2.

Preferably, the pH regulator in step 6 is hydrochloric acid.

Preferably, the pH of the glycine-hydrochloric acid buffer in step 6 is 2.

The invention has the beneficial effects that: the invention has large one-time preparation amount, the recovery rate is higher than 60%, the method is economic, simple and convenient, the popularization is easy, the titer is 2.2 multiplied by 10 degrees u/mg protein, the content of interferon in the PIFN-A accounts for 82 percent of the recovered interferon, the specific activity is higher, and the specific activity of IIFN is lower (5 multiplied by 10' u/mg protein).

Detailed Description

The following further describes embodiments of the present invention with reference to examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Step 1, separating a lime-yellow layer: collecting 100ml blood into a plastic bag containing anticoagulant, centrifuging, separating blood plasma to absorb a lime-yellow layer, and placing the absorbed lime-yellow layer in a refrigerator at 4 ℃ for 8 hours;

step 2, ammonium chloride treatment: adding 30ml of buffer saline into each gray yellow layer, adding 9 times of cold 0.83% ammonium chloride solution, mixing, standing at 40 deg.C for 10min, centrifuging at 4 deg.C (8,000 rpm) for 20min, discarding hemolysis supernatant, adding appropriate amount of buffer saline, collecting precipitated cells, making into suspension, treating with 9 times of 0.83% ammonium chloride solution once again, dissolving residual erythrocytes, taking precipitated leukocytes, suspending in culture solution, placing in ice bath, sampling for viable cell counting, and diluting with pre-warmed culture solution to contain 107 viable cells per ml;

step 3, starting induction: adding leukocyte interferon into the diluted cell suspension to make the final concentration of the leukocyte interferon 100u/ml, and stirring and culturing in 37 ℃ water bath for 2 h;

step 4, formal induction: the Sendai virus is added into the started white blood cells, the final concentration is 100-;

and 5, harvesting: centrifuging the culture (2, 500r/min) for 30min, sucking the supernatant to obtain crude interferon, sampling for aseptic test and determining titer, wherein each buffy coat can be used for preparing 100 ten thousand units of purified interferon;

and 6, purification: adding potassium thiocyanate into crude human leukocyte interferon to 0.5mol L, adjusting pH to 3-53.5 with 2mol L hydrochloric acid, centrifuging to remove supernatant, adding 1/5 amount of 90-96% cold ethanol into precipitate A, centrifuging to remove precipitate, adjusting pH to 5.5 with hydrochloric acid into supernatant A, centrifuging to remove precipitate, adjusting pH to 5.8, centrifuging to obtain supernatant B and precipitate B, adding 1/50 amount of glycine-hydrochloric acid buffer solution into precipitate B, dissolving, and detecting to obtain interferon.

And 6, adding hydrochloric acid into the supernatant B to reduce the pH value to 3, centrifuging to obtain A precipitate C, adding pH8 with the original volume of 1/5,500 and 0.1mol/L PBS to dissolve the precipitate C, adding NaOH to adjust the pH of 7-7.5, dialyzing the PBS, standing for 12 hours, centrifuging, collecting the supernatant, and detecting to obtain the PIEN-A with the interferon content of 82%.

And 6, regulating the pH value of the supernatant B to 8, centrifuging to remove the supernatant to obtain a precipitate D, adding 0.1mol/LPBSO.5mol/L potassium thiocyanate of the original volume of 1/50 into the precipitate D for dissolution, reducing the pH to 5.2, centrifuging to obtain a supernatant C and a precipitate E, adding 1/2 of the precipitate E, dissolving 500 of pH8 and 0.1mol L PBS into the precipitate E, regulating the pH to 7-7.5, dialyzing the PBS, standing for 12 hours, centrifuging, collecting the supernatant, and detecting to obtain the PIFN-B with the interferon content of PIFN-B.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.