Preparation and verification of interferon

Preparation and verification of interferon

(1) Principle Dry resistance is a type of highly active, multifunctional protein produced by interferon inducing agents acting on relevant biological cells. After it is produced and released from the cells, it acts on the corresponding other cells of the same kind, so that it can obtain anti-virus and anti-tumor immunity.

The so-called interferon inducer refers to a class of substances that can induce the production of interferon by the relevant biological cells. Those that can induce related biological cells to produce alpha and beta interferons are called class A interferon inducers, such as various animal viruses, intracellular parasitic microorganisms, etc .; those that can induce T cells to produce interferon gamma are called class B interferon Raw materials, such as lipopolysaccharide, streptococcal toxin, enterotoxin A, etc.

In actual work, two methods are used to prepare interferon. One is to use some interferon inducing agents to induce certain biological cells to produce interferon, which can be used after extraction, purification and verification. The cells used in this method are mostly peripheral blood leukocytes. The second is to use genetic engineering method for production, that is, the interferon gene is introduced into E. coli, and the interferon is produced by cultivating E. coli. Currently, large-scale production of interferon mainly uses genetic engineering methods. The following describes the preparation method of interferon only by using interferon inducing agent to prepare human leukocyte interferon as an example.

(2) Materials and methods

1 Material cell culture equipment (such as culture flask, multi-well culture plate, incubator, microscope, rotary incubator, etc.), water bath box, etc.

2 Method

(1) Preparation of inducing agent: using the attenuated strain of NDVF series, stored in chicken embryo allantoic fluid at -20 ℃,

The hemagglutination titer is stable between 1: 640 and 1: 1280. During mass reproduction, dilute 100-1000 times with 05% hydrolyzed milk protein, inoculate the allantoic cavity of 9-day-old chicken embryos, and incubate at 37 ℃ for 72h. Harvest allantoic fluid. Bacterial inspection should be qualified.

(2) Preparation of induced cells: Aseptically take human peripheral blood (multi-purpose human umbilical cord blood, or blood stored in a blood bank), place in a sterile bottle containing heparin, and store at 4 ℃ for no more than 24h. It is not extracted separately and replaced with whole blood.

(3) Preparation of crude interferon:

â‘  Add inducing agent, add 1ml of anticoagulant whole blood and 02ml of inducing agent (that is, NDVF allantoic fluid, its blood clot titer is not less than 1: 640);

② Warm the adsorption, place the anticoagulant whole blood added with the inducing agent in the 37 ° C water bath for 1h, shake it every 15min to make the NDVF adsorb on the leukocytes. Then centrifuge at 1000r / min for 20min, discard the supernatant and leave the sediment;

③ Add nutrient solution to incubate and induce, add Eagle nutrient solution to the above sediment in the amount of 1 to 2 times of anticoagulated whole blood, mix well, and place in a 35-36 ° C incubator for 18-20 hours;

④ Centrifuge and acid treatment, centrifuge the above culture at 2000r / min for 30min, take the supernatant, adjust its pH value to 20 with 6mol / L hydrochloric acid, put it in 4 ℃ refrigerator for 5 days to inactivate NDV;

⑤ Neutralization. After acidification for 5 days, adjust the pH to 72-74 with 6mol / L sodium hydroxide, which is crude interferon.

(4) Preparation of high-quality interferon:

â‘ KCNS precipitation, take the above crude interferon, add KCNS and adjust the pH to 35 with 2mol / L HCl, then centrifuge at 2 000r / min for 30min to take the precipitation;

②Alcohol extraction, dissolve the precipitate in 95% alcohol (pre-cooled to -20 ℃), adjust the pH to 42 with 2mol / L NaOH, centrifuge at 2 000r / min for 30min to take the supernatant; adjust the pH with 2mol / L HCl To 35, take the supernatant after centrifugation, then adjust the pH to 5.6, take the supernatant after centrifugation, and finally adjust the pH to 71, and take the precipitate after centrifugation;

③Precipitation of sodium periodate, dissolve the precipitate in PBS, add sodium periodate, adjust the pH to 4.5, dilute with 50% ethanol 10 times, take the supernatant after centrifugation, adjust the supernatant to 03mol / L ( NH4) 2CO3 (pH 76) was dialyzed overnight at 4 ° C.

Sephacryl S200 column chromatography: Sephacryl S200 is treated as required and packed into a column (4-5 × 100 cm column). After equilibration with PBS, the sample is added (ie, the above supernatant) and eluted with the washing solution. During the elution, continuous detection with a nucleic acid protein analyzer, collecting the corresponding peak is the refined interferon. Sampling for potency determination, dilution according to the results, sub-packaging and lyophilization.

(3) Verification

1 Determination of potency

(1) Preparation of attack virus: after vesicular stomatitis virus (VSV) is passaged on chicken embryo fibroblasts,

Then upload it to pig cells (IBRS) for 3 to 5 generations, so that it has a good pathogenic effect on IBRS, and its TCID50 should be stable (generally between 10-6 and 10-7).

(2) Prepare to measure cells: well-grown young IBRS monolayer cells.

(3) Determination: Take the above monolayer cells into several groups, add different dilutions of interferon to each group, and incubate at 37 ℃ for 20-24h

Then, each tube was challenged with 100 VSV of TCID50. After incubating at 37 ℃ for 48-72h, the results were observed. At the same time set up a cell control group and a virus control group. Virus control group CPE> 75%, normal cell control group CPE = 0, that is, the determination system is considered effective. The interferon criterion is based on the reciprocal of the highest dilution of interferon that can protect half of the cells from attacking viruses as the unit of interferon.

2 Determination of pH value Take 10 of this product, dissolve it with water, and accurately measure the pH value should be 6 ~ 75.

3 Moisture is measured according to the sulfur-sulfur solution method, which shall not exceed 3%.

4 Safety test Dissolve this product with water, and inject it into the tail vein of the mouse. There shall be no death within 48 hours.

5 Pyrogen check Take 1 bottle of this product, add water to dissolve it, check according to law, and it should meet the regulations.

6 Take 3 strains of this product, dissolve them in sterile water, and inoculate them separately into the culture medium for aerobic bacteria, anaerobic bacteria and molds. Incubate at 37 ℃ for 1 week, and they should be grown aseptically.

7 Hypersensitivity reaction Take 6 healthy guinea pigs and inject an appropriate amount of this product intraperitoneally for 3 consecutive times. After 20 days, inject the appropriate amount of this product into the ear snorkeling. There should be no allergic reactions.

(Four) matters needing attention

1 When preparing the NDVF series weakly toxic inducer, the seed poison should pass the sterility inspection and the titer should be above 1: 640. When collecting the poison, the contaminated chicken embryo should be discarded.

2 It is not necessary to extract white blood cells from the blood, because red blood cells have a nutritional effect on the production of interferon by white blood cells. The purified leukocytes produce interferon titers not necessarily high.

3 The purpose of acidification is to kill the inducing agent NDV, and at a pH of 20, the interferon is stable.

4 At normal temperature, the half-life of interferon is very short. Therefore, various operations should be carried out in a low-temperature environment, the action should be rapid, and the reagents used for purification should be pre-cooled. Interferon crude products and fine products should be stored at a low temperature in time. When determining the potency, the interferon should be dissolved immediately before use.