SU519138A3 - The method of growing shchura virus - Google Patents

Description

(54) METHOD OF CULTIVATION OF THE VIRUS OF THE VASCINE

In a fermentation tank prepared for cuptivirovaiin, a virus is prepared containing the required proportion of the nutrient medium.

As a nutrient medium, a physiological solution with a pH of 7.4-7.6 of the following composition is used, g: NaC 720, K Ct 18, CaC-t -bHaO 18, MgWa..09, NaWa, O ID.ANSOz 200 Waa, HP04 , 5Hj048,06, glucose YuO peptone 24O, kasayngrolizat ZOO, DL - methionine 12.8, DL - trypto: fan 8.0, I) L- TpeoHJ-ra 2.5, L-- cystine HCi 5.2, .. p thyroxin O.OOOOEDSa pantothenate O, O32, niacin OD6, chloramphenicol 1O, penicillin 32, neomycin sulfate 1O, polymyxin B 0.5I j nystatin 0.75. 1 In addition, the medium contains 0, 02% hemeinum solution 3.6 ml, insulin 80 36. and 10 O l of distilled water.

Initial mixture for growing after adding a suspension of tissue with viruses | ycTai-mocks using gpikokol buffer ka | pH value 7.4-7.6 and stirred at and from 25 rpm.

Glycol-Buffer has the following srstav, g: gl1 {coca 158, Na, C-l-4, NaOH eljr, as well as distilled water 100 (5 ml.

At the same time, sterile filtered pure oxygen (2 m / min) is introduced into the initial mixture for growth. The pH of the initial mixture is continuously controlled and ,; if necessary, adjust the addition of gl-i cocoa buffer to a pH value of 7.4-7.6.

After 18-24 hours, depending on the type of virus, the virus is stopped and the suspension is cooled to 4 ° C. Then the tissues containing the scar (tissues of the rumen and epithelial cells) are crushed using a COLLOID mill to get the cells in the cells. The weaved mass thus opened together with the medium containing the airs for growing is extracted, slowly stirring (35), for 1 hour at 4 ° C. mercury suspension is added to the suspension (TIM-, Rosal}, with co-centration of 1: 40OOO; or 0.2 i with 1% chloroform .-.,

After extracting the suspension, the virus ev, the centN is trifuged in a continuously operating center: tr fugue. The thus obtained clear extract of viruses after quantitatively decoupling its antigenic content of viruses, can be used as an antigen for the manufacture of a vaccine against schur.

Determination of content of Bfusa, namely the content of infectious antigen and complex, the spectrum of antigen binding, occurs quantitatively.

The content of infectious anti-pandehyde is determined in mice as follows.

Every 10 to 7 day old mice are given under ether anesthesia.

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dilution of viruses 1O, Yu, Yu 10, 1 p intravenous injection. Shaving continues for 7 days. Deaths prior to expiration of 48 hours after infection are not taken into account, since the incubation period lasts 48 hours.

The content of the complement-binding antigen is quantified serologically and the reaction of complement binding using spectrophotometric analysis.

Example.

15 kg of scar tissue and 15 of the tongue epithelial cells, purified by irradiation with ultraviolet rays, are collected in an isotonic, antibiotic-containing, saline solution and crushed in a miter.

The tissue mass thus obtained is washed with saline and then shaken for 1 hour with an isotonic, antibiotic-containing saline. Then the saline solution is separated by decanting and the tissue mass is infected with 30 kg of a suspension of scurvy virus, a South American type of virus C, the virus titer of which is KV 10

After adsorption for 20 minutes, the infected tissue mass under sterile conditions is placed in a fermenter filled with 240 kg of nutrient medium. The nutrient medium is first heated to 37 ° C. The initial mixture is applied with 25 O g g picoco / Buffer to pH 7.5 and stirred at 7 ° C and 25 rpm. Throughout the entire process, sterile filtered pure oxygen is passed into the tissue suspension (2 L / min). The pH value is constantly monitored: after 5 hours and 8 hours after infection, it is additionally set by adding 300 ml each, and 14 hours after infection, adding 400 ml of glycol buffer for pH 7.4.

After 12 hours of infection (at intervals of 2 hours) to 22 hours after infection of the van, infections are taken to quantify the content of infectious and complexgly-binding antigens. The cultivation is stopped after 22 hours, and the initial mixture is cooled to 4 ° C. Parts of the fabric of the initial mixture for cultivation using a colloid mill are opened for the final extraction of the virus.

The tissue mass thus treated together with the virus-containing growth medium is extracted for 1 hour at 4 ° C, slowly placing / 35 rpm /. After extraction, the virus suspension is centrifuged. Used ceaseless centrifuge. To the thus obtained clear suspension of the virus, mertags are added to a concentrate of 1: 40,000. The virus titer and the camper-titer of the antigen are developed within 22 hours of the process / table 1/. Example 2. According to example 3. Suspension is obtained from epithelial cells of cattle and scar tissue, and they are infected with South American shchura virus O, subtype O - Canipcb (iHTp virus Mi) l.l} 5Q). : The cultivation of the virus is stopped after; 20 hours The development of the virus titer and the titer of the com1 tribe-binding antigen passes; as follows (Table 2). Example 3. According to example 1, tissue suspension is made from the tissue of the rumen and bovine tongue epithelial cells and infected with South American type A virus, subtype A, (titer, iMbLDyolCT i. Virus: After 24 hours, the cultivation is stopped and the virus is harvested The development of the virus titer and the titer of the binding antigean binding is as follows (Table 3). J The virus suspensions obtained in Examples 1, 2, 3 are used as the antigen, the schivirus - ITV shura trivalent vaccine; Vaccine protects cattle against non-artificial the use of three types of O Cawpos jj and C viruses. (The proposed method allows using, for example, scurf virus, {European O, A and C, South American O, A and C, African SAT 1, 2 and 3, as well as | Asia7h: cue types, for example LbSh 1, and all under | types of the named main types of virus Table 1

table 2

Claims (1)

The invention formula of Spesob is the cultivation of a shura virus, including its cultivation on a suspension of surviving cells of the mucous membrane of the stomach of ruminants, predominantly of cattle, followed by release, about t i and-v 510138 8 Tables n 3 due to the fact that, in order to achieve a shade of the yield of the target product, the cultivation of the virus is carried out on the specified suspension, mixed with a suspension of surviving epithelial cells of the cattle language.