Experimental principle Stem resistance is a kind of highly active, multifunctional protein produced by interferon inducing agent 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 interferons. 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.
Experimental equipment Cell culture equipment (such as culture flask, multi-well culture plate, incubator, microscope, rotary incubator, etc.), water bath box, etc.
Experimental procedure 1. Preparation of inducing agent: using the attenuated strain of NDVF strain, stored in chicken embryo allantoic fluid at -20 ℃,
The hemagglutination titer is stable between 1: 640 ～ 1: 1 280. During mass reproduction, dilute 100-1,000 times with 0.5% hydrolyzed milk protein and inoculate the allantoic cavity of 9-day-old chicken embryos. After incubating at 37 ° C for 72h, harvest allantoic fluid. The titer should be greater than 1: 640. Bacterial inspection should be qualified.
2. Preparation of induced cells: Aseptically take human peripheral blood (multi-purpose human umbilical cord blood, or blood bank storage blood), place in a sterile bottle containing heparin, store at 4 ℃ for no more than 24h, the induced cells (leukocytes) are not Extracted separately and replaced with whole blood.
3. Preparation of crude interferon:
1) Add inducing agent, add 1ml of anticoagulant whole blood plus 0.2ml of inducing agent (that is, NDVF allantoic fluid, its blood clot titer is not less than 1: 640);
2) Adsorption by heating, place anticoagulant whole blood added with inducing agent in a 37 ° C water bath for 1 hour, shaking once every 15 minutes to make NDVF adsorb on 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 an amount of 1 to 2 times of anticoagulated whole blood, mix well, and place it in a 35-36 ° C incubator for 18-20 hours;
4) Centrifugation and acid treatment, the above culture was centrifuged at 2000r / min for 30min, the supernatant was taken, and its pH value was adjusted to 2.0 with 6mol / L hydrochloric acid, and the NDV was inactivated for 5 days in a 4 ° C refrigerator;
5) Neutralization. After acidification for 5 days, adjust the pH to 7.2 to 7.4 with 6 mol / L sodium hydroxide, which is crude interferon.
4. Preparation of high-quality interferon:
1) KCNS precipitation, take the above crude interferon, add KCNS and adjust the pH value to 3.5 with 2mol / L HCl, then centrifuge at 2 000r / min for 30min to take the precipitate;
2) Alcohol extraction, dissolve the precipitate in 95% alcohol (pre-cooled to -20 ℃), adjust the pH to 4.2 with 2mol / L NaOH, centrifuge at 2 000r / min for 30min to take the supernatant; adjust the pH with 2mol / L HCl Value to 3.5, take the supernatant after centrifugation, then adjust the pH to 5.6, take the supernatant after centrifugation, and finally adjust the pH to 7.1, take the precipitate after centrifugation;
3) Precipitate with sodium periodate, dissolve the precipitate in PBS, add sodium periodate, adjust pH to 4.5, dilute with 50% ethanol 10 times, take the supernatant after centrifugation, adjust the supernatant to 0.3mol / L (NH4) 2CO3 (pH 7.6) was dialyzed overnight at 4 ° C.
4) Sephacryl S200 column chromatography: Sephacryl S200 is processed according to the requirements and then packed into a column (4 ~ 5 × 100cm column). After equilibrating with PBS, the sample (that is, the above supernatant) is added 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.
5. Verification
1) Determination of potency
a. Preparation of attack virus: after vesicular stomatitis virus (VSV) is passaged in 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).
b. Prepare to measure cells: well-grown young IBRS monolayer cells.
c. Measurement: Take the above monolayer cells into several groups, add different dilutions of interferon to each group, incubate at 37 ℃ for 20-24 hours, then each tube is challenged with 100 TCID50 VSV and set at 37 ℃ for 48-72h After incubation, observe the results. 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 the measurement 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
Take 10 of this product, dissolve it with water, and accurately measure the pH value to be 6.0 to 7.5.
3) Moisture determination
According to the sulfur sulfur solution method, it shall not exceed 3%.
4) Safety test
Take this product and dissolve it with water, and the mouse is injected into the tail vein, and there should be no death within 48 hours.
5) Pyrogen inspection
Take 1 bottle of this product, dissolve it with water, and check it according to law, it should meet the regulations.
6) Bacterial examination
Take 3 bottles of this product, dissolve them in sterile water, and inoculate them on the culture medium for aerobic bacteria, anaerobic bacteria and molds respectively, incubate at 37 ℃ for 1 week, and they should grow aseptically.
7) Hypersensitivity
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.
Matters needing attention 1. When preparing the NDVF series attenuated inducing agent, the seed poison should pass the sterility inspection and the titer is 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. The interferon half-life is very short at room temperature. 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.
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