WO1991005052A1 - Method for obtaining a polypeptide with human-interleukin-2 activity, secreted by yeast cells, saccharomyces cerevisiae - Google Patents

Abstract

The invention relates to biotechnology and, in particular, to genetic engineering methods for obtaining recombinant proteins in yeast cells. The aim of the invention is to create a yeast strain, Saccharomyces cerevisiae, capable of effective synthesis and secretion of a polypeptide with human-interleukin-2 activity, as well as to provide for the possibility of effectively controlling such a synthesis by varying the conditions for the strain cultivation, which is necessary for the stable maintenance of the strain and for the possibility of its industrial cultivation. A method is described for obtaining yeast strain producers secreting a polypeptide with human-interleukin-2 activity, a construction and a method of construction of a recombinant plasmid providing for the synthesis and secretion of interleukin-2. The increased stability and productivity of these strains as compared with those known up to now is achieved by the use, in the plasmid construction, of a hybrid yeast promoter. The yeast strain cells carrying such plasmids secrete into a cultural medium a protein having the human-interleukin-2 activity in a quantity of about several milligrams per litre of yeast culture. The specific biological activity of said protein practically equals that of natural interleukin-2.

Description

 1

SPΟSΟB ПΟΛУЧΕΗИЯ ПΟΛИПΕПΤИΛΑ WITH ΑΚΤИΒΗΟСΤЬУ ИΗΤΕΡΛΕЙΚИΗΑ-2 ЧΕΛΟΒΕΚΑ, СΕΚΡΕΤИΡУΕΜΟГΟ

ΚΛΕΤΚΑΜИ and ΔΡΟЖЖЖЫЙ δасгаготтусеδ segeνϊδϊае. ν

5 Technology area

Izοbρeτenie οτnοsiτsya κ biοτeχnοlοgii and in chasτnοsτi, κ gennοy inzheneρii and πρedsτavlyaeτ sοbοy sποsοb ποlucheniya ποliπeπτida with aκτivnοsτyu inτeρleyκina-2 chelοveκa, seκρeτiρuemοgο κleτκami 10 dροzhzhey, sποsοb κοnsτρuiροvaniya ρeκοmbinanτnοy πlazmidy, οbesπechivayuschey sinτez and seκρetsiyu inτeρleyκina-2 and shτamm dροzhzhey δassagοtusez segeνϊδϊae - seκρeτορny Human Resources Interleukin-2.

15 ADVANCED TECHNOLOGY

Interleukin-2 is related to a group of proteins called lymphokines. These squirrels play a key role in the regulation of the immune system of the body and are considered extremely emergency.

20 effective medications for the treatment of certain forms of immunodeficiency, and also in oncology.

For the most part, the radiopharmaceutical is used for the preparation of purified preparations of Interleukin-2 and is used as a non-food source.

25 strains of microorganisms - producers of an interleukin-2 person.

A number of the genetically engineered by the strains of bacteria and other products of human interleukin-2 were known (Table 1). However, all the producer strains listed in Table 1 are free from other essential disadvantages. Intraleukin-2 production and distribution

30 pacts of bacteria have a potential problem with the following results from the presence of bacteria of endotoxins. Secondly, the intracellular synthesis of interleukin-2 is in both the car battery, and in the case of other car cords, it is inappropriate to consume the device present in the native interleukin-2.

Table 1.

FROM ΒΕΒΕΗΤΑΜΜΗΕ ΙPΤΑΜΜY-ΜIΚΡΟΟΡGΑΗIZΟ, FROM ΟΔ ^ ΙΤDZHESHCHIΕ ANDΗΤΕΡΛΕЙΚИΗ-2 ЧΕΛΟΒΕΚΑ

(The levels of biosynthesis in units used by the authors are shown, in the case of accidents - the approximate conversion in mg / l of the crop)

[1] Κϊϊаηο Κ-, Ριушюϊο δ-, Εвріпеейсский патентт Α2 0 194 818 (1986).

25 [2] Μagk ϋ-Ρ. ea, U.S. Patent No. δ 4,518,584 (1985). [3] Letters. ea, International Patent

85/03723 (1985). [4] ea, European Patent Α 0 1 0 091 539 (1984). [5] Explanators ΑΗ. And dρ. Please refer to the decision by auto. Application No. 4392922/13 (1988).

thirty

Biοsinτez inτeρleyκina-2 denaτuρiροvannοy φορme, οbuslavlivaeτ το, chτο for egο sοlyubilizatsii neοbχοdimο πρimenenie silnyχ denaτuρanτοv τiπa dοdetsilsulφaτa naτρiya or guanidine gidροχlορida and eτο in svοyu οcheρed, zaτρudnyaeτ ποsleduyuschuyu οchisτκu inτeρleyκina-2. Otherwise, the biological activity of the interleukin-2 is significantly lower than that of the same protein, which has correctly closed disulfide bonds. A dangerous method of eliminating the listed disadvantages is the creation of strains

5 of the producers of Interleukin-2, in other biosynthesis of this protein, it is associated with its secretion in the cultural environment. In this case, squirrels containing disulfide bonds are found, as a rule, in the native form and, consequently, in the active and biological systems. Otherwise, the protein and the clean squirrel will not be necessary for the destruction

10 cells, and a lot of it is much less expensive, because there are a lot of useful proteins in the cultural environment is much less than inside the cell. Izvestay shτammy dροzhzhey δassagοtuseδ segeνϊδϊae - seκρeτορnye προdutsenτy inτeρleyκina 2 chelοveκa [ΟδYta T. ea, Eurasian Patent ΕΡ 0 171 000 Α2 (1986); Βagg Ρ., Ea, \\ Yu

15 85/02200]. However, plasmids ρΥP - 241 and ραΙΙ-2, described in these patents, have a significant deficiency, and it is known that the expression of the gene for interleukin-2 is inadequate As a result of this, and also toxicity for other Interleukin-2, it is convenient to use

20 systems, is an unstable producer strain. In addition, for the selection of the transformed cells and the plasmid plasmid 24Υ-241, the gene ΤΚ.Ρ1 is used, which allows the use of It is known that the yeast plasmids of this type have a comparatively low

25 stability and support in other cells in a small number of copies. The cited applications do not provide quantitative data on the relative level of production of interleukin-2 and the stability of the strains. However, from the data of the elec- tric analysis of the cultural environment, strain ΙΑ221 / ρΥП-241, available from [ΟδЫт

30 Τ. ea, the European Patent ΕΡ 0 171 000 Α2 (1986)], it can be seen that the content of interleukin-2 is small and makes up the value of the order of several percentages of the entire DISCLOSURE OF INVENTION

Β οsnοvu nasτοyaschegο izοbρeτeniya ποlοzhena task sοzdaniya shτamma dροzhzhey δassagοtuseδ segeνϊδϊae, sποsοbnοgο κ eφφeκτivnοmu sinτezu and seκρetsii ποliπeπτida with aκτivnοsτyu inτeρleyκina-2 chelοveκa and τaκzhe οbesπecheniya vοzmοzhnοsτi eφφeκτivnο ρeguliροvaτ τaκοy sinτez on account changes uslοvy κulτiviροvaniya shτamma, chτο neοbχοdimο for sτabilnοgο ποddeρzhaniya shτamma and vοzmοzhnοsga egο large-scale cultivation.

10

Solving a technical task.

The task posed is achieved by improving the design of a recombinant plasmid that ensures biosynthesis

15 polipestida and the polipeptida se with active interleukin-2 cells of a friend, and it is personal, using a hybrid burner. In this case, a regulated part of the burnt house is used;

20, )05), and the parts of the industry that share the burden of the crisis, as well as a part of the region (region). An essential essential condition for solving the posed problem is the inclusion in the composition of the recombinant plasmid of elements that ensure its accumulation in

25 κleτκaχ dροzhzhey in vysοκοm including κοπy and ποvyshayuschiχ sτabilnοsτ ποddeρzhaniya πρi κulτiviροvanii - ρeπliκaτορa dvumiκροnnοy ΔΗΚ and deφeκτnοgο gene ΕGG2 of izvesτnοy πlazmidy ρL Β207 [Βe§§δ Lλ: Οeηe s1οπt§ ϊη ueaδϊ. Ιη "Οеηеϊϊс еη§ϊηеегϊη§",

Κ. EUZ, νοϊ. 2, Αsaάetϊs ρgeδδ, Lοηάοη (1981)]. Suitable for τρ

30 useful plasmids are the strains of the other are strains that are segregated that carry a non-neurological mutation in the L gene. Brief Description of the Figures

Β Further, the invention is explained in a convenient description of the examples of its implementation with reference to the accompanying drawings:

5

Φig. 1. Illustrates the structure of the recombinant plasmid ρΙϋΒ (ΑΑΡΗΟΡΗΟ-), which ensures the biosynthesis and secretion of interleukin-2 from the cells of the friend.

Φig. 2. An illustration of the method of concomitant plasmid 10 ρΑC137 carrying the hybrid ΡΗ05- * ΜΡα1 προмοτορ.

Φig. 3. Illustrates the ways of the plasmid ρΙϋΒ (LBΡΗΟΙ) conversion.

fifteen

PREFERRED EMBODIMENTS OF THE INVENTION

The plasmid RL5Β (ΑΙ-ΡΗΟP-,), the conversion will allow you to receive another strain with the listed properties, consists of the following 20 elements:

- φρagmenτ πlazmidnοy ΔΗΚ biφunκtsiοnalnοgο baκτeρialnο- dροzhzhevοgο veκτορa ρL) Β207, οgρanichenny ρesτρiκtsiοnnymi sayτami ΗϊηsShΙ and δaϊϊ, ρazmeροm 6.3 τl.0-, vκlyuchayuschy baκτeρialny κ gene usτοychivοsτi amπitsillinu, baκτeρialnuyu initiation οblasτ

25 replication, the reproductive gene Ι –SH, as well as a fragment of the two-sided plasmid, which ensures the replication of the plasmid in the cell of the other;

- a fragment of the plasminid plasmid ρυС19, limited by the restricted sites δаϊϊ and ΗΙatΗΙ, with a size of 20 π.ο .;

30 - Sat-δаиЗи ρ ρ аг д аг аг аг аг аг аг аг аг аг Α Α

0 5 τ .ο _^ Contributing party responsible for the regulation of activity of non-corporate activity;

- Community 47 is a gene fragment of the other, containing part of the territory and the risk of the participation of this gene, ρmeasurement 0.45 tl;

- С £ г13Ι-δа1Ι φ plasmid plasmid ρΑΑ12.13-23 [Conv. ΤL. and dρ. The second certificate of the CCC on the application Ν 4140988/3113, (1987)], which contains the coding part of the gene of the interleukin-2 person,

5 0.56 T.l .;

- δtaΙ-ΗϊηάGP φρagment of plasmid ρΜδ46 [анинstanin ΚЗ. et al., Biological and cell τ.4, Ν 4, str. 224-231 (1988)], which contains the terminology of the gene for ΡΗ05 with a different size of 0.4 T.

Total ρmesh π plasmids 8.1 τl.ο. (molecular weight 5β Μд),

10 plasmid structure is illustrated in FIG. 1.

In order to achieve the goal, we use the methods of plasma plasmid rL-) Β (ΑΑΡΗΟП-.), Which is included in this case, which means that the

I-, Ceϊϊ 30, ρρ. 933-943 (1983)], containing the ΜΡχΙ gene, (ρ size ρ φ pagment 1.6

15 T. o.) they are cloning on the Russian site of plasmid ρυС19. From the obtained such plasmid ρυС (ΜΡ) -2, 1.1 t are isolated. The ΔΗΚ group, limited by the restricted sites δаϊϊ and ΗϊШПП and containing the right to start the coding area of the gene иαΙ, and is liable to use the public,

20 weeks τρ) Β207 [§е§§δ W: Ιη "СеηеΙιс еηβϊηееπηβ",

Κ. eά, νοϊ. 2, ΑΑάάϊϊ ρgeδδ, 1, -οηάάοη (1981)], resulting in the plasmid ΑΑЗЗΙЗ as a result. Next, the plasmid ρυС (ΜΡ) -2 hydrates the process of processing 47, treats the "maple" phase I, hydrates the process, and

25 ΔΗΚ ρmeasurement ο 0.51 tl.ο. This fragment is ligated with the whole part of plasmid rPZZZZ, hydrated by process δt δ and ΗϊηάΙΙΙ, receiving plasmid ρΑС313-1. Then the plasmid ρΑС313-1 hydrolyzes the reactive cat and ligates from 0 5 T. ΒatΗΙ-δaiиΑ Α ρ ρ ρ ρ лаз ρ plasmid ΔΥΙ [plasmid ΥΙ ΥΙ ΥΙ [ et al., Bioplimimers and cell τ.4, Ν

30 4, p. 224-231 (1988)], bearing the regulatory site of the burnt gene ΡΗ05. Τaκim οbρazοm κοnsτρuiρueτsya πlazmida ρΑS137 in κοτοροy sοdeρzhiτsya nachalο κοdiρuyuschey οblasτi ΜΡαΙ gene (sοοτveτvuyuschee πρeπρο-οblasτi belκοvοgο προduκτa eτοgο gene) ποd κοnτροlem "gibρidnοgο" προmοτορa, aκτivnοsτ κοτοροgο in ρeguliρueτsya 7

dependencies on the content of a non-urban site in a cultural environment.

The interleukin-2 gene isolated 5πσg in the composition C £ g13 г-Сιг13Ι of the ΔΙ plasmid fragment ρΑΑ12.13-23. This item processes the "maple"

5 φρagmenτοm ΔΗΚ-I and ποlimeρazy ligiρuyuτ with gidροlizοvannοy ρesτρiκτazοy ΗϊηάSh and οbρabοτannοy "κlenοvsκim" φρagmeiτοm ΔΗΚ- ποlimeρazy I πlazmidοy ρΑS137 (ποluchaya πlazmidu ρΑSP-,). Further, in order to merge the obtained operation with the other thermostats from the plasmid ρΑSP. highlight 1 tl.ο. δаΙΙ-ΧЬаΙ φρagment

10 ΔΗΚ. This fragment is a one-day league with 0.9 t, ο. ΙЬΙΙ-ΗϊηάШ φρagment ΔΗΚ of the plasmid ρΜδΙЬ [Explained Α-Η. and dρ. Resolved. application No. 4392922/13 (1988)] and the majority of the plasmid RL) Β207. Obtained by the aforementioned method, a definitive genetically engineered structure (plasmid RL (S) (LNP.)), Are merged into

15 A single reading frame of the coding parts of the ΜΡαΙ genes (part of the gene that corresponds to the protein protector) and the gene is non-compliant. The ex- fusion of the squirrel is located in front of the “hybrid” controller ΡΗ05-ΜΡα1 at the moment, in κοτορο

20 ρegulyaτορny uchasτοκ gene ΡΗ05 οbesπechivaeτ vοzmαzhnοsτ egο ρeπρessii neορganichesκim φοsφaτοm and Z'-gene κοntsevοy uchasτσκ προmοτορa ΜΡαΙ οπρedelyaeτ τοchκu initiation τρansκρiπtsii and κοdiρueτ πρiροdnuyu sgρuκτuρu lideρnοy οblasτi mΡΗΚ. Efficient termination of transcription is ensured by the presence of plasmid

25 transcriptional terminology of gene ΡΗ05. The basic stages of plasmid conversion are: Л (ΑЫΗΟΙЬ) are illustrated in FIG. and 3. The essential complication of plasmid rL) Β (LBP-) is that the fusion of the coding genes of the ΜΡαΙ gene is not detected.

30 protein squirrel alpha factor. Pοsκοlκu προtsessing sliτyχ τaκim οbρazοm belκοv οbychnο προχοdiτ analοgichnο προtsessingu πρiροdnοgο belκa-πρedshesτvenniκa alφa φaκτορa, seκρeτiρuemy in κulτuρalnuyu sρedu ποliπeπτid imeeτ sτρuκτuρu πρiροdnοgο belκa. ΔTo achieve the goal, use the Strain for 8

е18-ρЛ) Β (Α-ΡΗΟΡΗΟ,,), obtained by the expansion of strain ΟΚΡ18 with plasmid ЛЛ) Β (ΑЬΡΗΟΙЬ). Βybορ shτamma ΟΚΡ18 in κachesτve shτamma-ρetsiπienτa οbuslοvlen τem, chτο eτοτ shτamm neseτ neρeveρτiρuyushuyu muτatsiyu gene G.ΕSH, chτο ποzvοlyaeτ οτbiρaτ 5 τρansφορmiροvannye πlazmidοy κleτκi and sτabilnο ποddeρzhivaτ shτamm SΚΡ18-ρLEΒ (Α1-ΡΗΟP--) on seleκτivnyχ sρedaχ not sοdeρzhaschiχ leucine. The content of interleukin-2 in the cultural environment of the strain ΟΚΡ18-ЛЛ) Β (ΑΑΡΗΟΡΗΟ) is about 1 mg / l.

10 Writing an experiment.

For example, 1.

Bacteria v. SOSI, containing plasmid ρ69Α, grow during the night in 500 ml of nutrient medium B с (1 prim., 03

15 προц. burnt extract, 1 percent. Chemotherapy), ampicillin was added to the concentration at a concentration of 50 mg / l. The batteries are centered (5,000 rpm, 10 minutes, 4 grams), they are suspended in 10 ml of lysis buffer (25 mg of hydrochloric acid, which is 50 mg).

20 incubate 10 min πρ and 4 deg. Then add 10 ml of 0.2Μ sodium hydroxide, containing 1%. dodecyl sulfate. After a quick stirring for about 1 min, the solution neutralizes 10 ml of acetate, and the 53- Δ further discharges 4 g. for 1 hour and costs 20,000 and 20,000

25 rpm (center 12-21, "Thekestaηη", 4 deg.). Κ The unit adds 0.6 volume of the product and separates the plant with a low centrifuge (5000 rpm) at 4 deg. It is dried in a vacuum, dispersed in 33 ml of water, added 5 g of cesium solution and 100 ml of a solution of etidium bromide (mg / ml) and

30 tsenτρiφugiρuyuτ πρi 50000 οb / min τechenie 12-16 hour tsenτρiφuge 01/05/50 ( 'Εesktaηη ") in ροτορe νP80. Pοsle tsenτρiφugiροvaniya οτbiρayuτ ποlοsu πlazmidnοy ΔΗΚ (bottom of dvuχ φlyuορestsiρuyuschiχ ποlοs in tsenτρe προbiρκi) twice eκsτρagiρuyuτ bροmisτy eτidy Equal volume of alcohol, dilutes the dissolution of cesium by the water in 2 times and planted ΔΗΚ with two volumes of ethyl alcohol. The plant, separated by centrifugation (10,000 rpm, 5 min) is washed with 70 percent ethanol, dried in a vacuum, and disposed of in water (500 μl). Κοntsenτρatsiyu πlazmidnοy ΔΗΚ απρedelyayuτ πο 5 οπτichesκοy πlοτnοsτi ρasτvορa πρi 260 nm (1 οπτichesκaya πlοτnοsτ sοοτveτsτvueτ κοntsenτρatsii mκg 50 / ml) chisτσgu πρeπaρaτa κοnτροliρuyuτ eleκτροφορezοm in agaροznοm gel (0.7 προts agaροzy in buφeρe ΤΒΕ -. ΟDΜ τρis-bορaτ, sοdeρzhaschy ΙmΜ EΔΤΑ and 1 mg / l of bromide etidium). 10 Gidροliz πlazmidι ρ69Α ρesτρiκτazοy ΕsοΚΙ προvοdyaτ in ΙΟmΜ τρis- gidροχlορidnοm buφeρe, sοdeρzhaschem 10 mΜ χlορisτy magnesium, 100 mΜ χlορisτy naτρy 1 mΜ diτiοτρeiτοl mκg and 100 / ml bychegο syvοροτοchnοgο albumin (buφeρ ΒS). Κ 50 mcg ΔΗΚ in a volume of 200 mc π add 100 units. Reactases and reacts at 30 degrees. at 15 for 2 hours The control panel for a complete hydrolysis is carried out with the help of an electrophoresis in an agar gel. Ρeaκtsiοnnuyu mixture vnοsyaτ in lunκu (2x50 mm) agaροznοgο gel ρyadοm with lunκοy for οchischaemοgο πρeπaρaτa ΔΗΚ vyρezayuτ lunκu (5x3 mm) for applying sτandaρτοv weight mοleκulyaρnοgο (ΔΗΚ φaga lambda gidροlizοvannaya ΡδϊΙ) and προvοdyaτ 20 eleκτροφορez πρi naπρyazhenii 5 V / cm in τechenie 2-3 hours The main area of interest is identified by elution, comparing it with the mobility of the Δ, standard hydraulics, which has been upgraded. Particularly sensitive area ΔΗΚ with a size of 1.6 t.o. cut a hole (3x60 mm) and place a dialysis membrane of the corresponding size into this hole 25 and fill it with a buffer. Electricity lasts for another 10-15 minutes, after which a buffer from the hole is removed, one is added to the battery and one or two more are added, it is added that it is added. ΔΗΚ Separate the centrifuge at 10,000 and 30 rpm for 10 minutes, wash the plant with ethanol, dry it in a vacuum and dispense 100 ml of water.

Hydrolysis of 10 μg of plasmid ρυС19, isolation of a similar analogous isolation of plasmid ρ69Α, with a capacity of 30 units. Researches of Coba in 50 mc of buffer C for 2 hours at 37 ° C.

10

The linear linear plasmid form is similar to that described above for the ΔΗΚ plasmid ρ69Α fragment.

Δ for plasmid irradiation, the designated рУС (ΜΡ) -2, the linear φοrm of the plasmid ρυС19 is ligated with the ΔΗΚ plasmid ρ69Α fragment carrying the gene

5 ΜΡαΙ. For this type of ΔΗΚ, the calculation shown above is mixed in quantities of 0.1 μg per 10 μl and 70 mu- Δ for adding the reaction of the league add 10 units. ΔΗΚ ligases of phage Τ4 and προ lead incubation to

10 nights πρ and 4 deg.

The resulting such a ligase mixture transports the cells of the U.S.I. ΗΒΙΟΙ. For this cage, it is protected in 100 ml of medium for 37 grams. and intensive stirring to achieve good optical performance at 600 nm 0.4-03- Cool

15 ice tsenτρiφugiρuyuτ πρi 3000 οb / min τechenie 10 min πρi 0 gρad., Ρesusπendiρuyuτ 50 ml 0DΜ χlορisτοgο κaltsiya, inκubiρuyuτ on ledyanοy bath for 40 min, ποvτορnο tsenτρiφugiρuyuτ πρi τeχ same uslοviyaχ, susπendyaρuyuτ 5 ml ρasτvορa χlορisτοgο κaltsiya, dοbavlyayuτ glitseρin up to 20 προц. Received by such a competitor

20 They distribute aliquots for 200 mcls and store frozen food at -70 degrees. for use .. For transports, compete cells in the ice pack are added to the ice bath, plus suspension of the cures for the treatment of lignazum. Next, the cradles are sending a quick shock to

25 for 2 min πρ and 42 deg., After which they are incubated in 13 ml of medium G. ππ and 37 deg. within 1 hour. The suspension of the cells is concentrated at a rate of 3,000 rpm for 10 minutes and increases the cup with the same good food. agar and 50 mg / l of ampicillin.

From 30 ποluchennyχ vysheοπisannym οbρazοm οτdelnyχ κlοnοv τρansφορmanτοv vydelyayuτ πlazmidnuyu ΔΗΚ meτοdοm, analοgichnym meτ allocation πlazmidy ρ69Α, with τem οτlichiem, chτο vyρaschivanie κleτοκ Ε.sο1ϊ veduτ in 10 ml πiτaτelnοy sρedy all οbemy ρasτvοροv sοοτveτsτvennο umenshayuτ 50 ρaz πο sρavneniyu with uκazannymi. Κροome eleven

Then, in addition to the last stage of the calculation (the center for processing cesium), the bank uses the processing unit. For this nucleic acid precipitate, obtained by precipitating from the panel, it grows in 100 microliters of buffer for testing and

5 Processing 10 ml of a solution of Baza (1 mg / ml) for 30 minutes at 37 ° C. Such a preparation is used for the structural analysis of the plasmid structure in individual cells. In the case of plasmid ρυС (ΜΡ) 2, the analysis of προ is as follows. For applications of 3 μl of the plasmid preparation, add for 7 μl of the UC buffer and further separate parts

10 treats the following combinations of experimental endonucleases (π 10 units): δΙΙΙΙ + ΗϊηάШ (the required plasmid yields fragments 1,1, 03 t.o.) and ΕсοΚΙ (фрagment) 1,6 τ. From the conventional method such as the transverse clone, which contains the plasmid ρυС (ΜΡ) -2, the receptive plasmid secrete plasmid Δа, as for Α plasmid.

15 In order to obtain the plasmid ρΑЗЗΙЗ, the plasmid ΔΗΚ рυС (ΜΡ) -2 (μg) is hydrolyzed with a mixture (πο 100 units) of δaϊϊ and ΗϊηάШ in 100 μl of ΒС buffer for 3 hours. Particle ΔΗΚ with a size of 1.1 T. o. Cleans the elec- trophoresis in an agar gel, as described above. By the methods of the drug, they clean the veterinary δаϊϊ-

20 ~ Ηϊηά Ш φρagment of plasmid ρЛ) Β207. By combining two of these phrases, by performing a transluc- For the purpose of π plasmid ρΑС313-1, π plasmid ΔΗΚ ρυС (ΜΡ) -2 (

25 mcg) hydrolyzes 100 units. Researches of Εсο47Ι in 100 mc of the ΒС buffer for 3 hours at 37 ° C. Further, the ΔΗΚ release is added to 100 μl of 100 m Μ of the hydrated buffer of, 7.4, containing 50 ис of pure magnesium, and all of which are free of impairment of the substance. "maple"

30 of the Δме-polymerase I. After incubation for an hour at a constant temperature, the ΔΗΚ-polymerase is inactivated by heating by 65 degrees. within 10 min., after which the hydrolysis of plasmid ΔΗΚ 100 units was carried out. ρesτρiκτazy ΗϊηάSh in τechenie 2 chasοv πρi 37 gρad. From the obtained hydro-group, a fragment ΔΗΚ with a size of 031 t. 12

electric gel in an agar gel, as described above. By means of methods, they produce and purify the effective δtΙ-ΗϊηάSh fragment of the plasmid plasmid. By dividing the two components, by entering the transformer on the mains plug and the structural analysis of the transverse part

5 by tests of δaΙΙ + ΗϊηάШ, KatΗΙ + Ρδύ, selects a clone containing plasmid ρΑС313-1.

The plasmid vector ρΑС137 is operated with the use of the above methods for the following scheme: from the plasmid ρУΥΙδ, the hydrolyzed Β atΗΙ, isolates 0.6. φρagment

10 ΔΗΚ, which additionally promotes the hydrolysis of the endonuclease inlet and from the obtained hydrolysis, cleans the fragment of the size 035 T. o. This fragment is ligated with a linearized test component of plasmid ΑΑC313-1 and after transmission, it is possible to use + analysis,

15 carrying plasmid ρΑС137.

Phase ΔΦ, which contains the coding part of the gene of interleukin-2, is isolated from the hydrolyzed processed product of plasmid ρΑΑ12.13- 23 and of the processed product (1). κ ο ο ο "" φ φ φ φ I. I. I. I. I. I. φ κ κ κ κ κ κ κ ’

20 PLEASURES A CLEANED LINEAR FORM OF THE VECTOR Α ,C137, a hydrated endoscopic ΗϊηάШ. By combining these two fractions, ΔΗΚ and by entering into the analysis of the bacteria, they take into account the data from the analysis (δΙΙ + ΙΙΙ, fragment 2), there is a difference

25 is located in the right direction for returning home.

In order to obtain a final operation - the plasmid expression of the process μΑ (--------) - the concurrent ligation is cleared: δаΙΙ-ΧЬаΙ of the plasmid plasmid ρΙСΙЬ, 0.9 tl.ο. Plasma plasmid agglomerate

30 ΜΜδГЬ, and the entire δΙΙΙΙ-ΗΗά φ plasmid fragment ρЛ) Β207, with a size of 63 T. τ. The structure of the plasmid rL Β (LIΡΗΟΙ) (Fig. 1) confirms the decompressive analysis using the decomposition enzyme, δϊϊ, ΒΒΗΙ and ΧΧΙΙ, as well as the other.

Further on, there will be a transfection of the vector experience of ρЛ) Β (ΑΑΡΗΟΙΡΗΟΙ) thirteen

burnt eggs, which is illustrated by the following example.

For example, 2.

5 In order ποlucheniya shτamma dροzhzhey δassagοtuseδ segeνϊδϊae, sinτeziρuyuschegο and seκρeτiρuyuschegο inτeρleyκin-2 chelοveκa, κleτκi dροzhzhey shτamma ΟΚΡ18 τρansφορmiρuyuτ πlazmidοy ρL Β (ΑΡΗ following οbρazοm: Δροzhzhevοy shτamm-ρetsiπienτ vyρaschivayuτ on sρe ΥΕΡϋ (2 προts πeπτοna 1 προts dροzhzhevοgο eκsτρaκτa.,. 2 προц.

10 glitches) to achieve a cultured optical density of 600 nano-2-4. The batteries are twice washed with water and one at a time for a sodium-citric buffer containing 1Μ of the output, they are suspended at the same time and processed for 150 minutes at a time. π and 30 grams of χ, and then 150 μl of glucurinidase within 30-

15 60 min. And the same temperature. Pοluchennye τaκim οbρazοm sφeροπlasgy τρizhdy προmyvayuτ οdnοmοlyaρnym sορbiτοm, susπendiρuyuτ in 0,01Μ τρis buφeρe, sοdeρzhaschem 0,01Μ χlορisτοgο κaltsiya and 1Μ sορbiτa and ποsle dοbavleniya 10 mκg ΔΗΚ πlazmidy ρL) Β (ΑΡΗΟP-.) Inκubiρuyuτ 30 min. ROOM TEMPERATURE. Then add the suspension of the plugs 44

20 percent of the growth rate of polyethylene glycol 4000 (December, Ρ выде), withstand it for 30 minutes. πρand 30 grad-, then 2 min. πρand 42 grad. and seeded with a deep seedbed for the medium δСЫδ (0.67 πρ с а 'с с "" ""), 2 ρ ц с "glucose, 50 mg / l of histidine), which contains 1 π.

25 In order to analyze προduκtsii κleτκami τρansφορmanτοv inτeρleyκina-2 iχ vyρaschivayuτ on sρede ΙGΕP (2 προts. Πeπτοna 2 προts. Glyuκοzy) dο dοsτizheniya sτatsiοnaρnοy φazy ροsτa and οτdelyayuτ κleτκi tsenτρiφugiροvaniem πρi 5000 οb / min τechenie 10 min. Β The agent determines the content of interleukin-2 by the immunoassay.

30 For the purpose of analyzing the products of interleukin-2 by the immunodeficiency method, the test methods are divided up to 15: 1 50 mb At the same payment, I place a range of dilutions of purified interleukin-2 (in the range of 2-100 ng). Δ further 14

Allows them to dry out during the night and at a room temperature. After the blocking of non-reactive payment sites, incubation of the cards with a single output of a bullish albumin of 37 gp. within 30 minutes and π ο мы washing φ φ бу,, in

5 lunκi dοbavlyayuτ ρasgvορ mοnοκlοnalnyχ myπshnyχ anτiτel κ inτeρleyκinu-2 ΜΚΑΤ-266-12 (ποluchennyχ in Belορussκοm ΗII gemaτοlοgii and πeρelivaniya κροvi) (πο 2 mκg on lunκu) and zaτem, ποsle still οdnοy προmyvκi, ρazvedenny 1200 κοnyugaτ vidοsπetsiφichesκiχ anτiτel, sπetsiφichnyχ κ mouse gamma globulins and

10 πеροκsidase (phiρmy "δϊ§ta"). Pοsle inκubatsii sφορmiροvannyχ τaκim οbρazοm κοmπleκοv with subsτρaτnym ρasτvοροm (0.0003 προts. Πeρeκis vοdοροda, 4 mg / ml in ορτο-φenilendiamina 0.1 Μ naτρy-atseτaτnοm buφeρe ρΗ 4,6) and οsτanοvκi ρeaκtsii dοbavleniem ρavnοgο οbema 2Μ seρnοy κislοτy, izmeρyayuτ οπτichesκuyu πlοτnοsτ πρ and 486 nm.

15 The concentration of interleukin-2 in the analyzed products is determined by comparing the value of absorption in the experimental samples with the experiment in the same way.

According to the received data, the cells of the other strain 18, which are transformed with the plasmid rP), are secured in

20 cultural medium, for example, 1 mg of interleukin-2 per liter of culture. The cultural environment is different from the rest of the strain (strain ΟΚΡ18, the transformed plasmid ЛL Β207), but this protein is not immune to the test.

25 The stability of the level of products of the Interleukin-2 program м-18--Л Л (ΑΑΡΗΟЬ) is subject to the following procedure. Freshly grown transplant is sown in 100 ml of the medium δСЫδ (for example, in this environment, the production of interleukin-2 is prevented by the high concentration of the non-absorbed water)

30 phases. From such a culture, 1 ml is sown in 100 ml of fresh δС medium with histidine and grows to a stable growth phase. In this way, there are four more investigative miscarriages. Further, 10 ml of yeast cells from each culture are separated by centrifugation and inoculated into 100 ml of PEP medium (in this case, the environment is consumed). fifteen

the synthesis of interleukin-2). In an immunoassay, the cultural environment after cultivating the cells of the various sources of the original culture does not detect differences in the level of the Internet. In general, the original level of production of this product

5 squirrel with the strain С-18-РЛ) Β (Α ---- ΡΗΟП--) is consumed by tension, at the very least, 20 generations.

The biological activity of the synthesis and the network of the strain S-18-RL) Β (ΑΙ - ΡΗΟ) is changed, the

10 international standard, amounted to about 10 million ΜΕ / mg of purified protein, which is practically the same as a specific activity of the second computer.

Summarizing the above, you can conclude that the received strain of the other

15 is consumed in a cultivated environment for interleukin-2 people at a level of about 1 mg / l; This strain of Interleukin-2 has a biological activity that is close to the active Interleukin-2.

20 Stamm of other δassbagotuseδ segevϊδϊе СΚΡ18-ρЛ) Β (ΑГЛΗΟП--)

- an associate of human intererleukin-2 - was disbursed in the All-Union collection of industrialized enterprises with a number of ΜPΜ-ΥY38.

Claims

16ΦΟΡΜУΛΑ OF ΟΟΡΕΤΕΗΡΕΤΕΗ.

1. Μeτοd ποlucheniya ποliπeπτida with aκτivnοsτyu inτeρleyκina-2 chelοveκa, seκρeτiρuemοgο κleτκami dροzhzhey δassagοtuseδ segeνϊδϊae with ποmοschyu κοnsτρuκtsy ρeκοmbinanτnοy ΔΗΚ, in κοτορyχ ποsledοvaτelnοsτi ΔΗΚ, κοdiρuyuschie sliτy ποliπeπτid πρeπροπeπτida α-φaκτορa and inτeρleyκina-2 chelοveκa, ποsτavleny ποd κοnτροl gibρidnοgο προmοτορa.

10

2. The method is in accordance with paragraph 1, in which the aforementioned hybrids are made up of a group of regulated and incinerated plants.

15 3. The method is in accordance with paragraph 2, in which the aforementioned regulated variable is the π05 gene.

4. The method is in accordance with paragraph 3, in the case of a hybrid appliance, the plasmid ρΑC137 is used.

20

5. The method is in accordance with paragraph 3, in which a hybrid agreement is taken into account by the merger of 0.45 T. o. QatSh-Εсο47Ш ρpropagment ΡΗ05 and 038 t.o. ΒδрΚΙ-ΗϊηάШ is a fragment of the gene ΜΡαΙ, including the part of the genera and the coding region of this gene.

25

6. The method according to paragraph 1, in the second prominent recombinant, is plasmid RL) Β (CHL).

7. The method of improving the productivity and stability of the strains

30 beneficiaries of heterologous proteins in other cities due to the use of hybrid products in accordance with paragraphs 3, 4, 5.

8. The non-recombinant plasmid ρL) Β (ΑΑΡΗΟΡΗΟ). 17

9. The Storm of the other δassагagotuseδ segeϊϊϊϊеΟΚΡΟΚΡ-----ρ)) Β (ΑΡΗΟΡΗΟ--.))), Which was introduced by the All-Union collection of industrialized means of labor.